Pharmaceutical composition for tumor vascular disruption

Abstract

A pharmaceutical composition for tumor vascular disruption, according to an exemplary embodiment, may include an alkaline anticancer agent, a cell metabolism regulator, and epinephrine. The composition can disrupt tumors by inducing apoptosis of vascular endothelial cells. For example, the composition can be topically administered to the tumor submucosal layer to maximize the drug's therapeutic effect.

Description

Pharmaceutical composition for destroying tumor blood vessels

[0001] The present invention relates to a pharmaceutical composition for destroying tumor blood vessels and can be used in the medical and pharmaceutical fields.

[0002] Systemic administration of anticancer drugs, either intravenously or orally, is widely used to treat solid tumors. However, this method has limitations: the drug may not sufficiently reach the tumor site or may be distributed to normal tissues, potentially causing serious systemic toxicity.

[0003] The tumor microenvironment (TME) refers to the surrounding environment of the tissue where a tumor grows. In addition to cancer cells, the tumor microenvironment includes cells such as blood vessels and immune cells, as well as various extracellular matrix components. The tumor microenvironment plays a crucial role in determining cancer growth, metastasis, and drug response. With growing interest in the tumor microenvironment, various studies targeting it are actively underway. For example, local drug delivery systems (LDDS) are a technology that can effectively control the tumor microenvironment.

[0004] Local drug delivery systems inject drugs locally into or around tumors to induce tumor tissue death. This method maximizes therapeutic efficacy and minimizes systemic side effects by directly delivering drugs to specific areas. Research is ongoing into various technologies to improve the efficiency and safety of local drug delivery systems.

[0005] Korean Patent No. 10-1698003 discloses an injectable composition for local administration for anticancer treatment, comprising a quinine hydrochloride suspension. However, direct injection of anticancer agents into tumors has limitations in effectively eliminating cancer cells, as the heterogeneity of tumor tissue and the pressure of solid bodies prevent the drug from evenly dispersing.

[0006] The purpose of the present invention is to provide a pharmaceutical composition for destroying tumor blood vessels with excellent anticancer effects and reduced side effects.

[0007] The purpose of the present invention is to provide a drug delivery system capable of maximizing the therapeutic effect of a drug while locally delivering the drug to the tumor base.

[0008] A pharmaceutical composition for destroying tumor blood vessels according to an exemplary embodiment may include an alkaline anticancer agent, a cell metabolism regulator, and epinephrine.

[0009] In one embodiment, the alkalinity may be pH 8 to 9.

[0010] In one embodiment, the alkaline anticancer agent may include vascular disrupting agents.

[0011] In one embodiment, the angiogenic agent may include one or more selected from Vadimezan or Fosbretabulin.

[0012] In one embodiment, the concentration of the angiogenic agent may be from 0.001 mg / mL to 20 mg / mL in the total composition.

[0013] In one embodiment, the alkaline anticancer agent may include at least one selected from a pyrimidine derivative, a taxane derivative, and an anthracene derivative.

[0014] In one embodiment, the epinephrine can constrict blood vessels surrounding tumor vascular endothelial cells, thereby inhibiting the diffusion of the alkaline anticancer agent and the cell metabolism regulator.

[0015] In one embodiment, the concentration of epinephrine may be from 0.001 to 0.2 mg / mL in the total composition.

[0016] In one embodiment, the cell metabolism modulator may include one or more selected from 2-deoxy-D-glucose (2-DG), dichloroacetic acid (DCA), and L-glucose.

[0017] In one embodiment, the cell metabolism regulator can induce tumor vascular endothelial cell apoptosis by increasing the expression of Fas receptor, Death Receptor 4, and Death Receptor 5.

[0018] In one embodiment, the concentration of the cell metabolism regulator may be from 0.1 mM to 500 mM in the total composition.

[0019] In one embodiment, the composition may be administered to the submucosal layer where tumor blood vessels are located.

[0020] In one embodiment, it may be administered by local injection into the submucosal layer.

[0021] In one embodiment, the tumor may be a solid tumor.

[0022] A solid tumor topical administration agent according to an exemplary embodiment may include the pharmaceutical composition for destroying tumor blood vessels.

[0023] The pharmaceutical composition for destroying tumor blood vessels according to the present invention can destroy tumors by inducing apoptosis of tumor vascular endothelial cells. For example, it can provide a drug delivery system that can maximize the therapeutic effect of a drug while locally delivering the drug to the tumor base.

[0024] The pharmaceutical composition for destroying tumor blood vessels according to the present invention can be applied to various solid cancers because it destroys blood vessels supplying blood to the tumor rather than the tumor itself.

[0025] Figure 1 shows the results of MTS analysis of the HUVEC killing effect according to the concentration of Vadimezan.

[0026] Figure 2 shows the results of confirming the short-term killing effect of HUVEC according to the concentration of Vadimezan using MTS.

[0027] Figure 3 shows the results of confirming the HUVEC killing effect according to Vadimezan concentration and pH using MTS.

[0028] Figure 4 shows the results of confirming the short-term killing effect of HUVEC according to Vadimezan concentration and pH using MTS.

[0029] Figure 5 shows the results of confirming the cell death effect of L-glucose on HUVEC according to the concentration of Vadimezan using MTS.

[0030] Figure 6 shows the results of MTS analysis of the cell death effect of HUVECs according to the concentration of Vadimezan and the concentration of L-glucose and pH.

[0031] Figures 7, 8a, and 8b show the results of confirming the spontaneous death of HUVEC according to the concentration of Vadimezan using confocal fluorescence microscopy.

[0032] Figure 9 shows the results of confirming the production of ROS (Reactive Oxygen Species) in HUVEC according to the concentration of Vadimezan.

[0033] Figure 10 shows the results of confirming the natural death mechanism of HUVEC by Vadimezan using western blot.

[0034] Figure 11 shows the results of confirming the pathological phenomena that appeared in the surrounding tissues when Vadimezan was injected around the tumor after xenografting human gastric cancer cells into nude mice.

[0035] Figures 12 and 13 show the results of an in-vivo experiment examining the response to anticancer drugs after xenografting human gastric cancer cells into nude mice.

[0036] Figure 14 shows the results of MTS analysis of the HUVEC killing effect according to the concentration of Gemcitabine.

[0037] Figure 15 shows the results of confirming the short-term killing effect of HUVEC according to the concentration of Gemcitabine using MTS.

[0038] Figure 16 shows the results of MTS analysis of the HUVEC killing effect according to Gemcitabine concentration and pH.

[0039] Figure 17 shows the results of MTS analysis of the short-term killing effect of HUVEC according to Gemcitabine concentration and pH.

[0040] Figure 18 shows the results of MTS analysis of the apoptotic effect of L-glucose on HUVEC according to the concentration of Gemcitabine.

[0041] Figure 19 shows the results of MTS analysis of the cell death effect of HUVECs according to the concentration of Gemcitabine and the concentration of L-glucose and pH.

[0042] Figure 20 shows the results of confirming the HUVEC death effect according to Taxol concentration using MTS.

[0043] Figure 21 shows the results of confirming the short-term killing effect of HUVEC according to Taxol concentration and pH using MTS.

[0044] Figure 22 shows the results of confirming the HUVEC death effect according to the concentration of 2-DG using MTS.

[0045] Figure 23 shows the results of confirming the short-term killing effect of HUVEC according to the concentration of 2-DG using MTS.

[0046] Figure 24 shows the results of confirming the HUVEC killing effect according to 2-DG concentration and pH using MTS.

[0047] Figure 25 shows the results of MTS analysis of the short-term killing effect of HUVEC according to 2-DG concentration and pH.

[0048] Figure 26 shows the results of MTS analysis of the HUVEC killing effect according to the concentration of 2-DG when 2-DG and 5-FU were mixed.

[0049] Figure 27 shows the results of confirming the spontaneous death of HUVEC according to the concentration of 2-DG using a confocal fluorescence microscope.

[0050] Figure 28 shows the results of Western blot analysis confirming the natural death mechanism of HUVECs by 2-DG.

[0051] Figures 29 and 30 show the results of an in-vivo experiment examining the response to anticancer drugs after xenografting human gastric cancer cells into nude mice.

[0052] Figure 31 shows the results of confirming the HUVEC death effect according to DCA concentration using MTS.

[0053] Figure 32 shows the results of confirming the short-term killing effect of HUVEC according to DCA concentration using MTS.

[0054] Figure 33 shows the results of confirming the HUVEC killing effect according to DCA concentration and pH using MTS.

[0055] Figure 34 shows the results of confirming the short-term killing effect of HUVEC according to DCA concentration and pH using MTS.

[0056] Figure 35 shows the results of MTS analysis of the HUVEC killing effect according to the DCA concentration when DCA and 5-FU were mixed.

[0057] Figures 36 and 37 show the results of an in-vivo experiment examining the response to anticancer drugs after xenografting human gastric cancer cells into nude mice.

[0058] Figure 38 shows the results of confirming the D-Glucose concentration in HUVEC cells according to the L-glucose concentration.

[0059] Figures 39a and 39b show the results of L-Glucose tracking observation in HUVEC cells following GLUT inhibitor treatment.

[0060] Figures 40a and 40b show the results of Western blot analysis confirming the natural death mechanism of HUVEC when mixed with L-glucose and 5-FU.

[0061] Figure 41 shows the results of analysis of CD31 and VEGF expression after xenotransplantation of human gastric cancer cells into nude mice.

[0062] Figure 42 shows the results of an in-vivo experiment examining the response to anticancer drugs after xenografting human gastric cancer cells into nude mice.

[0063] According to an exemplary embodiment, a pharmaceutical composition for destroying tumor blood vessels (hereinafter, referred to as the composition) comprises an alkaline anticancer agent, a cell metabolism regulator, and epinephrine. For example, the composition may induce the death of tumor vascular endothelial cells by including the alkaline anticancer agent. For example, the composition may inhibit the diffusion of the alkaline anticancer agent and the cell metabolism regulator by constricting blood vessels surrounding tumor vascular endothelial cells by including epinephrine. For example, the cell metabolism regulator may be introduced into tumor vascular endothelial cells via GLUT1 or MCT (Monocarboxylate Transporters), thereby inducing tumor vascular endothelial cell death.

[0064] Hereinafter, compositions according to exemplary embodiments of the present invention will be described in detail with reference to the drawings and examples. However, the drawings and examples are merely exemplary and the present invention is not limited thereto.

[0065] A composition according to an exemplary embodiment may include an alkaline anticancer agent.

[0066] Alkaline anticancer agents can destroy tumor blood vessels and inhibit angiogenesis, thereby blocking or inhibiting the supply of nutrients or oxygen to cancer cells and inducing their death.

[0067] In one embodiment, the alkaline anticancer agent may include a vascular disrupting agent. In one embodiment, the vascular disrupting agent may include one or more selected from vadimezan (DMXAA) and fosbretabulin (CA4P). Vadimezan is preferred.

[0068] In one embodiment, the concentration of the angioclast may be from 0.001 mg / mL to 20 mg / mL in the total composition. For example, the concentration may be from 0.005 mg / mL to 15 mg / mL, from 0.01 mg / mL to 10 mg / mL, from 0.1 mg / mL to 5 mg / mL, from 0.5 mg / mL to 2.5 mg / mL, or from 1 mg / mL to 2 mg / mL in the total composition, but is not limited thereto.

[0069] In one embodiment, among the angiogenic agents, Vadimezan can induce apoptosis of endothelial cells by mediating TNF-alpha, and fosbretabulin can destroy endothelial cells by inhibiting polymerization of microtubules.

[0070] In another embodiment, the alkaline anticancer agent may comprise one or more selected from a pyrimidine derivative, a taxane derivative, and anthracene derivative. For example, when the alkaline anticancer agent is locally injected into the base of tumor tissue, it may induce the death of vascular endothelial cells supplying blood to the tumor, destroying blood vessels and causing tumor necrosis.

[0071] Pyrimidine derivatives can induce apoptosis by interfering with nucleic acid synthesis in cells. Examples of pyrimidine derivatives include 5-fluorouracil (5-FU), gemcitabine, capecitabine, trifluridine, and floxuridine. Preferred examples include 5-fluorouracil and gemcitabine. For example, 5-fluorouracil can induce apoptosis by increasing the expression of caspase-3 and decreasing the expression of Bcl-2.

[0072] Taxane derivatives can disrupt the breakdown of cellular microtubules, thereby halting cell division and leading to cell death. Taxane derivatives include, for example, paclitaxel, docetaxel, and cabazitaxel. Paclitaxel, commercially known as Taxol, is preferred.

[0073] Anthracene derivatives can induce cell death by causing DNA damage and inhibiting topoisomerase II. Examples of anthracene derivatives include doxorubicin, daunorubicin, and epirubicin. Doxorubicin is preferred.

[0074] For example, the concentration of a pyrimidine derivative, e.g., 5-fluorouracil, in the total composition can be, but is not limited to, 0.1 mM to 600 mM, 0.1 mM to 500 mM, 0.5 mM to 390 mM, 1 mM to 350 mM, 10 mM to 300 mM, 50 mM to 250 mM, or 100 mM to 200 mM.

[0075] For example, the concentration of the pyrimidine derivative, for example, gemcitabine, in the total composition may be, but is not limited to, 1 nM to 100 μM, 5 nM to 50 μM, 10 nM to 25 μM, 50 nM to 10 μM, 100 nM to 5 μM or 500 nM to 2 μM.

[0076] For example, the concentration of the taxane derivative and the anthracene derivative may be, for example, 0.1 nM to 100 μM, 1 nM to 50 μM, 5 nM to 25 μM, 10 nM to 10 μM, 50 nM to 5 μM or 100 nM to 1 μM in the total composition, but is not limited thereto.

[0077] Alkaline anticancer agents can enhance their anticancer effects when mixed with alkaline solvents. For example, they may provide superior anticancer effects when mixed with alkaline solvents than with neutral or acidic solvents.

[0078] The tumor microenvironment, such as tumor vascular endothelial cells, is acidic due to rapid cancer cell growth and metabolic activity (particularly anaerobic fermentation), which produces large amounts of lactic acid. This acidic environment facilitates cancer cell invasion and metastasis and can lead to drug resistance by preventing drugs from entering the tumor cells. In this case, the pH of the drug administered locally is crucial. Drug intracellular transport is more active under alkaline pH conditions. Therefore, alkaline anticancer agents can target endothelial cells within cancer tissues and induce apoptosis. Alkaline solvents can enhance the endothelial cell killing effect compared to neutral or acidic conditions.

[0079] In some embodiments, the alkaline anticancer agent may be in the form of the anticancer agent dissolved in an alkaline solvent. The alkaline solvent may be, for example, alkaline water, saline solution, etc.

[0080] In one embodiment, the alkalinity may be a pH of 8.0 to 9.0, for example, 8.0 to 9.0, 8.2 to 9.0, or 8.4 to 9.0, but is not limited thereto.

[0081] A composition according to an exemplary embodiment may comprise epinephrine.

[0082] In one embodiment, epinephrine can inhibit the diffusion of the alkaline anticancer agent and the cell metabolism regulator by constricting the blood vessels surrounding tumor vascular endothelial cells. For example, epinephrine can inhibit bleeding by constricting the blood vessels at the injection site (e.g., subcutaneously or within the submucosal tissue), and can reduce side effects by inhibiting the systemic absorption of the alkaline anticancer agent. Therefore, the anticancer effect can be maximized by synergizing with the alkaline anticancer agent's effect on vascular endothelial cells.

[0083] In one embodiment, the concentration of epinephrine may be 0.001 to 0.2 mg / mL in the total composition. For example, it may be 0.001 to 0.1 mg / mL, or for example, it may be 0.002 mg / mL to 0.2 mg / mL. For example, when 200 mg of 10 cc of an alkaline anticancer agent is injected into the submucosal layer of the normal stomach wall immediately adjacent to cancer cells, the epinephrine dosage may be 0.001 mg / mL to 0.2 mg / mL.

[0084] In another embodiment, the epinephrine concentration of 0.1 mg / mL may be expressed as 1:10,000. The 1:10,000 may mean that the epinephrine is diluted 1:10,000 in a solution (e.g., saline solution). For example, the administrable concentration of epinephrine may be from 1:10,000 to 1:1,000,000.

[0085] A composition according to an exemplary embodiment may include a cell metabolism modulator.

[0086] In one embodiment, the cell metabolism regulator may include one or more selected from 2-deoxy-D-glucose (2-DG), dichloroacetic acid (DCA), and L-glucose. The cell metabolism regulator may induce apoptosis targeting tumor vascular endothelial cells. In some embodiments, the cell metabolism regulator may be dissolved in an alkaline solvent. The alkaline solvent may be, for example, alkaline water, physiological saline, or the like.

[0087] In one embodiment, the cell metabolism regulator can be introduced into tumor vascular endothelial cells through GLUT1 (L-glucose, 2DG) or MCT (DCA). In one embodiment, the cell metabolism regulator can induce tumor vascular endothelial cell apoptosis by increasing the expression of Fas receptor, Death Receptor 4, and Death Receptor 5. Increasing the expression of Fas receptor, Death Receptor 4, and Death Receptor 5 may be a natural death mechanism that appears when cells are severely malnourished. Even if L-glucose is introduced into tumor vascular endothelial cells through GLUT1, it is not broken down by enzymes and thus cannot be used as an energy source, thereby increasing the natural death of tumor vascular endothelial cells. Therefore, it can maximize the anticancer effect by exerting a synergistic effect with an alkaline anticancer agent.

[0088] In one embodiment, the concentration of the cell metabolism regulator may be 0.1 to 500 mM, 0.1 to 200 mM, or 1 to 100 mM in the total composition.

[0089] For example, the concentration of 2-deoxy-D-glucose in the total composition may be, but is not limited to, 0.1 mM to 200 mM, 0.5 mM to 150 mM, 1 mM to 100 mM, 5 mM to 80 mM, 10 mM to 60 mM, or 20 mM to 50 mM.

[0090] For example, the concentration of dichloroacetic acid can be selected without limitation by those skilled in the art. For example, it can be 0.1 mM to 100 mM, 0.5 mM to 80 mM, 1 mM to 60 mM, 5 mM to 50 mM, 10 mM to 40 mM, or 20 mM to 30 mM in the total composition, but is not limited thereto.

[0091] For example, the L-glucose concentration in the total composition may be, but is not limited to, 10 mM to 500 mM, 20 mM to 250 mM, 25 mM to 150 mM, 30 mM to 120 mM, or 50 mM to 100 mM.

[0092] In one embodiment, the composition may be a sustained-release formulation. The longer the exposure time to tumor vascular endothelial cells, the greater the apoptotic effect. To provide the composition as a sustained-release formulation, the composition may further include capric acid or a physiologically acceptable salt thereof, and a poloxamer.

[0093] Capric acid or a physiologically acceptable salt thereof, and poloxamer have low toxicity, are biocompatible, and exhibit superior in vivo stability. For example, by mixing capric acid or a physiologically acceptable salt thereof, and poloxamer, viscosity can be increased and the sol-gel transition temperature of the poloxamer can be raised. For example, the sol-gel transition temperature can be between room temperature and body temperature, for example, 20 to 40°C or 25 to 37°C. Within this range, the sol is convenient for storage, transportation, and use at room temperature. When acting in the body, it is easily diffused in tissues in a gel state.

[0094] In some embodiments, the weight ratio of capric acid and poloxamer may be 0.25 to 3.4:30, 1 to 3.4:30, 2 to 3:30, and specifically, the weight ratio of capric acid and poloxamer may be 2.75 to 3.3:30, 2.8 to 3.2:30. In the above range, the sol-gel transition of the carrier is likely to occur at 25 to 36°C.

[0095] Capric acid is a type of saturated fatty acid. Physiologically acceptable salts can be prepared using capric acid and a relatively non-toxic acid or base. Physiologically acceptable salts can be, for example, metal salts or acid addition salts.

[0096] The metal salt may be a sodium, potassium, or calcium salt. The metal salt may be prepared using a base. For example, an alkali metal or alkaline earth metal salt may be obtained by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and / or drying the filtrate.

[0097] Acid addition salts can be prepared from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous or phosphorous acids and non-toxic organic acid salts such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. These physiologically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propylates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexanoate-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, It may include terephthalate, benzenesulfonate, tert-butyl sulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0098] A poloxamer may be a terpolymer comprising a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) structure.

[0099] The molecular weight of the poloxamer may be, for example, a weight average molecular weight of 1,000 to 100,000, 10,000 to 100,000, 10,000 to 20,000, or 1,0000 to 15,000. The molecular weight may be appropriately selected by a person skilled in the art depending on the substance to be delivered.

[0100] Poloxamers are typically designated by a numbering system that indicates the approximate molecular weight of the poloxamer and the percentage of polyoxyethylene content, and are also referred to by the trade name pluronic. For example, Poloxamer 407 and the trade name Pluronic F-127 are interchangeable.

[0101] For example, the above poloxamer is poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, It may be poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407, etc.

[0102] The above poloxamer is a type of surfactant that can form micelles in an aqueous environment to load substances. A hydrogel composed of poloxamer can be used as a delivery vehicle that forms a matrix and locally and sustainably releases substances. The poloxamer hydrogel may include cross-linking between poloxamers. The poloxamer can be used in various molecular weights and ratios.

[0103] The composition according to some embodiments may contain one or more active ingredients exhibiting the same or similar function with respect to the treatment of the tumor.

[0104] The composition according to some embodiments may additionally contain a compound that maintains or increases the solubility and / or absorbability of the active ingredient.

[0105] In one embodiment, the tumor may comprise a solid tumor.

[0106] In some embodiments, the tumor may be, for example, gastric cancer, liver cancer, pancreatic cancer, osteosarcoma, skin cancer, lung cancer, neuroblastoma, uterine cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, biliary tract cancer, bladder cancer, ovarian cancer, brain tumor, cervical cancer, testicular cancer, penile cancer, genitourinary cancer, esophageal cancer, laryngeal cancer, gastrointestinal cancer, keratinocyte cancer, follicular carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, thyroid cancer, papillary cancer, biliary tract cancer, bone cancer, bone marrow disorder, hairy cell carcinoma, oral cancer, lip cancer, tongue cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, rectal cancer, vulvar cancer, endometrial cancer, central nervous system cancer, peritoneal cancer, hepatocellular carcinoma, head cancer, cervical cancer, etc., but is not limited thereto.

[0107] The formulation of the above composition may be prepared as an oral or parenteral formulation. For example, the formulation may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration. Alternatively, the formulation may be suitable for administration by inhalation or insufflation.

[0108] The composition may be administered in an injectable form. It does not form precipitates in biological environments, such as blood, and can be administered using a thin injection needle. The composition is preferably administered in an injectable form.

[0109] In one embodiment, the composition may be administered by local injection around the tumor.

[0110] For example, a composition can be delivered to the tumor's base via an injector using an endoscope. Specifically, the composition can be injected into the submucosa of the normal stomach wall immediately adjacent to the tumor, toward the tumor's basal layer. The drug, delivered to the vascular endothelial cells of the blood vessels supplying the tumor, can induce endothelial cell death or inhibit angiogenesis.

[0111] The above local injection can deliver a large amount of drug to the surrounding lymph nodes as well as the submucosal layer around the tumor compared to intravenous administration of general anticancer drugs, and can reduce the side effects of anticancer drugs compared to systemic administration.

[0112] According to one embodiment, the composition may be administered in a pharmaceutically effective amount. The effective dosage level may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, route and excretion rate, treatment duration, concurrent medications, and other factors well known in the medical field.

[0113] A topical solid tumor administration agent according to an exemplary embodiment may include the composition described above. Specific examples of solid tumors are as described above.

[0114] In some embodiments, the composition may be administered as an individual treatment or in combination with other treatments. The components included in the anticancer combination formulation may be administered sequentially or simultaneously, and may be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum efficacy with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0115] For example, the dosage of the composition may vary greatly depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The appropriate dosage may vary, for example, depending on the amount of drug accumulated in the patient's body and / or the specific efficacy of the carrier of the present invention used. For example, it may be 0.01 ㎍ to 1 g per 1 kg of body weight, and may be administered once or several times per unit period, such as daily, weekly, monthly, or yearly. Alternatively, it may be administered continuously for a long period of time using an infusion pump. The number of repeated administrations is determined by considering the time the drug remains in the body, the drug concentration in the body, etc. Depending on the progress of the disease treatment, the composition may be administered for relapse even after treatment has been completed.

[0116] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0117] Example 1: Toxicity of Vadimezan to Human Umbilical Vein Endothelial Cells (HUVEC) According to Concentration, Time & pH

[0118] (1) HUVEC death effect according to vadimezan (DMXAA) concentration

[0119] Changes in HUVEC toxicity according to vadimezan concentration were confirmed. HUVEC cells were seeded in 96-well plates at a density of 1x10 in Endothelial Cell Growth Medium containing 2% serum and cell growth factors. 4After culturing the dogs / well, the next day, 0.5 mg / ml, 5 mg / ml, 50 mg / ml, and 500 mg / ml of Vadimezan were added to 100 μl of the culture medium and treated. After 24 hours, 2.5 μl of CellTiter 96 AQueous One Solution Reagent (Promega Corporation), an MTS sample, was added per well, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 1 to 4 hours. The absorbance was measured at 490 nm using a 96-well plate reader (Spark, TECAN, CA, USA). The results are shown in Figure 1. Referring to Figure 1, the viability of HUVEC cells decreased depending on the concentration of Vadimezan.

[0120] (2) Short-term killing effect of HUVEC according to vadimezan concentration

[0121] To investigate the short-term cell death effect of vadimezan on HUVECs according to the concentration of vadimezan, an MTS cell proliferation assay was performed. The short-term cell death effect of vadimezan on HUVECs according to the concentration of vadimezan was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 1 x 10 4Cells were seeded at a density of 10 cells / well and cultured for 24 hours. The cells were then cultured with HUVEC medium at various Vadimezan concentrations for 1, 4, and 8 hours, respectively. Then, 2.5 μl of CellTiter 96 AQueous One Solution Reagent (Promega Corporation), an MTS sample, was added per well, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 1 to 4 hours. The absorbance was measured at 490 nm using a 96-well plate reader (Spark, TECAN, CA, USA). The results are shown in Figure 2. Referring to Figure 2, the viability of HUVEC cells decreased with time and concentration changes.

[0122] (3) HUVEC death effect according to vadimezan concentration and pH

[0123] To investigate the apoptotic effect of vadimezan on HUVECs according to the concentration and pH, an MTS cell proliferation assay was performed. The apoptotic effect of vadimezan on HUVECs according to the concentration and pH was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 1x10 4Cells were seeded at a density of 10 cells / well and cultured for 24 hours. Then, the cells were cultured with HUVEC medium at various Vadimezan concentrations and pH conditions for 24 hours. Afterwards, 2.5 ㎕ of CellTiter 96 AQueous One Solution Reagent (Promega Corporation), an MTS sample, was added per well, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 1 to 4 hours. The absorbance was measured at 490 nm using a 96-well plate reader (Spark, TECAN, CA, USA). The results are shown in Figure 3. Referring to Figure 3, the viability of HUVEC cells changed depending on the concentration and pH of Vadimezan.

[0124] (4) Short-term killing effect of HUVEC according to vadimezan concentration and pH

[0125] To investigate the short-term cell death effect of vadimezan on HUVECs according to the concentration and pH, an MTS cell proliferation assay was performed. The cell death effect on HUVECs according to the concentration and pH of vadimezan was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 1x10 4Cells were seeded at a density of 10 cells / well and cultured for 24 hours. The cells were then cultured with HUVEC medium at various Vadimezan concentrations and pH conditions for 1 hour, respectively. Then, 2.5 μl of CellTiter 96 AQueous One Solution Reagent (Promega Corporation), an MTS sample, was added per well, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 1 to 4 hours. The absorbance was measured at 490 nm using a 96-well plate reader (Spark, TECAN, CA, USA). The results are shown in Figure 4. Referring to Figure 4, HUVEC cell viability changed depending on pH.

[0126] (5) Apoptotic effect of L-glucose on HUVEC according to vadimezan concentration

[0127] To investigate the apoptotic effect of L-glucose on HUVECs according to the concentration of vadimezan when mixed with L-glucose, an MTS cell proliferation assay was performed. The cytotoxicity of L-glucose on HUVECs according to the concentration of vadimezan was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 1x10 4Cells were seeded at a density of 10 cells / well and cultured for 24 h. The cells were then cultured with HUVEC medium in the presence of various Vadimezan and L-glucose concentrations for 24 h. The medium was then replaced with 100 μl of fresh medium, and 2.5 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) was added. After incubation at 37°C for 1 to 4 h in a humidified 5% CO2 atmosphere, the absorbance was measured at 490 nm using a BIO-RAD model 680 microplate reader (Spark, TECAN, CA, USA). The results are shown in Figure 5. Referring to Figure 5, Vadimezan decreased cell viability at both 0.1 mg / ml and 0.5 mg / ml. When L-glucose was added, cell viability decreased further.

[0128] (6) Apoptotic effects of HUVECs on L-glucose concentration and pH according to vadimezan concentration

[0129] To investigate the effect of L-glucose concentration and pH on HUVEC cell death when mixing vadimezan and L-glucose, MTS cell proliferation assay was performed. Cytotoxicity of L-glucose concentration and pH on HUVEC according to vadimezan concentration was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 1x10 4Cells were seeded at a density of 10 cells / well and cultured for 24 h. Next, the cells were cultured with HUVEC medium at various Vadimezan concentrations and pHs and with 200 mM L-glucose for 24 h. The medium was then replaced with 100 μl of fresh medium, and 2.5 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) was added. After incubation at 37°C for 1 to 4 h in a humidified 5% CO2 atmosphere, the absorbance was measured at 490 nm using a BIO-RAD model 680 microplate reader (Spark, TECAN, CA, USA). The results are shown in Figure 6. Referring to Figure 6, cell viability further decreased with the addition of 200 mM L-glucose and changes in pH.

[0130] (7) Confirmation of spontaneous death of HUVEC according to the concentration of Vadimezan using confocal fluorescence microscopy.

[0131] The Apoptosis Detection Kit (blue, green) (ab176749) can be used to simultaneously monitor apoptosis and healthy cells. The Phosphatidylserine (PS) sensor can detect apoptosis by exhibiting green fluorescence (Ex / Em = 490 / 525 nm) when bound to cell membrane PS, and healthy cells can be identified using CytoCalcein Violet 450 (Ex / Em = 405 / 450 nm), a dye for labeling living cytoplasm. 5x10 vascular endothelial cells were cultured in a slide culture chamber. 4Cells were treated with Vadimezan at various concentrations (0 to 0.5 mg / ml) for 24 hours. Afterwards, the cells were washed once or twice with 100 μL assay buffer by pipetting the buffer and incubated with 200 μL assay buffer containing Apopxin / CytoCalcein at room temperature for 30 to 60 minutes. After washing the cells twice with 100 μL assay buffer, replace the cells with 100 μL assay buffer and transfer them to the stage of a confocal microscope. The halogen lamp column was tilted back, the slide was placed on the stage, and the analysis software was launched. In the Acquire tab, the imaging parameters were set to XYZ mode, 1024x1024 pixels, 600 Hz, Zoom 1x, average 4, accumulation 1, and rotation 0, and the sample was placed on the microscope and focused by fluorescence. After focusing, set the beam path to select the laser to be used, specify the light intensity, select the PMT, select the pseudocolor, and set the detection band in that order. When you press the live button, a preview scan will be performed and the captured image will be shown in the right window. After checking the image while increasing the gain and intensity values, turn the dial (Z position) to readjust the focus position and finally capture the image, save it, and analyze it. The results are shown in Figs. 7, 8a, and 8b. Referring to Figs. 7, 8a, and 8b, healthy cells decreased and apoptotic cells increased in proportion to the concentration of Vadimezan.

[0132] (8) Changes in ROS production in HUVECs according to vadimezan concentration

[0133] The effect of Vadimezan on the production of reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) was measured using the DCF-DA staining method. 10,000 HUVECs were cultured in 96-well microplates with dark, clear bottoms and treated with 0.005 mg / ml, 0.01 mg / ml, 0.025 mg / ml, and 0.05 mg / ml of Vadimezan for 3 hours. The culture medium was removed, and 100 μL / well of Hanks' Balanced Salt solution (HBSS) buffer was added and washed. 100 μL / well of diluted DCFDA was added, and the HBSS buffer and stained cells were removed. The cells were then cultured with the diluted DCFDA solution for 45 minutes at 37°C in the dark. 100 μL of HBSS buffer from which the DCFDA solution was removed was added, and the plate was immediately measured in a fluorescence plate reader at Ex / Em = 485 / 535 nm. The results are shown in Figure 9. Referring to Figure 9, ROS increased in proportion to the Vadimezan concentration.

[0134] (9) Confirmation of the natural death mechanism of HUVECs by Vadimezan using western blot

[0135] Western blot was performed to confirm the expression of apoptosis markers activated when natural death of human umbilical vein endothelial cells (HUVECs) occurs due to Vadimezan.

[0136] HUVECs were treated with Vadimezan 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, and 0.25 mg / ml after adjusting the pH to 7.0 and 9.0, respectively. After 3 hours, HUVEC cells grown on culture dishes were lysed in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet p-40, 0.5% sodium deoxycholate (SDC), 0.1% sodium dodecyl sulfate (SDS), and 1X Protease inhibitor cocktail. After cooling on ice for 30 minutes, the cells were hastily centrifuged at 14,000 g for 20 minutes, and the supernatant was separated.

[0137] Quantification was performed using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). 20 μg of protein solution was electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. The electrophoresed membrane was incubated with blocking milk for 1 hour, and then incubated with primary antibodies against TNF-alpha, cleaved caspase 3, and cleaved caspase 8 for 1 hour at room temperature. After washing three times at 15-minute intervals in Tris buffer containing 0.1% Tween 20, the membrane was incubated with secondary antibodies against the primary antibodies for 1 hour at room temperature, and detection was performed using a chemiluminescent agent (Amersham Life Science, Arlington Heights, IL, USA). Proteins extracted from untreated HUVECs were used as a negative control.

[0138] The quantitative results of HUVEC according to the concentration of Vadimezan are shown in Figure 10. Referring to Figure 10, it was confirmed that as the concentration of Vadimezan increased, the expression of apoptosis markers (TNF-alpha, cleaved caspase 3, cleaved caspase 8) increased.

[0139] (10) Confirmation of pathological phenomena in surrounding tissues when Vadimezan was injected around the tumor after xenografting human gastric cancer cells into nude mice.

[0140] The animals used in the experiment were 6 male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 grams. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells were administered to each nude mouse. 6 Cells / 100㎕ (PBS) were implanted into the dorsal subcutaneous fat layer. The tumor size was measured periodically, and when it reached a diameter of approximately 1 cm, 200 ug of vadimezan was administered to the tumor base (pH 9). After 3 hours, tissues including muscle were collected, stained with H&E, and pathological changes in the muscle layer were observed. The results are shown in Fig. 11. Referring to Fig. 11, fibrin-like material is deposited and narrows the blood vessels. It can be seen that the blood vessel walls are destroyed and red blood cells leak out into the tissue. As shown in A and B, fibrin-like material accumulates inside the blood vessels, narrowing the lumen. As shown in C and D, the blood vessel walls are damaged, allowing blood to flow out into the surrounding tissue. However, as shown in E, the underlying muscle tissue was not damaged.

[0141] (11) In-vivo experiment to observe the response to anticancer drugs after xenotransplanting human gastric cancer cells into nude mice

[0142] The animals used in the experiment were four male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 g. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells (Korea Cell Line Bank_N87 cell line) were used per mouse. 6 Cells / 100 μl (PBS) were implanted via subcutaneous injection into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached approximately 1 cm.

[0143] Drug administration was as follows: Group 1. 2 mg / ml Vadimezan, 1% HPMC, 1: 100,000 epinephrine (pH 8.4); Group 2. 5 mg / ml Vadimezan, 1% HPMC, 1: 100,000 epinephrine (pH 8.4); Group 3. 7 mg / ml Vadimezan, 5% L-glucose, 1: 100,000 epinephrine (pH 9.0). 0.1 cc was administered subcutaneously once a week for 4 weeks at a point right next to the tumor. The long and short diameters of the tumors growing on the dorsal epidermis were measured at 7-day intervals for 4 to 5 weeks after the start of drug administration, and the size before and after drug administration was calculated as Mean Tumor Volume = (long diameter χ short diameter 2 ) / 2(mm 3 ) were measured and compared. The photo results are shown in the graph in Fig. 12 and Fig. 13. Referring to Figs. 12 and 13, it was confirmed that the tumor size was significantly reduced.

[0144] Example 2: Gemcitabine toxicity to human umbilical vein endothelial cells (HUVEC) according to concentration, time, and pH.

[0145] (1) HUVEC apoptosis effect according to gemcitabine concentration

[0146] Changes in HUVEC toxicity according to gemcitabine concentration were confirmed. The procedure was the same as (1) of Example 1, except that gemcitabine 0.005 μM, 0.01 μM, 0.025 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, and 1 μM were added to 100 μl of culture medium for treatment. The results are shown in Figure 14. Referring to Figure 14, the viability of HUVEC cells decreased depending on the gemcitabine concentration.

[0147] (2) Short-term killing effect of HUVEC according to gemcitabine concentration

[0148] To investigate the short-term cell death effect of gemcitabine on HUVECs according to the concentration, an MTS cell proliferation assay was performed in the same manner as in (2) of Example 1. The results are shown in Figure 15. Referring to Figure 15, the survival rate of HUVEC cells decreased over time.

[0149] (3) HUVEC death effect according to gemcitabine concentration and pH

[0150] To investigate the apoptotic effect of Gemcitabine on HUVECs according to the concentration and pH, an MTS cell proliferation assay was performed in the same manner as in (3) of Example 1. The results are shown in Fig. 16. Referring to Fig. 16, it can be confirmed that the survival rate of HUVEC cells changes depending on the concentration and pH of Gemcitabine.

[0151] (4) Short-term killing effect of HUVEC according to gemcitabine concentration and pH

[0152] To investigate the short-term cell death effect of gemcitabine on HUVECs according to concentration and pH, an MTS cell proliferation assay was performed.

[0153] The apoptotic effect of Gemcitabine on HUVECs according to the concentration and pH was performed in the same manner as in Example 1 (4) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Referring to Figure 17, it can be confirmed that the survival rate of HUVEC cells changes depending on the concentration and pH of Gemcitabine.

[0154] (5) Apoptotic effect of HUVECs on L-glucose according to gemcitabine concentration

[0155] To investigate the apoptotic effect of L-glucose on HUVECs according to the concentration of gemcitabine when mixing gemcitabine and L-glucose, an MTS cell proliferation assay was performed in the same manner as in (5) of Example 1. The results are shown in Fig. 18. Referring to Fig. 18, gemcitabine significantly reduced cell viability at both 0.01 uM and 0.1 uM. Furthermore, cell viability decreased further with the addition of L-glucose.

[0156] (6) Apoptotic effects of HUVECs on L-glucose concentration and pH according to gemcitabine concentration

[0157] To investigate the apoptotic effect of HUVECs on L-glucose concentration and pH according to Gemcitabine concentration when mixing Gemcitabine and L-glucose, MTS cell proliferation assay was performed in the same manner as in Example 1 (6). The results are shown in Fig. 19. Referring to Fig. 19, Gemcitabine significantly reduced cell viability at both 0.01 uM and 0.1 uM. In addition, cell viability further decreased with the addition of L-glucose and changes in pH.

[0158] Example 3: Toxicity of Taxol to Human Umbilical Vein Endothelial Cells (HUVEC) According to Concentration, Time & pH

[0159] (1) HUVEC death effect according to Taxol concentration

[0160] Changes in HUVEC toxicity according to Taxol concentration were confirmed. The procedure was the same as (1) of Example 1, except that Taxol was added to 100 μl of culture medium at concentrations of 0.1 μM, 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM. The results are shown in Fig. 20. Referring to Fig. 20, the viability of HUVEC cells decreased depending on the concentration of Gemcitabine.

[0161] (2) Short-term killing effect of HUVEC according to Taxol concentration and pH

[0162] To investigate the short-term cell death effect of Taxol on HUVECs according to concentration and pH, an MTS cell proliferation assay was performed in the same manner as in (4) of Example 1. The results are shown in Fig. 21. Referring to Fig. 21, it can be confirmed that the survival rate of HUVEC cells changes depending on pH.

[0163] Example 4: Toxicity of 2-Deoxy-D-glucose (2-DG) to Human Umbilical Vein Endothelial Cells (HUVEC) According to Concentration, Time & pH

[0164] (1) HUVEC apoptosis effect according to 2-DG concentration

[0165] Changes in HUVEC toxicity according to 2-DG concentration were confirmed. The procedure was the same as (1) of Example 1, except that 2-DG 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM were added to 100 μl of culture medium for treatment. The results are shown in Fig. 22. Referring to Fig. 22, it can be confirmed that the viability of HUVEC cells decreases depending on the 2-DG concentration.

[0166] (2) Short-term killing effect of HUVEC according to 2-DG concentration

[0167] To investigate the short-term cell death effect on HUVECs according to the concentration of 2-DG, an MTS cell proliferation assay was performed in the same manner as in (2) of Example 1. The results are shown in Fig. 23. Referring to Fig. 23, HUVEC viability decreased with the concentration of 2-DG and the passage of time.

[0168] (3) Apoptotic effect on HUVEC according to 2-DG concentration and pH

[0169] To investigate the apoptotic effect of 2-DG on HUVECs according to the concentration and pH of 2-DG, an MTS cell proliferation assay was performed in the same manner as in (3) of Example 1. The results are shown in Fig. 24. Referring to Fig. 24, it can be confirmed that the survival rate of HUVEC cells changes according to the concentration and pH of 2-DG.

[0170] (4) Short-term apoptotic effect on HUVECs according to 2-DG concentration and pH

[0171] To investigate the short-term cell death effect of 2-DG on HUVECs according to the concentration and pH of 2-DG, an MTS cell proliferation assay was performed in the same manner as in (4) of Example 1. The results are shown in Fig. 25. Referring to Fig. 25, it can be confirmed that the survival rate of HUVEC cells changes depending on the concentration and pH of 2-DG.

[0172] (5) HUVEC apoptosis effect according to 2-DG concentration when 2-DG and 5-FU are mixed

[0173] To investigate the apoptotic effect of 2-DG and 5-FU on HUVECs according to the concentration of 2-DG, an MTS cell proliferation assay was performed in the same manner as in (5) of Example 1. The results are shown in Fig. 26. Referring to Fig. 26, 2-DG decreased cell viability at both 0.1 mM and 0.5 mM. Cell viability further decreased with the addition of 5-FU.

[0174] (6) Spontaneous death of HUVECs according to the concentration of 2-DG was confirmed using confocal fluorescence microscopy.

[0175] 2-DG was added at different concentrations (0 to 2 mM) in the same manner as in Example 1 (7). The results are shown in Fig. 27. Referring to Fig. 27, it can be confirmed that healthy cells decrease and apoptotic cells increase in proportion to the concentration of 2-DG. The results are shown in Fig. 27. Referring to Fig. 27, healthy cells decrease and apoptotic cells increase in proportion to the concentration of 2-DG.

[0176] (7) Confirmation of the natural death mechanism of HUVECs by 2-DG using western blot

[0177] HUVECs were cultured in culture dishes for 24 hours. Then, the cells were cultured with HUVEC medium at various concentrations of 2-DG and 5-FU for 3 hours. The procedure was then repeated under the same conditions as in (9) of Example 1. The results are shown in Figure 28. Referring to Figure 28, it was confirmed that as the concentration of 2-DG increased, the expression of apoptosis markers (TNF-alpha, cleaved caspase 3, cleaved caspase 8) increased.

[0178] (8) In-vivo experiment to observe the response to anticancer drugs after xenotransplantation of human gastric cancer cells into nude mice

[0179] The animals used in the experiment were three male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 g. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells (Korea Cell Line Bank NCI-N87 cell line) were used per mouse. 6 Cells / 100 μl (PBS) were implanted via subcutaneous injection into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached approximately 1 cm.

[0180] Drug administration consisted of 5% 2-DG, 50 mg / ml 5-FU, and 1:100,000 epinephrine (pH 9.0) administered subcutaneously next to the tumor at a dose of 0.1 cc once a week for a total of 3 weeks. After the start of administration, the long and short diameters of the tumor growing on the dorsal epidermis were measured at weekly intervals for 4 weeks, and the size before and after drug administration was calculated as mean tumor volume = (long diameter × short diameter) 2 ) / 2(mm 3 ) were measured and compared. The results are shown in Figs. 29 and 30. Referring to Figs. 29 and 30, it was confirmed that the tumor size was significantly reduced.

[0181] Example 5: Toxicity of sodium dichloroacetate (DCA) to human umbilical vein endothelial cells (HUVEC) according to concentration, time, and pH.

[0182] (1) HUVEC death effect according to DCA concentration

[0183] Changes in HUVEC toxicity according to DCA concentration were confirmed. The procedure was the same as (1) of Example 1, except that 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM were added to 100 μl of culture medium. The results are shown in Figure 31. Referring to Figure 31, the viability of HUVEC cells decreased depending on the DCA concentration.

[0184] (2) Short-term killing effect of HUVEC according to DCA concentration

[0185] To investigate the short-term cell death effect on HUVECs according to DCA concentration, an MTS cell proliferation assay was performed in the same manner as in (2) of Example 1. The results are shown in Fig. 32. Referring to Fig. 32, the survival rate of HUVEC cells decreased over time according to the DCA concentration and time.

[0186] (3) HUVEC death effect according to DCA concentration and pH

[0187] To investigate the apoptotic effect on HUVEC according to DCA concentration and pH, an MTS cell proliferation assay was performed in the same manner as in (3) of Example 1. The results are shown in Fig. 33. Referring to Fig. 33, it can be confirmed that the survival rate of HUVEC cells changes depending on DCA concentration and pH.

[0188] (4) Short-term killing effect of HUVEC according to DCA concentration and pH

[0189] To investigate the short-term cell death effect on HUVEC according to DCA concentration and pH, an MTS cell proliferation assay was performed in the same manner as in (4) of Example 1. The results are shown in Fig. 34. Referring to Fig. 34, it can be confirmed that the survival rate of HUVEC cells changes depending on DCA concentration and pH.

[0190] (5) HUVEC apoptosis effect according to DCA concentration when DCA and 5-FU are mixed

[0191] To investigate the apoptotic effect on HUVECs according to the concentration of DCA when mixing DCA and 5-FU, an MTS cell proliferation assay was performed in the same manner as in (5) of Example 1. The results are shown in Fig. 35. Referring to Fig. 35, DCA significantly reduced cell viability at both 25 mM and 50 mM. Cell viability further decreased with the addition of 5-FU.

[0192] (6) In-vivo experiment to observe the response to anticancer drugs after xenotransplanting human gastric cancer cells into nude mice

[0193] The animals used in the experiment were four male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 g. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells (Korea Cell Line Bank NCI-N87 cell line) were used per mouse. 6 Cells / 100 μl (PBS) were implanted via subcutaneous injection into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached approximately 1 cm.

[0194] Drug administration was performed by mixing 5-FU 50 mg / ml and 0.5% DCA under alkaline conditions (pH 9), adding epinephrine at a ratio of 1:100,000, and injecting 0.1 cc three times at 7-day intervals right next to the tumor. The long and short diameters of the tumor growing on the dorsal epidermis were measured for 6 weeks after the start of drug administration, and the size before and after drug administration was calculated as mean tumor volume = (long diameter × short diameter 2 ) / 2(mm 3) were measured and compared. The results are shown in Figs. 36 and 37. Referring to Figs. 36 and 37, it was confirmed that the tumor size was significantly reduced.

[0195] Example 6: Identification of receptors associated with intracellular transport of L-glucose into HUVECs

[0196] (1) Confirmation of D-Glucose concentration in HUVEC cells according to L-glucose concentration

[0197] To investigate the effect of L-glucose on the intracellular transport of D-glucose, L-glucose was incubated with 2-NBDLG, a D-glucose tracer, at various concentrations in HUVECs for 20 minutes, and the changes in marker concentration were observed. The results are shown in Fig. 38. It was found that the intracellular concentration of D-glucose decreased as the concentration of L-glucose increased, and it was confirmed that the intracellular uptake of L-glucose inhibited the intracellular transport of D-glucose. Therefore, it can be seen that high concentrations of L-glucose can inhibit HUVEC metabolism.

[0198] (2) Observation of L-Glucose in HUVEC cells following GLUT inhibitor treatment

[0199] To determine whether L-glucose enters HUVECs, L-glucose tracer was incubated with HUVECs for 2 hours. To determine which receptor was involved, phloretin, BAY876, and cytochalasin B were added. Confocal fluorescence microscopy results are shown in Figures 39a and 39b. The intracellular transport of L-glucose was inhibited by BAY876 and cytochalasin B, but not by phloretin, canagliflozin, or mizagliflozin. Therefore, it was confirmed that L-glucose enters HUVECs primarily through GLUT1.

[0200] (3) Western blot confirms the natural death mechanism of HUVECs when mixed with L-glucose and 5-FU.

[0201] HUVECs were cultured in culture dishes for 24 hours. Then, the cells were cultured for 3 hours with HUVEC medium in various mixtures of L-glucose and 5-FU. The same conditions as in (9) of Example 1 were followed. The results are shown in Figures 40a and 40b. When L-glucose was treated alone, the expression of FAS, DR4, and DR5 increased, and the effect was even greater when mixed with 5-FU. The expression of FAS, DR4, and DR5 is a natural death mechanism that appears when cells are severely malnourished. Since L-glucose is not broken down by enzymes and therefore cannot serve as an energy source, it can be confirmed that the higher the concentration, the more natural death due to malnourishment increases.

[0202] (4) Analysis of CD31 and VEGF expression after xenotransplantation of human gastric cancer cells into nude mice

[0203] The animals used in the experiment were four male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 g. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells (Korea Cell Line Bank NCI-N87 cell line) were used per mouse. 6 Cells / 100 μl (PBS) were implanted subcutaneously into the dorsal subcutaneous fat layer, and tumors were allowed to grow for 4 weeks. CD31 immunohistochemical staining, which stains vascular endothelial cells brown, was performed. 5 mg of 5-FU, 5% L-glucose, and 1:100,000 epinephrine were injected into the tumor base (pH 9). After 4 hours, pathological changes were observed in the blood vessels at the tumor base. The results are shown in Figure 41.

[0204] Observations revealed that endothelial cell destruction caused damage to the blood vessel wall, leading to red blood cell leakage into surrounding tissues. Furthermore, fibrin-like substances were found to cause vascular occlusion.

[0205] (5) In-vivo experiment to observe the response to anticancer drugs after xenotransplanting human gastric cancer cells into nude mice

[0206] The animals used in the experiment were four male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old and weighing an average of 30 g. They were used after a one-week inspection period at the laboratory. 5x10 human gastric cancer cells (Korea Cell Line Bank NCI-N87 cell line) were used per mouse. 6 Cells / 100 μl (PBS) were implanted via subcutaneous injection into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached approximately 1 cm.

[0207] The drug was administered at a ratio of 5% L-glucose, 5 mg 5-FU, and 1:100,000 epinephrine, and 0.1 cc was injected immediately adjacent to the tumor three times at 7-day intervals (pH 9). Tumor growth on the dorsal epidermis was observed for 4 weeks after the start of drug administration. The results are shown in Figure 42. After 4 weeks, the tumor volume was significantly reduced by approximately 95% (P <0.05). There was no recurrence even after 4 weeks, demonstrating an excellent tumor treatment effect. In contrast, tumor recurrence occurred when only 5 mg 5-FU was used.

Claims

1. A pharmaceutical composition for destroying tumor blood vessels, comprising an alkaline anticancer agent, a cell metabolism regulator, and epinephrine.

2. A pharmaceutical composition for destroying tumor blood vessels, wherein the alkalinity is pH 8 to 9 in claim 1.

3. A pharmaceutical composition for destroying tumor blood vessels, wherein the alkaline anticancer agent according to claim 1 comprises a vascular disrupting agent.

4. A pharmaceutical composition for destroying tumor blood vessels, wherein the angiogenic agent according to claim 3 comprises at least one selected from Vadimezan and Fosbretabulin.

5. A pharmaceutical composition for destroying tumor blood vessels, wherein the concentration of the angiogenic agent in claim 3 is 0.001 mg / mL to 20 mg / mL in the entire composition.

6. A pharmaceutical composition for destroying tumor blood vessels, wherein the alkaline anticancer agent according to claim 1 comprises at least one selected from a pyrimidine derivative, a taxane derivative, and an anthracene derivative.

7. A pharmaceutical composition for destroying tumor blood vessels, wherein the epinephrine constricts blood vessels surrounding tumor vascular endothelial cells, thereby inhibiting the diffusion of the alkaline anticancer agent and the cell metabolism regulator.

8. A pharmaceutical composition for destroying tumor blood vessels, wherein the concentration of epinephrine in the entire composition is 0.001 to 0.2 mg / mL according to claim 1.

9. A pharmaceutical composition for destroying tumor blood vessels, wherein the cell metabolism regulator according to claim 1 comprises at least one selected from 2-deoxy-D-glucose (2-DG), dichloroacetic acid (DCA), and L-glucose.

10. A pharmaceutical composition for destroying tumor blood vessels, wherein the cell metabolism regulator according to claim 1 induces tumor blood vessel endothelial cell apoptosis by increasing the expression of Fas receptor, Death Receptor 4, and Death Receptor 5.

11. A pharmaceutical composition for destroying tumor blood vessels, wherein the concentration of the cell metabolism regulator in claim 1 is 0.1 mM to 500 mM in the entire composition.

12. A pharmaceutical composition for destroying tumor blood vessels, wherein the composition is administered to the submucosal layer where tumor blood vessels are located, according to claim 1.

13. A pharmaceutical composition for destroying tumor blood vessels, administered by local injection into the submucosal layer according to claim 12.

14. A pharmaceutical composition for destroying tumor blood vessels, wherein the tumor comprises a solid tumor according to claim 1.

15. A local administration agent for solid tumors, comprising a pharmaceutical composition for destroying tumor blood vessels according to claim 1.

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