Pharmaceutical composition for preventing and treating vascular disease caused senescencing of endothelial progenitor cells comprising stemregenin-1

Abstract

The present invention relates to the prevention, improvement, or treatment of vascular diseases caused by aging of vascular endothelial progenitor cells containing Stemregenin-1. More specifically, Stemregenin-1 has excellent effects in protecting vascular endothelial progenitor cells from stress and aging experienced during various in vitro cultures while maintaining the cell-specific characteristics, proliferative ability, motility, and differentiation ability of vascular endothelial progenitor cells, and thus can be applied to the prevention, improvement, or treatment of vascular diseases caused by aging of vascular endothelial progenitor cells.

Description

Technology Field

[0001] The present invention relates to the use of a vascular disease caused by aging of vascular endothelial progenitor cells containing stemregenin-1 (SR1) for the prevention, improvement, or treatment of such disease. Background Technology

[0002] Cellular senescence is a phenomenon in which normal somatic cells are unable to divide further after a certain number of divisions; it contributes to the aging of individuals and tissues and is an important mechanism for inhibiting abnormal cell proliferation and cancer formation. Cellular senescence occurs when telomeres, the ends of chromosomes, shorten due to repeated division of somatic cells, or it can also be caused by increased activity of oncogenes or tumor suppressor genes, excessive oxidative stress, exposure to ultraviolet rays or radiation, cytotoxic substances such as anticancer drugs, and inflammatory responses.

[0003] Senescent cells have morphological characteristics such as becoming larger and flattened, having increased heterochromatin in the nucleus, and having many vacuoles in the cytoplasm. Along with this, SA-β-gal (senescence-associated β-galatosidase) activity increases, the amount of proteins that inhibit cell growth, such as p53, p16INK4, and p21 increases, and they secrete various inflammatory proteins such as insulin-like growth factor binding proteins (IGBPs), interleukin-6, transforming growth factor-β (TGF-β), and interferon.

[0004] Cellular senescence not only contributes to the aging of individuals or tissues but also plays a significant role in the etiology of various diseases. Senescent cells are frequently observed in inflammatory tissue lesions such as those associated with rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage, and atherosclerotic vascular tissue. Additionally, cellular senescence is observed in conditions such as prostatic hyperplasia, hepatitis, and liver cancer. When senescent cells accumulate in this manner, they fail to divide effectively, which prevents the proper repair of damaged tissues. Furthermore, by secreting enzymes that degrade surrounding tissues or inflammatory cytokines, they accelerate tissue damage, thereby contributing to the etiology of aging-related diseases.

[0005] Among these, SIRT1, a factor related to the inhibition of cellular senescence, has recently been attracting attention as a new target for methods to inhibit the aging of vascular endothelial cells. Sirtuin 1 is involved in chromatin modification and gene expression by deacetylating histones and is involved in life extension induced by dietary restriction (Autiero, Ida, Susan Costantini, and Giovanni Colonna. PloS one 4.10 (2009): e7350) It is involved in in vivo regulatory systems such as cell survival and death, inflammation, DNA damage response, insulin metabolism, liver and lipid metabolism, cell differentiation, stem cells, microRNA, and cancer development through the deacetylation of various transcription factors.

[0006] In addition, it is known that during the natural aging process of human vascular endothelial cells, the gene expression of sirtuin 1 is reduced and p53, known as a cell growth regulator, is acetylated, thereby inducing natural aging; it has already been reported that sirtuin 1 has the activity of deacetylating p53 (Cho, Si-Young, et al. Aging (Albany NY) 5.3 (2013): 174.).

[0007] Meanwhile, StemRegenin 1 (SR1) is a purine derivative and an antagonist of the aryl hydrocarbon receptor (AHR). While prior research results regarding this have disclosed patents for its use in radiation protection, there have been no reports to date regarding the efficacy of StemRegenin 1 in inhibiting vascular endothelial progenitor cell aging.

[0008] Accordingly, the inventors of the present invention confirmed that Stemregenin-1 has an excellent effect of protecting vascular endothelial progenitor cells from stress and aging experienced during various in vitro cultures while maintaining the cell-specific characteristics, proliferative ability, motility, and differentiation ability of vascular endothelial progenitor cells, and based on the idea that it can be applied to the prevention, improvement, or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells, they came to complete the present invention. The problem to be solved

[0009] The present invention has been devised in consideration of the above-mentioned problems, and the objective of the present invention is to provide a composition for the prevention, improvement, or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising Stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] The problems that the present invention aims to solve are not limited to the problem(s) mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0011] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising StemRegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Vascular diseases caused by the aging of the above-mentioned vascular endothelial progenitor cells may be one or more selected from the group consisting of stroke, kidney disease, arteriosclerosis, and cardiovascular disease.

[0013] The concentration of the above-mentioned stemregenin-1 or its pharmaceutically acceptable salt may be 0.01 to 1.5 μM.

[0014] The above-mentioned Stemregenin-1 or a pharmaceutically acceptable salt thereof may have characteristics selected from the ability to enhance proliferation of vascular endothelial progenitor cells, the ability to migrate of vascular endothelial progenitor cells, and the ability to enhance angiogenesis.

[0015] The above-mentioned stemlegenin-1 or pharmaceutically acceptable salt thereof may induce the expression of one or more angiogenesis-related markers selected from the group consisting of angiopoietin-1 (Ang1), basic fibroblast growth factor (b-FGF), and interleukin-8 (IL-8).

[0016] The above-mentioned Stemregenin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells.

[0017] The above-mentioned Stemregenin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells by inhibiting SA-β-galactosidase activity.

[0018] The above-mentioned Stemregenin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells by inhibiting the expression of one or more oxidative stress-inducing factors selected from the group consisting of reactive oxygen species (ROS) and CYP1A1 (Cytochrome P450 1A1).

[0019] The above-mentioned stemgenin-1 or a pharmaceutically acceptable salt thereof may inhibit the expression of one or more aging-related markers selected from the group consisting of SMP30 (Senescence marker protein 30), p21, p53, interleukin-6 (IL-6), and interleukin-1α (IL-1α).

[0020] In addition, the present invention provides a health functional food composition for the prevention or improvement of vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] In addition, the present invention provides a cosmetic composition for preventing or improving vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient. Effects of the invention

[0022] According to the present invention, a composition for the prevention, improvement, or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells can be provided, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0023] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention. Brief explanation of the drawing

[0024] Figure 1 is an in vitro ( in vitro As a result of evaluating cell viability and characteristic preservation ability, Fig. 1a is a graph confirming cell viability according to SR-1 treatment concentration; Fig. 1b is an image confirming the morphology of cells according to SR-1 (1 μM) treatment; and Fig. 1c is a flow cytometry analysis result confirming the expression level of intrinsic surface markers of vascular endothelial progenitor cells according to SR-1 (1 μM) treatment. Figure 2 shows an in vitro ( in vitro This is the result of flow cytometry analysis confirming the degree of senescence of serially cultured vascular endothelial progenitor cells. Figure 3 shows the in vitro ( in vitro As a result of evaluating the proliferative capacity of vascular endothelial progenitor cells, Fig. 3a is a graph showing the accumulated number of cells that can be obtained when cultured with SR-1 for a long period, and a graph showing the morphological image of the cell confirmed in the cultured cells and the result of quantifying the size (Cell area); Fig. 3b is a cell image confirming the proliferative capacity of vascular endothelial progenitor cells when cultured with SR-1 for a long period using an EdU experiment and a graph showing the result of quantifying it. Figure 4 shows the in vitro ( in vitro As a result of evaluating the aging characteristics of vascular endothelial progenitor cells, Fig. 4a is an image confirming the aging characteristics of vascular endothelial progenitor cells according to SR-1 treatment through β-Galactosidase staining and a graph quantifying the same; Fig. 4b is an image confirming the aging characteristics of vascular endothelial progenitor cells according to SR-1 treatment through the expression levels of SMP30, p21, and p53 via Western blotting; and Fig. 4c is a graph confirming the aging characteristics of vascular endothelial progenitor cells according to SR-1 treatment through the expression levels of Interleukin-6 (IL-6) and Interleukin-1α (IL-1α) at ​​the mRNA level. Figure 5 shows the in vitro ( in vitroAs the results of evaluating the migration and angiogenic performance of vascular endothelial progenitor cells, Fig. 5a shows the image and quantified graph confirming the migration ability of SR-1-treated vascular endothelial progenitor cells using the scratch wound healing technique; Fig. 5b shows the image and quantified graph confirming the migration ability of SR-1-treated vascular endothelial progenitor cells using the transwell migration technique; Fig. 5c shows the image and quantified graph confirming the angiogenic performance of SR-1-treated vascular endothelial progenitor cells using the tube formation assay; and Fig. 5d shows the graph confirming the angiogenic performance of SR-1-treated vascular endothelial progenitor cells at the mRNA level through the expression of Angiopoietin-1 (Ang1), basic fibroblast growth factor (b-FGF), and Interleukin-8 (IL-8). Figure 6 shows the in vitro ( in vitro As a result of evaluating the inhibition of oxidative stress in vascular endothelial progenitor cells, Fig. 6a is a graph showing the mRNA levels of CYP1A1, CYP1A2, and CYP1B1, which are members of the CYP1 family, in SR-1-treated vascular endothelial progenitor cells; Fig. 6b is an image of the flow cytometry results confirming the amount of reactive oxygen species in SR-1-treated vascular endothelial progenitor cells in an oxidative stress environment using the H2DFFDA staining technique, and a graph quantifying the result; Fig. 6c is an image of the results confirming gammaH2AX, a marker capable of measuring damaged DNA in SR-1-treated vascular endothelial progenitor cells in an oxidative stress environment, using immunofluorescence staining and Western blotting, and a graph quantifying the result. Figure 7 shows the in vitro ( in vitroAs a result of evaluating the inhibition of oxidative stress in vascular endothelial progenitor cells in an aging environment, Figure 7a is the result of flow cytometry analysis using the H2DFFDA staining technique, which can measure the amount of reactive oxygen species in vascular endothelial progenitor cells in an environment where oxidative stress and reactive oxygen species have accumulated; Figure 7b is a graph analyzing the mRNA expression level of CYP1A1; and Figure 7c is an image of the result confirming the amount of reactive oxygen species in cells when treated with SR-1 for a long period and a graph quantifying it. Figure 8 shows the in vivo ( in vitvo As a result of evaluating the inhibition of oxidative stress in the aging environment of vascular endothelial progenitor cells, Fig. 8a is an image of matrigel collected 6 days after matrigel injection; Fig. 8b is an image of matrigel collected 6 days after matrigel injection and an image stained with CD31, an endothelial cell marker, by performing immunofluorescence staining of matrigel, and a graph quantifying the results. Specific details for implementing the invention

[0025] Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be interpreted as being limited to the embodiments below. These embodiments are provided to more fully explain the present invention to those with average knowledge in the art. Accordingly, the shapes of the elements in the drawings have been exaggerated to emphasize clearer explanations.

[0026] The present invention provides a pharmaceutical composition for the prevention or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising StemRegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0027] The above-mentioned Stemlegenin-1 (CAS 1227633-49-9) is a purine derivative and an antagonist of the aryl hydrocarbon receptor (AHR), and may be a compound represented by the following chemical formula 1. In addition, the Stemlegenin-1 in the present invention may include various derivatives thereof, but the scope of the present invention is not limited thereto. In the present invention, Stemlegenin-1 and its derivatives may be compounds isolated from nature, chemically synthesized compounds, or commercially available ones.

[0028] [Chemical Formula 1]

[0029]

[0030] Vascular diseases induced by the aging of the above-mentioned vascular endothelial progenitor cells are not specifically limited to diseases that can typically develop due to vascular aging and / or a decrease in elasticity, but specifically, they may be one or more selected from the group consisting of stroke, kidney disease, arteriosclerosis, and cardiovascular disease [Bloom, SI, Islam, MT, Lesniewski, LA et al. Mechanisms and consequences of endothelial cell senescence. Nat Rev Cardiol 20, 38-51 (2023). ( https: / / doi.org / 10.1038 / s41569-022-00739-0 ) reference]

[0031] According to one embodiment of the present invention, the concentration of Stemregenin-1 or a pharmaceutically acceptable salt thereof may be 0.01 to 1.5 μM, 0.01 to 1.0 μM, 0.05 to 1.0 μM, 0.1 to 1.0 μM, or 0.5 to 1.0 μM, but is not limited thereto. Within the above concentration range, there is no cytotoxicity or very low cytotoxicity, resulting in excellent cell viability, and it may be advantageous to maximize the activity of inhibiting the aging of vascular endothelial progenitor cells.

[0032] According to one embodiment of the present invention, the stem-genin-1 or a pharmaceutically acceptable salt thereof may have a characteristic selected from the ability to enhance the proliferation of vascular endothelial progenitor cells, the ability to migrate vascular endothelial progenitor cells, and the ability to enhance angiogenesis. In particular, it may have the above characteristics while maintaining the intrinsic morphology of vascular endothelial progenitor cells.

[0033] According to one embodiment of the present invention, the stem-genin-1 or a pharmaceutically acceptable salt thereof may induce the expression of one or more angiogenesis-related markers selected from the group consisting of angiopoietin-1 (Ang1), basic fibroblast growth factor (b-FGF), and interleukin-8 (IL-8).

[0034] According to one embodiment of the present invention, the stem-genin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells.

[0035] According to one embodiment of the present invention, the stem-genin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells by inhibiting SA-β-galactosidase activity.

[0036] In addition, the above-mentioned Stemlegenin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells by inhibiting the expression of one or more oxidative stress-inducing factors selected from the group consisting of reactive oxygen species (ROS) and CYP1A1 (Cytochrome P450 1A1). Specifically, the above-mentioned Stemlegenin-1 or a pharmaceutically acceptable salt thereof may inhibit the aging of vascular endothelial progenitor cells by inhibiting reactive oxygen species and CYP1A1 proteins in vascular endothelial progenitor cells exposed to oxidative stress.

[0037] In addition, the above-mentioned stemgenin-1 or a pharmaceutically acceptable salt thereof may inhibit the expression of one or more aging-related markers selected from the group consisting of SMP30 (Senescence marker protein 30), p21, p53, interleukin-6 (IL-6), and interleukin-1α (IL-1α).

[0038] As mentioned above, Stemregenin-1 can be highly effective in protecting vascular endothelial progenitor cells from stress and aging experienced during various in vitro cultures while maintaining the cell-specific characteristics, proliferative ability, motility, and differentiation ability of vascular endothelial progenitor cells.

[0039] In addition, the present invention is in a test tube ( in vitro ) or in vitro ( ex vivo A method for inhibiting the aging of vascular endothelial progenitor cells is provided, comprising the step of contacting vascular endothelial progenitor cells with Stemregenin-1 or a pharmaceutically acceptable salt thereof.

[0040] In addition, the present invention provides a method for preventing or treating vascular disease caused by aging of vascular endothelial progenitor cells, comprising the step of administering Stemregenin-1 or a pharmaceutically acceptable salt thereof to an individual.

[0041] In addition, the present invention provides a method for inhibiting the aging of vascular endothelial progenitor cells, comprising the step of administering Stemregenin-1 or a pharmaceutically acceptable salt thereof to an individual.

[0042] In the above method for inhibiting the aging of vascular endothelial progenitor cells or the method for preventing or treating vascular diseases, the concentration of Stemregenin-1 or a pharmaceutically acceptable salt thereof that comes into contact with vascular endothelial progenitor cells or is administered to an individual may be 0.01 to 1.5 μM, 0.01 to 1.0 μM, 0.05 to 1.0 μM, 0.1 to 1.0 μM, or 0.5 to 1.0 μM, but is not limited thereto. Within the above concentration ranges, there is no cytotoxicity or very low cytotoxicity, resulting in excellent cell viability, and it may be advantageous to maximize the activity of inhibiting the aging of vascular endothelial progenitor cells.

[0043] The term "prevention" as used in this invention refers to any act of inhibiting the aging of vascular endothelial progenitor cells or delaying the progression of a disease caused by the administration of the composition of this invention.

[0044] As used in the present invention, the term "treatment" refers to any act of inhibiting the aging of vascular endothelial progenitor cells or improving or beneficially altering a disease caused by the administration of the composition of the present invention, and refers to an attempt to obtain useful or desirable results, including clinical results. Useful or desirable clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction of the disease range, stabilization of the disease state, inhibition of disease onset, inhibition of disease spread, delay or slowing of disease progression, delay or slowing of disease onset, improvement or alleviation of the disease state, and decline (partially or entirely), whether detectable or not. Additionally, "treatment" may mean the extension of a patient's survival beyond what would be predicted in the absence of treatment. Furthermore, "treatment" may mean inhibition of disease progression or temporary slowing of disease progression, and more preferably relates to permanently stopping the progression of the disease. In the present invention, it may mean improving the survival of a patient by promoting one or more treatments selected from the group consisting of vascular diseases, specifically stroke, kidney disease, arteriosclerosis, and cardiovascular disease.

[0045] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the formulation is prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with Stemregenin-1 according to the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, for example, wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0046] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to treatment, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of a person skilled in the art, and generally, the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is from 0.1 mg / kg / day to 1000 mg / kg / day. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0047] The pharmaceutical composition of the present invention may be administered to mammals, such as rats, livestock, and humans, by various routes. All modes of administration are expected, for example, orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebrovascularly.

[0048] In the present invention, a pharmaceutical composition for preventing or treating vascular diseases caused by the aging of vascular endothelial progenitor cells may additionally include, in addition to the active ingredient, any compound or natural extract known to have activity whose safety has already been verified to enhance and reinforce the inhibition of vascular endothelial progenitor cell aging.

[0049] In addition, the present invention provides a food composition or a health functional food composition for preventing or improving vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0050] The above food or health functional food composition may further include an additive selected from the group consisting of flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, natural carbohydrates, nutritional agents, vitamins, thickeners, pH adjusters, preservatives, and mixtures thereof.

[0051] The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives. Food compositions of the above types can be prepared in various forms according to conventional methods known in the art.

[0052] For example, as a health food, the above composition itself may be prepared in the form of tea, juice, or drink for consumption, or it may be consumed after being granulated, encapsulated, or powdered. In addition, as a functional food, the extract may be added to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). Furthermore, to use the composition of the present invention as a food additive, it may be prepared and used in the form of a powder or a concentrate.

[0053] The preferred content of Stemlegenin-1 in the food composition of the present invention may be 0.001 to 50% with respect to the total weight of the food composition, and preferably may be contained in the range of 0.01 to 30%.

[0054] In one embodiment of the present invention, the health functional food composition of the present invention may be manufactured in general formulations such as tablets, pills, granules, powders, liquids, hard capsules, soft capsules, etc., and may be manufactured in any form such as porridge, bread, beverages, bars, chocolates, cookies, teas, drinks, vitamin complexes, meat, sausages, candy, noodles, jelly, etc.

[0055] To manufacture various formulations or forms as described above, food-grade acceptable carriers or additives such as the excipients mentioned above may be used, and any carrier or additive known to be available in the art for manufacturing the formulation or form to be manufactured may be used.

[0056] In addition, the present invention provides a feed composition for preventing or improving vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0057] When the Stemlegenin-1 of the present invention is provided in the form of a feed composition, the feed composition may additionally include known feed aids, food additives, or feed additives, and may be manufactured in the form of fermented feed, compound feed, pellets, silage, etc.

[0058] In addition, the present invention provides a cosmetic composition for preventing or improving vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0059] When Stemregenin-1 of the present invention is provided as a cosmetic composition, the cosmetic composition may include, without limitation, conventionally accepted ingredients in addition to the active ingredient, such as conventional auxiliary agents like antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers. The cosmetic composition may be interpreted to mean including various materials for skin health without limitation.

[0060] In addition, the present invention provides an adjuvant for treating vascular diseases caused by the aging of vascular endothelial progenitor cells, comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0061] As used herein, the term "adjuvant for the treatment of vascular disease" refers to a composition that, when applied in conjunction with treatment with a pharmaceutical composition according to the present invention, prevents side effects caused by said pharmaceutical composition and synergistically increases the therapeutic effect of the vascular disease. Accordingly, said adjuvant for the treatment of vascular disease can be administered together with, simultaneously with, or sequentially with said pharmaceutical composition.

[0062] In addition, the present invention provides a pharmaceutical composition for inhibiting the aging of vascular endothelial progenitor cells comprising stemlegenin-1 or a pharmaceutically acceptable salt thereof as an active ingredient. Through the pharmaceutical composition for inhibiting the aging of vascular endothelial progenitor cells, vascular diseases caused by the aging of vascular endothelial progenitor cells can be prevented, improved, or treated.

[0064] The above description explains the technical concept of the present invention using one embodiment, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential characteristics of the present invention. Accordingly, the embodiments described in this invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by such embodiments. The scope of protection of the present invention shall be interpreted by the claims, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

[0065] The present invention will be explained in more detail below through examples.

[0067] Example 1. In vitro (according to SR-1 treatment) in vitro Evaluation of cell viability and characteristic preservation ability of vascular endothelial progenitor cells

[0068] To determine the concentration of SR-1, the cell viability of vascular endothelial progenitor cells according to the treatment concentration of SR-1 and the ability to preserve cell characteristics according to treatment with SR-1 were evaluated, and the results are shown in Figures 1a to 1c. At this time, the control group is (untreated group).

[0069] Figure 1a shows the results of confirming cell viability for setting the concentration of SR-1. After treating vascular endothelial progenitor cells with SR-1 at different concentrations, cell viability was confirmed by measuring absorbance using a microplate reader with a cell counting kit-8 assay. Referring to Figure 1a, it can be seen that cell viability decreases at concentrations of 2 μM or higher; accordingly, subsequent verifications were carried out at a concentration of 1 μM.

[0070] Figure 1b is an image confirming the morphology of vascular endothelial progenitor cells according to SR-1 treatment using an optical microscope. Referring to Figure 1b, it can be confirmed that there are no abnormalities in the morphology of vascular endothelial progenitor cells when culture is carried out with SR-1 at a concentration of 1 μM.

[0071] Figure 1c shows the results of confirming the expression of intrinsic surface markers of vascular endothelial progenitor cells following SR-1 treatment through flow cytometry. Referring to Figure 1c, 1 μM It can be confirmed that the expression of the intrinsic surface markers of vascular endothelial progenitor cells is maintained even after treatment with a concentration of SR-1.

[0072] In summary, through Figure 1, it was verified that treating vascular endothelial progenitor cells with SR-1 at a concentration of 1 μM did not impair the characteristics of the cells.

[0074] Example 2. In vitro (according to SR-1 treatment) in vitro Evaluation of aging characteristics of serially cultured vascular endothelial progenitor cells

[0075] SR-1 from young vascular endothelial progenitor cells with limited passages (The surface markers were compared with cells that were long-term subcultured from passage 6 to passage 14 by continuously treating with SR-1 (1 μM / day) and confirmed through flow cytometry, and the results are shown in Figure 2. At this time, SR-1 was treated at a concentration of 1 μM, and the control group was (untreated group).

[0076] Referring to Figure 2, it can be seen that the expression of vascular endothelial progenitor cell surface markers is maintained at a younger cell level in cells treated with SR-1 compared to the control.

[0078] Example 3. In vitro (following long-term SR-1 treatment) in vitro Evaluation of the proliferative capacity of vascular endothelial progenitor cells

[0079] The proliferative capacity of vascular endothelial progenitor cells following long-term treatment with SR-1 was evaluated, and the results are shown in Figures 3a and 3b [x-axis in Figure 3b: passage number]. In each experiment, long-term treatment with SR-1 was performed at a concentration of 1 μM / day, and the control group was (untreated group).

[0080] Figure 3a is a graph showing the accumulated number of cells obtained by treating SR-1 for a long period from passage 6 to passage 20 and a graph showing the results of quantifying the cell area by photographing the morphological images of the cultured cells with an optical microscope. Referring to Figure 3a, it can be verified that SR-1 is effective in increasing the cell yield compared to the control group and further maintaining the intrinsic morphology of vascular endothelial progenitor cells.

[0081] Figure 3b is an EdU experiment that confirms proliferation ability through the principle of binding to DNA that replicates during proliferation, verifying that the proliferation ability of vascular endothelial progenitor cells cultured with SR-1 for a long period (from passage 6 to passage 14) is higher than that of the control group.

[0083] Example 4. In vitro (according to SR-1 treatment) in vitro Evaluation of aging characteristics of vascular endothelial progenitor cells

[0084] The aging characteristics of vascular endothelial progenitor cells were evaluated according to SR-1 treatment, and the results are shown in Figures 4a to 4c. In each experiment, SR-1 was treated at a concentration of 1 μM, and the control group was the untreated group.

[0085] Figure 4a shows the results of measuring the degree of aging using β-Galactosidase staining, which is one of the aging markers. Referring to Figure 4a, it can be verified that aging progresses less when SR-1 (1 μM / day) is applied to the culture for a long period from passage 6 to passage 18.

[0086] Figure 4b shows the results of measuring the expression of SMP30, p21, and p53, known as aging-related proteins, using the Western blotting method. Referring to Figure 4b, it can be verified that the degree of aging is reduced in the aged group treated with SR-1.

[0087] Figure 4c shows that when the expression of Interleukin-6 (IL-6) and Interleukin-1α (IL-1α), which are senescence-associated secretory phenotypes (SASP) whose secretion increases with aging, is examined at the mRNA level, it can be verified that the SASP expression of SR-1-treated vascular endothelial progenitor cells is significantly lower.

[0089] Example 5. In vitro (according to SR-1 treatment) in vitro Evaluation of the migratory ability and angiogenic capacity of vascular endothelial progenitor cells

[0090] The migratory and angiogenic capabilities of vascular endothelial progenitor cells were evaluated following SR-1 treatment, and the results are shown in Figures 5a to 5d. In each experiment, SR-1 was applied at a concentration of 1 μM, and the control group was the untreated group.

[0091] Figure 5a shows a scratch wound healing technique to verify cell migration ability, and it can be seen that the migration ability of vascular endothelial progenitor cells treated with SR-1 is superior compared to the control group.

[0092] Figure 5b shows the transwell migration technique used to verify cell migration ability, which confirms that the migration ability of vascular endothelial progenitor cells treated with SR-1 is superior to that of the control group.

[0093] Figure 5c is an image of the results of a tube formation assay that can confirm angiogenesis ability and a graph quantifying it. Referring to Figure 5c, it can be seen that the angiogenesis ability of vascular endothelial progenitor cells treated with SR-1 is high.

[0094] Figure 5d is a graph showing the results of confirming the expression of Angiopoietin-1 (Ang1), basic fibroblast growth factor (b-FGF), and Interleukin-8 (IL-8), which are known as angiogenesis-related markers, at the mRNA level. Referring to Figure 5d, it can be seen that the expression is significantly higher in vascular endothelial progenitor cells treated with SR-1.

[0096] Example 6. In vitro (according to SR-1 treatment) in vitro Evaluation of oxidative stress inhibition in vascular endothelial progenitor cells

[0097] The effects of SR-1 on vascular endothelial progenitor cells in an environment inducing oxidative stress were confirmed, and the results are shown in Figures 6a to 6c. In each experiment, SR-1 was treated at a concentration of 1 μM, and the control group was untreated.

[0098] Figure 6a confirmed that when the mRNA levels of CYP1A1, CYP1A2, and CYP1B1, which are CYP1 family members capable of inducing transcription by the signaling pathway of AhR, including AhR, the target receptor of SR-1, were examined, a tendency for CYP1A1-specific expression to be suppressed was observed.

[0099] Figure 6b shows the result image and quantified graph of flow cytometry analysis performed using the H2DFFDA staining technique, which can measure the amount of reactive oxygen species in cells under oxidative stress conditions. Referring to Figure 6b, it can be confirmed that the amount of reactive oxygen species in SR-1-treated vascular endothelial progenitor cells is lower.

[0100] Figure 6c shows the results of confirming gammaH2AX, a marker capable of measuring intracellular damaged DNA that can be induced by oxidative stress, through immunofluorescence staining and Western blotting. Referring to Figure 6c, it can be verified that there is less DNA damage in vascular endothelial progenitor cells treated with SR-1.

[0102] Example 7. In vitro (according to SR-1 treatment) in vitro Evaluation of Oxidative Stress Inhibition in Vascular Endothelial Progenitor Cells in an Aging Environment

[0103] The effect of reducing the expression of CYP1A1 against oxidative stress identified in Figure 6 and protecting against reactive oxygen species was confirmed in an aging environment related to this, and the results are shown in Figures 7a to 7c. In each experiment, SR-1 was treated at a concentration of 1 μM, and the control group was untreated.

[0104] Figure 7a shows the results of flow cytometry analysis using the H2DFFDA staining technique, which can measure the amount of reactive oxygen species in vascular endothelial progenitor cells in an environment where oxidative stress and reactive oxygen species have accumulated. Referring to Figure 7a, it can be confirmed that the amount of reactive oxygen species in SR-1-treated vascular endothelial progenitor cells is lower.

[0105] Referring to Figure 7b, it can be confirmed that even in an aging environment, mRNA expression of CYP1A1 is also low in SR-1-treated vascular endothelial progenitor cells in proportion to the amount of reactive oxygen species.

[0106] Figure 7c is an image taken using an automated microscope and a graph quantifying the results of confirming the amount of reactive oxygen species in cells when SR-1 was treated for a long period from passage 6 to passage 17 based on the verification of Figures 6 and 7a to 7b. By referring to Figure 7c, it can be verified that the amount of reactive oxygen species in cells is low when SR-1 is treated.

[0108] Example 8. In vivo (according to SR-1 treatment) in vivo Evaluation of Oxidative Stress Inhibition in Vascular Endothelial Progenitor Cells in an Aging Environment

[0109] Based on the confirmation of the effects of SR-1 in oxidative stress and aging environments in in vitro culture, the effects of SR-1 in various stress environments experienced by cells during in vivo injection were confirmed using matrigel plug experiments, and the results are shown in Figures 8a and 8b. In each experiment, SR-1 was treated at a concentration of 1 μM, and the control group was untreated.

[0110] Figure 8a is an image of the Matrigel collected 6 days after injection. Referring to Figure 8a, it can be seen that the SR-1 treated group exhibits superior angiogenesis ability compared to the control group even under stressful conditions.

[0111] Figure 8b shows an image of matrigel stained with CD31, an endothelial cell marker, and a graph quantifying the results. Referring to Figure 8b, it was confirmed that the formation of vascular structures within matrigel in vascular endothelial progenitor cells treated with SR-1 was high.

[0113] As seen above, it was confirmed that SR-1 has an excellent effect in protecting vascular endothelial progenitor cells from stress and aging experienced during various in vitro cultures while maintaining the cell-specific characteristics, proliferative ability, motility, and differentiation ability of vascular endothelial progenitor cells.

[0114] Accordingly, a composition comprising SR-1 or a pharmaceutically acceptable salt thereof according to the present invention can be applied for the purpose of inhibiting the aging of vascular endothelial progenitor cells or for the prevention, improvement, or treatment of vascular diseases caused by the aging of vascular endothelial progenitor cells.

[0116] The above detailed description is illustrative of the present invention. Furthermore, the foregoing describes preferred embodiments of the present invention, and the present invention may be used in various other combinations, modifications, and environments. That is, modifications or alterations are possible within the scope of the concept of the invention disclosed herein, the scope equivalent to the disclosed content, and / or the scope of the art or knowledge. The described embodiments describe the best state for implementing the technical concept of the present invention, and various modifications required in specific fields of application and uses of the present invention are possible. Accordingly, the above detailed description of the invention is not intended to limit the present invention to the disclosed embodiments. Additionally, the appended claims should be interpreted as including other embodiments.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of stroke or arteriosclerosis comprising StemRegenin-1 as an active ingredient, wherein the StemRegenin-1 is included at a concentration of 0.01 to 1.5 μM. Claim 2 delete Claim 3 delete Claim 4 A pharmaceutical composition according to claim 1, wherein the Stemregenin-1 has one or more characteristics selected from the ability to enhance proliferation of vascular endothelial progenitor cells, the ability to migrate of vascular endothelial progenitor cells, and the ability to enhance angiogenesis. Claim 5 A pharmaceutical composition according to claim 4, wherein the above-mentioned stemlegenin-1 induces the expression of one or more angiogenesis-related markers selected from the group consisting of angiopoietin-1 (Ang1), basic fibroblast growth factor (b-FGF), and interleukin-8 (IL-8). Claim 6 delete Claim 7 A pharmaceutical composition according to claim 1, wherein the Stemregenin-1 inhibits the aging of vascular endothelial progenitor cells by inhibiting SA-β-galactosidase activity. Claim 8 A pharmaceutical composition according to claim 1, wherein the Stemregenin-1 inhibits the aging of vascular endothelial progenitor cells by inhibiting the expression of one or more oxidative stress-inducing factors selected from the group consisting of reactive oxygen species (ROS) and CYP1A1 (Cytochrome P450 1A1). Claim 9 A pharmaceutical composition according to claim 1, wherein the stemgenin-1 inhibits the expression of one or more aging-related markers selected from the group consisting of SMP30 (Senescence marker protein 30), p21, p53, interleukin-6 (IL-6), and interleukin-1α (IL-1α). Claim 10 delete Claim 11 delete

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