Pharmaceutical composition for destroying tumor blood vessels

Abstract

A pharmaceutical composition for destroying tumor blood vessels according to an exemplary embodiment may include alkaline 5-fluorouracil (5-FU) and epinephrine. Epinephrine may enhance the anticancer effect by exerting synergy with the apoptotic effect of alkaline 5-fluorouracil on tumor vascular endothelial cells.

Description

Technology Field

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

[0002] 5-fluorouracil (5-FU) is a widely used anticancer chemotherapy agent for cancer treatment. 5-fluorouracil is a uracil analog present in the human body and exerts anticancer effects by inhibiting DNA synthesis and repair through the inhibition of thymidylate synthase, as well as by inhibiting RNA function.

[0003] 5-fluorouracil is mainly administered intravenously and must be administered directly in a hospital, and because it has the side effect of thrombosis, an oral 5-FU prodrug has recently been developed.

[0004] Although the effects of 5-fluorouracil are significant, it can cause myelosuppression or gastrointestinal toxicity due to phosphorylation in the gastrointestinal tract. In addition, cardiotoxicity, a serious, life-threatening side effect, has been reported in, for example, 1.5 to 18% of cases.

[0005] Therefore, research is actively underway to reduce side effects while maintaining the anticancer effect of 5-fluorouracil. For example, Korean published patent No. 10-2016-0049312 aims to effectively improve anticancer drug side effects caused by 5-fluorouracil, such as inflammatory response, intestinal damage, and myelosuppression, by using cyanidin-3-glucoside. Prior art literature

[0006] Korean Published Patent No. 10-2016-0049312 The problem to be solved

[0007] The objective of the present invention is to provide a pharmaceutical composition for tumor angiogenesis that has excellent anticancer effects and reduced side effects. means of solving the problem

[0008] A pharmaceutical composition for destroying tumor blood vessels according to an exemplary embodiment may include alkaline 5-fluorouracil (5-FU) and epinephrine.

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

[0010] In one embodiment, the alkaline 5-fluorouracil may be in the form of 5-fluorouracil dissolved in an alkaline solvent.

[0011] In one embodiment, the alkaline 5-fluorouracil can increase thrombospondin-1 protein expression in tumor vascular endothelial cells, thereby causing the vascular endothelial cells to die.

[0012] In one embodiment, the concentration of the alkaline 5-fluorouracil may be 0.1 to 600 mM in the total composition.

[0013] In one embodiment, the epinephrine can induce apoptosis of tumor vascular endothelial cells.

[0014] In one embodiment, the concentration of the epinephrine may be 0.001 to 0.2 mg / mL in the total composition.

[0015] In one embodiment, the composition may further comprise one or more selected from glucose, a nitric oxide production inhibitor, bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0016] 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).

[0017] In one embodiment, the composition may further include one or more selected from cisplatin, carboplatin, cyclophosphamide, paclitaxel, tamoxifen, toremifene, fulvestrant, diindolylmethane, exemestane, raloxifene, aromatase inhibitor, doxorubicin, oxaliplatin, vincristine, gemcitabine, anthracycline, taxane, irinotecan, docetaxel, and eribulin.

[0018] In one embodiment, the cancer may include a solid tumor.

[0019] In one embodiment, the composition may be a sustained-release formulation.

[0020] In one embodiment, the composition may be administered by local injection around a tumor.

[0021] In one embodiment, the area surrounding the tumor may be the submucosal layer where the tumor blood vessels are located.

[0022] A local administration agent for solid tumors according to an exemplary embodiment may include the pharmaceutical composition for tumor angiogenesis. Effects of the invention

[0023] The pharmaceutical composition for destroying tumor blood vessels according to the present invention may include alkaline 5-fluorouracil and epinephrine.

[0024] For example, injecting alkaline 5-fluorouracil into the base of a tumor can destroy the tumor by inducing apoptosis of tumor vascular endothelial cells. In addition, it can inhibit tumor angiogenesis.

[0025] For example, injecting epinephrine into the base of a tumor can induce apoptosis of tumor vascular endothelial cells. It can enhance the anticancer effect by synergizing with the apoptotic effect of alkaline 5-fluorouracil on tumor vascular endothelial cells. Additionally, it can suppress bleeding by constricting blood vessels at the epinephrine injection site. Therefore, by inhibiting the systemic absorption of 5-fluorouracil, it can help induce apoptosis of tumor vascular endothelial cells while sustaining the effect and reducing side effects.

[0026] The pharmaceutical composition for destroying tumor blood vessels according to the present invention can be applied to various solid tumors because it destroys the blood vessels supplying blood to the tumor rather than the tumor itself. Brief explanation of the drawing

[0027] FIG. 1 is an example of administering a pharmaceutical composition for destroying tumor blood vessels according to one embodiment by local injection around a tumor. Figures 2a and 2b show the results of confirming the HUVEC cell death effect according to epinephrine concentration using MTS. Figures 3a and 3b show the results of confirming the HUVEC cell death effect according to epinephrine concentration using Western blot. Figure 4 shows the results of confirming DNA lamination in gel electrophoresis according to epinephrine concentration. Figures 5a to 5e show the results of confirming the HUVEC cell death effect according to epinephrine concentration using a confocal fluorescence microscope. Figure 6a is a graph showing the size (volume) of tumors when 5-FU was administered or 5-FU and epinephrine were administered together after xenografting human gastric cancer cells into nude mice. 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. Figure 7a is a graph showing the size (volume) of tumors when 5-FU and epinephrine were administered together and when 5-FU, epinephrine, and L-glucose were administered together after xenografting human gastric cancer cells into nude mice. 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. Specific details for implementing the invention

[0028] A pharmaceutical composition for destroying tumor blood vessels according to an exemplary embodiment (hereinafter, the composition) comprises alkaline 5-fluorouracil (5-FU) and epinephrine. For example, the composition may include epinephrine to induce apoptosis of tumor vascular endothelial cells, thereby exhibiting synergy with the apoptotic effect of alkaline 5-fluorouracil on tumor vascular endothelial cells and significantly enhancing the anticancer effect.

[0029] Hereinafter, compositions according to exemplary embodiments of the present invention will be described in detail with reference to the drawings and embodiments. However, the drawings and embodiments are merely illustrative and the present invention is not limited thereto.

[0030] A composition according to an exemplary embodiment may include alkaline 5-fluorouracil (5-FU) and epinephrine.

[0031] Alkaline 5-fluorouracil produces an anticancer effect by destroying tumor blood vessels and inhibiting neovascularization, thereby blocking or inhibiting the supply of nutrients or oxygen to cancer cells.

[0032] In one embodiment, the alkaline 5-fluorouracil can increase the expression of thrombospondin-1 (TSP-1) protein in tumor vascular endothelial cells, thereby causing the death of vascular endothelial cells. Since this is a result of the destruction of blood vessels supplying blood to the tumor rather than the tumor itself, it can be applied to various solid tumors.

[0033] The anticancer effect of 5-fluorouracil can be enhanced when mixed with an alkaline solvent. For example, it may provide a superior anticancer effect when mixed with an alkaline solvent compared to neutral or acidic solvents. For example, alkaline 5-fluorouracil can target vascular endothelial cells within cancer tissue to induce apoptosis. For example, injecting alkaline 5-fluorouracil into the base of tumor tissue can induce apoptosis of vascular endothelial cells supplying blood to the tumor, thereby destroying blood vessels and causing necrosis of the tumor. In this case, the pH of 5-fluorouracil is an important factor in inducing apoptosis of tumor vascular endothelial cells, and when the pH is slightly alkaline, e.g., 8.4 to 9.0, it can significantly increase the apoptosis of vascular endothelial cells compared to neutral or acidic 5-fluorouracil.

[0034] In one embodiment, the alkaline 5-fluorouracil may be in the form where 5-fluorouracil is dissolved in an alkaline solvent. The alkaline solvent may be, for example, alkaline water, physiological saline solution, etc.

[0035] In one embodiment, the alkalinity may have a pH of 8.0 to 9.0. For example, it may be 8.0 to 9.0, 8.2 to 9.0, 8.4 to 9.0, or 8.4 to 8.8. However, it is not limited thereto.

[0036] In one embodiment, the alkaline 5-fluorouracil concentration may be 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 in the total composition. However, it is not limited thereto.

[0037] Epinephrine can induce apoptosis in tumor vascular endothelial cells. Therefore, it can maximize the anticancer effect by synergizing with the vascular endothelial cell death effect caused by 5-FU. In addition, it can suppress bleeding by constricting blood vessels at the epinephrine injection site (e.g., subcutaneous or submucosal tissue) and reduce side effects by inhibiting systemic absorption of 5-FU. The duration of the effect of 5-FU with inhibited absorption can be extended, which may also enhance the vascular endothelial cell death effect.

[0038] In one embodiment, the concentration of the 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, and for example, 0.002 mg / mL to 0.2 mg / mL. For example, when 10 cc of 200 mg alkaline 5-fluorouracil is injected into the submucosal layer of the normal gastric wall right next to the cancer cells, the epinephrine dosage may be 0.001 mg / mL to 0.2 mg / mL.

[0039] The above epinephrine concentration of 0.1 mg / mL can be expressed as 1:10,000. The above 1:10,000 may mean that epinephrine is diluted 1:10,000 in a solution (e.g., physiological saline). For example, the administerable concentration of epinephrine may be from 1:10,000 to 1:1,000,000.

[0040] In one embodiment, the composition may further comprise one or more selected from glucose, a nitric oxide production inhibitor, bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0041] In some embodiments, the glucose may include one or more selected from D-glucose and L-glucose.

[0042] For example, high concentrations of D-glucose can delay endothelial cell replication (replicative delay) and promote apoptosis. For instance, exposure to high concentrations (e.g., 25 mM or higher) for more than 48 hours can increase DNA fragmentation and increase the expression of thrombospondin-1, thereby inducing endothelial cell death. High concentrations of glucose can increase reactive oxygen species within tumor vascular endothelial cells, thereby heightening oxidative stress. In other words, the increase in reactive oxygen species caused by high concentrations of glucose shares the same mechanism as the endothelial toxicity caused by alkaline 5-fluorouracil.

[0043] For example, L-glucose inhibits the metabolism of tumor vascular endothelial cells, so it can induce apoptosis.

[0044] For example, the glucose may be included in the total composition in an amount of 10 mM to 500 mM, 20 mM to 250 mM, 25 mM to 150 mM, 30 mM to 120 mM, and 50 mM to 100 mM.

[0045] 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).

[0046] Nitrile oxide is synthesized and secreted by NO synthase in vascular endothelial cells, dilates blood vessels, and protects cells by inhibiting the progression of vascular endothelial cells to apoptosis caused by external stimuli such as lipopolysaccharide (LPS), angiotensin II, caspase-3 overexpression, and TNF-alpha.

[0047] Nitric oxide inhibitors can constrict tumor blood vessels and promote the development of thrombosis within tumor blood vessels damaged by 5-fluorouracil. For example, tumor vascular endothelial cells may be damaged or destroyed by alkaline 5-fluorouracil, leading to the formation of blood clots within the tumor blood vessels. Nitric oxide inhibitors can promote the occlusion of these tumor blood vessels, thereby inducing tumor necrosis more effectively.

[0048] In some embodiments, the nitric oxide formation inhibitor is preferably L-NMMA (N G It may contain -Methyl-L-arginine).

[0049] L-NMMA is a nonspecific NO synthase inhibitor that effectively raises blood pressure when administered intravenously. Under physiological conditions, inhibition of NO synthase by L-NMMA alone does not lead to platelet activation in vivo; however, if damage occurs to endothelial cells in tumor blood vessels, it can induce platelet activation and thrombosis. In other words, it can induce damaged vascular endothelial cells to form blood clots. Therefore, using L-NMMA in combination with 5-fluorouracil can provide excellent anticancer effects by inducing both tumor vascular endothelial cell apoptosis and tumor blood vessel occlusion.

[0050] For example, L-NMMA may be included in an amount of 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. However, it is not limited thereto.

[0051] As another example, the concentration of the nitric oxide production inhibitor may be 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. However, it is not limited thereto.

[0052] Bevacizumab is a recombinant humanized monoclonal antibody designed to inhibit vascular endothelial growth factor (VEGF), which promotes angiogenesis. Typically, bevacizumab can be used as an anticancer agent, either alone or in combination with other drugs. It has traditionally been administered primarily by intravenous injection, and it is known that, for example in cases of advanced gastric cancer, combining it with fluoropyrimidine-cisplatin therapy improves progression-free survival and overall response rate. However, in some embodiments, bevacizumab is provided in a sustained-release form in the composition to inhibit neovascularization of cancer cells, thereby more effectively eliminating blood vessels distributed within the tumor.

[0053] In one embodiment, on the other hand, the composition may be a sustained-release formulation. The longer the exposure time to tumor vascular endothelial cells, the greater the apoptotic effect may be. To provide the composition as a sustained-release formulation, the composition may further comprise capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0054] Capric acid or its physiologically acceptable salt and poloxamer have low toxicity, are biocompatible, and have superior in vivo stability. For example, by mixing capric acid or its physiologically acceptable salt and poloxamer, viscosity can be increased and the sol-gel transition temperature of poloxamer can be raised. For example, the sol-gel transition temperature may be between room temperature and body temperature, for example, 20 to 40°C or 25 to 37°C. Within this range, storage, transport, and use in a sol state at room temperature are convenient. When acting in the body, diffusion in tissues is also easy in the gel state.

[0055] In some embodiments, the weight ratio of capric acid to poloxamer may be 0.25 to 3.4:30, 1 to 3.4:30, or 2 to 3:30, and specifically, the weight ratio of capric acid to poloxamer may be 2.75 to 3.3:30 or 2.8 to 3.2:30. Within the above range, the sol-gel transition of the carrier is likely to occur between 25 and 36°C.

[0056] Capric acid is a compound represented by the following chemical formula 1 and is a type of saturated fatty acid.

[0057] [Chemical Formula 1]

[0058]

[0059] Physiologically acceptable salts may be salts prepared using capri acid and relatively non-toxic acids or bases. Physiologically acceptable salts may be, for example, metal salts or acid addition salts.

[0060] Metal salts can be sodium, potassium, or calcium salts. Metal salts can be prepared using a base. For example, alkali metal or alkaline earth metal salts can be obtained by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and / or drying the filtrate.

[0061] Acid addition salts can be prepared from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromide, hydroiodide, nitrous acid, or phosphoric acid, and non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkandioates, aromatic acids, and 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, propyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, malieates, butin-1,4-dioate, nucleus-1,6-dioate, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, It may include terephthalate, benzenesulfonate, ethylsulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, 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 amount of aqueous acid solution and precipitating the salt using a hydrated organic solvent, such as methanol, ethanol, acetone, or acetonitrile.

[0062] 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.

[0063] [Chemical Formula 2]

[0064]

[0065] The molecular weight of the poloxamer may, for example, be 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 above molecular weight may be appropriately selected by a person skilled in the art depending on the material to be delivered.

[0066] Poloxamer is generally designated by a numbering system that indicates the approximate molecular weight of the poloxamer and the percentage of polyoxyethylene content, and is also referred to by the trade name Pluronic. For example, Poloxamer 407 and the trade name Pluronic F-127 are interchangeable.

[0067] For example, the above poloxamers are 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.

[0068] The above poloxamer is a type of surfactant and can load substances by forming micelles in an aqueous environment. A hydrogel composed of poloxamer can be used as a carrier to form a matrix and release substances locally and continuously. The above poloxamer hydrogel may include cross-linking between poloxamers. The above poloxamer can be used in various molecular weights and ratios.

[0069] In one embodiment, the composition may further include known anticancer agents. For example, it may include cisplatin, carboplatin, cyclophosphamide, paclitaxel, tamoxifen, toremifene, fulvestrant, diindolylmethane, exemestane, raloxifene, aromatase inhibitors, doxorubicin, oxaliplatin, vincristine, gemcitabine, anthracycline, taxane, irinotecan, docetaxel, and eribulin, etc. However, it is not limited thereto. The above components may additionally include other carriers, etc., and may be formulated together with the carriers, and the appropriately selected by a person skilled in the art considering the type of physiologically active substance, the route of administration of the carrier, etc. The additional components may additionally include other carriers, etc., and may be formulated together with the carriers, and the appropriately selected by a person skilled in the art considering the type of physiologically active substance, the route of administration of the carrier, etc.

[0070] A composition according to some embodiments may contain one or more active ingredients that exhibit the same or similar functions in relation to the treatment of tumors.

[0071] Compositions according to some embodiments may additionally contain compounds that maintain or increase the solubility and / or absorption of the active ingredient.

[0072] In one embodiment, the tumor may include a solid tumor.

[0073] 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, colorectal cancer, biliary tract cancer, bladder cancer, ovarian cancer and brain tumor, cervical cancer, prostate cancer, testicular cancer, penile cancer, genitourinary cancer, testicular tumor, esophageal cancer, laryngeal cancer, gastrointestinal cancer, keratosporoma, 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 carcinoma, cholangiocarcinoma, kidney, bone cancer, bone marrow disorder, hair cell carcinoma, 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 cancer, cervical cancer It may be cancer, etc. However, it is not limited to this.

[0074] 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 the cheek and under the tongue), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration. Or it may be a formulation suitable for administration by inhalation or insufflation.

[0075] The formulation of the above composition may be an injectable. It does not form a precipitate in the biological environment, blood, etc., and can be administered with a thin needle. It is preferable that the formulation of the above composition be an injectable formulation.

[0076] In one embodiment, the composition may be administered by local injection around a tumor.

[0077] Referring to FIG. 1, for example, the composition can be delivered to the base of the tumor through an injector using an endoscope. Specifically, the composition can be injected into the submucosa zone of the normal gastric wall right next to the tumor, toward the basal layer of the tumor. The drug delivered to the vascular endothelial cells of the blood vessels supplying blood to the tumor tissue may produce a death effect on the vascular endothelial cells or an inhibitory effect on the synthesis of new blood vessels.

[0078] Compared to the intravenous administration of conventional anticancer drugs, the above-mentioned local injection can deliver a large amount of drug not only to the submucosal layer surrounding the tumor but also to the surrounding lymph nodes, and can reduce the side effects of anticancer drugs compared to systemic administration.

[0079] The composition according to one embodiment may be administered in a pharmaceutically effective amount. The effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0080] A local administration agent for solid tumors according to an exemplary embodiment may include the above composition. Specific examples of solid tumors are the same as those described above.

[0081] In some embodiments, the composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents. The components included in the anticancer combination formulation may be administered sequentially or simultaneously, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0082] For example, the dosage of the above composition varies widely depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. 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 delivery vehicle of the present invention used. For example, it may be 0.01 μg to 1 g per kg of body weight and may be administered in divided doses of one to several times per unit period, on a daily, weekly, monthly, or yearly basis. Alternatively, it may be administered continuously over a long period using an infusion pump. The number of repeated administrations is determined by considering the time the drug remains in the body and the drug concentration in the body. Depending on the course of disease treatment, the composition may be administered for recurrence even after treatment has been achieved.

[0083] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0084] <Example>

[0085] Example 1: Confirmation of HUVEC apoptosis effect according to epinephrine concentration using the MTS method

[0086] Cytotoxicity of human umbilical vein endothelial cells (HUVECs) according to epinephrine concentration was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were placed in a 96-well plate at a rate of 5 x 10⁶ 3Cells were seeded at a cell / well density and cultured for 24 hours. Subsequently, cells were cultured in DMEM medium at various epinephrine concentrations for 24 hours. Then, the cells were rinsed twice with PBS, 100 µl of fresh growth DMEM medium was added, and the cells were cultured for an additional 48 hours. After replacing the medium with 100 µl of fresh growth DMEM medium, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) was added. After an additional 2 hours of culture, 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.

[0087] In Figure 2a, HUVEC cell viability decreased in proportion to the concentration of epinephrine. In Figure 2b, when 100 µm of 5-FU was administered together, HUVEC cell viability decreased in proportion to the concentration of epinephrine. Therefore, it can be seen that epinephrine exerts a synergistic effect on the apoptosis of 5-FU.

[0088] Example 2: Confirmation of the mechanism of HUVEC apoptosis by epinephrine using Western blot

[0089] Western blot was performed to confirm the expression of FAS, cleaved caspase 3, and cleaved caspase 9, which are activated when natural death of HUVECs occurs due to epinephrine.

[0090] HUVEC cells cultured in a petri dish 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 a 1X protease inhibitor cocktail. Subsequently, the cells were cooled on ice for 30 minutes, centrifuged at 14,000 g for 10 minutes, and the supernatant was separated. Quantification was performed using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA); 30 μg of the protein solution was transferred to a nitrocellulose membrane after electrophoresis on a 10% SDS-polyacrylamide gel. The electrophoresed membrane was incubated with block milk for 1 hour, followed by incubation with primary antibodies against cleaved caspase 3, cleaved caspase 9, and FAS at room temperature for 1 hour. The samples were washed three times at 15-minute intervals in Tris buffer containing 0.1% Tween 20, reacted with a secondary antibody against the primary antibody at room temperature for 1 hour, and detected using a chemiluminescent agent (Amersham Life Science, Arlington Heights, IL, USA). The results are shown in Figure 3a.

[0091] Proteins extracted from HUVECs were used as a positive control. The expression results of FAS, cleaved caspase 3, and cleaved caspase 9 in HUVECs according to the concentration of epinephrine are shown in Figure 3b.

[0092] 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.

[0093] Example 3: Confirmation of DNA laddering induced by epinephrine in gel electrophoresis

[0094] Characteristic DNA lamination was confirmed during the electrophoresis process of Example 2 and is shown in Figure 4. It can be seen that DNA fragmentation occurred as apoptosis of HUVECs was induced by epinephrine.

[0095] Referring to Figure 4, it can be seen that as the concentration of epinephrine increases, DNA fragmentation occurs more frequently due to the apoptosis of HUVECs, and the DNA migration speed increases.

[0096] Example 4: Confirmation of natural death of HUVECs according to epinephrine concentration using confocal fluorescence microscopy

[0097] Using the apoptosis detection kit (blue, green) (ab176749), apoptosis and healthy cells can be monitored simultaneously. The PS sensor can confirm apoptosis by exhibiting green fluorescence (Ex / Em = 490 / 525 nm) when bound to the cell membrane PS. Healthy cells can be identified using CytoCalcein Violet 450 (Ex / Em = 405 / 450 nm), a dye for labeling living cytoplasm.

[0098] 5x10 vascular endothelial cells in a slide culture chamber 4After treating cells with epinephrine at various concentrations (0–200 μM) for 24 hours, wash the cells 1 to 2 times with 100 μL of analysis buffer by pipetting the buffer up and down. Incubate with 200 μL of analysis buffer containing apopxin / cytocalcein at room temperature for 30 to 60 minutes. Wash the cells twice with 100 μL of analysis buffer, replace the buffer with 100 μL of analysis buffer, and transfer to the stage of a confocal microscope. Tilt the halogen lamp column back, place the slide on the stage, and run the analysis software. In the Acquire tab, set the imaging parameters to XYZ mode, 1024x1024 pixels, 600 Hz, Zoom 1x, average 4, accumulation 1, and rotation 0. Then, place the sample on the microscope and focus with fluorescence. After focusing, the Beam Path Setting is configured in the following order: selection of laser to be used simultaneously, specification of light intensity, selection of PMT, selection of pseudocolor, and setting of detection band. When the Live button is pressed, a preview scan is performed and the captured image is displayed in the right window. After checking the image while increasing the Gain and intensity values, the dial (Z position) is rotated to readjust the final focus position, and the image is captured, saved, and analyzed. The results are shown in Figures 5a to 5e.

[0099] Referring to Figures 5a to 5e, it can be seen that healthy cells decrease and apoptosis cells increase in proportion to the concentration of epinephrine.

[0100] Example 5: Confirmation of toxicity of 5-FU and epinephrine on vascular endothelial cells in tumor tissue In-vivo experiment 1

[0101] The animals used in the experiment were six 5-week-old male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, weighing approximately 30 grams, which were used after undergoing a one-week inspection period at the laboratory. 5 x 10 human gastric cancer cells per nude mouse 6 Cells / 100 µl (PBS) were transplanted into the dorsal subcutaneous fat layer. The size of the tumor was measured periodically, and when it reached a diameter of 1 cm, a drug administration experiment was performed.

[0102] The experimental animals were divided into three groups. The first group served as the control and was administered only physiological saline; the second group was administered 0.2cc of 5mg pH 8.4 5-FU; and the third group was administered a total of 0.2cc of 5mg pH 8.4 5-FU and epinephrine 1:200,000. The injections were administered four times at weekly intervals right next to the tumor. After one month, the major and minor axes of the tumors growing on the dorsal epidermis were measured, and the size before and after drug administration was calculated as mean tumor volume = (major axis × minor axis). 2 ) / 2(mm 3 It was measured and compared using ). The results are shown in Fig. 6a.

[0103] 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.

[0104] Additionally, the third group is shown in Figure 6b with photographs of the tumor size taken before and after drug administration. Referring to Figure 6b, it can be seen that the tumor size decreased significantly over time.

[0105] Example 6: Confirmation of toxicity of 5-FU and epinephrine on vascular endothelial cells in tumor tissue In-vivo experiment 2

[0106] The animals used in the experiment were 38 male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, 5 weeks old, with an average weight of approximately 30 grams, and were used after undergoing a one-week inspection period at the laboratory. 5 x 10 human gastric cancer cells per nude mouse 6 Cells / 100 µl (PBS) were transplanted into the dorsal subcutaneous fat layer. The size of the tumor was measured periodically, and when it reached a diameter of 1 cm, a drug administration experiment was performed.

[0107] When 5 mg of 5-FU at pH 8.4 was administered to 26 of the 38 prepared mice, 6 mice (about 23.1%) experienced rapid weight loss or death.

[0108] When 0.2 cc of epinephrine 1:200,0000 was injected along with 5 mg of pH 8.4 5-FU into the remaining 12 mice, only one mouse experienced weight loss and death, confirming that the toxicity of the drug was reduced.

[0109] Example 7: In-vivo experiment confirming the toxicity of 5-FU, epinephrine, and L-glucose to vascular endothelial cells in tumor tissue

[0110] The animals used in the experiment were six 5-week-old male nude mice (Crj: BALB / c-nu / nu mice, male) produced by Orient, weighing approximately 30 grams, which were used after undergoing a one-week inspection period at the laboratory. 5 x 10 human gastric cancer cells per nude mouse 6 Cells / 100 µl (PBS) were transplanted into the dorsal subcutaneous fat layer. The size of the tumor was measured periodically, and when it reached a diameter of 1 cm, a drug administration experiment was performed.

[0111] 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 epinephrine 1:200,000, while the other group was administered a total of 0.2 cc of 5 mg pH 8.4 5-FU, epinephrine 1:200,000, and 5% L-glucose. The injections were administered four times at weekly intervals right next to the tumor. After one month, the major and minor axes of the tumors growing on the dorsal epidermis were measured, and the size before and after drug administration was calculated as mean tumor volume = (major axis × minor axis). 2 ) / 2(mm 3 It was measured and compared using ). The results are shown in Fig. 7a.

[0112] Referring to Figure 7a, when 5-FU, L-glucose, and epinephrine at pH 8.4 were injected, the tumors were almost completely killed compared to when 5-FU and epinephrine were injected.

[0113] In addition, in the case where 5-FU, L-glucose, and epinephrine at pH 8.4 were injected, photographs of the tumor size before and after drug administration are shown in Fig. 7b.

[0114] Referring to Figure 7b, when 5-FU, L-glucose, and epinephrine at pH 8.4 were injected, the size (volume) of the tumor was significantly reduced compared to before the drug injection.

Claims

Claim 1 A pharmaceutical composition for treating solid tumors comprising 5-fluorouracil and epinephrine, wherein the 5-fluorouracil is in a form dissolved in a solvent having a pH of 8.0 to 9.

0. Claim 2 delete Claim 3 delete Claim 4 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the 5-fluorouracil increases thrombospondin-1 protein expression in tumor vascular endothelial cells to cause vascular endothelial cell death. Claim 5 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the 5-fluorouracil concentration is 10 to 600 mM in the total composition. Claim 6 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the epinephrine induces apoptosis of tumor vascular endothelial cells. Claim 7 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the concentration of epinephrine is 0.001 to 0.2 mg / mL in the total composition. Claim 8 A pharmaceutical composition for treating solid tumors according to claim 1, further comprising glucose. Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the composition is a sustained-release formulation. Claim 13 A pharmaceutical composition for treating solid tumors according to claim 1, wherein the composition is administered by local injection around a tumor. Claim 14 A pharmaceutical composition for treating solid tumors according to claim 13, wherein the tumor surrounding area is a submucosal layer where tumor blood vessels are located. Claim 15 A local administration agent for solid tumors comprising a pharmaceutical composition for treating solid tumors according to claim 1.

Images