KR20240034170A - Pharmaceutical composition for inhibiting angiogenesis comprising steppogenin as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for inhibiting angiogenesis containing stepogenin as an active ingredient. Stepogenin of the present invention can inhibit cell migration or proliferation of vascular endothelial cells by inhibiting DLL4 expression, thereby inhibiting angiogenesis, It is expected to be useful as a treatment or anticancer adjuvant for various diseases in which angiogenesis is abnormally regulated, such as macular degeneration.

Description

Pharmaceutical composition for inhibiting angiogenesis comprising steppogenin as an active ingredient}

The present invention relates to a pharmaceutical composition for inhibiting angiogenesis containing stepogenin as an active ingredient.

Angiogenesis is a biological process that creates new blood vessels in tissues or organs. Under normal physiological conditions, humans or animals create new blood vessels only under very limited circumstances. This angiogenesis is a stage in which blood vessels are reorganized and new capillaries are created through the decomposition of the vascular basement membrane by proteolytic enzymes and the formation of tubes (vasculature) by migration, proliferation, and vascular endothelial cell differentiation of vascular endothelial cells forming the vascular wall. It occurs through a series of sequential steps including:

In addition, the process of angiogenesis is strictly regulated by various negative and positive regulators (Folkman and Cotran., Int. Rev. Exp. Patho., 16, 207-248, 1976), and this angiogenesis is normally If not controlled, it can cause pathological disorders such as diabetic retinopathy, rheumatoid arthritis, inflammation, endometriosis, age-related vision loss, psoriasis, and hemangioma.

Diseases related to angiogenesis can be roughly divided into inflammatory diseases such as arthritis, eye diseases such as diabetic retinopathy, dermatological diseases such as psoriasis, and cancer. Eye diseases caused by angiogenesis include macular degeneration, diabetic retinopathy, a complication of diabetes in which capillaries in the retina invade the vitreous body and eventually lead to blindness, retinopathy of premature infants, and neovascular glaucoma. There are diseases such as, and these diseases cause blindness to millions of people around the world every year. In addition, arthritis is caused by autoimmune abnormalities, but it is known that chronic inflammation in the synovial cavity induces angiogenesis during the course of the disease, and it is a disease that occurs when new capillaries invade the joint and cartilage is destroyed. In addition, psoriasis is a chronic proliferative disease that occurs in the skin, and since a lot of blood must be supplied for rapid proliferation, angiogenesis is bound to occur actively. Therefore, the discovery of substances that inhibit the formation of new blood vessels can be widely used in the treatment of diseases related to new blood vessel formation, such as diabetic retinopathy, rheumatoid arthritis, inflammation, endometriosis, age-related loss of vision, psoriasis, and hemangioma. Currently, academia and industry are continuously conducting research and development on new materials with these functions.

In addition, abnormal angiogenesis plays a role in supplying nutrients and oxygen necessary for tumor growth and proliferation, and new capillaries that have infiltrated the tumor provide an opportunity for metastatic cancer cells to enter the blood circulation system, which can lead to metastasis. Let it happen. Therefore, research on the mechanism of angiogenesis and the development of substances that can inhibit it have become the focus of interest in the prevention and treatment of cancer. Recently, tumor angiogenesis has been inhibited in animal cancer models and human clinical trials. As it has been proven that it can effectively inhibit tumor growth and development and extend the life of patients, research on the development of angiogenesis inhibitors is actively underway.

About 200 types of angiogenesis inhibitors have been reported that have been developed to date. These angiogenesis inhibitors include mechanisms that reduce the activity of specific angiogenesis-promoting factors, mechanisms that inhibit the growth or induce death of vascular endothelial cells, and mechanisms that promote angiogenesis. It can be broadly classified into four types, which play a role in mechanisms that inhibit the action of indirect factors that regulate stimulating factors or endothelial cell survival factors and mechanisms that increase the activity of angiogenesis inhibitors present in the body, especially angiostatin. , endostatin, PK5, prothrombin kringle 2, and Avastin (VEGF antibody) approved by the US FDA are well known (O'Relly, M.S. et al. Cell., 79, 315 ~328, 1994; Lee. T.H., Biol. Che., 273, 28805~28812, 1998; Shih, Mikhail V. Blagosklonny, Cancer Biology & Therapy, 4:12, 1307-1310, 2005). However, even though various angiogenesis inhibitors are already known, it is difficult to maintain excellent activity for a long time, their pharmaceutical properties are low and they can be easily denatured, so the development of new angiogenesis inhibitors is required. there is.

Meanwhile, delta like 4 (Dl4) or Delta Like Ligand 4 (DLL4) (hereinafter referred to as “DLL4”) is a ligand that uses the Notch protein overexpressed in vascular endothelial cells as a receptor. As a member of the delta class, it is known to be a major factor in regulating angiogenesis. DLL4 specifically binds to Notch 1 or Notch 4 receptors overexpressed on vascular endothelium. DLL4 is also expressed in normal blood vessels, but is known to be highly overexpressed in cancer blood vessels (Reinacher-Schick A et al., Nat Clin Pract Gastroenterol Hepatol 2008;5(5):250-67). In tumors, angiogenesis occurs by angiogenic factors such as VEGF to receive oxygen and nutrients in the hypoxic area of the cancer tissue. In tumors, angiogenesis is known to play an important role not only in tumor growth but also in metastasis. Blocking Notch signaling by DLL4 in tumors can inhibit cancer growth because angiogenesis becomes poorly regulated.

Republic of Korea Patent No. 10-1644440

The present inventors conducted extensive research to find a substance with angiogenesis inhibitory activity, and as a result, it was confirmed that stepogenin suppresses cell migration or proliferation of vascular endothelial cells by suppressing the expression of DLL4, which is a major factor regulating angiogenesis. By doing this, the present invention was completed.

The purpose of the present invention is to provide a pharmaceutical composition for inhibiting angiogenesis containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide an anticancer supplement containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing and improving angiogenesis-related diseases containing steppogenin or a foodologically acceptable salt thereof as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting angiogenesis containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing and improving angiogenesis-related diseases containing steppogenin or a foodologically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the stepogenin may inhibit the expression of the DLL4 gene or reduce the level of the protein expressed by the DLL4 gene, but is not limited thereto.

In another embodiment of the present invention, the composition may inhibit cell migration or proliferation of vascular endothelial cells, but is not limited thereto.

In another embodiment of the present invention, the composition can prevent or treat diseases caused by angiogenesis, but is not limited thereto.

In another embodiment of the present invention, the disease includes arthritis, rheumatoid arthritis, chronic inflammation, osteoarthritis, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, corneal disease caused by neovascularization, and macular degeneration. macular degeneration, macular degeneration, pterygium, retinal degeneration, erythroderma, proliferative retinopathy, retrophakic fibroplasia, granular conjunctivitis, psoriasis, telangiectasia, psoriasis, seborrheic dermatitis, and acne. It may be one, but is not limited to this.

In another embodiment of the present invention, the composition may further include an angiogenesis inhibitor, but is not limited thereto.

In addition, the present invention provides an anticancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient, which is characterized by being capable of inhibiting angiogenesis.

In one embodiment of the present invention, the stepogenin may inhibit the expression of the DLL4 gene or reduce the level of the protein expressed by the DLL4 gene, but is not limited thereto.

In another embodiment of the present invention, the anticancer adjuvant may be administered simultaneously or sequentially with the anticancer agent, but is not limited thereto.

In another embodiment of the present invention, the anti-cancer adjuvant may be administered in combination with an anti-cancer agent, but is not limited thereto.

Additionally, the present invention provides a method for preventing or treating angiogenesis-related diseases, comprising administering a composition containing stepogenin or a pharmaceutically acceptable salt thereof to an individual in need thereof.

Additionally, the present invention provides the use of a composition containing stepogenin or a pharmaceutically acceptable salt thereof for the prevention or treatment of angiogenesis-related diseases.

Additionally, the present invention provides the use of stepogenin or a pharmaceutically acceptable salt thereof to produce a drug for preventing or treating angiogenesis-related diseases.

Additionally, the present invention provides a combination therapy for preventing or treating cancer, comprising administering a composition containing stepogenin or a pharmaceutically acceptable salt thereof along with an anticancer agent to an individual in need thereof.

Additionally, the present invention provides an auxiliary anticancer use of a composition containing stepogenin or a pharmaceutically acceptable salt thereof in the prevention or treatment of cancer.

Stepogenin of the present invention can inhibit cell migration or proliferation of vascular endothelial cells by inhibiting DLL4 expression, and is therefore used as a treatment for various diseases in which angiogenesis is abnormally regulated, such as inhibition of angiogenesis and macular degeneration, and as an angiogenesis agent such as Avastin. It is expected that it can be usefully used as an anti-cancer adjuvant through its inhibitory function.

Figure 1 is a diagram showing the results of confirming the cytotoxicity of 70 candidate compounds.
Figure 2a is a diagram showing the results of confirming the HIF-1α promoter reporter activity of 70 candidate compounds (*p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic control group).
Figure 2b is a diagram showing the results of confirming whether compounds selected as potential HIF-1α inhibitors among 70 candidate compounds inhibit HIF-1α protein expression (H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic control group; $$p < 0.01 vs. normoxic control group).
Figure 2c is a diagram showing the results of confirming whether compounds that inhibit HIF-1α protein expression among compounds selected as potential HIF-1α inhibitors inhibit DLL4 protein expression (*p < 0.05, **p < 0.005, and ***p < 0.001 vs. VEGF treated control; $$p < 0.01 vs. untreated control).
Figure 3a is a diagram showing the results of confirming cell viability for stepogenin (H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxia control group).
Figure 3b is a diagram showing the results of confirming the cytotoxicity of stepogenin (H: hypoxia).
Figure 3c is a diagram showing the results of confirming the HRE-luciferase reporter activity of stepogenin (H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxia control group).
Figure 3d is a diagram showing the results of confirming IC ₅₀ for the HIF-1α activity of stepogenin (H: hypoxia).
Figure 3e is a diagram showing the results of confirming the DLL4-luciferase reporter activity of stepogenin (*p < 0.05, **p < 0.005, and ***p < 0.001 vs. VEGF-treated control group).
Figure 3f is a diagram showing the results of confirming IC ₅₀ for DLL4 activity of stepogenin.
Figure 4a is a diagram showing the results of confirming whether stepogenin inhibits HIF-1α protein expression in HEK293T human embryonic kidney epithelial cells, A549 human lung cancer cells, and ARPE19 human retinal pigment epithelial cells under hypoxic conditions (H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic control group; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. normoxic control group).
Figure 4b is a diagram showing the results of immunofluorescence analysis confirming the effect of stepogenin on the nuclear expression of HIF-1α under hypoxic conditions (NOR: normal oxygen, HYPO or H: hypoxic).
Figure 4c is a diagram showing the results confirming the effect of stepogenin on the mRNA expression of HIF-1α target genes VEGF, GLUT1, CXCR4, and CA9 under hypoxic conditions (H: hypoxia; *p < 0.05, **p < 0.005 , and ***p < 0.001 vs. hypoxic control group; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. normoxic control group).
Figure 4d is a diagram showing the results confirming the effect of stepogenin on the protein levels of HIF-1α target genes VEGF, CXCR4, and CA9 under hypoxic conditions (H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic control; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. normoxic control).
Figure 5a is a diagram showing the results confirming the effect of stepogenin on DLL4, Notch1, and NICD protein levels under VEGF treatment (*p < 0.05, **p < 0.005, and ***p < 0.001 vs. VEGF treated control; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. untreated control).
Figure 5b is a diagram showing the results of confirming the effect of stepogenin on DLL4 expression under hypoxic conditions through immunofluorescence analysis (NOR: normoxia, HYPO or H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic controls; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. normoxic controls).
Figure 5c is a diagram showing the results confirming the effect of stepogenin on cell migration under hypoxic conditions (NOR: normoxia, HYPO or H: hypoxia; *p < 0.05, **p < 0.005, and ***p < 0.001 vs. hypoxic control; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. normoxic control).
Figure 5d is a diagram showing the results confirming the effect of stepogenin on the germination ability of EA.hy926 (HUVEC) spheroids under VEGF treatment (*p < 0.05, **p < 0.005, and ***p < 0.001 vs. VEGF treated control; $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. untreated control).
Figure 6a is a diagram showing the results of confirming the effect of stepogenin on tumor growth in a tumor animal model (*p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001 vs. control group, same hereinafter).
Figure 6b is a diagram showing the results of confirming the change in body weight of a tumor animal model after administration of stepogenin.
Figure 6c is a diagram showing the results confirming the effect of stepogenin on hypoxic areas and HIF-1α expression in tumors of a tumor animal model (hypoxic areas: green, HIF-1α: red). Scale bar = 100 μm.
Figure 6d is a diagram showing the results of confirming the effect of stepogenin on angiogenesis through microvessel density analysis using CD34 antibody in a tumor animal model (CD34: red).
Figure 6e is a diagram showing the results of immunofluorescence analysis confirming the effect of stepogenin on CD3+, CD4+, and CD8+ T cells in a tumor animal model.

The present inventors conducted extensive research to find a substance with angiogenesis inhibitory activity, and as a result, it was confirmed that stepogenin suppresses cell migration or proliferation of vascular endothelial cells by suppressing the expression of DLL4, which is a major factor regulating angiogenesis. By doing this, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for inhibiting angiogenesis containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present specification, “steppogenin” may be represented by the following formula (1), (2S)-2-(2,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4 It can have an IUPAC name of -one. Additionally, the molecular weight may be 288.25 and the chemical formula may be C ₁₅ H ₁₂ O ₆ . In addition, the stepogenin can be chemically synthesized by a method known in the field to which the present invention pertains, or a commercially available material can be used, and it can be obtained by extraction and separation, but is not limited thereto.

[Formula 1]

The present invention may also include a pharmaceutically acceptable salt of steppogenin as an active ingredient. As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid. , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excessive amount of aqueous acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. It can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering the precipitated salt.

Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excessive amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to produce sodium, potassium or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

The content of steppogenin or a pharmaceutically acceptable salt thereof in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 based on the total weight of the composition. It may be from 99.9% by weight, or from 0.001 to 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount with the solvent removed.

According to one embodiment of the present invention, the stepogenin or a pharmaceutically acceptable salt thereof inhibits the expression of the DLL4 and HIF-1α (Hypoxia-inducible factor-1 alpha) genes, or inhibits the expression of the DLL4 and HIF-1α genes. Cell migration of vascular endothelial cells by reducing the level of proteins expressed by Notch1 and NICD, and inhibiting the expression of target genes VEGF, GLUT1, CXCR4, and CA9 of HIF-1α. Alternatively, it can inhibit proliferation and inhibit angiogenesis and tumor growth, so it can be useful as a composition for preventing, improving, or treating angiogenesis-related diseases and as an anticancer adjuvant.

In the present invention, “expression” includes all steps in the process of turning a gene into a protein in vitro or within a cell, for example, transcription from gene to mRNA and translation from mRNA to protein.

“Disease caused by angiogenesis” or “angiogenesis-related disease”, which is a disease to be prevented, improved, or treated by the composition of the present invention, refers to a disease caused by abnormal progress of new blood vessel formation. Within this specification, “angiogenesis-related disease” and “disease caused by angiogenesis” can be described interchangeably.

In the present invention, the angiogenesis-related diseases include, for example, angiogenesis-dependent cancer, benign tumor, arthritis, rheumatoid arthritis, chronic inflammation, osteoarthritis, diabetic retinopathy, retinopathy of prematurity, and neovascular glaucoma. , corneal disease caused by new blood vessels, macular degeneration, macular degeneration, pterygium, retinal degeneration, erythroderma, proliferative retinopathy, posterior lens fibroplasia, granular conjunctivitis, psoriasis, telangiectasia, pyogenic granuloma , psoriasis, erythrosis, seborrheic dermatitis, or acne, but is not limited thereto, and may include all diseases caused by abnormal new blood vessel formation.

In the present invention, the composition of the present invention may further include a known anticancer agent or angiogenesis inhibitor in addition to stepogenin or a pharmaceutically acceptable salt thereof as an active ingredient, and other known treatments for the treatment of these diseases. It can be used in combination with. Other treatments may include, but are not limited to, chemotherapy, radiation therapy, hormone therapy, bone marrow transplantation, stem-cell replacement therapy, other biological treatments, and immunotherapy.

Anticancer agents that may be additionally included in the pharmaceutical composition of the present invention include, for example, 17-AAG (tanespimycin), DNA alkylating agents such as mechloethamine, chlorambucil, and phenylalanine. ), mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan ( busulfan), thiotepa, cisplatin and carboplatin; Anti-cancer antibiotics include dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, and plicama. plicamycin, mitomycin and C Bleomycin; and plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, and It may include, but is not limited to, iridotecan.

Angiogenesis inhibitors that may be additionally included in the pharmaceutical composition of the present invention include, for example, angiostatin (plasminogen fragment); Anti-angiogenic antithrombin III angiozyme; ABT-627; Bay 12-9566; benefin; bevacizumab; BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagen XVIII fragment); fibronectin sections; Gro-beta; Halofuginone; heparinase; Heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; interferon alpha/beta/gamma; interferon-inducible protein (IP-10); interleukin-12; Kringle 5 (plasminogen fragment); marimastat; dexamethasone; metalloproteinase inhibitor (TIMP); 2-methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitors; plasminogen activator inhibitor; platelet factor-4 (PF4); prinomastat; Prolactin 16 kD fragment; proliferin-related protein (PRP); PTK 787/ZK 222594; retinoid sorimastat; squalamine; SS 3304; SU 5416; SU6668; SU11248; tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP-1); TNP-470; Transforming growth factor-beta (TGF-b); vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD6474; farnesyl transferase inhibitor (FTI); and bisphosphonates (e.g., alendronate, etidronate, pamidronate, risedronate, ibandronate, zoledronate, olpadronate, icandronate, or Neri) It includes, but is not limited to, neridronate).

In this specification, “active ingredient” refers to an ingredient that can exhibit the desired activity alone or in combination with a carrier that is inactive on its own.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, paste preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

Additionally, the present invention provides a method for preventing or treating angiogenesis-related diseases, comprising administering a composition containing stepogenin or a pharmaceutically acceptable salt thereof to an individual in need thereof.

Additionally, the present invention provides the use of a composition containing stepogenin or a pharmaceutically acceptable salt thereof for the prevention or treatment of angiogenesis-related diseases.

Additionally, the present invention provides the use of stepogenin or a pharmaceutically acceptable salt thereof to produce a drug for preventing or treating angiogenesis-related diseases.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, administration means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed.

In addition, in another aspect of the present invention, the present invention provides a health functional food composition for preventing and improving angiogenesis-related diseases containing steppogenin or a foodologically acceptable salt thereof as an active ingredient.

In the present invention, “improvement” means any action that reduces parameters related to the desired disease, such as the degree of symptoms, by administering the composition according to the present invention. At this time, the health functional food composition can be used simultaneously or separately with drugs for treatment before or after the onset of angiogenesis-related diseases or cancer in order to prevent or improve angiogenesis-related diseases or cancer.

In this specification, “health functional food” is the same term as food for special health use (FoSHU), and refers to food with high medical and medical effects that has been processed to efficiently exhibit bioregulatory functions in addition to supplying nutrients. This means that the food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to achieve useful effects in preventing or improving angiogenesis-related diseases or cancer.

The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

In addition, since angiogenesis plays an important role in tumor growth and metastasis, the present invention provides, in another aspect of the present invention, an anti-cancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient, Provided is an anti-cancer adjuvant characterized by the ability to inhibit angiogenesis. The specific description of stepogenin is the same as the above description.

The anticancer adjuvant can enhance the efficacy of anticancer drugs by inhibiting angiogenesis. Additionally, the anticancer adjuvant may be administered simultaneously or sequentially with the anticancer agent. When administered sequentially, the anticancer agent may be administered after the anticancer adjuvant, or the anticancer adjuvant may be administered after the anticancer adjuvant, and may be administered alone or in combination with the anticancer agent, but is not limited thereto. The administration method may be changed to improve anticancer efficacy.

In the present invention, the cancer may be a solid cancer, and the solid cancer includes, for example, colon cancer, liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, lung cancer, glioblastoma, head and neck cancer, metastatic cancer, melanoma, and uterine cancer. , ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, perianal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, kidney It may be selected from the group consisting of cellular carcinoma, renal pelvic carcinoma, and central nervous system tumor, but is not limited thereto.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Experimental example]

Experimental Example 1. Materials

The 70 compounds used (isolated from various natural herbs) were provided by Professor Namin Baek from Kyung Hee University's nature compound library (College of Life Science, Kyung Hee University, Korea). Before the experiment, 70 compounds were randomly numbered, and each compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock concentration of 10 mM. Information on the 70 compounds used was not disclosed except for steppogenin (compound #57), and tanespimycin (17-AAG) was purchased from Selleckchem (Pittsburgh, PA, US).

Experimental Example 2. Cell culture and hypoxic conditions

Human embryonic kidney epithelial cells (HEK293T; KCLB, Seoul, Korea) and HUVEC cell lines (human umbilical vein EC; EA.hy926; ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (Hyclon, Logan, UT, USA). Human lung cancer cells (A549; ATCC) were cultured in RPMI-1640 (Hyclon), and human retinal pigment epithelial cells (ARPE-19) were cultured in 1% antibiotics (100-units/mL penicillin and 100-mg/mL streptomycin). ; Invitrogen, Carlsbad, CA, USA) and 10% FBS (fetal bovine serum) were cultured in DME/F-12 media (Hyclon). HUVEC (passaged 2 to 8 times) were gelatin-coated (0.1% gelatin; Sigma-2) in EBM-2 (Lonza, Basel, Switzerland) supplemented with EGM-2 Kit, 10% FBS and 1% antibiotics. Aldrich) was grown in a culture dish. All cells were cultured under normoxic conditions (5% CO ₂ and 95% air), and cells were cultured under hypoxic conditions (5% CO ₂ , 1% O ₂ and 94% N ₂ ) in hypoxia chambers (ASTEC, Fukuoka, Japan). exposed to.

Experimental Example 3. Animal model

Six-week-old adult male C57BL/6J mice (SLC, Japan) were used and housed in a specific pathogen-free facility with an individual ventilation system at Kyungpook National University. Animal handling and experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (protocol numbers: KNU 2014-0189 and KNU 2017-0145) notified by the Animal Hospital Ethics Committee of Kyungpook National University.

Experimental Example 4. Cell counting Kit-8 assay

HEK293T cells were seeded in a 96-well plate at a density of 5×10 ³ cells/well and cultured at 37°C for 24 hours. After 24 hours, the natural compound was diluted in the medium according to the corresponding concentration and replaced. Cell viability was confirmed after exposure to hypoxic conditions for 24 hours using Cell Counting Kit-8 (DOJINDO, Rockville, MD, USA) according to the manufacturer's instructions, and measured using a microplate reader (Infinite M200 Pro; TECAN, Mannedorf) at 450 nm. , Switzerland) was used.

Experimental Example 5. Dual-luciferase assay

To perform the HRE-luciferase assay, HEK293T cells were seeded in a 96-well plate at a density of 5×10 ³ cells/well and cultured at 37°C. Then, cells were co-transfected with pGL3-HRE-luciferase plasmid and pRL-SV40 Renilla plasmid containing five copies of the HRE of the human VEGF gene using Vivamagic transfection reagent (Vivagen, Seongnam, Korea) (Kwon et al ., 2015). After 24 hours, the cells were pretreated with various concentrations of natural compounds for 1 hour and then incubated in hypoxic conditions for 24 hours. To perform the DLL4-luciferase assay, EA.hy926 cells were seeded in a 24-well plate at a density of 4×10 ⁴ cells/well and cultured at 37°C for 24 hours. Cells were co-transfected with pGL3-DLL4 luciferase and pRL-SV40 Renilla plasmids using Lipofectamine 2000 (Invitrogen) and cultured at 37°C for 24 h. Then, they were incubated with various concentrations of natural compounds for 1 hour and then incubated in the presence or absence of VEGF (10 ng/mL) for 24 hours. The luminescence of HRE- and DLL4-promoter activity was detected using a microplate reader and Dual-Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The luciferase activity of the natural compound was normalized to Renilla luciferase activity.

Experimental Example 6. Immunofluorescence staining

Cells were seeded on coverslips in a 4-well plate at a density of 2-3 × 10 ⁴ cells/well and cultured for 24 hours. For HIF-1α staining, A549 cells were treated with various compounds and incubated under normoxic or hypoxic conditions for 4 hours. For Ki-67 and DLL4 staining, EA.hy926 cells were treated with the same compounds and maintained under VEGF (20 ng/mL) treatment. Then, the cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100. The fixed cells were blocked with phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) and then incubated with primary antibodies overnight. After incubation, cells were incubated with secondary antibodies for 1 h and nuclei were stained using DAPI. Stained cells were visualized using a confocal microscope (Leica TCS SP5 II Dichroic/CS, Leica, Germany).

Experimental Example 7. Western blot analysis

Proteins were extracted from cultured cells using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA). Protein extracts were separated using SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Maidstone, England). Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature. After blocking, the membrane was incubated with specific primary antibodies at 4°C overnight and then with secondary antibodies for 1 hour at room temperature. Proteins were detected using the Enhanced Chemiluminescence Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. HIF-1α (BD Biosciences, San Diego, CA, USA), VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CXCR4 (Abcam, Cambridge, UK), CA9 (Abcam), DLL4 (Santa Cruz Biotechnology), Primary antibodies against NOTCH1 (Thermo Scientific) and β-actin (Santa Cruz Biotechnology) were used.

Experimental Example 8. Lactate dehydrogenase cytotoxicity assay

HEK293T cells were seeded in a 96-well plate at 2×10 ³ cells/well and cultured at 37°C for 24 hours. Cells were then treated with various concentrations of stepogenin for 24 hours under hypoxic conditions. Lactate dehydrogenase (LDH) release values were evaluated using the Cytotoxicity LDH Assay Kit-WST (DOJINDO) according to the manufacturer's instructions. Cytotoxicity was measured using a microplate reader (Infinite M200 Pro; TECAN) at 490 nm.

Experimental Example 9. RNA isolation and quantitative reverse-transcription polymerase chain reaction

Total RNA was isolated using TRIzol Reagent (Invitrogen). The isolated RNA was used to synthesize cDNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. The synthesized cDNA was mixed with gene-specific primers. Amplification real-time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). Thermal cycling proceeded as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s denaturation at 95 °C, and 1 min annealing at 60 °C. Mean cycle threshold values were used to calculate mRNA expression with normalization to β-actin as an internal control. The sequences of specific primers for real-time PCR are shown in Table 1.

Experimental Example 10. Wound-healing migration assay

EA.hy926 cells were seeded in a 12-well plate at a density of 4×10 ⁴ cells/well and incubated for 24 hours. Cells were starvated in serum-free medium overnight and treated with mitomycin C (0.5 μg/mL) for 1 hour. At confluence, a wound was made with a pipette tip and washed with serum-free medium to remove cell debris. The plates were then treated with conditioned medium collected from HEK293T cell cultures under hypoxic conditions treated with stepogenin. Wound closure was monitored and photographs were taken at 0h and 8h. Cell migration was measured by calculating the wound area between 0 h and 8 h.

Experimental Example 11. Spheroid sprouting assay

EA.hy 926 cell suspension (5 × 10 ³ cells) was distributed on the lid of a 100 mm dish using the hanging-drop method and maintained for 24 hours. The next day, spheroids were harvested and mixed with Methocel stock solution containing 20% FBS. Spheroids containing Methocel solution were then seeded in 24-well plates. After incubation at 37°C for 30 minutes, VEGF or stepogenin was added to the polymerized Methocel solution and incubated for 2 days. Finally, germinated endothelial cells (EC) were counted and graphed.

Experimental Example 12. Development of tumor model and treatment regimen

To prepare the LLC (Lewis lung carcinoma) allograft tumor model, LLC cell suspension (5 × 10 ⁵ cells in 200 μL of serum-free culture medium) was implanted subcutaneously into the dorsal flank of 6-week-old male C57BL/6J mice. . Mice were administered 17-AAG (25 mg/kg) or stepogenin (2 mg/kg) via intraperitoneal injection at various time points after LLC-cell inoculation. Then, the tumor volume was measured according to the following formula: V = 0.5 × A × B ² , where A and B refer to the longest and shortest dimensions, respectively. Finally, the mice were anesthetized and tissues were collected for further analysis.

Experimental Example 13. Immunohistochemical and histological analyzes

For immunohistochemical analysis, collected tumor or tissue samples were fixed in 4% paraformaldehyde and embedded in tissue freezing medium (Leica, Wetzlar, Germany). Frozen blocks were cut into 10 μm sample sections, blocked with 3% BSA in PBST (0.3% Triton X-100 in PBS), and incubated with appropriate primary antibodies overnight at 4°C. Primary antibodies were: anti-CD34 (Boster Bio, CA, USA), anti-DLL4 (R&D Systems, MN, USA), anti-pimonidazole (Hypoxyprobe-1, HPI, MA, USA), anti-CD3. (Abcam), anti-CD4 (Invitrogen), and anti-CD8 (Invitrogen). After several washes, the samples were incubated with the recommended fluorophore-tagged secondary antibodies for 1 hour at room temperature. Then, cell nuclei were stained using Antifade Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA). Immunofluorescence images were acquired using a Zeiss confocal microscope (Carl Zeiss, Germany). Then, pimonidazole hydrochloride (60 mg/kg; Hypoxyprobe-1, HPI) was injected intravenously through the mouse's tail vein, and the mouse was anesthetized 90 minutes later. Finally, tumor tissue was collected for further analysis.

Experimental Example 14. Statistical Analysis

All data were expressed as the mean ± standard deviation (SD) of at least three samples, and a P value <0.05 was considered statistically significant. Data were analyzed using Student's t test and ANOVA.

[Example]

Example 1. Screening of potential HIF-1α and DLL4 inhibitors among 70 natural compounds

To test the inhibitory activity of 70 natural compounds on HIF-1 and DLL4 activity, we first tested their cytotoxic effects on immortalized normal cells under hypoxic conditions. In particular, under these conditions, HEK293T cells were treated with compounds at various concentrations (0.1 to 10 μM). As a result, as shown in Figure 1, two natural compounds (#31 and #61) with low cell survival rate under hypoxia were excluded from further analysis.

To select potential HIF-1α inhibitors, the effect of 70 natural compounds was tested on HIF-1α promoter reporter activity using a dual-luciferase assay. As a result, as shown in Figure 2a, 12 natural compounds (#17, #18, #34, #42, #47, #49, #50, #51, #52, #56, #57, and # 70) showed HRE-luciferase activity of <70% compared to the hypoxic vehicle (DMSO) control, and was selected as a potential HIF-1α inhibitor.

To confirm the effect of selected natural compounds on HIF-1α protein expression, Western blot was performed on HEK293T cells. As a result, as shown in Figure 2b, five natural compounds (#51, #52, #56, #57, and #70) inhibited HIF-1α protein expression. Tip Since DLL4 expression in Endothelial Cells (EC) is induced by VEGF (Blanco and Gerhardt, 2013), simultaneous targeting of HIF-1α in hypoxic tumor cells and DLL4 in vascular EC promotes tumor growth, sprouting, and angiogenesis. This was effectively suppressed. Therefore, the effects of five compounds with HIF-1α inhibitory activity on DLL4 reporter activity in EC were also investigated. As a result, as shown in Figure 2c, the DLL4 protein level and DLL4 luciferase activity induced by VEGF were significantly inhibited by treatment with compound #57. Therefore, compound #57 was selected as a potential inhibitor of HIF-1α and DLL4.

Example 2. HIF-1α and DLL4 inhibitory activity of stepogenin

Natural compound #57 was identified as stepogenin extracted from the root bark of Morus alba L. As a result of further testing, as shown in Figures 3a and 3b, it was confirmed that dose-dependent cell viability and cytotoxicity under hypoxic conditions were not changed by stepogenin treatment compared to vehicle treatment. However, as shown in Figure 3c, HRE-luciferase reporter activity was significantly inhibited by 0.1 to 10 μM stepogenin in a dose-dependent manner. Additionally, as shown in Figure 3D, the half maximal inhibitory concentration (IC ₅₀ ) for HIF-1α activity was 0.56 ± 0.043 μM. DLL4-luciferase reporter activity was also significantly reduced by 0.3 - 10.0 μM stepogenin, as shown in Figure 3e. As shown in Figure 3f, the IC ₅₀ for DLL4 activity was 8.46 ± 1.08 μM in EA.hy926 cells.

Example 3. Stepogenin inhibits expression of HIF-1α in cancer cells under hypoxic conditions

As shown in Figure 4A, stepogenin significantly suppressed HIF-1α protein levels in a dose-dependent manner in HEK293T human embryonic kidney epithelial cells, A549 human lung cancer cells, and ARPE19 human retinal pigment epithelial cells under hypoxic conditions ( Maximum dose: 3 μM). As a result of immunofluorescence analysis, as shown in Figure 4b, the nuclear expression of HIF-1α increased in hypoxia compared to the normoxic control group. However, similar to 17-AAG treatment, stepogenin (#57) treatment decreased the nuclear expression of HIF-1α under hypoxic conditions.

The transcription factor HIF-1α regulates the expression of various target genes that can overcome low oxygen levels or are involved in angiogenesis, cell survival, metastasis, and glucose metabolism (Semenza, 2007). To confirm the inhibitory effect of stepogenin on HIF-1α activity, the mRNA levels of HIF-1α target genes were examined using real-time qRT-PCR. As a result, as shown in Figure 4c, stepogenin suppressed the mRNA expression of VEGF, GLUT1, CXCR4, and CA9 under hypoxic conditions. Similarly, as shown in Figure 4D, under hypoxic conditions, stepogenin suppressed protein levels of VEGF, CXCR4, and CA9 compared to levels detected in vehicle controls. Therefore, it was confirmed that stepogenin inhibits HIF-1α activity and its target gene expression.

Example 4. Stepogenin Inhibits DLL4 Activity and Budding Angiogenesis in EC

Stepogenin dose-dependently reduced VEGF-induced DLL4, Notch1, and NICD protein levels in HUVEC, as shown in Figure 5A. Because the DLL4-NOTCH signaling pathway in ECs is important for tip and stalk cell fate during sprouting angiogenesis, we evaluated the angiogenic efficacy of stepogenin in vitro. Interestingly, immunofluorescence analysis showed that DLL4 expression in EC was significantly increased under hypoxic conditions, as shown in Figure 5b. However, stepogenin treatment dramatically inhibited DLL4 expression under these conditions. The level of Ki-67, a cell proliferation marker, increased under hypoxic conditions, but decreased by stepogenin treatment. Further analysis of HUVECs showed that the number of migrating cells and wound closure were significantly reduced in the presence of stepogenin under hypoxic conditions, as shown in Figure 5C. Additionally, as shown in Figure 5D, in 3D EC spheroid cultures, sprouting ability (determined by counting the number of sprouts) was increased by VEGF, but was significantly inhibited by stepogenin treatment. Therefore, it was found that stepogenin acts as an inhibitor of HIF-1α and DLL4 and inhibits angiogenesis in vitro.

Example 5. Stepogenin Inhibits Tumor Growth and Angiogenesis

17-AAG (25 mg/kg), stepogenin (2 mg/kg), or vehicle (control) was administered to LLC on days 7, 9, 11, 13, and 15 after inoculation with the LLC cell suspension of Experimental Example 12. The in vivo effect of stepogenin was tested by treating an allograft tumor model. As a result, as shown in Figure 6a, both 17-AAG and stepogenin significantly inhibited tumor growth. However, as shown in Figure 6b, there was no significant difference in mean body weight between groups during the administration period. As a result of immunohistochemical staining, as shown in Figure 6c, the hypoxic area and HIF-1α expression level in the tumor were significantly reduced by treatment with stepogenin or 17-AAG compared to the effect in the control group. Additionally, as shown in Figure 6D, microvessel density analyzed using CD34 antibody (a tip-cell marker in tumor tissue) was significantly reduced by stepogenin compared to that observed in control tissue. Abnormal vasculature creates a hypoxic microenvironment that polarizes inflammatory cells toward immunosuppression (Palazon et al., 2012). To determine whether stepogenin induces vascular normalization, T-cell infiltration was examined in tumor tissue. As a result, as shown in Figure 6e, stepogenin significantly increased the infiltration of CD3+ T cells, and the cytotoxicity (CD8+) vs. Reduced the proportion of regulatory T (CD4+) cells. These results imply that stepogenin inhibits tumor growth by normalizing tumor blood vessels by suppressing active sprouting in HIF-1α and EC in tumor tissues.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (11)

A pharmaceutical composition for inhibiting angiogenesis comprising steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.
According to paragraph 1,
A pharmaceutical composition wherein the stepogenin inhibits the expression of the DLL4 gene or reduces the level of the protein expressed by the DLL4 gene.
According to paragraph 1,
A pharmaceutical composition characterized in that the composition inhibits cell migration or proliferation of vascular endothelial cells.
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition can prevent or treat diseases caused by angiogenesis.
According to paragraph 4,
The diseases include arthritis, rheumatoid arthritis, chronic inflammation, osteoarthritis, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, corneal disease caused by neovascularization, macular degeneration, spot degeneration, Pharmacology, characterized in that it is any one disease selected from the group consisting of pterygium, retinal degeneration, erythroderma, proliferative retinopathy, retrophakic fibroplasia, granular conjunctivitis, psoriasis, telangiectasia, psoriasis, seborrheic dermatitis, and acne. enemy composition.
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition further comprises an angiogenesis inhibitor.
An anticancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient, characterized in that it is capable of inhibiting angiogenesis.
In clause 7,
The stepogenin is an anti-cancer adjuvant, characterized in that it inhibits the expression of the DLL4 gene or reduces the level of the protein expressed by the DLL4 gene.
In clause 7,
An anticancer adjuvant, characterized in that the anticancer adjuvant can be administered simultaneously or sequentially with an anticancer agent.
In clause 7,
The anticancer adjuvant is characterized in that the anticancer adjuvant is administered in combination with an anticancer agent.
A health functional food composition for preventing and improving angiogenesis-related diseases, comprising steppogenin or a foodologically acceptable salt thereof as an active ingredient.