Pharmaceutical composition for disruption of tumor blood vessels

Abstract

A pharmaceutical composition for disruption of tumor blood vessels according to an exemplary embodiment may include alkaline 5-fluorouracil (5-FU) and epinephrine. Epinephrine can synergize with the tumor endothelial cell death effect induced by alkaline 5-fluorouracil, thereby enhancing the anticancer effect.

Description

Pharmaceutical composition for destroying tumor blood vessels

[0001] The present invention relates to a pharmaceutical composition for destroying tumor blood vessels.

[0002] 5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent for cancer treatment. 5-Fluorouracil is a naturally occurring uracil analogue. It exerts its anticancer effects by inhibiting thymidylate synthase, thereby inhibiting DNA synthesis and repair, and by suppressing RNA function.

[0003] 5-Fluorouracil must be administered directly in a hospital, mainly by intravenous administration, and has the side effect of thrombosis, so an oral 5-FU prodrug has recently been developed.

[0004] While 5-fluorouracil is highly effective, it can cause myelosuppression and gastrointestinal toxicity due to phosphorylation within the gastrointestinal tract. Furthermore, serious, potentially life-threatening side effects, such as cardiotoxicity, have been reported in 1.5 to 18% of patients.

[0005] Therefore, active research is underway to maintain the anticancer effects of 5-fluorouracil while reducing its side effects. For example, Korean Patent Publication No. 10-2016-0049312 utilizes cyanidin-3-glucoside to effectively alleviate anticancer side effects such as inflammation, intestinal damage, and myelosuppression caused by 5-fluorouracil.

[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] A pharmaceutical composition for destroying tumor blood vessels according to an exemplary embodiment may comprise alkaline 5-fluorouracil (5-FU) and epinephrine.

[0008] In one embodiment, the alkalinity may be pH 8 to 9.

[0009] In one embodiment, the alkaline 5-fluorouracil may be in the form of 5-fluorouracil dissolved in an alkaline solvent.

[0010] In one embodiment, the alkaline 5-fluorouracil can kill endothelial cells by increasing the expression of thrombospondin-1 protein in tumor endothelial cells.

[0011] In one embodiment, the concentration of the alkaline 5-fluorouracil may be from 0.1 to 600 mM in the total composition.

[0012] In one embodiment, the epinephrine can induce apoptosis of tumor vascular endothelial cells.

[0013] In one embodiment, the concentration of epinephrine may be from 0.001 to 0.2 mg / mL in the total composition.

[0014] In one embodiment, the composition may further comprise one or more selected from glucose, a nitric oxide formation inhibitor, bevacizumab, capric acid or a physiologically acceptable salt thereof, and a poloxamer.

[0015] In one embodiment, the nitric oxide formation inhibitor is L-NMMA(N G -Methyl-L-arginine), L-NAME(N G - It may include one or more selected from -Nitro-L-arginine methyl ester), L-NA (Nitro Arginine), and 7NI (Nitroindazole).

[0016] In one embodiment, the composition may further comprise one or more selected from cisplatin, carboplatin, cyclophosphamide, paclitaxel, tamoxifen, toremifene, fulvestrant, diindolimethane, exemestane, raloxifene, an aromatase inhibitor, doxorubicin, oxaliplatin, vincristine, gemcitabine, anthracyclines, taxanes, irinotecan, docetaxel, and eribulin.

[0017] In one embodiment, the cancer may comprise a solid cancer.

[0018] In one embodiment, the composition may be a sustained-release formulation.

[0019] In one embodiment, the composition may be administered by local injection around the tumor.

[0020] In one embodiment, the tumor periphery may be the submucosal layer where tumor blood vessels are located.

[0021] A solid tumor topical administration agent according to an exemplary embodiment may include the pharmaceutical composition for destroying tumor blood vessels.

[0022] The pharmaceutical composition for destroying tumor blood vessels according to the present invention may include alkaline 5-fluorouracil and epinephrine.

[0023] For example, injection of alkaline 5-fluorouracil into the tumor base can induce apoptosis of tumor vascular endothelial cells, destroying the tumor. It can also inhibit tumor angiogenesis.

[0024] For example, epinephrine injected into the tumor base can induce apoptosis of tumor endothelial cells. This synergistic effect with the apoptotic effect of alkaline 5-fluorouracil on tumor endothelial cells can enhance anticancer effects. Furthermore, epinephrine can constrict blood vessels at the injection site, suppressing bleeding. Therefore, by inhibiting the systemic absorption of 5-fluorouracil, side effects can be reduced while maintaining its effectiveness, contributing to the apoptosis of tumor endothelial cells.

[0025] 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.

[0026] Figure 1 is an example of administering a pharmaceutical composition for destroying tumor blood vessels by local injection around a tumor according to one embodiment.

[0027] Figures 2a and 2b show the results of confirming the HUVEC cell death effect according to the epinephrine concentration using MTS.

[0028] Figures 3a and 3b show the results of Western blot analysis of the HUVEC cell death effect according to the concentration of epinephrine.

[0029] Figure 4 shows the results of DNA laddering confirmed by gel electrophoresis according to epinephrine concentration.

[0030] Figures 5a to 5e show the results of confirming the HUVEC cell death effect according to the epinephrine concentration using a confocal fluorescence microscope.

[0031] Figure 6a is a graph showing the size (volume) of tumors when 5-FU or 5-FU and epinephrine were administered together after xenografting human gastric cancer cells into nude mice.

[0032] Figure 6b is a photograph showing the size (volume) of a tumor when 5-FU and epinephrine were administered together after xenografting human gastric cancer cells into nude mice.

[0033] Figure 7a is a graph showing the size (volume) of tumors when 5-FU and epinephrine were administered together after xenografting human gastric cancer cells into nude mice, and when 5-FU, epinephrine, and L-glucose were administered together.

[0034] Figure 7b is a photograph showing the size (volume) of a tumor when 5-FU, epinephrine, and L-glucose were administered together after xenografting human gastric cancer cells into nude mice.

[0035] According to an exemplary embodiment, a pharmaceutical composition for destroying tumor blood vessels (hereinafter, "composition") comprises alkaline 5-fluorouracil (5-FU) and epinephrine. For example, the composition may induce apoptosis of tumor vascular endothelial cells by including epinephrine, thereby synergistically enhancing the anticancer effect with the tumor vascular endothelial cell killing effect of alkaline 5-fluorouracil.

[0036] 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.

[0037] A composition according to an exemplary embodiment may comprise alkaline 5-fluorouracil (5-FU) and epinephrine.

[0038] Alkaline 5-fluorouracil exerts its anticancer effect by destroying tumor blood vessels and inhibiting angiogenesis, thereby blocking and inhibiting the supply of nutrients or oxygen to cancer cells.

[0039] In one embodiment, the alkaline 5-fluorouracil can kill tumor endothelial cells by increasing the expression of the thrombospondin-1 (TSP-1) protein in the tumor. Since this is a result of destruction of blood vessels supplying the tumor, rather than the tumor itself, it can be applied to various solid tumors.

[0040] 5-Fluorouracil can enhance its anticancer effects when mixed with an alkaline solvent. For example, it can provide superior anticancer effects when mixed with an alkaline solvent than with a neutral or acidic solvent. For example, alkaline 5-Fluorouracil can target vascular endothelial cells within cancer tissue and induce apoptosis. For example, injecting alkaline 5-Fluorouracil into the base of a tumor tissue can induce apoptosis of the vascular endothelial cells supplying blood to the tumor, destroying the blood vessels and causing tumor necrosis. In this case, the pH of 5-Fluorouracil is a critical factor in inducing apoptosis of tumor vascular endothelial cells. A slightly alkaline pH, such as 8.4 to 9.0, can significantly increase endothelial cell death compared to neutral or acidic 5-Fluorouracil.

[0041] In one embodiment, the alkaline 5-fluorouracil may be in the form of 5-fluorouracil dissolved in an alkaline solvent. The alkaline solvent may be, for example, alkaline water, saline solution, etc.

[0042] 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, 8.4 to 9.0, or 8.4 to 8.8, but is not limited thereto.

[0043] In one embodiment, the alkaline 5-fluorouracil concentration 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.

[0044] Epinephrine can induce apoptosis in tumor vascular endothelial cells. Therefore, it can maximize anticancer effects by synergizing with the endothelial cell-killing effect of 5-FU. Furthermore, epinephrine can constrict blood vessels at the injection site (e.g., subcutaneously or within the submucosal tissue) to suppress bleeding and reduce side effects by inhibiting the systemic absorption of 5-FU. Furthermore, the duration of action of 5-FU with inhibited absorption can be extended, potentially enhancing the endothelial cell-killing effect.

[0045] 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 10 cc of 200 mg of alkaline 5-fluorouracil is injected into the submucosal layer of the normal stomach wall immediately adjacent to the cancer cells, the epinephrine dosage may be 0.001 mg / mL to 0.2 mg / mL.

[0046] The above epinephrine concentration of 0.1 mg / mL can be expressed as 1:10,000. The above 1:10,000 can mean that epinephrine is diluted 1:10,000 in a solution (e.g., saline solution). For example, the administrable concentration of epinephrine can be from 1:10,000 to 1:1,000,000.

[0047] In one embodiment, the composition may further comprise one or more selected from glucose, a nitric oxide formation inhibitor, bevacizumab, capric acid or a physiologically acceptable salt thereof, and a poloxamer.

[0048] In some embodiments, the glucose may comprise one or more selected from D-glucose and L-glucose.

[0049] For example, high concentrations of D-glucose can delay replicative delay and promote apoptosis in endothelial cells. For example, exposure to high concentrations (e.g., 25 mM or higher) for more than 48 hours can increase DNA fragmentation and upregulate thrombospondin-1 expression, inducing endothelial cell death. High concentrations of glucose can also increase reactive oxygen species (ROS) within tumor vascular endothelial cells, thereby enhancing oxidative stress. In other words, the increase in ROS caused by high concentrations of glucose shares a mechanism with the endothelial cell toxicity induced by alkaline 5-fluorouracil.

[0050] For example, L-glucose can induce apoptosis by inhibiting the metabolism of tumor vascular endothelial cells.

[0051] For example, the glucose may be included in the total composition at 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.

[0052] In one embodiment, the nitric oxide formation inhibitor is L-NMMA(N G -Methyl-L-arginine), L-NAME(N G - It may include one or more selected from -Nitro-L-arginine methyl ester), L-NA (Nitro Arginine), and 7NI (Nitroindazole).

[0053] Nitric oxide is synthesized and secreted by NO synthase in vascular endothelial cells, dilates blood vessels, and protects cells by inhibiting apoptosis of vascular endothelial cells caused by external stimuli such as lipopolysaccharide (LPS), angiotensin II, caspase-3 overexpression, and TNF-alpha.

[0054] Nitric oxide production inhibitors can constrict tumor blood vessels and promote thrombosis within tumor vessels damaged by 5-fluorouracil. For example, alkaline 5-fluorouracil can damage or destroy tumor vascular endothelial cells, leading to thrombosis within tumor vessels. Nitric oxide production inhibitors can promote occlusion of these tumor vessels, more effectively inducing tumor necrosis.

[0055] In some embodiments, the nitric oxide formation inhibitor is preferably L-NMMA(N G -Methyl-L-arginine) may be included.

[0056] L-NMMA is a nonspecific NO synthase inhibitor that effectively increases blood pressure when administered intravascularly. Under physiological conditions, inhibition of NO synthase alone by L-NMMA does not induce platelet activation in vivo. However, damage to the endothelial cells of tumor blood vessels can induce platelet activation and thrombosis. In other words, damaged vascular endothelial cells can induce thrombosis. Therefore, when L-NMMA is used in combination with 5-fluorouracil, it can provide excellent anticancer effects by simultaneously inducing tumor endothelial cell death and tumor vessel occlusion.

[0057] For example, L-NMMA may be included in a dose of, but not limited to, 0.5 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg, or 4 mg / kg to 6 mg / kg, based on the weight of the subject being treated with the drug.

[0058] For another example, the concentration of the nitric oxide production inhibitor may be, but is not limited to, 0.1 mM to 300 mM, 0.1 mM to 250 mM, 1 mM to 200 mM, or 10 mM to 200 mM in the total composition.

[0059] Bevacizumab is a recombinant humanized monoclonal antibody that inhibits vascular endothelial growth factor (VEGF), which promotes angiogenesis. Typically, bevacizumab can be used as an anticancer agent alone or in combination with other drugs. Previously, it was primarily administered intravenously. For example, in the case of advanced gastric cancer, it has been reported to improve progression-free survival (PFS) and overall response rates when combined with fluoropyrimidine-cisplatin therapy. However, in some embodiments, bevacizumab can be provided in a sustained-release form in the composition to inhibit angiogenesis of cancer cells, thereby more effectively destroying blood vessels distributed within the tumor.

[0060] 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.

[0061] 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.

[0062] 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 between 25 and 36°C.

[0063] Capric acid is a type of saturated fatty acid and is a compound represented by the following chemical formula 1.

[0064] [Chemical Formula 1]

[0065]

[0066] Physiologically acceptable salts may be salts prepared using caprylic acid and a relatively non-toxic acid or base. Physiologically acceptable salts may be, for example, metal salts or acid addition salts.

[0067] 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.

[0068] Acid addition salts can be prepared from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous or phosphorous acid and non-toxic organic acids 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. For example, an acid addition salt of a compound represented by formula 1 can be obtained by dissolving the compound in an excess aqueous acid solution and precipitating the salt using a hydratable organic solvent such as methanol, ethanol, acetone or acetonitrile.

[0069] Poloxamer may be a terpolymer comprising a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) structure. For example, it may be represented by the following chemical formula 2.

[0070] [Chemical Formula 2]

[0071]

[0072] 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.

[0073] 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.

[0074] 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.

[0075] The above poloxamer is a type of surfactant that forms micelles in an aqueous environment, allowing the loading of 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.

[0076] In one embodiment, the composition may further include a known anticancer agent. For example, the composition may include cisplatin, carboplatin, cyclophosphamide, paclitaxel, tamoxifen, toremifene, fulvestrant, diindolimethane, exemestane, raloxifene, an aromatase inhibitor, doxorubicin, oxaliplatin, vincristine, gemcitabine, anthracyclines, taxanes, irinotecan, docetaxel, and eribulin. However, the composition is not limited thereto. The component may further include other carriers, etc., and may be formulated together with the carrier, which may be appropriately selected by a person skilled in the art in consideration of the type of the bioactive substance, the route by which the carrier is administered, etc. The additional component may further include other carriers, etc., and may be formulated together with the carrier, and may be appropriately selected by a person skilled in the art in consideration of the type of the bioactive substance, the route by which the carrier is administered, etc.

[0077] 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.

[0078] The composition according to some embodiments may additionally contain a compound that maintains or increases the solubility and / or absorbability of the active ingredient.

[0079] In one embodiment, the tumor may comprise a solid tumor.

[0080] In some embodiments, the tumor is, 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, and brain tumor, cervical cancer, prostate cancer, testicular cancer, penile cancer, genitourinary tract cancer, seminoma, esophageal cancer, laryngeal cancer, gastrointestinal cancer, keratoacanthoma, follicular carcinoma, melanoma, small cell lung carcinoma, non-small cell lung carcinoma (NSCLC), lung adenocarcinoma, squamous cell carcinoma of the lung, colon cancer, thyroid cancer, papillary cancer, biliary tract cancer, kidney, bone cancer, bone marrow disorder, hairy cell cancer, oral and pharyngeal (oral) cancer, lip cancer, tongue cancer, oral cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, colon cancer, rectal cancer, vulvar cancer, thyroid cancer, endometrial cancer, central nervous system cancer, peritoneal cancer, hepatocellular carcinoma, head and neck cancer It could be cancer, cervical cancer, etc., but it is not limited to these.

[0081] 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.

[0082] 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.

[0083] In one embodiment, the composition may be administered by local injection around the tumor.

[0084] Referring to Figure 1, for example, a composition can be delivered to the tumor base via an injector using an endoscope. Specifically, the composition can be injected into the submucosa zone of the normal gastric 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 blood to the tumor tissue can induce endothelial cell death or inhibit the synthesis of new blood vessels.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0091] <Example>

[0092] Example 1: Confirmation of the apoptotic effect of HUVECs according to epinephrine concentration using the MTS method.

[0093] Cytotoxicity of human umbilical vein endothelial cells (HUVECs) according to epinephrine concentration was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were seeded in 96-well plates at a density of 5x10 3Cells were seeded at a density of 10 cells / well and cultured for 24 h. The cells were then cultured with DMEM medium at various epinephrine concentrations for 24 h. The cells were then rinsed twice with PBS, 100 μl of fresh growth DMEM medium was added, and the cells were cultured for an additional 48 h. The medium was replaced with 100 μl of fresh growth DMEM medium, and 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) was added. After an additional 2 h of incubation, the absorbance was measured at 490 nm using a BIO-RAD Model 680 microplate reader (Spark, TECAN, CA, USA). The results are shown in Figures 2a and 2b.

[0094] In Figure 2a, HUVEC cell viability decreased in proportion to the concentration of epinephrine. In Figure 2b, when 100 μm of 5-FU was co-administered, HUVEC cell viability decreased in proportion to the concentration of epinephrine. Therefore, it can be seen that epinephrine exerts a synergistic effect on 5-FU's apoptosis.

[0095] Example 2: Confirmation of the natural death mechanism of HUVECs by epinephrine using Western blot.

[0096] Western blot was performed to confirm the expression of FAS, cleaved caspase 3, and cleaved caspase 9, which are activated when HUVEC undergoes apoptosis induced by epinephrine.

[0097] 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. The cells were then chilled on ice for 30 min, centrifuged at 14,000 g for 10 min, and the supernatant was separated. Quantification was performed using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA), and 30 μg of protein solution was electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The electrophoresed membrane was reacted with blocking milk for 1 hour, and then incubated with primary antibodies against cleaved caspase 3, cleaved caspase 9, and FAS for 1 hour at room temperature. The membrane was washed three times at 15-minute intervals in Tris buffer containing 0.1% Tween 20, and incubated with secondary antibodies against the primary antibodies for 1 hour at room temperature, followed by detection using a chemiluminescent agent (Amersham Life Science, Arlington Heights, IL, USA). The results are shown in Fig. 3a.

[0098] Proteins extracted from HUVECs were used as a positive control. The expression of FAS, cleaved caspase 3, and cleaved caspase 9 in HUVECs according to the concentration of epinephrine is shown in Figure 3b.

[0099] In Figures 3a and 3b, it was confirmed that as the concentration of epinephrine increased, the expression levels of FAS, cleaved caspase 3, and cleaved caspase 9 increased.

[0100] Example 3: Confirmation of DNA laddering by epinephrine as revealed by gel electrophoresis.

[0101] In the electrophoresis process of Example 2, characteristic DNA laddering was confirmed and is shown in Figure 4. It can be seen that DNA fragmentation occurred as a result of HUVEC apoptosis caused by epinephrine.

[0102] Referring to Figure 4, it can be seen that as the concentration of epinephrine increases, DNA fragmentation occurs more frequently due to apoptosis of HUVECs, and the DNA movement speed increases.

[0103] Example 4: Confirmation of spontaneous death of HUVECs according to the concentration of epinephrine using confocal fluorescence microscopy.

[0104] The Apoptosis Detection Kit (Blue, Green) (ab176749) allows simultaneous monitoring of apoptotic and healthy cells. The PS sensor, upon binding to cell membrane PS, fluoresces green (Ex / Em = 490 / 525 nm), indicating apoptosis. Healthy cells can be identified using CytoCalcein Violet 450 (Ex / Em = 405 / 450 nm), a dye used to label live cytoplasm.

[0105] Endothelial cells were plated 5x10 in a slide culture chamber. 4After treating cells with epinephrine at various concentrations (0-200uM) for 24 hours, wash the cells once or twice with 100μL assay buffer by pipetting the buffer up and down. Incubate the cells with 200μL assay buffer containing Apopxin / CytoCalcein at room temperature for 30-60 minutes. Wash the cells twice with 100μL assay buffer, then replace with 100μL assay buffer and transfer to the stage of a confocal microscope. Tilt back the halogen lamp column, place the slide on the stage, launch the analysis software, and set the imaging parameters in the Acquire tab to XYZ mode, 1024x1024 pixels, 600Hz, Zoom 1x, average 4, accumulation 1, rotation 0, then place the sample on the microscope and focus by fluorescence. After focusing, set the Beam Path Setting to select the laser to be used, specify the light quantity, select the PMT, select the pseudocolor, and set the detection band in that order. Press the Live button to perform a preview scan and display the captured image 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 Figures 5a to 5e.

[0106] Referring to FIGS. 5A to 5E, it can be seen that healthy cells decrease and apoptotic cells increase in proportion to the concentration of epinephrine.

[0107] Example 5: In-vivo Experiment 1: Confirmation of the Toxicity of 5-FU and Epinephrine to Vascular Endothelial Cells in Tumor Tissue

[0108] 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 μl (PBS) were transplanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached 1 cm.

[0109] The experimental animals were divided into three groups. The first group was the control group and was administered only saline, the second group was administered only 0.2 cc of 5 mg pH 8.4 5-FU, and the third group was administered a total of 0.2 cc of 5 mg pH 8.4 5-FU and epinephrine 1:200,000. The injections were performed four times at one-week intervals right next to the tumor. After one month, the long and short diameters of the tumors growing on the dorsal epidermis were measured 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 Fig. 6a.

[0110] Referring to Figure 6a, when alkaline 5-FU and epinephrine were administered together, the tumor size was significantly reduced compared to the control group and when alkaline 5-FU was injected alone.

[0111] Additionally, the third group is shown in Figure 6b, which shows photographs of tumor size before and after drug administration. Referring to Figure 6b, it can be confirmed that the tumor size significantly decreased over time.

[0112] Example 6: In-vivo Experiment 2: Confirmation of the Toxicity of 5-FU and Epinephrine on Vascular Endothelial Cells in Tumor Tissue

[0113] The animals used in the experiment were 38 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 μl (PBS) were transplanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached 1 cm.

[0114] Among 38 prepared mice, 26 mice were administered 5 mg of 5-FU at pH 8.4, and 6 mice (approximately 23.1%) showed rapid weight loss or death.

[0115] When the remaining 12 mice were injected with 0.2 cc of epinephrine 1:200,0000 together with 5 mg of pH 8.4 5-FU, only 1 mouse showed weight loss and died, confirming that the toxicity of the drug was reduced.

[0116] Example 7: In-vivo experiment to confirm the toxicity of 5-FU, epinephrine, and L-glucose on vascular endothelial cells in tumor tissue.

[0117] 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 μl (PBS) were transplanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were conducted when the diameter reached 1 cm.

[0118] The experimental animals were divided into two groups. One group was administered a total of 0.2 cc of 5 mg pH 8.4 5-FU and 1:200,000 epinephrine, and the other group was administered a total of 0.2 cc of 5 mg pH 8.4 5-FU, 1:200,000 epinephrine, and 5% L-glucose. The injections were performed four times at one-week intervals right next to the tumor. One month later, the long and short diameters of the tumors growing on the dorsal epidermis were measured, 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 Fig. 7a.

[0119] Referring to Figure 7a, when 5-FU, L-glucose, and epinephrine at pH 8.4 were injected, the tumor was almost killed compared to when 5-FU and epinephrine were injected.

[0120] Additionally, in the case of injection of 5-FU, L-glucose, and epinephrine at pH 8.4, photographs of tumor size before and after drug administration are shown in Figure 7b.

[0121] Referring to Figure 7b, when 5-FU, L-glucose, and epinephrine at pH 8.4 were injected, the tumor size (volume) was significantly reduced compared to before drug injection.

Claims

1. A pharmaceutical composition for destroying tumor blood vessels, comprising alkaline 5-fluorouracil and epinephrine.

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

3. A pharmaceutical composition for destroying tumor blood vessels according to claim 1, wherein the alkaline 5-fluorouracil is in a form in which 5-fluorouracil is dissolved in an alkaline solvent.

4. A pharmaceutical composition for destroying tumor blood vessels according to claim 1, wherein the alkaline 5-fluorouracil increases the expression of thrombospondin-1 protein in tumor vascular endothelial cells, thereby killing the vascular endothelial cells.

5. A pharmaceutical composition for destroying tumor blood vessels according to claim 1, wherein the concentration of alkaline 5-fluorouracil in the entire composition is 10 to 600 mM.

6. A pharmaceutical composition for destroying tumor blood vessels, wherein the epinephrine induces natural death of tumor vascular endothelial cells in claim 1.

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

8. A pharmaceutical composition for destroying tumor blood vessels, further comprising at least one selected from glucose, a nitric oxide production inhibitor, bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer, according to claim 1.

9. In claim 8, the nitric oxide formation inhibitor is L-NMMA (N G -Methyl-L-arginine), L-NAME(N G - A pharmaceutical composition for destroying tumor blood vessels, comprising at least one selected from -Nitro-L-arginine methyl ester, L-NA (Nitro Arginine), and 7NI (Nitroindazole).

10. A pharmaceutical composition for destroying tumor blood vessels, further comprising at least one selected from cisplatin, carboplatin, cyclophosphamide, paclitaxel, tamoxifen, toremifene, fulvestrant, diindolimethane, exemestane, raloxifene, an aromatase inhibitor, doxorubicin, oxaliplatin, vincristine, gemcitabine, anthracyclines, taxanes, irinotecan, docetaxel, and eribulin, in claim 1.

11. A pharmaceutical composition for destroying tumor blood vessels according to claim 1, wherein the tumor includes a solid cancer.

12. A pharmaceutical composition for destroying tumor blood vessels, wherein the composition is a sustained-release preparation according to claim 1.

13. A pharmaceutical composition for destroying tumor blood vessels, wherein the composition according to claim 1 is administered by local injection around the tumor.

14. A pharmaceutical composition for destroying tumor blood vessels according to claim 13, wherein the area surrounding the tumor is a submucosal layer where tumor blood vessels are located.

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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