Novel adiponectin-derived peptide derivatives and their applications

Abstract

The present invention relates to a novel peptide derivative, which is a partially modified adiponectin receptor peptide and has higher stability and superior p-AMPK activity than existing adiponectin receptor peptides, and has the advantage of being advantageous for drug formulation due to improved physical properties and activity. Therefore, the peptide derivative of the present invention can be used for preventing or treating skin inflammatory diseases, wounds, hair loss, fibrosis, metabolic diseases, or cancer, and for preventing or improving aging, sensitive skin, wrinkles, or moisture.

Description

[Technical Field]

[0001] This invention relates to novel adiponectin-derived peptide derivatives and their applications. [Background technology]

[0002] Skin aging is generally caused by external and internal processes related to the increase of wrinkles and sagging of the skin. External aging is mainly caused by repeated exposure to ultraviolet rays (UV) and is therefore commonly called "photoaging." Naturally aged skin is smooth, pale, and has fine wrinkles, while photoaged skin develops deep wrinkles, hyperpigmentation, and telangiectasia (dilated capillaries).

[0003] Subcutaneous fat plays a crucial role in maintaining energy homeostasis by secreting hormones and adipokines that regulate the metabolism of other tissues. Recently, it has been found that ultraviolet radiation, an environmental factor that induces various diseases such as photoaging, inflammation, immunosuppression, and cancer, reduces the fat content of human subcutaneous fat tissue, decreases adiponectin, a fat-derived product, in aged skin, and induces an increase in MMP-1 and a decrease in collagen.

[0004]

[0005] The human body has over 1.3 million hairs, with scalp hairs numbering between 100,000 and 150,000. Each hair has a different cycle, going through three stages: the growth phase (anagen, the active growth phase), the regression phase (catagen, the apoptotic regression phase), and the resting phase (telogen, the resting phase) to grow, be maintained, and fall out. This cycle repeats every 3 to 6 years, resulting in an average of 50 to 100 hairs falling out each day. Generally, "hair loss" refers to a condition where there are fewer or no hairs than normal due to any cause.

[0006] Today, it is known that hair loss can be caused by internal factors such as the effects of male hormones, or external factors such as mental stress in daily life and the accumulation of lipid peroxides on the scalp, with these factors interacting in complex ways to cause hair loss symptoms. Recently, in addition to male pattern baldness, the number of women experiencing hair loss is also increasing due to changes in diet and increased stress from the social environment. As a result, the number of people suffering from the above-mentioned abnormal symptoms of the scalp and hair is gradually increasing, and the age of those affected is also getting younger.

[0007] Currently available hair growth products lack clear efficacy and have side effects. They must be taken continuously, and can induce side effects such as decreased sexual function, allergies, and depression.

[0008]

[0009] Metabolic diseases are syndromes characterized by the coexistence of risk factors such as obesity due to excessive nutrient accumulation in the body and lack of exercise, diabetes, hypertension, arteriosclerosis, and nonalcoholic fatty liver disease (NAFLD). More recently, they have been officially named metabolic syndrome or insulin resistance syndrome through the Adult Treatment Program III established by the World Health Organization and the Heart, Lung and Blood Institute of the National Institutes of Health in the United States. Furthermore, according to the ATP (Abdominal Threshold Power) published by the US NCEP (National Cholesterol Education Program) in 2001, if a patient has three or more of the following five risk factors: abdominal obesity with a waist circumference of 40 inches (102 cm) or more for men and 35 inches (88 cm) or more for women; triglycerides of 150 mg / dL or more; HDL cholesterol of 40 mg / dL or less for men and 50 mg / dL or less for women; blood pressure of 130 / 85 mmHg or more; and fasting glucose of 110 mg / dL or more, it is judged to be a metabolic disease. For East Asians, abdominal obesity is defined as a waist circumference of 90 cm or more for men and 80 cm or more for women. Recent research reports indicate that when these regulations are applied, approximately 25% of the Korean population exhibits symptoms of metabolic syndrome.

[0010]

[0011] On the other hand, adiponectin, a type of adipokine—a protein hormone specifically secreted by fat cells—is known to play a crucial role in regulating cardiovascular diseases such as hyperglycemia, hyperinsulinemia, obesity, and arteriosclerosis by enhancing insulin function, suppressing insulin resistance, blocking inflammation, and preventing fat accumulation in blood vessels. Furthermore, adiponectin has the function of suppressing cancer cell metastasis and inflammatory responses, and in addition to promoting the proliferation of keratinocytes, it can perform functions such as wound healing, inhibition of fibrosis, improvement of skin wrinkles, and moisturizing by promoting the expression of filaggrin, hyaluronic acid, and extracellular matrix in the skin.

[0012] Adiponectin is composed of 244 amino acids and consists of a signal sequence, a collagen-like domain located at the N-terminal, and a C1q-like globular domain located at the C-terminal. The hexamer and the 400 kDa high molecular weight complex (HMW complex) are the main oligomers, and the HMW complex is known to be more active than the low molecular weight complex (LMW complex).

[0013] The development of peptides derived from modified adiponectin, which has traditionally been known to possess various physiological activities, has been a target for many researchers and pharmaceutical companies both domestically and internationally. However, the difficulty in polymer formation within the body has made the likelihood of ultimate success relatively low.

[0014]

[0015] Against this backdrop, the inventors developed a short peptide derivative that can be applied to the skin by improving the physical properties and activity of existing adiponectin-derived peptides, which are difficult to formulate into dosage forms due to their poor usability. The inventors then completed the present invention by confirming effects such as increased adiponectin expression, hair growth promotion, and suppression of triglycerides. [Overview of the Initiative] [Problems that the invention aims to solve]

[0016] The object of the present invention is to provide novel peptide derivatives.

[0017] Another object of the present invention is to provide a composition for increasing adiponectin expression, which contains the peptide derivative as an active ingredient.

[0018] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, or cancer, comprising the peptide derivative as an active ingredient.

[0019] Another object of the present invention is to provide a cosmetic composition for preventing or improving skin inflammatory diseases, wounds, hair loss, fibrosis, metabolic diseases, or cancer, which contains the peptide derivative as an active ingredient.

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or improving aging, sensitive skin, wrinkles, or moisturization, which contains the peptide derivative as an active ingredient.

[0021] Another object of the present invention is to provide a cosmetic composition for preventing or improving aging, sensitive skin, wrinkles, or moisturization, which contains the peptide derivative as an active ingredient.

[0022] However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and further problems not mentioned will be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0023] In order to solve the above problems, the present invention provides a peptide derivative represented by the following [Chemical Formula 1]. [Chemical Formula 1]

[0024]

Chem.

[0025] In the above [Chemical Formula 1],

[0026] R1 is any one or more selected from the group consisting of an amino group (NH2), an acetylamino group (Ac-NH), NH2-(CH2CH2O) m -CH2CONH, C 15 -C 20 amide groups, and combinations thereof, where m is an integer from 1 to 5, preferably 3, and the amide group is preferably a C 16 amide group, that is, a palmitamide group.

[0027] R2 is an amino group or a hydroxyl group (OH),

[0028] R3 is hydrogen (H),

[0029] R4 is one or more selected from the group consisting of a C1-C6 chain-type alkyl group, a C2-C6 alkylcarboxyl group, a C1-C6 alkylamide group, an imidazoylmethyl group, and combinations thereof.

[0030] R5 and R6 are either identical or different, and are independently a benzyl group, a phenylethyl group, or an indolylmethyl group, wherein the benzyl group is either unsubstituted or one or more selected from the group consisting of a hydroxyl group, a trifluoromethyl group (CF3), a halogen group, a cyano group, a nitro group, a C1-C6 chain-type alkyl group, a C1-C6 alkoxy group, and combinations thereof. Replaced the law of nature,

[0031] R7 is a benzyl group or a C1-C6 alkylamino group, and the benzyl group is either unsubstituted or has one or more selected from the group consisting of a hydroxyl group, a halogen group, a cyano group, a nitro group, a C1-C6 linear alkyl group, a C1-C6 alkoxy group, and a combination thereof. Replaced Ruka,

[0032] R3 and R7 are connected to each other and C 15 -C 20 Forms a heterocycloalkene group,

[0033] R8, R9, and R 10 These are either identical or different from each other, and each is independently hydrogen or a C1-C6 alkyl group.

[0034] n is either 0 or 1.

[0035] When n is 0, R4 is a C2-C6 alkyl carboxyl group, and R5, R6, and R7 are benzyl groups.

[0036] However, R1 is NH2, R2 is OH, R3 is hydrogen, R4 is an isobutyl group, both R5 and R6 are hydroxybenzyl groups, R7 is an unsubstituted benzyl group, and R8, R9, and R 10 are all hydrogen, except when n is 1. That is, NH2-GLYYF-OH, which is known as the APN5 peptide, is excluded.

[0037] As an embodiment of the present invention, the above R4 is

[0038]

Chemical formula

[0039] As another embodiment of the present invention, the above R5 and R6 are the same as or different from each other, and each independently

[0040]

Chemical formula

[0041]

Chemical formula

[0042] As another embodiment of the present invention, the above C 15 -C 20 heterocycloalkene group is

[0043]

Chemical formula

[0044] [ka] It is one or more selected from the group consisting of combinations thereof, but is not limited to these.

[0045] As another embodiment of the present invention, the peptide derivative is selected from, but is not limited to, one or more peptide derivatives represented by the following [Chemical Formula 1-1] to [Chemical Formula 1-56] (Table 1).

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] [Table 1] TIFF0007852055000008.tif191149 TIFF0007852055000009.tif201149 TIFF0007852055000010.tif191149 TIFF0007852055000011.tif174149 TIFF0007852055000012.tif174149 TIFF0007852055000013.tif196149 TIFF0007852055000014.tif191149 TIFF0007852055000015.tif196149 TIFF0007852055000016.tif196149 TIFF0007852055000017.tif191149 TIFF0007852055000018.tif148149

[0058]

[0059] As another example of the present invention, the peptide derivative is obtained by modifying one or more peptide sequences selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NOs: 16, but is not limited thereto (Table 2).

[0060] [Table 2] TIFF0007852055000020.tif134149

[0061]

[0062] Furthermore, the present invention provides a composition for increasing adiponectin expression, which contains the peptide derivative as an active ingredient.

[0063]

[0064] Furthermore, the present invention provides one or more preventive or therapeutic pharmaceutically active ingredients comprising the peptide derivative as an active ingredient, selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer.

[0065] Furthermore, the present invention provides one or more methods for the prevention or treatment of skin inflammatory diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, selected from the group comprising the step of administering the peptide derivative to an individual.

[0066] Furthermore, the present invention provides uses for the peptide derivatives for the production of one or more preventive or therapeutic agents selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer.

[0067] Furthermore, the present invention provides a cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, which contains the peptide derivative as an active ingredient.

[0068] Furthermore, the present invention provides a food composition for the prevention or improvement of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, which contains the peptide derivative as an active ingredient.

[0069]

[0070] Furthermore, the present invention provides a pharmaceutical composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient.

[0071] Furthermore, the present invention provides one or more methods for preventing or improving aging, sensitive skin, wrinkles, and moisturizing, selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which include the step of administering the peptide derivative to an individual.

[0072] Furthermore, the present invention provides uses for the peptide derivatives for the production of one or more preventive or ameliorative agents selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing.

[0073] Furthermore, the present invention provides a cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient.

[0074] Furthermore, the present invention provides a food composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient. [Effects of the Invention]

[0075] A peptide derivative according to one embodiment of the present invention is a modified adiponectin receptor peptide and has the advantage of having even higher stability and superior p-AMPK activity than existing adiponectin receptor peptides, as well as improved physical properties and activity, which is advantageous for drug formulation.

[0076] Therefore, the peptide derivatives of the present invention can be used for the prevention or treatment of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, or cancer, as well as for the prevention or improvement of aging, sensitive skin, wrinkles, or moisturizing.

[0077] The effects of the peptide derivative according to one embodiment of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawing]

[0078] [Figure 1] Figure 1 is a graph showing the effect of the peptide derivatives of the present invention (DS-1 to DS-10), represented by [Chemical Formula 1-1] to [Chemical Formula 1-10], on AMPK phosphorylation activity in mouse adipocytes. Here, V is the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0079] [Figure 2]Figure 2 is a graph showing the effect of the peptide derivatives of the present invention (DS-11, DS-12), represented by [Chemical Formula 1-11] and [Chemical Formula 1-12], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0080] [Figure 3] Figure 3 is a graph showing the effect of the peptide derivatives of the present invention (DS-17 to DS-20), represented by [Chemical Formulas 1-13] to [Chemical Formulas 1-16], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0081] [Figure 4] Figure 4 is a graph showing the effect of the peptide derivatives of the present invention (DS-27 to DS-30), represented by [Chemical Formulas 1-17] to [Chemical Formulas 1-20], on phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0082] [Figure 5] Figure 5 is a graph showing the effect of the peptide derivatives of the present invention (DS-35 to DS-38), represented by [Chemical Formulas 1-21] to [Chemical Formulas 1-24], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0083] [Figure 6]Figure 6 shows a graph of the effects of the peptide derivatives of the present invention (DS-39 to DS-42, DS-44, DS-45), represented by [Chemical Formulas 1-25] to [Chemical Formulas 1-30], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0084] [Figure 7] Figure 7 shows a graph of the effects of the peptide derivatives of the present invention (DS-48 to DS-56), represented by [Chemical Formulas 1-31] ​​to [Chemical Formulas 1-39], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0085] [Figure 8] Figure 8 shows a graph of the effects of the peptide derivatives of the present invention (DS-57 to DS-63), represented by [Chemical Formulas 1-40] to [Chemical Formulas 1-46], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0086] [Figure 9] Figure 9 shows a graph illustrating the effect of the peptide derivatives of the present invention (DS-68, DS-70, DS-72 to DS-75), represented by [Chemical Formulas 1-47] to [Chemical Formulas 1-52], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0087] [Figure 10]Figure 10 is a graph showing the effect of the peptide derivatives of the present invention (DS-76 to DS-79), represented by [Chemical Formulas 1-53] to [Chemical Formulas 1-56], on AMPK phosphorylation activity in mouse adipocytes. Here, V represents the vehicle treated with DMSO, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group.

[0088] [Figure 11] Figure 11 shows whether the increased AMPK phosphorylation activity by the peptide derivatives of the present invention (DS-4, DS-5, DS-6, DS-8, DS-28, DS-43, DS-52), represented by [Chemical Formula 1-4], [Chemical Formula 1-5], [Chemical Formula 1-6], [Chemical Formula 1-8], [Chemical Formula 1-9], [Chemical Formula 1-18], and [Chemical Formula 1-35] decreases upon adiponectin receptor 1 knockdown. Here, NC is scrambled siRNA, and R1 is adiponectin receptor 1 siRNA. V is DMSO-treated as the vehicle, and P5 is the APN5 peptide, which has an NH2-GLYYF-OH sequence, as the positive control group. DS-43 is adiporon, an agonist of adiponectin receptor 1.

[0089] [Figure 12]Figure 12 shows whether the increased AMPK phosphorylation activity by the peptide derivatives of the present invention (DS-2, DS-7, DS-17, DS-18, DS-27, DS-29, DS-48, DS-51), represented by [Chemical Formula 1-2], [Chemical Formula 1-7], [Chemical Formula 1-13], [Chemical Formula 1-14], [Chemical Formula 1-17], [Chemical Formula 1-19], [Chemical Formula 1-31], and [Chemical Formula 1-34] decreases upon adiponectin receptor 1 knockdown. Here, NC is scrambled siRNA, and R1 is adiponectin receptor 1 siRNA. V is DMSO-treated as the vehicle, and P5 is the APN5 peptide as the positive control group, which has an NH2-GLYYF-OH sequence. DS-43 is adiporon, an agonist of adiponectin receptor 1.

[0090] [Figure 13] Figure 13 shows the cytotoxicity of the peptide derivatives of the present invention, represented by [Chemical Formulas 1-6], against four cell lines (3T3-L1 adipocytes, RD muscle cells, keratinocytes, and fibroblasts). Here, Veh represents the vehicle treated with DMSO.

[0091] [Figure 14] Figure 14 shows the cytotoxicity of the peptide derivatives of the present invention, represented by [Chemical Formulas 1-8], against four cell lines (3T3-L1 adipocytes, RD muscle cells, keratinocytes, and fibroblasts). Here, Veh represents the vehicle treated with DMSO.

[0092] [Figure 15]Figure 15 shows the cytotoxicity of the peptide derivatives of the present invention, represented by [Chemical Formula 1-18], against four cell lines (3T3-L1 adipocytes, RD muscle cells, keratinocytes, and fibroblasts). Here, Veh represents the vehicle treated with DMSO.

[0093] [Figure 16] Figure 16 shows the cytotoxicity of the peptide derivatives of the present invention, represented by [Chemical Formula 1-31], against four cell lines (3T3-L1 adipocytes, RD muscle cells, keratinocytes, and fibroblasts). Here, Veh represents the vehicle treated with DMSO.

[0094] [Figure 17] Figure 17 shows whether the peptide derivatives of the present invention represented by [Chemical Formula 1-6], [Chemical Formula 1-18], and [Chemical Formula 1-31] ​​increase AMPK phosphorylation activity in a concentration-dependent manner.

[0095] [Figure 18] Figure 18 shows the hair growth efficacy of the peptide derivatives of the present invention represented by [Chemical Formulas 1-8], [Chemical Formulas 1-18], and [Chemical Formulas 1-31]. Here, V is the vehicle, EtOH:polyethylene glycol = 30:70, v / v; P5 is the positive control group, APN5 peptide, which has an NH2-GLYYF-OH sequence; and Mnx is the positive control group, minoxidil, a well-known hair growth agent.

[0096] [Figure 19] Figure 19 shows the skin aging improvement efficacy of the peptide derivatives of the present invention represented by [Chemical Formula 1-8], [Chemical Formula 1-18], and [Chemical Formula 1-31]. Here, Veh is the vehicle treated with DMSO, and P5 is the APN5 peptide as the positive control group.

[0097] [Figure 20A] Figure 20A shows the neutral fat removal efficacy of the peptide derivatives of the present invention represented by [Chemical Formula 1-8], [Chemical Formula 1-5], [Chemical Formula 1-6], [Chemical Formula 1-18], [Chemical Formula 1-2], [Chemical Formula 1-7], [Chemical Formula 1-31], and [Chemical Formula 1-50]. [Figure 20B] Figure 20B shows the differentiation and triglyceride-inhibiting efficacy of the peptide derivatives of the present invention represented by [Chemical Formulas 1-6], [Chemical Formulas 1-8], [Chemical Formulas 1-18], and [Chemical Formulas 1-31]. Here, V is the vehicle treated with DMSO, P5 is the APN5 peptide as the positive control group, and has an NH2-GLYYF-OH sequence. ND is cultured in non-differentiation medium, and D is cultured in differentiation medium.

[0098] [Figure 21] Figure 21 shows the sensitive skin improvement efficacy of the peptide derivatives of the present invention represented by [Chemical Formula 1-8], [Chemical Formula 1-18], and [Chemical Formula 1-31]. Here, Veh is the vehicle treated with DMSO, LA is lactic acid, and P5 is the APN5 peptide as the positive control group. [Modes for carrying out the invention]

[0099] This invention relates to the synthesis of novel peptide derivatives based on the adiponectin-derived peptide APN5, and more specifically, to peptides in which some amino acids have been modified to improve the physical properties and activity of existing adiponectin peptides. The peptide derivatives in this invention have improved physical properties and activity, making them advantageous for drug formulation.

[0100]

[0101] Herein, the present invention provides a peptide derivative represented by the following [Chemical Formula 1]. [Chemical formula 1]

[0102] [ka]

[0103] In the above [Chemical Formula 1],

[0104] R1 is an amino group (NH2), an acetylamino group (Ac-NH), or NH2-(CH2CH2O). m -CH2CONH, C 15 -C 20 One or more selected from the group consisting of amide groups and combinations thereof, where m is an integer from 1 to 5.

[0105] R2 is an amino group or a hydroxyl group (OH),

[0106] R3 is hydrogen (H),

[0107] R4 is one or more selected from the group consisting of a C1-C6 chain-type alkyl group, a C2-C6 alkylcarboxyl group, a C1-C6 alkylamide group, an imidazoylmethyl group, and combinations thereof.

[0108] R5 and R6 are either identical or different, and are independently a benzyl group, a phenylethyl group, or an indolylmethyl group, wherein the benzyl group is either unsubstituted or one or more selected from the group consisting of a hydroxyl group, a trifluoromethyl group (CF3), a halogen group, a cyano group, a nitro group, a C1-C6 chain-type alkyl group, a C1-C6 alkoxy group, and combinations thereof. Replaced the law of nature,

[0109] R7 is a benzyl group or a C1-C6 alkylamino group, and the benzyl group is either unsubstituted or has one or more selected from the group consisting of a hydroxyl group, a halogen group, a cyano group, a nitro group, a C1-C6 linear alkyl group, a C1-C6 alkoxy group, and a combination thereof. Replaced Ruka,

[0110] R3 and R7 are connected to each other and C 15 -C 20 Forms a heterocycloalkene group,

[0111] R8, R9, and R 10 These are either identical or different from each other, and each is independently hydrogen or a C1-C6 alkyl group.

[0112] n may be 0 or 1.

[0113]

[0114] In the present invention, the term "substitution" refers to a reaction in which an atom or group of atoms contained in a molecule of a compound is replaced with another atom or group of atoms.

[0115] In this invention, the term "chain-type" refers to a molecule having a chain-type structure, which is a chemical structure in which carbon atoms are linked together in a chain, and can be a straight chain or a branched shape.

[0116] In this invention, the term "ring-shaped" refers to a structure in which the ends of a chain of organic compound skeletons are connected to form a ring.

[0117] In the present invention, the term "chain-type or ring-type alkyl group" means a monovalent linear, branched, or ring-type saturated hydrocarbon residue having 1 to 12 carbon atoms and composed solely of carbon and hydrogen atoms. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, cyclobutyl group, cyclopentyl group, and cyclohexyl group.

[0118] In the present invention, the term "halogen group" may refer to elements belonging to Group 17 of the periodic table, such as florin (F), chloride (Cl), bromine (Br), or iodine (I).

[0119] In the present invention, the term "alkoxy group" means the atomic group CnH2n+1O- formed by bonding an oxygen source to an alkyl group, and includes, but is not limited to, methoxy, ethoxy, propoxy, or butoxy.

[0120] In this invention, the term "cycloalkene group" also refers to a ring-type alkene group, which is a ring group in which all ring components are carbon atoms and have one or more double bonds (but is not aromatic). The term "heterocycloalkene group" refers to a cycloalkene group which contains at least one heteroatom of N, O, or S. The cycloalkene group or heterocycloalkene group each contains one or more ring structures, which may be, for example, a single ring, a double ring, a triple ring, etc.

[0121]

[0122] Furthermore, the present invention provides one or more preventive or therapeutic pharmaceutically active ingredients comprising the peptide derivative as an active ingredient, selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer.

[0123] Furthermore, the present invention provides a pharmaceutical composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient.

[0124] In the present invention, the term "prevention" means all actions that suppress or delay the onset, spread, or recurrence of the disease by administering the composition of the invention, and "treatment" means all actions that improve or modify the symptoms of the disease by administering the composition of the invention.

[0125] In this invention, the term "improvement" means all actions that at least reduce the severity of symptoms or improve or modify a condition, such as reducing the severity of symptoms or improving the condition.

[0126] In the present invention, the term "pharmaceutical composition" means a product manufactured for the purpose of preventing or treating the aforementioned disease, and each may be used in various dosage forms by conventional methods. For example, it can be made into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be used in the form of topical preparations, suppositories, and sterile injection solutions.

[0127] In this invention, "contained as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to achieve the desired biological effect. In actual application, the amount to be included as an active ingredient is determined as an amount to treat the target disease, taking into consideration factors that do not cause other toxicity, and may vary depending on various factors such as the disease or condition being treated, the form of the composition administered, the size of the subject, or the severity of the disease or condition. A person skilled in the art to which this invention belongs can empirically determine the effective amount of an individual composition without excessive testing.

[0128] Furthermore, the pharmaceutical compositions of the present invention may contain one or more additional pharmaceutically acceptable carriers in addition to the active ingredients described above, depending on the dosage form.

[0129] The pharmaceutically acceptable carrier may be a mixture of one or more of the following: saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and its components, and may further contain other common additives such as antioxidants, buffers, and bacteriostatic agents as needed. Diluents, dispersants, surfactants, binders, and lubricants may also be added to form injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, the formulation may be preferably prepared according to the disease or components, using appropriate methods in the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).

[0130] The compositions of the present invention can be administered orally or parenterally in pharmaceutically effective amounts by the intended method, and the term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat a disease with a reasonable gain / risk ratio applicable to medical treatment and without causing side effects, and the effective dose level may be determined by factors including the patient's health status, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, and elimination ratio, duration of treatment, drugs used in combination or concurrently, and other factors known in the medical field.

[0131] In the present invention, "inflammatory skin disease" refers to inflammation occurring in the outer layer of the skin that often induces itching, blisters, redness, swelling, exudation, scabbing, and peeling. Non-limiting examples include, but are not limited to, psoriasis, atopic dermatitis, eczema, contact dermatitis, erythroderma, chronic lichen simplex, nummular dermatitis, seborrheic dermatitis, and stasis dermatitis.

[0132] In the present invention, "metabolic disease" refers to a syndrome in which risk factors such as obesity due to excessive nutrient accumulation in the body and lack of exercise, diabetes, hypertension, arteriosclerosis, and nonalcoholic fatty liver disease (NAFLD) appear together. Non-limiting examples include, but are not limited to, diabetes, hypertension, hyperlipidemia, cardiovascular disease, thrombosis, dyslipidemia, stroke, arteriosclerosis, and hyperinsulinemia.

[0133]

[0134] Furthermore, the present invention provides one or more methods for the prevention or treatment of skin inflammatory diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, selected from the group comprising the step of administering the peptide derivative to an individual.

[0135] Furthermore, the present invention provides one or more methods for preventing or improving aging, sensitive skin, wrinkles, and moisturizing, selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which include the step of administering the peptide derivative to an individual.

[0136] In the present invention, the term "individual" is not limited to any mammal, such as livestock or humans, for which prevention, treatment, and / or diagnosis of the aforementioned disease is necessary, but preferably it may be a human.

[0137] In this invention, the term "administration" means providing a predetermined substance to a patient by any suitable method. The pharmaceutical compositions of this invention are formulated into various dosage forms for administration to an individual, with injectable dosage forms being typical for parenteral administration, preferably isotonic aqueous solutions or suspensions. Injectable dosage forms can be manufactured by art well known in the industry using appropriate dispersants or wetting and suspending agents. For example, each component can be dissolved in saline or buffer solution to form an injectable dosage form. Oral dosage forms include, for example, ingestible tablets, buccal preparations, lozenges, capsules, elixirs, suspensions, syrups, and wafers. These dosage forms may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine) and lubricants (e.g., silica, talc, stearic acid, and their magnesium or calcium salts and / or polyethylene glycol). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and may further contain disintegrants such as starch, agar, alginic acid, or their sodium salts, absorbents, colorants, flavorings and / or sweeteners as desired. The dosage form may be manufactured by granulation or coating of a conventional mixture.

[0138] Furthermore, the pharmaceutical composition of the present invention may further contain adjuvants such as preservatives, wettable powders, emulsifiers, salts or buffers for osmotic pressure regulation, and other therapeutically useful substances, and can be formulated by conventional methods.

[0139] The pharmaceutical compositions according to the present invention may be administered via various routes, including orally, transdermally, subcutaneously, intravenously, intranasally, intraperitoneally, or intramuscularly, and the dosage of the active ingredient may be appropriately selected based on various factors such as the route of administration, the patient's age, sex, weight, and the severity of the patient's condition. Furthermore, the compositions of the present invention may be administered in parallel with well-known compounds that can enhance the desired effect.

[0140]

[0141] Furthermore, the present invention provides a cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, which contains the peptide derivative as an active ingredient.

[0142] Furthermore, the present invention provides a cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient.

[0143] The cosmetic composition may contain, for example, the peptide derivative or a cosmetically acceptable salt thereof as an active ingredient and be manufactured together with dermatologically acceptable excipients in the form of basic cosmetic compositions (lotions, creams, essences, facial cleansers such as cleansing foams and cleansing waters, packs, body oils), color cosmetic compositions (foundations, lipsticks, mascaras, makeup bases), hair product compositions (shampoos, rinses, hair conditioners, hair gels), and soaps.

[0144] The excipients may include, for example, emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. More specifically, examples include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene glycol, water, and ethanol.

[0145]

[0146] Furthermore, the present invention provides a food composition for the prevention or improvement of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, which contains the peptide derivative as an active ingredient.

[0147] Furthermore, the present invention provides a food composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, which contains the peptide derivative as an active ingredient.

[0148] The food composition may contain, for example, the peptide derivative or a pharmaceutically acceptable salt thereof as an active ingredient. When the peptide derivative is used as an additive to the food composition, it can be added as is or used together with other foods or food ingredients, and can be used appropriately by conventional methods. Generally, the composition of the present invention is added to the raw materials in an amount of 15% by weight or less, preferably 10% by weight or less, during the manufacture of food or beverages. However, for long-term intake for health and hygiene purposes, or for health regulation purposes, the amount may be less than the above range, and since there are no safety issues, the active ingredient may be used in amounts greater than the above range. That is, the amount of active ingredient mixed can be determined appropriately according to each purpose of use, such as prevention, health, or treatment.

[0149] The dosage form of the food composition may be in the form of powder, granules, rings, tablets, or capsules, as well as in the form of general food or beverage.

[0150] The food product of the present invention can be manufactured by methods commonly used in the art, and may be manufactured by adding raw materials and components commonly used in the art during such manufacturing. Specifically, it may contain proteins, carbohydrates, fats, nutrients, seasonings, and flavorings, and examples of carbohydrates include, but are not limited to, glucose, fructose, maltose, sucrose, oligosaccharides, dextrin, cyclodextrin, xylitol, sorbitol, erythritol, saccharin, or synthetic flavorings.

[0151]

[0152] The terms used in the embodiments are for illustrative purposes only and are not intended to be limiting. Singular expressions include plural expressions unless, in context, they have a clearly different meaning. In this specification, terms such as “includes” or “has” indicate the presence of features, figures, stages, operations, components, parts, or combinations thereof described in the specification, and should be understood not to preemptively exclude the possibility of the presence or addition of one or more other features, figures, stages, operations, components, parts, or combinations thereof.

[0153] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as those commonly understood by a person of ordinary skill in the art to which this embodiment belongs. Commonly used predefined terms should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not as ideal or overly formal unless expressly defined herein.

[0154] Furthermore, when describing the components of an embodiment, terms such as First, Second, A, B, (a), (b), etc., may be used. Such terms are used to distinguish a component from other components, and the terms do not limit the nature, order, or sequence of the component. When it is stated that any component is “connected,” “joined,” or “connected” to another component, it should be understood that while that component may be directly connected to or connected to that different component, further components may be “connected,” “joined,” or “connected” between each component.

[0155]

[0156] The embodiments will be described in detail below with reference to the attached drawings. However, since the embodiments can be modified in various ways, the scope of the patent application will not be limited or restricted by these embodiments. All modifications, equivalents, or substitutions to the embodiments should be understood to be included within the scope of the patent.

[0157] Furthermore, when explaining with reference to the attached drawings, the same reference numerals will be assigned to the same components regardless of the reference numerals in the drawings, and redundant explanations will be omitted. In the description of the embodiments, if it is determined that a specific explanation of related prior art would unnecessarily obscure the gist of the embodiment, such detailed explanation will be omitted.

[0158]

[0159] The present invention can be modified in various ways and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following description. However, this is not intended to limit the present invention to specific embodiments, but rather to include all modifications, equivalents, or substitutes that fall within the spirit and technical scope of the present invention. Where a specific description of relevant prior art would obscure the gist of the invention, such detailed description will be omitted.

[0160] [Modes for carrying out the invention] Embodiment 1. Production of the peptide derivative of the present invention

[0161] The peptide derivatives of the present invention were prepared using conventional amino acid synthesis methods (Umbarger.HE, Ann.Rev.Biochem.1978, 47, 533-606).

[0162] C-terminal initiation is achieved by using Wang resin (100-200 mesh, Novabiochem®, CAS No: 65307-53-1) to produce peptides with a common COOH carboxyl group at the C-terminus, and by using Rink amide AM resin (100-200 mesh, Novabiochem®, CAS No: 65307-53-1) to produce peptides with a CONH2 amide terminus at the C-terminus.

[0163]

[0164] Embodiment 1.1. When the N-terminus is an amino group and the C-terminus is a carboxyl group

[0165] For the production of peptides consisting of the C-terminal carboxyl group, dried Wang resin (100 mg, 0.5-1.3 mmol / g) was stirred in dichloromethane (DCM) solvent for 60 minutes using a Torviq reaction vessel polypropylene syringe, and then the solvent was removed to initiate the first step of the reaction. In the initial reaction, hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBt), diisopropylcarbodiimide (N,N'-Diisopropylcarbodiimide, DIC), 4-Dimethylaminopiridine (DMAP), and the first amino acid residue Fmoc-Phe-OH were stirred for 2 hours in a solvent mixture of dichloromethane (DCM) and dimethylformamide (DMF) in a 3:2 ratio. The resin was then washed by repeating the washing process twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF). The second step in peptide monomer synthesis involves reacting 1 mL of dimethylformamide (DMF) with 20% (v / v) piperidine at room temperature for 10 minutes twice to remove the Fmoc protecting group. Then, the resin is washed by repeating the washing process twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF). After that, hydroxybenzotriazole (1 -Hydroxybenzotriazole (HOBt), N-[(dimethylamino-)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU), N,N-diisopropylethylamine (N,N-diisopropylethylamine, DIEA), and Fmoc-Tyr(OtBu)-OH were reacted and stirred for 2 hours under the solvent of dimethylformamide (DMF).The reacted resin was washed twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process. The next step, peptide monomer synthesis, was carried out using the same process as the second monomer synthesis step. After the monomer synthesis of each step was complete, the peptides were finally subjected to a reaction to remove the Fmoc protecting group of the last peptide in the resin. This involved reacting 1 mL of dimethylformamide (DMF) with 20% (v / v) piperidine at room temperature for 10 minutes each, twice, followed by washing twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process. The peptides were purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity verification and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS.

[0166]

[0167] Embodiment 1.2. When the N-terminus is an amino group and the C-terminus is an amide group

[0168] For the production of peptides consisting of a C-terminal amide group, dried Rink amide AM resin (100 mg, 0.9 mmol / g) was stirred for 60 minutes in dimethylformamide (DMF) solvent using a Torviq reaction vessel polypropylene syringe, and then the solvent was removed to initiate the first step of the reaction. For the removal of the Fmoc protecting group in the resin, Fmoc-Rink amid MBHA was used. The resin was reacted twice at room temperature for 10 minutes each with 1 mL of dimethylformamide (DMF) supplemented with 20% (v / v) piperidine. Then, the resin was washed twice each using dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF). To introduce the first amino acid residue, hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBt), N-[(dimethylamino-)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU), N,N-diisopropylethylamine (N,N-diisopropylethylamine, DIEA), and Fmoc-Phe-OH were reacted and stirred for 2 hours under the solvent of dimethylformamide (DMF). The reacted resin was washed twice each using dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process.The second step in peptide monomer synthesis involves reacting 1 mL of dimethylformamide (DMF) with 20% (v / v) piperidine at room temperature for 10 minutes twice to remove the Fmoc protecting group. Then, the resin is washed by repeating the washing process twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF). After that, hydroxybenzotriazole (1 -Hydroxybenzotriazole (HOBt), N-[(dimethylamino-)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU), N,N-diisopropylethylamine (N,N-diisopropylethylamine, DIEA), and Fmoc-Tyr(OtBu)-OH were reacted and stirred for 2 hours under the solvent of dimethylformamide (DMF). The reacted resin was washed twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF). The peptide monomer synthesis in the next step was carried out using the same procedure as the monomer synthesis step described above. Finally, in the Fmoc protecting group removal reaction of the last peptide in the resin, 1 mL of dimethylformamide (DMF) with 20% (v / v) piperidine was used, and the reaction was carried out twice at room temperature for 10 minutes each. Then, the washing process was repeated twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and the final dimethylformamide (DMF) to complete the reaction. The peptide was purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity confirmation and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS.

[0169]

[0170] Embodiment 1.3. N-terminus acetyl protection

[0171] The N-terminal unprotected peptide synthesized in Embodiment 1.1 or 1.2 was reacted and stirred with acetic anhydride and N,N-diisopropylethylamine (DIEA) under dimethylformamide (DMF) for 2 hours. The reacted resin was washed twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to synthesize a peptide with an acetyl group protecting the N-terminus. The peptide was purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity confirmation and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS.

[0172]

[0173] Embodiment 1.4. N-terminus PAL protection

[0174] For the synthesis of PAL (Palmitic acid)-protected peptides from the N-terminal unprotected peptides synthesized in Embodiment 1.1 or 1.2, hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBt), N-[(dimethylamino-)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU), N,N-diisopropylethylamine (N,N-diisopropylethylamine, DIEA), and PEG (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid) or PAL (Palmitic acid) were reacted and stirred for 2 hours under the solvent of dimethylformamide (DMF). The reacted resin was washed twice each using dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process.

[0175] The crude peptide obtained by the above method was reacted with a separation solution of trifluoroacetic acid (TFA):H2O:triisopropylsilane (TIPS) (95:2.5:2.5 vol. / vol.) for 2-3 hours to remove the protecting group and separate the peptide from the resin. The peptide was then precipitated with chilled diethyl ether. The peptide was purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity confirmation and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS.

[0176]

[0177] Embodiment 1.5. PEG protection of the N-terminus

[0178] For the synthesis of peptides protected with (PEG)3(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid) to the N-terminal unprotected peptides synthesized in Embodiment 1.1 or 1.2, hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBt), N-[(dimethylamino-)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU), N,N-diisopropylethylamine (N,N-diisopropylethylamine, DIEA), and PEG(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid) or PAL (Palmitic acid) were reacted and stirred for 2 hours under the solvent of dimethylformamide (DMF). The reacted resin was washed twice each using dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process.

[0179] The crude peptide obtained by the above method was reacted with a separation solution of trifluoroacetic acid (TFA):H2O:triisopropylsilane (TIPS) (95:2.5:2.5 vol. / vol.) for 2-3 hours to remove the protecting group and separate the peptide from the resin. The peptide was then precipitated with chilled diethyl ether. The peptide was purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity confirmation and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS.

[0180]

[0181] Embodiment 1.6. Synthesis of ring-shaped peptides

[0182] Peptides were synthesized using the method of Embodiment 1.2, specifically a peptide production method for peptides consisting of the C-terminal amide group. The first and last amino acids were synthesized linearly using Fmoc-Allyl-Gly-OH in the same manner as in Embodiment 1.2. The resin, with its N-terminus protected by Fmoc, was then dissolved in dimethylformamide (DMF) under dichloromethane (DCM) solvent to produce LiCl (0.4M), and Grubbs' catalyst was added. The mixture was then reacted at 100°C for 1-2 hours using microwaves (2.5 GHz, 300 W). The reacted resin was then washed twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and finally dimethylformamide (DMF) to complete the process. Finally, in the Fmoc protecting group removal reaction of the last peptide in the resin, 1 mL of dimethylformamide (DMF) with 20% (v / v) piperidine was used to react the peptide twice at room temperature for 10 minutes each. Then, the washing process was repeated twice each with dimethylformamide (DMF), methyl alcohol (MeOH), dichloromethane (DCM), and the final dimethylformamide (DMF) to complete the reaction. The crude peptide obtained by the above method was reacted with a separation solution of trifluoroacetic acid (TFA):H2O:triisopropylsilane (TIPS) (95:2.5:2.5 vol. / vol.) for 2-3 hours to remove the protecting group and separate the peptide from the resin. The peptide was then precipitated with chilled diethyl ether. The peptide was purified using high-performance liquid chromatography (Prep-HPLC, Gilson) with an acetonitrile-water gradient solvent composition. For purity confirmation and analysis, peptides with a purity of 98% or higher were obtained using RP-HPLC and LC-MS (J.Pept.Sci.2007;13:280-285).

[0183]

[0184] The sequence and molecular weight of the peptide derivative according to one embodiment of the present invention are shown in Table 3 below.

[0185] [Table 3]

[0186] [Table 3] TIFF0007852055000023.tif128149

[0187] In Table 3 above, each amino acid constituting the peptide derivative of the present invention can have the following structure.

[0188] [ka]

[0189]

[0190] Test Example 1. Verification of in vitro AMPK phosphorylation activity

[0191] The effects of the peptide derivatives of the present invention on adiponectin expression and receptor activity in mouse adipocytes, which are the main site of adiponectin production, were investigated. Mouse adipocyte cell line 3T3-L1 was treated with each peptide derivative of the present invention at a concentration of 10 μM and cultured for 24 hours before cells were obtained. Subsequently, proteins were extracted, and their effects on AMPK phosphorylation, which is responsible for the main subsignaling of adiponectin, were examined by Western blot.

[0192] As a result, the peptide derivatives of the present invention increased AMPK phosphorylation activity related to adiponectin receptor activity in mouse adipocytes (Figures 1 to 10). In particular, the peptide derivatives represented by [Chemical Formula 1-2], [Chemical Formula 1-4] to [Chemical Formula 1-9], [Chemical Formula 1-13], [Chemical Formula 1-14], [Chemical Formula 1-17] to [Chemical Formula 1-19], [Chemical Formula 1-31], [Chemical Formula 1-34], and [Chemical Formula 1-35] were confirmed to have a significant effect in increasing AMPK phosphorylation activity compared to APN5, a conventionally known adiponectin-derived peptide (Table 4).

[0193] [Table 4]

[0194]

[0195] Test Example 2. Verification of whether or not AMPK phosphorylation activity is lost after in vitro adiponectin receptor 1 knockdown.

[0196] To verify whether the peptide derivatives of the present invention bind to adiponectin receptor 1, mouse adipocyte cell line 3T3-L1 was treated with adiponectin receptor 1 siRNA to knock down the cells. The cells were then treated with the peptide derivatives of the present invention at a concentration of 10 μM and cultured for 24 hours before the cells were obtained. Subsequently, proteins were extracted, and Western blotting was used to confirm whether the increased AMPK phosphorylation activity due to the peptide derivatives disappeared.

[0197] As a result, it was confirmed that the peptide derivatives of the present invention, including [Chemical Formula 1-2], [Chemical Formula 1-5] to [Chemical Formula 1-8], [Chemical Formula 1-13], [Chemical Formula 1-14], [Chemical Formula 1-17] to [Chemical Formula 1-19], [Chemical Formula 1-31], [Chemical Formula 1-34], and [Chemical Formula 1-35], reduce AMPK phosphorylation activity after knockdown of adiponectin receptor 1 (Figures 11 and 12).

[0198] Adiponectin acts by binding to adiponectin receptors, and when adiponectin receptors are activated, it is known to increase the phosphorylation of AMPK and the activity of PPARα / PPARγ, which are lower signaling pathways (Crystal structures of the human adiponectin receptors. Nature. 2015 Apr16;5207547:312-6. doi:10.1038 / nature14301). Furthermore, it is known that adiponectin and its receptors have decreased expression in photoaged skin and UV-irradiated skin. This decrease in adiponectin expression can alter the extracellular matrix by regulating the expression of MMP-1 and procollagen, which are regulators of skin aging, and thus worsen photoaging (UV-induced inhibition of adipokine production in subcutaneous fat aggravates dermal matrix degradation in human skin. 2016. Scientific Reports. 10;6:25616. doi:10.1038 / srep25616).

[0199] Therefore, the peptide derivatives of the present invention increase the activity of AMPK phosphorylation related to the activity of the adiponectin receptor, thereby increasing the expression of adiponectin in the body and playing a major role in protecting against skin aging.

[0200]

[0201] Test Example 3. Verification of in vitro cytotoxicity

[0202] Using four cell lines (3T3-L1 adipocytes, RD muscle cells, keratinocytes, and fibroblasts), each cell was placed in a 24-well plate at a rate of 5 × 10⁶ cells per well. 4After dispensing into individual cells, each cell was cultured for 24 hours under its own culture conditions. The culture medium was discarded, washed with PBS, and replaced with fresh medium without 10% FBS. Each cell was then treated with the peptide derivative of the present invention at concentrations of (0, 0.1, 1, 10, 100, and 500 μM) and cultured for 24 hours. The medium was again carefully removed and washed with PBS. The MTT reagent was added according to the manufacturer's method, and after reacting at room temperature for 30 minutes, the absorbance was measured at 450 nm.

[0203] As a result, the peptide derivatives of the present invention, including [Chemical Formula 1-6], [Chemical Formula 1-8], [Chemical Formula 1-18], and [Chemical Formula 1-31], were confirmed to be non-toxic in multiple cell lines (Figures 13-16).

[0204]

[0205] Test Example 4. Verification of the concentration dependence of AMPK phosphorylation activity in vitro.

[0206] Mouse adipocyte cell line 3T3-L1 was treated with the peptide derivative of the present invention at a series of concentrations (0, 0.01, 0.1, 1, 10, 100 μM) and cultured for 24 hours, after which the cells were obtained. Subsequently, proteins were extracted, and the concentration-dependent effect of the peptide derivative on AMPK phosphorylation was confirmed by Western blotting.

[0207] As a result, it was confirmed that the peptide derivatives of the present invention, including [Chemical Formula 1-6], [Chemical Formula 1-18], and [Chemical Formula 1-31], increase AMPK phosphorylation activity in a concentration-dependent manner (Figure 17).

[0208]

[0209] Test Example 5. Solubility Test

[0210] The PRISMA HT solution was diluted with distilled water at a ratio of 1:40, and the pH was adjusted to 7.4 to be used as the test buffer. In addition, the reference substances (Diclofenac stock 50 mM, Phenazopyridine stock 25 mM) used for accuracy in general solubility tests, and the peptide derivative of the present invention (50 mM) were prepared in DMSO and used as the test standard solution.

[0211] A 10 mL sample of the prepared test substance was diluted in 190 μL of isopropyl alcohol. Additionally, 75 mL of the test solution and 70 mL of isopropyl alcohol were mixed to create a blank sample, and 5 mL of the diluted test substance was added to the blank sample. After mixing the diluted samples, the absorbance was measured and used as the initial test value. For the solubility test, 1 mL of the test solution and 10 mL of the DMSO standard test solution were mixed and reacted at room temperature for 24 hours. Afterward, the precipitate was removed from the sample using a filter plate, and 75 mL of isopropyl alcohol was added to the filtered 74 mL sample. The absorbance was then measured and used as the sample value.

[0212] The solubility values ​​of the test substances were determined by measuring the absorbance of each sample via a microplate reader, followed by analysis using msol Explorer (Solubility Explorer program). The mean and standard deviation of three repeated test values ​​(n=3) were calculated using an Excel program.

[0213] As a result, the peptide derivatives of the present invention showed excellent solubility in DMSO (Table 5).

[0214] [Table 5]

[0215]

[0216] Test Example 6. Plasma Stability Test

[0217] Two types of plasma (human and rat) were treated with the peptide derivative of the present invention at a concentration of 1 μM. The samples were placed in separate tubes and cultured at 37°C for different time intervals (0, 30, 60, 120, and 240 minutes). At each time interval, the tubes containing the plasma were removed, an acetonitrile solution containing an internal standard (chlorpropamide) was added, the mixture was vortexed for 5 minutes, and the mixture was centrifuged for 5 minutes (14,000 rpm, 4°C). The upper layer was then injected into an LC-MS / MS system to analyze the drug at each time interval, thereby evaluating the plasma stability of the peptide derivative. Procaine and Enalapril were used as reference substances for accuracy in general plasma stability tests.

[0218] The amount of drug remaining in each tube after the reaction was analyzed by LC-MS / MS using the Shimadzu Nexera XR system and TSQ vantage (Thermo). A Luna C18 column (2.0 × 50 mm, 3 μm particle size; Phenomenex, US) was used as the HPLC column, and the mobile phases were 0.1% formic acid-containing distilled water (A) and 0.1% formic acid-containing acetonitrile (B). Data analysis was performed using Xcalibur (version 1.6.1). The results analysis shows the plasma stability of each peptide derivative in %remaining for each time period compared to uncultured samples.

[0219] As a result, the peptide derivative of the present invention exhibited excellent plasma stability, and in particular, it was confirmed that its plasma stability was significantly improved compared to APN5, a conventionally known adiponectin-derived peptide (Table 6).

[0220] [Table 6]

[0221]

[0222] Test Example 7. Test of metabolic stability

[0223] Three types of liver microsomes (Human, Rat, Mouse, 0.5 mg / ml), 0.1 M phosphate buffer solution (pH 7.4), and the peptide derivative of the present invention were added at a concentration of 1 μM and pre-cultured at 37°C for 5 minutes. Then, NADPH Regeneration System solution was added and cultured at 37°C for 30 minutes. Subsequently, to terminate the reaction, an acetonitrile solution containing an internal standard (chlorpropamide) was added, and the mixture was centrifuged for 5 minutes (14,000 rpm, 4°C). The supernatant was then injected into an LC-MS / MS system, and the metabolic stability of the substrate drug was evaluated by analysis. Here, verapamil was used as a reference substance for accuracy, which is commonly used in plasma stability tests.

[0224] The amount of substrate remaining after the above reaction was analyzed by LC-MS / MS using an Agilent 1290 infinity series pump system (Agilent, USA) and a Triple Quad 5500 LC-MS / MS system (Applied Biosystems, USA). A Kinetex C18 column (2.1 × 100 mm, 1.7 μm particle size; Phenomenex, USA) was used as the HPLC column, and the mobile phase consisted of distilled water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). TurboSpray Ionization was used as the ion source for MS / MS, and a Triple Quadruple type mass spectrometer was used. The generated metabolites were quantified using the MRM (multiple reaction monitoring) quantitative mode, and data analysis was performed using Analyst software (version 1.6.1).

[0225] As a result, the peptide derivative of the present invention exhibited excellent metabolic stability, and in particular, its metabolic stability was confirmed to be significantly improved compared to APN5, a conventionally known adiponectin-derived peptide (Table 7).

[0226] [Table 7]

[0227]

[0228] Test Example 8. CYP Inhibition Analysis

[0229] Human liver microsomes (0.25 mg / ml), 0.1 M phosphate buffer solution (pH 7.4), a substrate drug cocktail of five drug-metabolizing enzymes (Phenacetin 50 μM, S-mephenytoin 100 μM, dextromethorphan 5 μM, midazolam 2.5 μM, diclofenac 10 μM), and the peptide derivative of the present invention were added at concentrations of 0 and 10 μM, respectively, and pre-cultured at 37°C for 5 minutes. Then, NADPH Regeneration System solution was added and cultured at 37°C for 15 minutes. Subsequently, an acetonitrile solution containing an internal standard (chlorpropamide) was added to terminate the reaction, and the mixture was centrifuged for 5 minutes (14,000 rpm, 4°C). The upper layer was then injected into an LC-MS / MS system to simultaneously analyze the metabolites of the substrate drugs, thereby evaluating the inhibitory activity of the peptide derivative on drug-metabolizing enzymes. Here, ketoconazole was used as a reference substance for accuracy when evaluating the inhibitory activity of drug-metabolizing enzymes in general.

[0230] Metabolites of each CYP enzyme-indicating drug generated via the above reaction were analyzed by LC-MS / MS using a Shimadzu Nexera XR system and TSQ vantage (Thermo). A Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size; Phenomenex, USA) was used for the HPLC column, and the mobile phases were 0.1% formic acid-containing distilled water (A) and 0.1% formic acid-containing acetonitrile (B). TurboSpray Ionization was used as the ion source for MS / MS, and a triple quadruple type mass spectrometer was employed. The generated metabolites were quantified using MRM (multiple reaction monitoring) quantitative mode, and data analysis was performed using Xcalibur (version 1.6.1).

[0231] As a result, the peptide derivatives of the present invention inhibit CYP enzymes, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (Table 8), suggesting that they do not cause fatal adverse reactions due to drug interactions.

[0232] [Table 8]

[0233]

[0234] Test Example 9. Evaluation of the efficacy of topical application for hair growth and improvement of skin aging in mice.

[0235] After shaving the backs of 7-week-old C57BL / 6 mice, the peptide derivatives of the present invention were dissolved in a vehicle (EtOH:polyethylene glycol = 30:70, v / v) at a concentration of 0.15 mM and applied to the surface. A solution equivalent to 200 μl per mouse was uniformly applied to the entire back daily for 35 days.

[0236] Furthermore, skin tissue was extracted after the final application, and mRNA was extracted. The effect on the expression of procollagen, a factor in skin aging, was then examined using real-time PCR.

[0237] As a result, the proportion of areas where hair grew back among the areas where hair was removed for the first time significantly increased in the group treated with the peptide derivative of the present invention. In particular, it was confirmed that hair follicle formation was promoted, similar to the group treated with minoxidil, which was used as the positive control group (Figure 18).

[0238] Furthermore, it was confirmed that procollagen, a factor in skin aging, was expressed in a similar or even higher amount than APN5, a conventionally known adiponectin-derived peptide, in the group treated with the peptide derivative of the present invention (Figure 19).

[0239]

[0240] Test Example 10. Verification of the efficacy of suppressing triglycerides in adipocytes.

[0241] Mouse adipocyte cell line 3T3-L1 was treated with a differentiation induction reagent to differentiate it into adipocytes, and after the cells were filled with triglycerides, the peptide derivatives of the present invention were treated at a series of concentrations (10 and 30 μM) and cultured for 24 hours. Cells were then obtained and their triglyceride content was measured. As a result, it was confirmed that treatment with the peptide derivatives of the present invention reduced the triglyceride content, which was produced in a concentration-dependent manner (Figure 20A).

[0242] On the other hand, mouse adipocyte cell line 3T3-L1 was treated with the peptide derivative of the present invention at a concentration of 10 μM along with a differentiation induction reagent before differentiation induction to induce adipocytes. After differentiation, the cells were obtained and the triglyceride content was measured. As a result, it was confirmed that treatment with the peptide derivative of the present invention suppressed the accumulation of triglycerides (Figure 20B).

[0243]

[0244] Test Example 11. Verification of the efficacy of muscle cells in improving sensitive skin.

[0245] After culturing the RD cell line, which is a human muscle cell line, when the cells are 90% confluent, they are switched to a serum-free medium and treated with 50 mM lactic acid (LA), which is a sensitive skin inducer, and 10 μM of the peptide derivative of the present invention. After 4 hours, the cells are collected and mRNA is extracted, and the effect on the expression of CGRP, which is a neurotransmitter of pain mediators, is observed.

[0246] As a result, when the peptide derivative of the present invention was treated, it was confirmed that the expression of pain mediators was significantly reduced compared to the case of treating with lactic acid, and it was similar to or lower than the level when neither was treated (Figure 21). That is, it can be seen that the peptide derivative of the present invention significantly suppresses the expression of genes related to skin sensitivity induced by lactic acid.

[0247]

[0248] As described above, the embodiments of the present invention have been described in detail with reference to the drawings. However, the present invention is not limited to the above-described embodiments, and those having ordinary knowledge in the technical field can apply various technical modifications and deformations based on the above. For example, the described technology may be executed in a different order from the described method, and / or the components such as the described system, structure, device, circuit, etc. may be combined or assembled in a different form from the described method, or replaced or substituted by other components or equivalents, and appropriate results can still be achieved.

[0249] Therefore, other realizations, other embodiments, and equivalents to the claims, etc. also fall within the scope of the claims described later.

[0250]

Industrial Applicability

[0251] The present invention relates to novel peptide derivatives, which are modified adiponectin receptor peptides and possess even higher stability and superior p-AMPK activity than existing adiponectin receptor peptides. These derivatives have improved physical properties and activity, making them advantageous for drug formulation. Therefore, the peptide derivatives of the present invention can be conveniently used in the fields of preventing or treating inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, or cancer, as well as preventing or improving aging, sensitive skin, wrinkles, or moisturizing.

Claims

1. A peptide derivative as shown in [Chemical Formula 1] below. [Chemical formula 1] 【Chemistry 1】 In the above [Chemical Formula 1], R １ is any one or more selected from the group consisting of an amino group (NH ２ ), an acetylamino group (Ac-NH), NH ２ -(CH ２ CH ２ O) ｍ -CH ２ CONH, C １５ -C ２０ amide groups, and any combination thereof (where m is an integer from 1 to 5), R ２ is an amino group or a hydroxyl group (OH), R ３ It is hydrogen (H), R ４ isobutyl group, C ２ -C ６ The alkylcarboxyl group, C １ -C ６ It is one or more selected from the group consisting of alkylamide groups and combinations thereof, R ５ and R ６ These are either identical or different, and each is independently a benzyl group, a phenylethyl group, or an indolemethyl group, wherein the benzyl group is unsubstituted or a hydroxyl group, a trifluoromethyl group (CF ３ ), halogen group, cyano group, nitro group, C １ -C ６ Chain-type alkyl group, C １ -C ６ It is substituted with one or more selected from the group consisting of alkoxy groups and combinations thereof, R ７ is a benzyl group or C １ -C ６ The alkylamino group is an alkylamino group, and the benzyl group is unsubstituted or a hydroxyl group, halogen group, cyano group, nitro group, C １ -C ６ Chain-type alkyl group, C １ -C ６ It is substituted with one or more of the alkoxy groups and combinations thereof selected from the group, R ３ and R ７ They are connected to each other and C １５ -C ２０ Forms a heterocycloalkene group, R ８ , R ９ , and R １０ They are either identical or different from each other, and each is independently hydrogen or C １ -C ６ It is an alkyl group, n is either 0 or 1, If n is 0, R ４ is C ２ -C ６ It is an alkylcarboxyl group, R ５ , R ６ , and R ７ It is a benzyl group, However, R １ NH ２ And R ２ OH is R ３ is hydrogen, R ４ is an isobutyl group, R ５ and R ６ All of them are hydroxybenzyl groups, R ７ R is an unsubstituted benzyl group, ８ , R ９ , and R １０ All of them are hydrogen, except when n is 1.

2. The aforementioned R ４ teeth, 【Chemistry 2】 The peptide derivative according to claim 1, characterized in that it is one or more selected from the group consisting of the following and combinations thereof.

3. The aforementioned R ５ and R ６ They are either identical or different from one another, and each is independent of the other. 【Transformation 3】 The peptide derivative according to claim 1, characterized in that it is one or more selected from the group consisting of the following and combinations thereof.

4. Said C １５ -C ２０ The heterocycloalkene group is 【Chemistry 4】 The peptide derivative according to claim 1, characterized in that it is one or more selected from the group consisting of the following and combinations thereof.

5. The peptide derivative according to claim 1, characterized in that the peptide derivative is one or more selected from the group consisting of peptide derivatives represented by the following chemical formulas [Chemical Formula 1-1] to [Chemical Formula 1-56]. 【Transformation 5】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】

6. The peptide derivative according to claim 1, characterized in that the peptide derivative is obtained by modifying one or more peptide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

16.

7. A composition for increasing adiponectin expression, comprising a peptide derivative according to any one of claims 1 to 6 as an active ingredient.

8. A pharmaceutical composition for the prevention or treatment of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, comprising a peptide derivative according to any one of claims 1 to 6 as an active ingredient.

9. A cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer, comprising a peptide derivative according to any one of claims 1 to 6 as an active ingredient.

10. A pharmaceutical composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, comprising a peptide derivative according to any one of claims 1 to 6 as an active ingredient.

11. A cosmetic composition for the prevention or improvement of one or more conditions selected from the group consisting of aging, sensitive skin, wrinkles, and moisturizing, comprising a peptide derivative according to any one of claims 1 to 6 as an active ingredient.

12. Use of a peptide derivative according to any one of claims 1 to 6 for the manufacture of one or more pharmaceuticals selected from the group consisting of inflammatory skin diseases, wounds, hair loss, fibrosis, metabolic diseases, and cancer for the prevention or treatment of a disease.

13. Use of a peptide derivative according to any one of claims 1 to 6 for the manufacture of a cosmetic composition for improving one or more of the following: aging, sensitive skin, wrinkles, and moisturizing.

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