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WO2024039183A1 - Composition pharmaceutique pour inhiber l'angiogenèse comprenant de la stépogénine en tant que principe actif - Google Patents

Composition pharmaceutique pour inhiber l'angiogenèse comprenant de la stépogénine en tant que principe actif Download PDF

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WO2024039183A1
WO2024039183A1 PCT/KR2023/012127 KR2023012127W WO2024039183A1 WO 2024039183 A1 WO2024039183 A1 WO 2024039183A1 KR 2023012127 W KR2023012127 W KR 2023012127W WO 2024039183 A1 WO2024039183 A1 WO 2024039183A1
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angiogenesis
stepogenin
acceptable salt
anticancer
pharmaceutically acceptable
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이유미
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Industry Academic Cooperation Foundation of KNU
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/08—Antiseborrheics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/30—Other Organic compounds

Definitions

  • 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:
  • 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.
  • Eye 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.
  • 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.
  • 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.
  • diseases related to new blood vessel formation such as diabetic retinopathy, rheumatoid arthritis, inflammation, endometriosis, age-related loss of vision, psoriasis, and hemangioma.
  • AMD diabetic retinopathy
  • 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.
  • 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.
  • 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).
  • angiogenesis occurs by angiogenic factors such as VEGF to receive oxygen and nutrients in the hypoxic area of the cancer tissue.
  • 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.
  • 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.
  • the present invention provides a pharmaceutical composition for inhibiting angiogenesis containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.
  • 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.
  • 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.
  • the composition may inhibit cell migration or proliferation of vascular endothelial cells, but is not limited thereto.
  • the composition can prevent or treat angiogenesis-related diseases, but is not limited thereto.
  • 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.
  • the composition may further include an angiogenesis inhibitor, but is not limited thereto.
  • the present invention is an anti-cancer supplement containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient,
  • the stepogenin or a pharmaceutically acceptable salt thereof provides an anti-cancer adjuvant characterized in that it can inhibit angiogenesis.
  • 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.
  • the anticancer adjuvant may be administered simultaneously or sequentially with the anticancer agent, but is not limited thereto.
  • the anti-cancer adjuvant may be administered in combination with an anti-cancer agent, but is not limited thereto.
  • 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.
  • 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.
  • the present invention provides the use of steppogenin or a pharmaceutically acceptable salt thereof for manufacturing a drug for treating angiogenesis-related diseases.
  • 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.
  • the present invention provides a method of improving or enhancing the anticancer efficacy of an anticancer agent comprising administering an anticancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof to an individual in need thereof.
  • the present invention provides a use of a composition containing steppogenin or a pharmaceutically acceptable salt thereof to improve or enhance the anticancer efficacy of an anticancer agent.
  • the present invention provides the use of steppogenin or a pharmaceutically acceptable salt thereof to produce a drug that improves or enhances the anticancer efficacy of an anticancer agent.
  • Stepogenin of the present invention can inhibit the proliferation and migration of vascular endothelial cells by inhibiting DLL4 expression, thereby preventing, improving, or treating angiogenesis-related diseases caused by abnormal new blood vessel formation, or inhibiting angiogenesis, etc. It is expected that it can be usefully applied to effective medicines, foods, and anti-cancer supplements.
  • 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-1 ⁇ 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-1 ⁇ inhibitors among 70 candidate compounds inhibit HIF-1 ⁇ protein expression (H: hypoxia; *p ⁇ 0.05, **p ⁇ 0.005, and ***p ⁇ 0.001 vs. hypoxic control group; $$p ⁇ 0.01 vs. normoxic control group).
  • Figure 2c is a diagram showing the results of confirming whether compounds that inhibit HIF-1 ⁇ protein expression among compounds selected as potential HIF-1 ⁇ inhibitors inhibit DLL4 protein expression (*p ⁇ 0.05, **p ⁇ 0.005, and ***p ⁇ 0.001 vs. VEGF treated control; $$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-1 ⁇ 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-1 ⁇ 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-1 ⁇ 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-1 ⁇ 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-1 ⁇ 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 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.
  • the present invention provides a pharmaceutical composition for inhibiting angiogenesis containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient.
  • 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.
  • the present invention may also include a pharmaceutically acceptable salt of steppogenin as an active ingredient.
  • pharmaceutically acceptable salt includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • acids examples 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.
  • a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile.
  • 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.
  • 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).
  • an appropriate silver salt eg, silver nitrate
  • 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.
  • the stepogenin or a pharmaceutically acceptable salt thereof inhibits the expression of the DLL4 and HIF-1 ⁇ (Hypoxia-inducible factor-1 alpha) genes, or inhibits the expression of the DLL4 and HIF-1 ⁇ genes.
  • DLL4 and HIF-1 ⁇ Hypoxia-inducible factor-1 alpha
  • 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.
  • 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.
  • 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:
  • the composition can prevent or treat angiogenesis-related diseases, but is not limited thereto.
  • 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 the abnormal progression of new blood vessel formation.
  • angiogenesis-related disease and “disease caused by angiogenesis” may be used interchangeably.
  • the angiogenesis-related diseases include, for example, angiogenesis-dependent cancer, benign tumor, arthritis, rheumatoid arthritis, chronic inflammation, osteoarthritis, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, and neovascularization.
  • corneal disease macular degeneration, macular degeneration, pterygium, retinal degeneration, erythema, proliferative retinopathy, posterior lens fibroplasia, granular conjunctivitis, psoriasis, telangiectasia, pyogenic granuloma, psoriasis, This may include, but is not limited to, erythema, seborrheic dermatitis, or acne, and may include all diseases caused by abnormal new blood vessel formation.
  • 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 plicamycin, mitomycin and C Bleomycin
  • plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, and Iridotecan may be included, but is not limited thereto.
  • 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);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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-
  • 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.
  • 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.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • 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.
  • 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.
  • 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.
  • the present invention provides the use of steppogenin or a pharmaceutically acceptable salt thereof for manufacturing a drug for treating angiogenesis-related diseases.
  • “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
  • administration means providing a given composition of the present invention to an individual by any appropriate method.
  • prevention refers to any action that suppresses or delays the onset of the desired disease
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides an anti-cancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof as an active ingredient,
  • stepogenin or a pharmaceutically acceptable salt thereof provides an anti-cancer adjuvant characterized in that it can inhibit angiogenesis.
  • the specific description of stepogenin is the same as the above description.
  • the present invention provides a method of improving or enhancing the anticancer efficacy of an anticancer agent comprising administering an anticancer adjuvant containing steppogenin or a pharmaceutically acceptable salt thereof to an individual in need thereof.
  • the present invention provides a use of a composition containing steppogenin or a pharmaceutically acceptable salt thereof to improve or enhance the anticancer efficacy of an anticancer agent.
  • the present invention provides the use of steppogenin or a pharmaceutically acceptable salt thereof to produce a drug that improves or enhances the anticancer efficacy of an anticancer agent.
  • the anticancer adjuvant according to the present invention containing stepogenin as an active ingredient can improve the efficacy of the anticancer agent by inhibiting angiogenesis.
  • 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.
  • the cancer may be a solid cancer
  • 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.
  • the 70 compounds used 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).
  • DMSO dimethyl sulfoxide
  • HUVEC cell lines human umbilical vein EC; EA.hy926; ATCC, Manassas, VA, USA
  • Dulbecco's modified Eagle's medium Hyclon, Logan, UT, USA
  • Human lung cancer cells A549; ATCC
  • RPMI-1640 Hyclon
  • ARPE-19 human retinal pigment epithelial cells
  • HUVEC human bovine serum
  • EBM-2 Lida, Basel, Switzerland
  • EGM-2 Kit 10% FBS and 1% antibiotics. Aldrich
  • 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.
  • 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.
  • 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.
  • Vivamagic transfection reagent Vivagen, Seongnam, Korea
  • 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.
  • Cells were seeded on coverslips in a 4-well plate at a density of 2 ⁇ 10 4 - 3 ⁇ 10 4 cells/well and cultured for 24 hours.
  • HIF-1 ⁇ staining A549 cells were treated with various compounds and incubated under normoxic or hypoxic conditions for 4 hours.
  • 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).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • 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-1 ⁇ (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 ⁇ -actin (Santa Cruz Biotechnology) were used.
  • 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.
  • LDH Lactate dehydrogenase
  • 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 ⁇ g/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.
  • 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.
  • EC germinated endothelial cells
  • LLC cell suspension (5 ⁇ 10 5 cells in 200 ⁇ L 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.
  • the mice were anesthetized and tissues were collected for further analysis.
  • HIF-1 ⁇ inhibitors To select potential HIF-1 ⁇ inhibitors, the effect of 70 natural compounds was tested on HIF-1 ⁇ 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-1 ⁇ inhibitor.
  • DMSO hypoxic vehicle
  • 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 ⁇ M stepogenin in a dose-dependent manner. Additionally, as shown in Figure 3D, the half maximal inhibitory concentration (IC 50 ) for HIF-1 ⁇ activity was 0.56 ⁇ 0.043 ⁇ M. DLL4-luciferase reporter activity was also significantly reduced by 0.3 - 10.0 ⁇ M stepogenin, as shown in Figure 3e. As shown in Figure 3f, the IC 50 for DLL4 activity was 8.46 ⁇ 1.08 ⁇ M in EA.hy926 cells.
  • Example 3 Stepogenin inhibits expression of HIF-1 ⁇ in cancer cells under hypoxic conditions
  • stepogenin significantly suppressed HIF-1 ⁇ 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 ⁇ M).
  • the nuclear expression of HIF-1 ⁇ increased in hypoxia compared to the normoxic control group.
  • stepogenin (#57) treatment reduced the nuclear expression of HIF-1 ⁇ under hypoxic conditions.
  • the transcription factor HIF-1 ⁇ 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).
  • the mRNA levels of HIF-1 ⁇ target genes were examined using real-time qRT-PCR.
  • stepogenin suppressed the mRNA expression of VEGF, GLUT1, CXCR4, and CA9 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-1 ⁇ activity and its target gene expression.
  • Example 4 Stepogenin Inhibits DLL4 Activity and Budding Angiogenesis in EC
  • DLL4-NOTCH signaling pathway in ECs is important for tip and stalk cell fate during sprouting angiogenesis.
  • immunofluorescence analysis showed that DLL4 expression in EC was significantly increased under hypoxic conditions, as shown in Figure 5b.
  • stepogenin treatment dramatically inhibited DLL4 expression under these conditions.
  • Ki-67 a cell proliferation marker, increased under hypoxic conditions, but decreased by stepogenin treatment.
  • stepogenin acts as an inhibitor of HIF-1 ⁇ and DLL4 and inhibits angiogenesis in vitro.
  • Example 5 Stepogenin Inhibits Tumor Growth and Angiogenesis
  • 17-AAG 25 mg/kg
  • stepogenin (2 mg/kg)
  • vehicle control
  • the in vivo effect of stepogenin was tested by treating an allograft tumor model.
  • both 17-AAG and stepogenin significantly inhibited tumor growth.
  • the hypoxic area and HIF-1 ⁇ expression level in the tumor were significantly reduced by treatment with stepogenin or 17-AAG compared to the effect in the control group.
  • microvessel density analyzed using CD34 antibody 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).
  • T-cell infiltration was examined in tumor tissue.
  • stepogenin significantly increased the infiltration of CD3+ T cells, and the cytotoxicity (CD8+) vs. Reduced the proportion of regulatory T (CD4+) cells.
  • Stepogenin of the present invention can inhibit the proliferation and migration of vascular endothelial cells by inhibiting DLL4 expression, thereby preventing, improving, or treating angiogenesis-related diseases caused by abnormal new blood vessel formation, or inhibiting angiogenesis, etc. It can be usefully applied to effective medicines, foods, anti-cancer supplements, etc., so it has industrial applicability.

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Abstract

La présente invention concerne une composition pour inhiber l'angiogenèse, la composition comprenant de la stépogénine en tant que principe actif. La stépogénine selon la présente invention peut inhiber la prolifération et la migration de cellules endothéliales vasculaires par inhibition de l'expression de DLL4 et, par conséquent, devrait être utile dans un médicament, un aliment ou un complément anticancéreux pour prévenir, améliorer ou traiter des maladies liées à l'angiogenèse causées par une angiogenèse anormale, ou présentant des bienfaits tels que l'inhibition de l'angiogenèse.
PCT/KR2023/012127 2022-08-19 2023-08-16 Composition pharmaceutique pour inhiber l'angiogenèse comprenant de la stépogénine en tant que principe actif Ceased WO2024039183A1 (fr)

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