Compositions for rejuvenating senescent cells or for delaying or preventing cellular senescence

Abstract

The composition of the present invention for rejuvenating senescent cells or delaying or preventing cellular senescence comprises an ATM inhibitor and a ROCK inhibitor, and includes one or more selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator.

Description

[Technical field]

[0001] The present invention relates to a composition for rejuvenating senescent cells or delaying or preventing cellular aging. More specifically, the present invention relates to a composition for rejuvenating senescent cells or delaying or preventing cellular aging through a reverse aging effect that returns senescent cells to a young cell state, such as by causing senescent cells that have reached the Hayflick limit to resume cell division or by reducing the accumulation of undesirable metabolic products associated with cellular aging, such as lipofuscin. [Background technology]

[0002] As life expectancy increases due to advances in medical technology, problems related to aging-related diseases are on the rise. For this reason, many researchers are working to elucidate the mechanisms of aging and use this knowledge to address problems related to aging, such as aging-related diseases.

[0003] Cellular senescence refers to the process of the degeneration of cellular characteristics and functions, as well as the associated cessation of proliferation, and generally refers to the loss of the ability to divide. Unlike cancer cells, normal cells reach the Hayflick limit, at which they can no longer divide, after 40 to 60 cell divisions. When cells senesce, unlike young cells, metabolic waste products such as lipofuscin accumulate, causing various degenerative diseases.

[0004] Various research is being conducted to solve the problem of cellular aging, and in the 2010s, through an experiment in which gene modification technology was used to remove only senescent cells in aged mice, it was discovered that removing senescent cells in an aged individual increases both the lifespan and healthspan of the aged individual, opening the door to the field of senotherapeutics. Since then, there has been active development of senolytics, compounds that remove senescent cells, and senomorphics, compounds that suppress the traits of senescent cells.

[0005] While it is true that anti-aging drugs can extend an individual's lifespan and slow down aging, to date the effects have not been so dramatic, and no anti-aging drugs have been reported that can help senescent cells surpass the Hayflick limit and divide at the same rate as young cells.

[0006] As the causes of aging are multifactorial, the cell signaling pathways involved are also diverse. Recently, research results have been reported showing that when cells are treated with an ATM (ataxia telangiectasia mutated) inhibitor (ATM kinase inhibitor) and a ROCK (Rho-associated, coiled-coil containing protein kinase) inhibitor, they resume cell division as if they had rejuvenated and become young cells. In particular, recent research results have shown that when ATM inhibitor KU60019 and ROCK inhibitor Y27632 are treated simultaneously, a synergistic effect is exerted on this rejuvenation effect.

[0007] The present inventors aimed to develop a technology that can more effectively solve the problem of cellular senescence by further developing such existing technology, and further prevent, ameliorate and treat diseases associated with such cellular senescence. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Yang Eun-jae “revitalization of senescent cells via sequential activation of FOXM1 and E2F1.”, Doctoral dissertation, Department of New Biology, Gyeongbuk Institute of Science and Technology, Daegu, 2022 Summary of the Invention [Problem to be solved by the invention]

[0009] The main object of the present invention is to effectively solve the problems associated with cellular aging by providing a composition that can rejuvenate aging cells through a reverse aging effect that returns aging cells to a young cell state, such as by causing aging cells that have reached the Hayflick limit to resume cell division or by reducing the accumulation of undesirable metabolic products associated with cellular aging, or by delaying or preventing cellular aging. [Means for solving the problem]

[0010] The present invention provides a composition for rejuvenating senescent cells or delaying or preventing cellular senescence, comprising an ATM inhibitor and a ROCK inhibitor, and one or more selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator. Effect of the Invention

[0011] The composition of the present invention can rejuvenate senescent cells that have reached the Hayflick limit, or delay or prevent cellular aging through its anti-aging effect of returning senescent cells to a young cell state, such as by causing senescent cells to resume cell division or by reducing the accumulation of undesirable metabolic products associated with cellular aging, such as lipofuscin. In particular, the composition of the present invention can provide a synergistic effect on rejuvenating senescent cells or delaying or preventing cellular aging by combining specific active ingredients. In addition, the composition of the present invention can effectively prevent, treat or improve cellular aging-related diseases based on the above-mentioned effects. In addition, the composition of the present invention can effectively prevent or improve skin aging, loss of skin elasticity, skin wrinkles, skin keratin, etc. based on the above-mentioned effects. [Brief description of the drawings]

[0012] [Figure 1] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Diagram 2] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Diagram 3] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 4] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Diagram 5] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 6] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 7] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 8] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 9] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 10] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 11] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 12] The results of an experiment using Hoechst analysis to compare the cell division ability of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1 to 7) with other cases (Comparative Examples 1 to 7) are shown. [Figure 13]The results of an experiment using β-galactosidase staining to compare the degree of senescence of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 4, and 7) with other cases (Comparative Example 2) are shown below. K, KU60019; Y, Y27632; A, ABT263; R, Resveratrol; M, Metformin; K+Y, Comparative Example 2; K+Y+A, Example 1; K+Y+A+R, Example 4; K+Y+A+R+M, Example 7. [Figure 14] The results of an experiment using β-galactosidase staining to compare the degree of senescence of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 4, and 7) with other cases (Comparative Example 2) are shown below. K, KU60019; Y, Y27632; A, ABT263; R, Resveratrol; M, Metformin; K+Y, Comparative Example 2; K+Y+A, Example 1; K+Y+A+R, Example 4; K+Y+A+R+M, Example 7. [Figure 15] The results of an experiment using β-galactosidase staining to compare the degree of senescence of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 4, and 7) with other cases (Comparative Example 2) are shown below. K, KU60019; Y, Y27632; A, ABT263; R, Resveratrol; M, Metformin; K+Y, Comparative Example 2; K+Y+A, Example 1; K+Y+A+R, Example 4; K+Y+A+R+M, Example 7. [Figure 16] The following shows the results of observing the morphology of senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 4, and 7) in comparison with other cases (Comparative Example 2). K, KU60019; Y, Y27632; A, ABT263; R, Resveratrol; M, Metformin; K+Y, Comparative Example 2; K+Y+A+R+M, Example 7. [Figure 17] The results of an experiment in which the amount of lipofuscin in senescent cells was compared when treated under conditions according to one embodiment of the present invention (Examples 1, 6 and 7) with other cases (Comparative Example 2) are shown. [Figure 18]The results of an experiment were shown in which the mRNA expression levels of MMP-1 (Figure 18) and MMP-3 (Figure 19) in senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 2, 3 and 7) were compared with other cases (Comparative Example 2). [Figure 19] The results of an experiment were shown in which the mRNA expression levels of MMP-1 (Figure 18) and MMP-3 (Figure 19) in senescent cells treated under conditions according to one embodiment of the present invention (Examples 1, 2, 3 and 7) were compared with other cases (Comparative Example 2). [Figure 20] The results of preclinical experiments on the effectiveness of administration of the composition of the present invention in improving skin keratin (FIG. 20) and wrinkles (FIG. 21) are shown. [Figure 21] The results of preclinical experiments on the effectiveness of administration of the composition of the present invention in improving skin keratin (FIG. 20) and wrinkles (FIG. 21) are shown. [Figure 22] FIG. 22 shows the results of an experiment confirming the tumorigenicity of senescent cells when treated under conditions according to one embodiment of the present invention (Example 7; combination of compounds), comparing the mRNA expression levels of Ki67 (FIG. 22) and CDKN2A (FIG. 23) with cancer cells. [Figure 23] FIG. 22 shows the results of an experiment confirming the tumorigenicity of senescent cells when treated under conditions according to one embodiment of the present invention (Example 7; combination of compounds), comparing the mRNA expression levels of Ki67 (FIG. 22) and CDKN2A (FIG. 23) with cancer cells. [Figure 24]1 shows the results of an experiment on the cell division ability of senescent cells when treated under conditions according to one embodiment of the present invention. With respect to the circles in the table in the figure, the circle in the "KU60019" item means that KU60019 was treated at a concentration of 0.5 μM, the circle in the "AZ32" item means that AZ32 was treated at a concentration of 0.5 μM, the circle in the "Y27632" item means that Y27632 was treated at a concentration of 2.5 μM, and the circle in the "ABT263" item means that ABT263 was treated at a concentration of 0.75 μM. The circle in the "ABT737" section means that ABT737 was treated at a concentration of 0.75 μM, the circle in the "linderalactone" section means that linderalactone was treated at a concentration of 0.5 μM, the circle in the "resveratrol" section means that resveratrol was treated at a concentration of 1.5 μM, and the circle in the "metformin" section means that metformin was treated at a concentration of 6 μM. [Diagram 25] 1 shows the results of an experiment on the cell division ability of senescent cells when treated under conditions according to one embodiment of the present invention. With respect to the circles in the table in the figure, the circle in the "KU60019" item means that KU60019 was treated at a concentration of 0.5 μM, the circle in the "AZ32" item means that AZ32 was treated at a concentration of 0.5 μM, the circle in the "Y27632" item means that Y27632 was treated at a concentration of 2.5 μM, and the circle in the "ABT263" item means that ABT263 was treated at a concentration of 0.75 μM. The circle in the "ABT737" section means that ABT737 was treated at a concentration of 0.75 μM, the circle in the "linderalactone" section means that linderalactone was treated at a concentration of 0.5 μM, the circle in the "resveratrol" section means that resveratrol was treated at a concentration of 1.5 μM, and the circle in the "metformin" section means that metformin was treated at a concentration of 6 μM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The composition of the present invention for rejuvenating senescent cells or delaying or preventing cellular senescence comprises an ATM inhibitor and a ROCK inhibitor, and is characterized by containing one or more selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator.

[0014] In the present invention, an ATM inhibitor refers to a substance that inhibits the action of ATM (ataxia telangiectasia mutated) kinase, for example, the Ser15 phosphorylation action of p53. In one embodiment, the ATM inhibitor is any compound conventionally known to inhibit ATM kinase. In a specific embodiment, the ATM inhibitor is "KU60019" (CAS925701-46-8) (chemical formula 1) or "AZ32" (CAS2288709-96-4) (chemical formula 2), or a derivative or analog thereof.

[0015] [ka]

[0016] [ka]

[0017] In the present invention, a ROCK inhibitor refers to a substance that inhibits the action of ROCK (Rho-associated protein kinase) (ROCK1 and / or ROCK2), for example, the binding action of ROCK to Rho GTPase and / or the action of mediating the activity of Rho GTPase. In one embodiment, the ROCK inhibitor is any compound conventionally known to inhibit ROCK. In a specific embodiment, the ROCK inhibitor is "Y27632" (CAS129830-38-2) (chemical formula 3) or a derivative or analog thereof.

[0018] [ka]

[0019] In the present invention, the Bcl-2 inhibitor is a Bcl-2 (B-cell lymphoma 2), such as by binding to the apoptotic effector Bax and Bak proteins. In one embodiment, the Bcl-2 inhibitor is any compound conventionally known to inhibit Bcl-2. In a particular embodiment, the Bcl-2 inhibitor is "ABT263" (CAS923564-51-6) (chemical formula 4), "ABT737" (CAS852808-04-9) (chemical formula 5), ​​or "linderalactone" (CAS728-61-0) (chemical formula 6), or a derivative or analog thereof.

[0020] [ka]

[0021] [ka]

[0022] [ka]

[0023] In the present invention, SIRT1 activator means a substance that activates the action of SIRT1 (sirtuin 1). In one embodiment, SIRT1 activator is any compound that is conventionally known to activate SIRT1. In a specific embodiment, SIRT1 activator is resveratrol or its derivative or analogue.

[0024] In the present invention, the AMPK activator refers to a substance that activates the action of AMPK (AMP-activated kinase). In one embodiment, the AMPK activator is any compound conventionally known to activate AMPK. In one embodiment, the AMPK activator is an MC1 (mitochondrial complex 1) inhibitor. In one embodiment, the MC1 inhibitor is any compound conventionally known to inhibit MC1. In a particular embodiment, the AMPK activator is metformin, which is also an MC1 inhibitor, or a derivative or analog thereof.

[0025] The compounds referred to herein, unless otherwise specified, are intended to include salt forms of the compounds, e.g., pharma- ceutically, edible and / or cosmetically acceptable salt forms.

[0026] As used herein, rejuvenation of senescent cells means restoring various characteristics of senescent cells to the same or similar characteristics of young cells. For example, it may mean reversing aging in which old cells that have reached the Hayflick limit and no longer proliferate resume vigorous proliferation like young cells, and / or reversing aging markers such as β-galactosidase staining and lipofuscin to the level of young cells (e.g., the expression level observed in young cells). In one embodiment, the composition of the present invention has one or more activities selected from the group consisting of cell proliferation activity increasing activity, lipofuscin accumulation reducing activity, and β-galactosidase activity reducing activity.

[0027] The rejuvenation of senescent cells can be distinguished from tumorigenesis or cancer cell transformation. For example, the rejuvenation of senescent cells can be distinguished from tumorigenesis or cancer cell transformation by regulating the expression of cell growth factors (e.g., Ki67) and / or cell cycle regulators (e.g., CDKN2A) within normal ranges (expression levels observed in normal cells, not expression levels observed in tumor or cancer cells).

[0028] The composition of the present invention can cause senescent cells that have reached the Hayflick limit to resume cell division, or can rejuvenate senescent cells through its anti-aging effect of returning senescent cells to a young cell state, such as reducing the accumulation of undesirable metabolic products associated with cellular aging, such as lipofuscin. In this context, the composition of the present invention can be a composition for inducing anti-aging.

[0029] In one embodiment of the invention, the cell is an animal, e.g., a mammalian, e.g., a human cell, and in a particular embodiment, a skin cell, e.g., a skin fibroblast, and in another particular embodiment, a colon cell, e.g., a colon fibroblast.

[0030] In one embodiment, the composition of the present invention comprises an ATM inhibitor, a ROCK inhibitor, and a Bcl-2 inhibitor. In another embodiment, the composition of the present invention comprises an ATM inhibitor, a ROCK inhibitor, and a SIRT1 activator. In another embodiment, the composition of the present invention comprises an ATM inhibitor, a ROCK inhibitor, and an AMPK activator.

[0031] In a preferred embodiment, the composition of the present invention comprises an ATM inhibitor, a ROCK inhibitor and a Bcl-2 inhibitor, and includes one or more selected from the group consisting of a SIRT1 activator and an AMPK activator.

[0032] In a more preferred embodiment, the composition of the present invention comprises all of an ATM inhibitor, a ROCK inhibitor, a Bcl-2 inhibitor, a SIRT1 activator and an AMPK activator.

[0033] In one embodiment, the composition of the present invention contains only the active ingredients listed, i.e., ATM inhibitor, ROCK inhibitor, Bcl-2 inhibitor (if included), SIRT1 activator (if included), and AMPK activator (if included), as active ingredients. In another embodiment, the composition of the present invention also contains other active ingredients other than the listed ingredients.

[0034] In order to obtain a higher expected effect of the present invention, the molar ratio of the ATM inhibitor to the ROCK inhibitor in the composition of the present invention is 1:2.5 to 20, more preferably 1:2.5 to 15, more preferably 1:3 to 10, more preferably 1:3.5 to 8, more preferably 1:4 to 7, and more preferably The ratio is usually 1:4.5~6.

[0035] In an embodiment in which a Bcl-2 inhibitor is included, in order to achieve a higher expected effect of the present invention, the molar ratio of the ATM inhibitor to the Bcl-2 inhibitor in the composition of the present invention is 1:0.25-6, more preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.1-3, more preferably 1:1.2-2.5, and even more preferably 1:1.3-2.

[0036] In embodiments in which a SIRT1 activator is included, in order to achieve a higher expected effect of the present invention, the molar ratio of ATM inhibitor to SIRT1 activator in the composition of the present invention is 1:1.5-6, more preferably 1:1.8-5.5, more preferably 1:2-5, more preferably 1:2.1-4.5, more preferably 1:2.3-4.3, more preferably 1:2.5-4.

[0037] In an embodiment in which an AMPK activator is included, in order to achieve a higher expected effect of the present invention, the molar ratio of the ATM inhibitor to the AMPK activator in the composition of the present invention is 1:6-24, more preferably 1:7-22, more preferably 1:8-20, more preferably 1:9-18, more preferably 1:9.5-16, more preferably 1:10-15.

[0038] The composition of the present invention may be in a form in which the active ingredient is dissolved or suspended in a medium, for example, in the form of an aqueous solution.

[0039] In one embodiment, the composition of the present invention is a composition provided for use without additional dilution or addition of additional substances. In another embodiment, the composition of the present invention is a composition provided for use by dilution or suspension in a suitable medium (e.g., a solvent, e.g., water).

[0040] In an embodiment of the composition provided for use without further dilution or addition of substances, in order to achieve a higher expected effect, the composition of the present invention contains the ATM inhibitor at a concentration of 0.125 μM or more, for example, 0.125 to 1.5 μM, more preferably at a concentration of 0.25 μM or more, for example, 0.25 to 1 μM, and based on this concentration, other active ingredients are present at concentrations in the indicated percentage ranges.

[0041] In the case of an embodiment of a composition that is provided to be used by dilution or suspension in a suitable vehicle, it may contain, for example, a concentration of 1.5 times (ATM inhibitor concentration 0.75 μM) or more, for example 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times the concentration of a 0.5 μM ATM inhibitor.

[0042] In one embodiment, the composition of the present invention is a pharmaceutical composition for the prevention or treatment of a cellular senescence associated disease.

[0043] In another embodiment, the composition of the present invention is a functional food composition for preventing or improving cellular aging-related diseases, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

[0044] In another embodiment, the composition of the present invention is a cosmetic composition for preventing or improving cellular aging-related diseases, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

[0045] In another embodiment, the composition of the present invention is for preventing or improving a cellular senescence-related disease, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or or a functional feed composition for preventing or improving skin keratin.

[0046] In the present invention, the cellular aging-associated disease may be a skin cellular aging-associated disease, for example, a disease selected from the group consisting of hair loss, gray hair, wrinkles, xeroderma, xerosis, keratin, melasma, age spot, delayed wound healing, dermatitis, psoriasis, scleroderma, vitiligo, and systemic lupus erythematosus.

[0047] In the present invention, the cellular senescence-associated disease may be a disease selected from the group consisting of lipofuscin accumulation-associated diseases, such as lipofuscinosis, macular degeneration, neuronal ceroid lipofuscinoses (NCL), Stargardt disease, neurofibrillary tangles, brown atrophy of the heart, acromegaly, denervation atrophy, lipid myopathy, and chronic obstructive pulmonary disease (COPD).

[0048] Other examples of the cellular senescence-related diseases in the present invention include progeria (Hutchinson-Gilford progeria syndrome or Werner syndrome), hunched back, osteoporosis, osteoarthritis, rheumatoid arthritis, benign prostatic hyperplasia, presbycusis, rhinitis, immunosenescence, post transplantation late and chronic solid organ rejection, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, Graves' disease, and other conditions. disease, polymyositis, sarcopenia, Addison disease, glomerulonephritis, pernicious anemia, asthma, cystic fibrosis, pulmonary fibrosis, inflammatory bowel diseases, autoimmune diabetes, type 2 diabetes mellitus, diabetic retinopathy, hypertension, arrhythmia, angina, chronic obstructive pulmonary diseases;COPD), ischemic heart disease, ischemia-reperfusion injury, post-angioplasty restenosis, atherosclerosis, coronary artery disease, gastrointestinal allergies allergies, cancer, frailty, small artery disease and graft-versus-host disease, conjunctivitis, glaucoma, cataract, presbyopia, stroke, dementia, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), but is not limited thereto, and may also include diseases known to those of ordinary skill in the art as diseases caused by cellular senescence.

[0049] The composition of the present invention can be formulated into an appropriate dosage form by using one or more pharma- ceutical , food- and / or feed-acceptable carriers, additives, etc. in combination with the active ingredient of the present invention.

[0050] The composition of the present invention can be administered orally or parenterally in clinical administration.When administered parenterally, it can be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine intradural injection, intracerebrovascular injection or intrathoracic injection, and can be used in the form of a general pharmaceutical preparation.

[0051] The compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy, and methods employing biological response modifiers.

[0052] The dosage of the composition of the present invention varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and can be, for example, about 0.0001 to 100 mg per kg of subject body weight per day.

[0053] The food composition of the present invention may be, for example, a health functional food including processed meat products, fish products, tofu, muk (a traditional Korean jelly-like food), porridge, noodles such as ramen and soba, seasoned foods such as soy sauce, miso, chili miso, mixed miso, sauces, confectioneries, dairy products such as fermented milk and cheese, pickled foods such as kimchi and pickles, fruits, vegetables, soy milk, beverage products such as fermented drinks, etc.

[0054] The feed compositions of the present invention also come in a variety of forms and may be feed compositions for farm animals (e.g., sheep, pigs, cows, horses, goats), laboratory test animals (e.g., mice, rabbits, rats and guinea pigs), and pets (e.g., dogs and cats).

[0055] In one embodiment, the composition of the present invention is a composition, for example a cosmetic composition, for preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

[0056] The cosmetic composition of the present invention can be formulated into a cosmetic formulation by using the active ingredient of the present invention and one or more cosmetically acceptable carriers, additives, etc. In this case, the carriers and additives may also include pharma- ceutically acceptable ones.

[0057] The cosmetic composition of the present invention may be formulated into various forms, such as a mist, a moisturizing cream, a nourishing lotion, a nourishing cream, a massage cream, a pack, and a nourishing essence.

[0058] In one embodiment, the composition of the invention is a cell culture medium composition or a medium additive composition.

[0059] The medium composition of the present invention may be in a form in which the active ingredient of the present invention is added with a normal medium component suitable for the cells to be cultured. For example, in the case of an embodiment of a medium composition for culturing human skin fibroblasts, the active ingredient of the present invention may be added to DMEM (Dulbecco's modified Eagle's medium) at the ratio shown.

[0060] The present invention also relates to a method for rejuvenating senescent cells, comprising the step of treating senescent cells with one or more selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator, together with an ATM inhibitor and a ROCK inhibitor.

[0061] The present invention also relates to a method for preventing, treating or ameliorating a cellular senescence-associated disease, the method comprising the step of administering to a subject in need thereof one or more agents selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator and an AMPK activator, together with an ATM inhibitor and a ROCK inhibitor.

[0062] The present invention also relates to a method for preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin, the method comprising the step of administering to a subject in need thereof one or more selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator, together with an ATM inhibitor and a ROCK inhibitor.

[0063] In one embodiment, the method of the present invention is to treat or administer an ATM inhibitor, a ROCK inhibitor and a Bcl-2 inhibitor. In another embodiment, the method of the present invention is to treat or administer an ATM inhibitor, a ROCK inhibitor and a SIRT1 activator. In another embodiment, the method of the present invention is to treat or administer an ATM inhibitor, a ROCK inhibitor and an AMPK activator.

[0064] In a preferred embodiment, the method of the present invention involves treating or administering one or more selected from the group consisting of a SIRT1 activator and an AMPK activator together with an ATM inhibitor, a ROCK inhibitor and a Bcl-2 inhibitor.

[0065] In a more preferred embodiment, the method of the present invention involves treating or administering all of an ATM inhibitor, a ROCK inhibitor, a Bcl-2 inhibitor, a SIRT1 activator and an AMPK activator.

[0066] For achieving a higher expected effect of the present invention, the molar ratio of the ATM inhibitor to the ROCK inhibitor in the method of the present invention is 1:2.5-20, more preferably 1:2.5-15, more preferably 1:3-10, more preferably 1:3.5-8, more preferably 1:4-7, more preferably 1:4.5-6.

[0067] In an embodiment in which a Bcl-2 inhibitor is treated or administered, in order to achieve a higher expected effect of the present invention, the molar ratio of the ATM inhibitor to the Bcl-2 inhibitor in the method of the present invention is 1:0.25-6, more preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.1-3, more preferably 1:1.2-2.5, and even more preferably 1:1.3-2.

[0068] In an embodiment in which a SIRT1 activator is treated or administered, for higher expected effects of the present invention, the molar ratio of the ATM inhibitor to the SIRT1 activator in the method of the present invention is 1:1.5 to 6, more preferably 1:1.8 to 5.5, more preferably 1:2 to 5, more preferably 1:2.1 to 4.5, more preferably 1:2.3 to 4.3, more preferably 1:2.5 to The answer is 4.

[0069] In an embodiment in which an AMPK activator is treated or administered, for higher expected effects of the present invention, the molar ratio of the ATM inhibitor to the AMPK activator in the method of the present invention is 1:6-24, more preferably 1:7-22, more preferably 1:8-20, more preferably 1:9-18, more preferably 1:9.5-16, more preferably 1:10-15.

[0070] The subject in the methods of the invention may be a mammal, such as humans, primates and livestock animals (e.g., sheep, pigs, cows, horses, goats), laboratory test animals (e.g., mice, rabbits, rats and guinea pigs), and pets (e.g., dogs and cats).

[0071] The administration in the method of the present invention is oral or parenteral. In the case of parenteral administration, it may be by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine intradural injection, intracerebrovascular injection or intrathoracic injection.

[0072] The methods of the invention can be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy, and methods using biological response modifiers.

[0073] In one embodiment, the method of the present invention provides for the ATM inhibitor to be administered at a single dose of 3 to 10 mg / kg, preferably 4 to 8 mg / kg, and more preferably 4.5 to 6 mg / kg.

[0074] The present invention will be described in more detail below with reference to examples. These examples are merely for illustrating the present invention, and therefore the scope of the present invention is not to be construed as being limited by these examples. EXAMPLES

[0075] cell culture The human dermal fibroblasts used in the experiment were samples taken from the skin of a newborn baby (PCS-201-010; ATCC). The cells were cultured in Dulbecco's modified Eagle's medium [25 mM glucose, 10% FBS (S001-01; Welgene), 100 units / mL penicillin, 10 μg / mL streptomycin, and 25 ng / mL amphotericin B (LS203-01; Welgene)] at 37°C and 5% CO. 2 The cells were cultured under the following conditions. Subculture was performed whenever the cell confluency (cell distribution rate on the bottom of the cell culture tank) did not exceed 80%. Subculture was continued by newly culturing only 1 / 4 of the cells collected from the first subculture of newborn skin up to the 48th subculture. From the 48th subculture, only 1 / 2 of the cells were subcultured. When the population doubling time (DT) exceeded 14 days and the proportion of SA-β exceeded 95%, the cells were regarded as sufficiently aged and experiments were carried out. In contrast, only cells that had been subcultured less than 10 times and had a population doubling time (DT) of 1 day or less were regarded as young cells and experiments were carried out.

[0076] The human breast skin fibroblast cells (CCD-986Sk) used in the experiment were samples taken from the breast of a 22-year-old black woman (CRL-1947; ATCC). The cells were cultured in Dulbecco's modified Eagle's medium [25 mM glucose, 10% FBS (S001-01; Welgene), 100 units / mL penicillin, 10 μg / mL streptomycin, and 25 ng / mL amphotericin B (LS20 3-01; Welgene)] culture medium at 37°C and 5% CO 2The cells were cultured under the following conditions. Subculture was performed whenever the cell confluency (cell distribution rate on the bottom of the cell culture vessel) did not exceed 80%. Subculture was continued by newly culturing only 1 / 4 of the cells collected from the first subculture to the 15th subculture. From the 15th subculture, subculture was continued by newly culturing 1 / 2 of the cells.

[0077] The experiment was performed on cells at passage 19. When the population doubling time (DT) exceeded 14 days and the proportion of SA-β exceeded 95%, the cells were considered to be sufficiently senescent and the experiment was carried out.

[0078] The human colon fibroblast cells (CCD-18Co) used in the experiment were samples (CRL-1459; ATCC) taken from the colon of a 2.5-month-old black female infant. The cells were cultured in Dulbecco's modified Eagle's medium [25 mM glucose, 10% FBS (S001-01; Welgene), 100 units / mL penicillin, 10 μg / mL streptomycin, and 25 ng / mL amphotericin B (LS203-01; Welgene)] at 37°C and 5% CO. 2 The cells were cultured under the following conditions. Subculture was performed whenever the cell confluency (cell distribution rate on the bottom of the cell culture tank) did not exceed 80%. Subculture was continued by newly culturing only 1 / 4 of the cells collected from the first subculture to the 10th subculture. From the 10th subculture onwards, only 1 / 2 of the cells were cultured. The experiment was performed on the 16th subculture cell. When the population doubling time (DT) exceeded 14 days and the proportion of SA-β exceeded 95%, the cells were considered to have aged sufficiently and the experiment was carried out.

[0079] Example 1 Senescent cells were prepared and treated with ATM inhibitors (KU60019 or AZ32) at a concentration of 0.5 μM, ROCK inhibitor (Y27632) at a concentration of 2.5 μM, and Bcl-2 inhibitors (ABT263, ABT737, or linderalactone) at concentrations of 0.5 to 2.5 μM in the medium, once every 4 days for a total of 4 treatments for 16 days.

[0080] Comparative Example 1 The same procedures as in Example 1 were carried out, except that no ATM inhibitor or ROCK inhibitor was added, and only a Bcl-2 inhibitor was added to the medium at a concentration of 0.1 to 10 μM.

[0081] Comparative Example 2 The same procedure as in Example 1 was carried out, except that no Bcl-2 inhibitor was added, and only the ATM inhibitor and the ROCK inhibitor were added to the medium.

[0082] Comparative Example 3 The same procedures as in Example 1 were carried out, except that a Bcl-2 inhibitor was added to the medium at a concentration of 0.1 μM or 5 to 10 μM.

[0083] Example 2 The same procedures as in Example 1 were carried out, except that a SIRT1 activator (resveratrol) was added to the medium at a concentration of 1 to 2 μM instead of the Bcl-2 inhibitor.

[0084] Comparative Example 4 The same procedures as in Example 2 were carried out, except that no ATM inhibitor and no ROCK inhibitor were added, and only the SIRT1 activator was added to the medium at a concentration of 0.1 to 50 μM.

[0085] Comparative Example 5 The same procedure as in Example 2 was carried out, except that the SIRT1 activator was added to the medium at a concentration of 4 to 10 μM.

[0086] Example 3 The same procedures as in Example 1 were carried out, except that an AMPK activator (metformin) was added to the medium at a concentration of 3 to 12 μM instead of the Bcl-2 inhibitor.

[0087] Comparative Example 6 The same procedure as in Example 3 was carried out, except that no ATM inhibitor or ROCK inhibitor was added, and only the AMPK activator was added to the medium at a concentration of 3 to 200 μM.

[0088] Comparative Example 7 The same procedure as in Example 3 was followed, except that the AMPK activator was added to the medium at a concentration of 24 μM.

[0089] Example 4 The same procedure as in Example 1 was carried out, except that a Bcl-2 inhibitor was added to the medium at a concentration of 0.5 to 0.75 μM, and a SIRT1 activator was further added to the medium at a concentration of 1.5 to 2 μM.

[0090] Example 5 The same procedure as in Example 4 was carried out, except that an AMPK activator was added to the medium at a concentration of 6 μM instead of the SIRT1 activator.

[0091] Example 6 The same procedure as in Example 2 was followed, except that an AMPK activator was added to the medium at a concentration of 6 μM.

[0092] Example 7 The same procedure as in Example 4 was carried out, except that an AMPK activator was further added to the medium at a concentration of 6 μM.

[0093] Examples 1 to 7 and Comparative Examples 1 to 7 are summarized in Table 1 below.

[0094] [Table 1]

[0095] Experimental example 1. Cell division confirmation experiment The cell division ability of senescent cells treated with the compounds in the above Examples and Comparative Examples was examined.

[0096] The cells used in the experiment were transplanted into well plates 3 days before the experiment. The compound-treated cells were stained with 10 μg / mL Hoechst 33342 for 30 minutes, washed twice with PBS (phosphate-buffered saline), and the amount of cells was quantified using a plate reader (FLUOstar Omega) at ex / em: 355 / 460 nm wavelength band.

[0097] 1 to 10 show the experimental results for each of the examples and comparative examples in which human dermal fibroblasts collected from the skin of a newborn were used as senescent cells.

[0098] Senescent cells that reach the Hayflick limit through subculture no longer undergo cell division. However, similar to previous research results, we confirmed that when treated with ATM inhibitors and ROCK inhibitors (Comparative Example 2), the cells resumed cell division as if they had been rejuvenated and become young cells. In addition, when treated with Bcl-2 inhibitors, SIRT1 activators, or AMPK activators at specific concentrations (Examples 1 to 3), the cell rejuvenation effect was more pronounced, and the effect was particularly good when treated with a Bcl-2 inhibitor (Example 1) (Figures 1 to 3).

[0099] On the other hand, when the Bcl-2 inhibitor, SIRT1 activator or AMPK activator was treated alone (Comparative Examples 1, 4 and 6), rejuvenation of senescent cells could not be induced (Figures 4 to 6).

[0100] Furthermore, the cell rejuvenation effect was more pronounced when a SIRT1 activator or an AMPK activator was added together with a Bcl-2 inhibitor (Examples 4 and 5) than when only a Bcl-2 inhibitor was added to a base of an ATM inhibitor and a ROCK inhibitor (Figure 7).

[0101] Furthermore, the cell rejuvenation effect was more pronounced when the SIRT1 activator and the AMPK activator were treated together (Example 6) than when only one of the SIRT1 activator and the AMPK activator was additionally treated with the ATM inhibitor and the ROCK inhibitor (Examples 2 and 3) (Figure 7).

[0102] When an ATM inhibitor and a ROCK inhibitor were combined with two selected from a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator (Examples 4 to 6), the effect was particularly good when a Bcl-2 inhibitor was included (Examples 4 and 5) (Figure 7).

[0103] Furthermore, the cell rejuvenation effect was superior when the ATM inhibitor, ROCK inhibitor, Bcl-2 inhibitor, SIRT1 activator and AMPK activator were all treated (Example 7) compared to the above cases, and the senescent cells treated in this manner showed characteristics that were indistinguishable from those of young cells (Figures 7 and 8).

[0104] The same results were obtained when AZ32 was used instead of KU60019 as the ATM inhibitor, and when ABT737 or linderalactone was used instead of ABT263 as the Bcl-2 inhibitor (Figures 8 to 10). In particular, AZ32 and linderalactone are compounds with a molecular weight of 500 Da or less, and have the advantage of being easily absorbed through the skin, which may be advantageous for application in cosmetics.

[0105] Figures 11 and 12 show the experimental results when other cells (human breast dermal fibroblasts (CCD-986Sk) and human colon fibroblasts (CCD-18Co) respectively) were used as senescent cells, and showed results similar to those shown above.

[0106] Experimental example 2. SA-β-gal staining experiment In order to examine the degree of senescence of the senescent cells treated with the compounds in the above Examples and Comparative Examples, β-galactosidase staining, which is an indicator of cellular senescence, was performed.

[0107] SA-β-gal staining was performed according to the method of X-gal-based Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860). Cells were transplanted 3 days before the experiment. The cells were washed with PBS, fixed with 4% formaldehyde for 3 minutes, and then washed twice with PBS. Then, the cells were fixed with β-gal solution (1 mg / mL X-gal, 5 mM K 4 Fe(CN) 6 , 5 mM K 3 Fe(CN) 6 , 2 mM MgCl 2 The cells were stained with PBS (150 mM NaCl, pH 6.0) for 24 hours in an incubator at 37° C. The next day, the cells were washed twice with PBS again, and the degree of staining of the cells was observed and counted using an Olympus CKX53 microscope.

[0108] 13 and 14 show the experimental results for each of the Examples and Comparative Examples when human dermal fibroblasts collected from the skin of a newborn were used as senescent cells.

[0109] Unlike young cells, senescent cells are stained blue by β-galactosidase staining, which is one of the biomarkers, but it was confirmed that the number of unstained cells increases when these senescent cells are treated with a compound. In particular, it was found that the effects were superior when ATM inhibitor + ROCK inhibitor + Bcl-2 inhibitor were treated (Example 1), ATM inhibitor + ROCK inhibitor + Bcl-2 inhibitor + SIRT1 activator were treated (Example 4), and ATM inhibitor + ROCK inhibitor + Bcl-2 inhibitor + Bcl-2 inhibitor + SIRT1 activator + AMPK activator were treated (Example 7) compared to when ATM inhibitor and ROCK inhibitor were treated (Comparative Example 2).

[0110] FIG. 15 shows the results of an experiment in which other cells (human colon fibroblasts (CCD-18Co)) were used as senescent cells, and the results were similar to those shown above.

[0111] Experimental example 3. Observation of cell morphology The senescent cells of each of the Examples and Comparative Examples in the state before staining in Experimental Example 2 were observed under a microscope.

[0112] FIG. 16 shows the experimental results for each of the Examples and Comparative Examples when human dermal fibroblasts collected from the skin of a newborn were used as senescent cells.

[0113] Senescent cells show a bulging and voluminous morphology, while young cells show a slimmer and smaller morphology, but when senescent cells are treated with a compound, they change to the typical slim and small morphology of young cells. In particular, it was found that the effect was superior when ATM inhibitor + ROCK inhibitor + Bcl-2 inhibitor + SIRT1 activator + AMPK activator were treated (K + Y + A + R + M, Example 7) compared to treatment with only ATM inhibitor and ROCK inhibitor (K + Y, Comparative Example 2).

[0114] This can also be confirmed from the results of the SA-β staining experiment in Experimental Example 2 (FIGS. 13 and 15).

[0115] Experimental Example 4. Lipofuscin quantification experiment The lipofuscin of senescent cells treated with the compounds in the above Examples and Comparative Examples was cin) was quantified.

[0116] The amount of lipofuscin per cell was quantified using a FACSCanto II flow cytometer. Because lipofuscin is autofluorescent, values ​​were measured at 488 nm without staining using a 530 / 30 nm bandpass filter (FITC channel).

[0117] FIG. 17 shows the experimental results for each of the Examples and Comparative Examples when human dermal fibroblasts collected from the skin of a newborn were used as senescent cells.

[0118] In senescent cells, lipofuscin, a type of metabolic waste product produced during the digestion or processing of various metabolites in the lysosomes within the cells, is produced. Lipofuscin, a yellowish-brown pigment granule, is not produced in young cells, where metabolic waste products are actively processed, but when produced in senescent cells, it causes various degenerative diseases.

[0119] It was confirmed that lipofuscin was reduced when senescent cells were treated with compounds. In particular, it was found that the effect was superior when Bcl-2 inhibitor was additionally treated (Example 1) and when SIRT1 activator and AMPK activator were additionally treated (Example 7) compared to when ATM inhibitor and ROCK inhibitor were treated (Comparative Example 2).

[0120] Experimental Example 5. Experiment to confirm the expression levels of MMP1 and MMP3 The expression levels of MMP1 and MMP3 in senescent cells treated with the compounds in the above Examples and Comparative Examples were confirmed by q-PCR. MMP1 and MMP3 are enzymes that degrade extracellular matrix and are highly related to skin elasticity and skin wrinkles.

[0121] Specifically, 10 μL of reagent (TOPreal qPCR 2X PreMIX), 1 μL of primer 1 (5-10 pmol / μL), 1 μL of primer 2 (5-10 pmol / μL), 1 μL of template RNA, and distilled water (RNase-free) were added to a microtube to make a total of 20 μL, and the reaction was carried out at 95°C for 10 minutes, after which 35 additional PCR reactions (95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 15 seconds) were performed.

[0122] The primers used for MMP1 and MMP3 are shown in Table 2 below.

[0123] [Table 2]

[0124] Figures 18 and 19 show the experimental results for each Example and Comparative Example when human dermal fibroblasts collected from the skin of a newborn were used as senescent cells. It was found that when senescent cells were treated with a compound, the mRNA expression levels of MMP1 and MMP3 were reduced. In particular, the effect of reducing mRNA expression of MMP1 and MMP3 was superior when treated with an ATM inhibitor and a ROCK inhibitor together with a Bcl-2 inhibitor (Example 1), a SIRT1 activator (Example 2) or an AMPK activator (Example 3), and when ATM inhibitor + ROCK inhibitor + Bcl-2 inhibitor + SIRT1 activator + AMPK activator were all treated together (Example 7), compared to when treated with only an ATM inhibitor and a ROCK inhibitor (Comparative Example 2). I found out that...

[0125] Experimental Example 6. Preclinical experiment on effectiveness in improving skin keratin and wrinkles A preclinical study was conducted on DBA mice (species and strain: DBA / 1J, producer and purchaser: Hana Shoji) to evaluate the effectiveness of the compound in improving skin keratin and wrinkles.

[0126] DBA mice are known to be suitable for analyzing aging characteristics and are widely used in metabolic index research through bioimage analysis, and were selected to confirm the degree of aging using analytical equipment. All mice were male, and 10 5-week-old mice were prepared as young mice and 20 16-month-old mice were prepared as old mice. When the animals were obtained, they were visually inspected and used for animal experiments after a 7-day acclimation period. During the experiment, when separating the animals into groups, individual identification was made on the tails of the animals using a permanent marker, and individual identification cards were attached to the breeding boxes. When separating the groups, the experimental group of old mice was randomly separated into two groups, with 10 mice per group, the day before the start of the experiment.

[0127] The ATM inhibitor (KU60019) 4.93 mg / kg, ROCK inhibitor (Y27632) 11.13 mg / kg, Bcl-2 inhibitor (ABT263) 13.16 mg / kg, SIRT1 activator (resveratrol) 6.16 mg / kg, and AMPK activator (metformin) 13.95 mg / kg were administered intravenously twice a week for a total of 8 weeks (drug treatment group).

[0128] For skin imaging, the animal was anesthetized by inhaling 3% isoflurane, and when it became motionless under anesthesia, it was placed in a camera equipped with a Skinscope (Visioscan® VC 20plus) and the images were taken, obtaining both keratin and wrinkle values ​​at the same time.

[0129] The results are shown in Figures 20 and 21.

[0130] Old mice had higher keratinocyte and wrinkle scores than young mice, but drug administration reduced the keratinocyte and wrinkle scores to almost the same level as young mice.

[0131] Experimental Example 7. Tumorigenicity Confirmation Experiment To confirm whether the cell rejuvenation effect was due to cell cancer or tumorigenesis, the expression levels of Ki67, a known cell growth factor, and CDKN2A, a known cell cycle regulator, were examined in comparison with cancer cells.

[0132] The same q-PCR method as in Experimental Example 5 was used, but the primers in Table 3 below were used.

[0133] [Table 3]

[0134] The results are shown in Figures 22 and 23. Here, the "combination of compounds" item refers to the case where ATM inhibitor (KU60019 treated at a concentration of 0.5 μM) + ROCK inhibitor (Y27632 treated at a concentration of 2.5 μM) + Bcl-2 inhibitor (ABT263 treated at a concentration of 0.75 μM) + SIRT1 activator (resveratrol treated at a concentration of 1.5 μM) + AMPK activator (metformin treated at a concentration of 6 μM) were treated together (Example 7). It was found that when senescent cells were treated with the compound, the expression of Ki67, known as a cell growth factor, increased within the normal range, and the expression of CDKN2A, known as a cell cycle regulator, also decreased within the normal range. This means that the substance combination according to the present invention can be used safely without worrying about cell tumorigenesis or cancer.

[0135] Experimental Example 8. Experiment on the effect of changing additional concentration Further experiments were carried out using a similar method to Example 1, but with more varied concentrations of the treatment substances.

[0136] Table 4 shows the experimental results when ATM inhibitor (KU60019) + ROCK inhibitor (Y27632) + Bcl-2 inhibitor (ABT263) + SIRT1 activator (resveratrol) + AMPK activator (metformin) were treated together, but the ATM inhibitor was treated at different concentrations of 0.125 to 1 μM, and the remaining substance (YARM) was treated at different concentrations of 0.25 to 1X. Each result is shown based on the cell division ability of the untreated group (100%). At this time, YARM 1X is 2.5 μM ROCK inhibitor, 0.75 μM Bcl-2 inhibitor, 1.5 μM SIRT1 activator, and 6 μM AMPK activator.

[0137] It was found that even when the concentration of the ATM inhibitor was changed within the molar ratio of the present invention, an excellent cell rejuvenation effect was observed.

[0138] [Table 4]

[0139] Table 5 shows the experimental results when ATM inhibitor (KU60019) + ROCK inhibitor (Y27632) + Bcl-2 inhibitor (ABT263) + SIRT1 activator (resveratrol) + AMPK activator (metformin) were treated together, ROCK inhibitor was treated at different concentrations of 1.25 to 10 μM, and the remaining substance (KARM) was treated at different concentrations of 0.25 to 1X. Each result is shown based on the cell division ability of the untreated group (100%). At this time, KARM 1X is 0.5 μM ATM inhibitor, 0.75 μM Bcl-2 inhibitor, 1.5 μM SIRT1 activator, and 6 μM AMPK activator.

[0140] It was found that even when the concentration of the ROCK inhibitor was changed within the molar ratio of the present invention, an excellent cell rejuvenation effect was observed.

[0141] [Table 5]

[0142] FIG. 24 shows the experimental results when ATM inhibitor+ROCK inhibitor+Bcl-2 inhibitor+SIRT1 activator+AMPK activator were treated together, but AZ32 was used as the ATM inhibitor.

[0143] It was also found that the use of AZ32 as an ATM inhibitor had an excellent cell rejuvenation effect.

[0144] FIG. 25 shows the experimental results when ATM inhibitor+ROCK inhibitor+Bcl-2 inhibitor+SIRT1 activator+AMPK activator were treated together, but linderalactone was used as the Bcl-2 inhibitor.

[0145] It was also found that using linderalactone as a Bcl-2 inhibitor had an excellent cell rejuvenation effect.

Claims

1. including an ATM inhibitor and a ROCK inhibitor, A composition for rejuvenating senescent cells or delaying or preventing cellular senescence, comprising one or more agents selected from the group consisting of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator.

2. comprising a Bcl-2 inhibitor, The composition of claim 1 , comprising one or more selected from the group consisting of a SIRT1 activator and an AMPK activator.

3. The composition of claim 1, comprising all of a Bcl-2 inhibitor, a SIRT1 activator, and an AMPK activator.

4. the molar ratio of the ATM inhibitor to the ROCK inhibitor is 1:2.5-20; the molar ratio of the ATM inhibitor to the Bcl-2 inhibitor is 1:0.25-6; the molar ratio of the ATM inhibitor to the SIRT1 activator is 1:1.5-6; The composition of claim 1, wherein the molar ratio of the ATM inhibitor to the AMPK activator is 1:6-24.

5. The composition according to claim 1, wherein the composition has one or more activities selected from the group consisting of an activity of increasing cell proliferation potential, an activity of reducing lipofuscin accumulation, and an activity of reducing β-galactosidase activity.

6. The composition according to claim 1 , wherein the composition is a pharmaceutical composition for preventing or treating a cellular senescence-related disease.

7. 2. The composition according to claim 1, which is a functional food composition for preventing or improving a cell aging-related disease, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

8. 2. The composition according to claim 1, which is a cosmetic composition for preventing or improving a cellular aging-related disease, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

9. The composition according to claim 1, which is a functional feed composition for preventing or improving a cell aging-related disease, preventing or improving skin aging, preventing or improving loss of skin elasticity, preventing or improving skin wrinkles, or preventing or improving skin keratin.

10. The composition according to claim 6 , wherein the composition is a pharmaceutical composition for preventing or treating a skin cell aging-related disease.

11. The composition according to claim 10, which is a pharmaceutical composition for preventing or treating a disease selected from the group consisting of hair loss, gray hair, wrinkles, xeroderma, senile xerosis, keratin, melasma, age spots, delayed wound healing, dermatitis, psoriasis, scleroderma, vitiligo, and systemic lupus erythematosus.

12. The composition according to claim 6, wherein the composition is a pharmaceutical composition for preventing or treating a lipofuscin accumulation-associated disease.

13. The composition is for use in treating lipofuscinosis, macular degeneration, neuronal ceroid lipofuscinoses (NCL), Stargardt disease, neurofibrillary tangles, brown atrophy of the heart, acromegaly, denervation atrophy, lipid storage myopathy, and chronic obstructive pulmonary disease.

13. The composition according to claim 12, which is a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of chronic obstructive pulmonary disease (COPD).

14. The composition is useful for the treatment of progeria (Hutchinson-Gilford progeria syndrome or Werner syndrome), kyphosis, osteoporosis, degenerative arthritis, rheumatoid arthritis, benign prostatic hyperplasia, presbycusis, rhinitis, immunosuppression, post transplantation late and chronic solid organ rejection, and osteoporosis, osteoarthritis, rheumatoid arthritis, benign prostatic hyperplasia, presbycusis, rhinitis, immunosuppression, post transplantation late and chronic solid organ rejection, osteoporosis, osteoarthritis, osteoporosis ... rejection, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, Graves disease, polymyositis, senile sarcopenia, Addison's disease, glomerulonephritis, pernicious anemia, asthma, cystic fibrosis, pulmonary fibrosis fibrosis), Inflammatory Bowel Diseases, Autoimmune Diabetes, Type 2 Diabetes mellitus), diabetic retinopathies, hypertension, arrhythmia, angina, chronic obstructive heart disease. pulmonary diseases (COPD), ischemic heart disease, ischemia-reperfusion injury, post-angioplasty restenosis, atherosclerosis, coronary artery disease, gastrointestinal allergies, cancer, frailty, small artery disease, and graft-versus-host disease.

7. The composition according to claim 6, which is a pharmaceutical composition for preventing or treating a disease selected from the group consisting of glaucoma, cataract, presbyopia, stroke, dementia, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS).

15. the ATM inhibitor is KU60019 or AZ32; the ROCK inhibitor is Y27632; the Bcl-2 inhibitor is ABT263, ABT737 or linderactone; The SIRT1 activator is resveratrol; The composition of claim 1 , wherein the AMPK activator is metformin.

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