Pharmaceutical composition for disrupting tumor blood vessels

Abstract

According to an exemplary embodiment, the pharmaceutical composition for disrupting tumor blood vessels may contain alkaline 5-fluorouracil and a nitric oxide production inhibitor. For example, by including alkaline 5-fluorouracil and a nitric oxide production inhibitor, it is possible to simultaneously achieve the effect of killing tumor vascular endothelial cells and the occlusion of tumor blood vessels, thereby providing an excellent anti-cancer effect.

Description

[Technical Field]

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

[0002] 5-Fluorouracil (5-FU) is an anti-cancer chemotherapy drug widely used in the treatment of various cancers. 5-Fluorouracil is an analogue of uracil, which is naturally present in the human body, and exerts its anti-cancer effect primarily by inhibiting thymidylate synthase, thereby inhibiting DNA synthesis and repair, and suppressing RNA function.

[0003] 5-Fluorouracil is primarily administered intravenously and must be administered directly in a hospital. Because of its side effects, such as thrombosis, an oral 5-FU prodrug form has recently been developed.

[0004] Like other anticancer drugs, 5-fluorouracil exerts its anticancer effect by inhibiting the synthesis of intracellular nucleic acids or by directly binding to nucleic acids and impairing their function. However, rather than acting selectively on cancer cells, it also damages normal cells, especially those in actively dividing tissues, frequently causing myelosuppression and gastrointestinal toxicity due to phosphorylation in the gastrointestinal tract. Furthermore, while cardiac toxicity is rare, potentially life-threatening side effects have been reported in 1.5–18% of cases.

[0005] Therefore, there is a need for research into anticancer agents that maintain the anticancer effects of existing 5-fluorouracil while reducing side effects. For example, Korean Patent Publication No. 10-2013-0074325 discloses a pharmaceutical composition for cancer treatment containing 5-fluorouracil, but it is unable to simultaneously provide reduced side effects and excellent anticancer effects. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a pharmaceutical composition for disrupting tumor blood vessels that has excellent anti-cancer effects and reduced side effects. [Means for solving the problem]

[0007] According to an exemplary embodiment, a pharmaceutical composition for disrupting tumor blood vessels may include alkaline 5-fluorouracil and an inhibitor of nitric oxide production.

[0008] In one embodiment, the nitric oxide production inhibitor is L-NMMA(N G -Monomethyl-L-arginine), L-NAME(N G The compound may contain one or more selected from L-nitro-L-arginine methyl ester), L-NA (nitro arginine), and 7NI (nitroindazole).

[0009] In one embodiment, the nitric oxide production inhibitor is L-NMMA(N G -Monomethyl-L-arginine).

[0010] In one embodiment, the alkalinity may be pH 8 to 9.

[0011] In one embodiment, the alkaline 5-fluorouracil (5-FU) may be in a form in which 5-fluorouracil is dissolved in an alkaline solvent.

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

[0013] In one embodiment, the alkaline 5-fluorouracil can increase the expression of thrombospondin-1 protein in tumor vascular endothelial cells, resulting in the death of vascular endothelial cells.

[0014] In one embodiment, the concentration of the nitric oxide production inhibitor may be 0.1 to 300 mM relative to the total composition.

[0015] In one embodiment, the nitric oxide production inhibitor can constrict tumor blood vessels and promote the development of thrombosis in tumor blood vessels damaged by 5-fluorouracil, resulting in vascular closure.

[0016] In one embodiment, the composition may further comprise one or more selected from bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0017] In one embodiment, the composition may further comprise one or more selected from a cytotoxic anti-cancer agent, a targeted anti-cancer agent, and an immune anti-cancer agent.

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

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

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

[0021] According to an exemplary embodiment, a topical administration agent for solid tumors may contain the pharmaceutical composition for disrupting tumor blood vessels. [Effects of the Invention]

[0022] The pharmaceutical composition for therapeutically disrupting tumor blood vessels according to the present invention may contain alkaline 5-fluorouracil and an inhibitor of nitric oxide production.

[0023] For example, alkaline 5-fluorouracil injected into the base of a tumor can effectively induce the death of vascular endothelial cells (apoptosis) and destroy the tumor. This is not due to the destruction of the tumor itself, but rather to the destruction of the blood vessels that supply the tumor, and therefore may be applicable to other cancers.

[0024] The pharmaceutical composition for destroying tumor blood vessels according to the present invention contains alkaline 5-fluorouracil and a nitric oxide production inhibitor, and thus provides both the killing effect of tumor vascular endothelial cells and the occlusion of tumor blood vessels, thereby providing excellent anti-cancer effects.

[0025] The pharmaceutical composition for disrupting tumor blood vessels according to the present invention can also reduce the side effects of anticancer drugs. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows an example in which a pharmaceutical composition for disrupting tumor blood vessels according to one embodiment is administered by local injection around a tumor. [Figure 2] 1 shows the results of a Western blot experiment confirming the expression of TSP-1 protein in HUVEC cell lines. [Figure 3] 1 shows the results of TSP-1 expression in HUVEC cells depending on the concentration and administration period of 5-FU. [Figure 4] The figure shows the results of measuring the amount of LDH released in HUVECs as a function of 5-FU concentration and exposure time. [Figure 5] 1 shows the results of confirming the cell killing effect of 5-FU on HUVECs depending on pH. [Figure 6] The results of CD31 staining of gastric cancer tumors (right) and the control group (left) are shown one week after alkaline 5-FU injection. [Figure 7] The results of VEGF staining of gastric cancer tumors (right) and the control group (left) are shown one week after alkaline 5-FU injection. [Figure 8] 1 shows photographs showing the size of tumors when human gastric cancer cells were xenografted into nude mice and alkaline 5-FU was administered. [Figure 9] 1 is a graph showing tumor size (volume) when human gastric cancer cells were xenografted into nude mice and then alkaline 5-FU was administered. [Figure 10A] After human gastric cancer cells were xenografted into nude mice, alkaline 5-FU was injected around the tumor, and pathological phenomena were observed in the surrounding tissue. [Figure 10B] After human gastric cancer cells were xenografted into nude mice, alkaline 5-FU was injected around the tumor, and pathological phenomena were observed in the surrounding tissue. [Figure 11] 1 shows the results of confirming the cell killing effect of a mixture of L-NMMA and alkaline 5-FU. [Figure 12] 1 shows photographs showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then L-NMMA and alkaline 5-FU were administered. [Figure 13A] 1 is a graph showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then L-NMMA and alkaline 5-FU were administered. [Figure 13B] 1 is a graph showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then L-NMMA and alkaline 5-FU were administered. [Figure 14] 1 shows photographs showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then bevacizumab was administered. [Figure 15] 1 is a photograph showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then bevacizumab and alkaline 5-FU were administered. [Figure 16] 1 is a graph showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then bevacizumab and alkaline 5-FU were administered. [Figure 17] 1 is a graph showing the viscosity of P407 as a function of temperature and viscosity. [Figure 18] 1 is a graph showing the results of a drug release experiment for 5-FU sol-gel. [Figure 19] 1 shows photographs showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then 30% P407 and alkaline 5-FU were administered. [Figure 20] 1 is a graph showing the size (volume) of the tumor when human gastric cancer cells were xenografted into nude mice and then 30% P407 and alkaline 5-FU were administered. DETAILED DESCRIPTION OF THE INVENTION

[0027] According to an exemplary embodiment, a pharmaceutical composition for disrupting tumor blood vessels (hereinafter referred to as the composition) contains alkaline 5-fluorouracil (5-FU) and a nitric oxide production inhibitor, which can enhance the killing of vascular endothelial cells near cancer cells and the occlusion of blood vessels, thereby providing excellent anti-cancer effects.

[0028] Hereinafter, compositions according to exemplary embodiments of the present invention will be described in detail with reference to the drawings and embodiments, but the drawings and embodiments are merely illustrative and the present invention is not limited thereto.

[0029] Compositions according to exemplary embodiments may include alkaline 5-fluorouracil (5-FU).

[0030] In one embodiment, the alkaline 5-fluorouracil can increase the expression of thrombospondin-1 (TSP-1) protein in tumor vascular endothelial cells, resulting in the death of vascular endothelial cells.

[0031] Mixing 5-fluorouracil with an alkaline solvent can produce a synergistic effect. For example, it can provide superior anticancer effects when mixed with an alkaline solvent compared to neutral or acidic solvents. For example, alkaline 5-fluorouracil can target vascular endothelial cells in cancer tissue and induce cell death. For example, injecting alkaline 5-fluorouracil into the base of tumor tissue can induce the death of vascular endothelial cells that supply blood to the tumor, destroying blood vessels and causing tumor necrosis. In this case, the pH of 5-fluorouracil is an important factor in inducing vascular endothelial cell death. A weakly alkaline pH, e.g., 8.4-9.0, can significantly increase vascular endothelial cell death compared to neutral or acidic 5-fluorouracil.

[0032] In other words, alkaline 5-fluorouracil can exert its anti-cancer effect by destroying blood vessels near cancer cells, inhibiting the formation of new blood vessels, and blocking and suppressing the supply of nutrients and oxygen to cancer cells. This is the result of the destruction of blood vessels that supply blood to tumors, rather than destroying the tumor itself, so it can be applied to other cancers as well. It can also reduce the side effects of anti-cancer drugs.

[0033] In one embodiment, the alkaline 5-fluorouracil may be in the form of 5-fluorouracil dissolved in an alkaline solvent, such as alkaline water or physiological saline.

[0034] 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 to these.

[0035] In some embodiments, the concentration of alkaline 5-fluorouracil may be selected without limitation by those skilled in the art, for example, 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 of the total composition, but is not limited thereto.

[0036] Compositions according to exemplary embodiments may include alkaline 5-fluorouracil (5-FU) and a nitric oxide production inhibitor.

[0037] The nitric oxide production inhibitor is, for example, L-NMMA(N G -Monomethyl-L-arginine), L-NAME(N G -Nitro-L-arginine methyl ester), L-NA (Nitro Arginine), 7NI (Nitroindazole), etc.

[0038] Nitric oxide is synthesized and secreted by vascular endothelial cells by NO synthase, dilating blood vessels and protecting vascular endothelial cells by inhibiting their death due to external stimuli such as lipopolysaccharide (LPS), angiotensin II, overexpression of caspase-3, and TNF-alpha.

[0039] In some embodiments, the nitric oxide production inhibitor can constrict tumor blood vessels and promote thrombosis in tumor blood vessels damaged by 5-FU. For example, tumor vascular endothelial cells can be damaged or destroyed by alkaline 5-fluorouracil, leading to thrombosis in tumor blood vessels. The nitric oxide production inhibitor can promote the closure of these tumor blood vessels and more effectively induce tumor necrosis.

[0040] In some embodiments, the nitric oxide production inhibitor is preferably L-NMMA (NG -Monomethyl-L-arginine).

[0041] L-NMMA, a nonspecific NO synthase inhibitor, effectively increases blood pressure when administered intravascularly. Under physiological conditions, L-NMMA alone inhibits NO synthase, but does not activate platelets in vivo. However, damage to tumor vascular endothelial cells can induce platelet activation and thrombosis. In other words, damaged vascular endothelial cells can be induced to form thrombi. Therefore, the use of L-NMMA in combination with 5-fluorouracil can simultaneously kill tumor vascular endothelial cells and occlude tumor vasculature, potentially providing excellent anticancer effects.

[0042] For 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 relative to the total composition.

[0043] As other examples, L-NMMA may be included at a dose 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 to be treated with the drug, but is not limited to these.

[0044] In one embodiment, the composition may further comprise one or more selected from bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0045] Bevacizumab is a recombinant humanized monoclonal antibody that inhibits vascular endothelial growth factor (VEGF), which promotes angiogenesis.

[0046] Bevacizumab can be used as an anticancer drug either alone or in combination with other drugs. It has traditionally been administered intravenously, but in the case of advanced gastric cancer, it has been shown to improve progression-free survival and overall response rates when administered in conjunction with fluoropyrimidine-cisplatin therapy.

[0047] However, in one embodiment, the composition may also contain bevacizumab, which may be provided in a sustained release form, to inhibit the formation of new blood vessels for cancer cells, thereby effectively eliminating blood vessels within the tumor.

[0048] Capric acid or its physiologically acceptable salt, and poloxamer have low toxicity, biocompatibility, and excellent in vivo stability. For example, mixing capric acid or its physiologically acceptable salt with poloxamer can increase the viscosity and raise the sol-gel transition temperature of the poloxamer. For example, the sol-gel transition temperature can be between room temperature and body temperature, e.g., 20°C to 40°C or 25°C to 37°C. Within the above ranges, the sol-gel transition temperature can be conveniently stored, transported, and used at room temperature, and can be easily diffused in tissues in the gel state when used in the body.

[0049] 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 more 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 these ranges, the sol-gel transition of the delivery form is likely to occur between 25°C and 36°C.

[0050] The capric acid is a compound represented by the following formula 1 and is a type of saturated fatty acid.

[0051] [ka]

[0052] The physiologically acceptable salts may be salts prepared using capric acid and relatively non-toxic acids or bases, such as metal salts or acid addition salts.

[0053] The metal salt may be a sodium, potassium, or calcium salt. Metal salts may be prepared using a base. For example, alkali metal or alkaline earth metal salts may be prepared by dissolving the compound in a solution of excess alkali metal hydroxide or alkaline earth metal hydroxide, filtering the undissolved compound salt, and evaporating and / or drying the filtrate.

[0054] Acid addition salts may be prepared from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such physiologically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dicarboxylate, hexane-1,6-dicarboxylate, benzoate ... The acid addition salts include benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate. For example, acid addition salts of the compound of Formula 1 can be obtained by dissolving the compound in an excess of aqueous acid and precipitating the salt using a miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile.

[0055] The poloxamer may be a terpolymer containing a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) structure, which may be represented, for example, by the following formula 2:

[0056] [ka]

[0057] The molecular weight of the poloxamer can 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 10,000 to 15,000. The molecular weight may be appropriately selected by those skilled in the art depending on the substance to be delivered.

[0058] Poloxamers are commonly designated by a numbering system that indicates the poloxamer's approximate molecular weight and 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 may be used interchangeably.

[0059] For example, the poloxamer can be 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, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407.

[0060] Poloxamers are a type of surfactant that can form micelles in an aqueous environment and load substances. Hydrogels made of poloxamers can form matrices and be used as delivery vehicles for localized and continuous substance release. The poloxamer hydrogels can contain cross-links between poloxamers. Poloxamers can be used in a variety of molecular weights and ratios.

[0061] In one embodiment, the composition may further comprise a known anti-cancer agent, for example, one or more selected from a cytotoxic anti-cancer agent, a targeted anti-cancer agent, and an immunological anti-cancer agent.

[0062] Cytotoxic anticancer drugs can be anticancer drugs that interfere with the metabolic pathways of cancer cells, inhibiting the synthesis and division of DNA or RNA, or damaging DNA itself to induce cell death. Cytotoxic anticancer drugs can refer to chemical anticancer drugs or chemical drug anticancer drugs. For example, they can be alkylating agents, platinum compounds, antimetabolites, plant-derived alkaloids, etc. These are not limited to those that provide anticancer effects through chemical drugs.

[0063] For example, an alkylating agent refers to a substance that can introduce an alkyl group R-CH2 into another compound. For example, the alkylating agent can be cyclophosphamide, ifosfamide, bendamustine, etc.

[0064] For example, a platinum compound refers to a substance containing platinum and forming an oxide, chloride, or complex, such as cisplatin, carboplatin, or oxaliplatin.

[0065] For example, an antimetabolite refers to a substance that inhibits cell growth and proliferation by competing with essential metabolites that are essential for tumor cell metabolism and growth. For example, an antimetabolite can be methotrexate, cladribine, fludarabine, pemetrexed, mercaptopurine, etc.

[0066] For example, plant-derived alkaloids refer to compounds containing basic nitrogen in plant extracts that have strong physiological effects on animals, such as docetaxel, cabazitaxel, paclitaxel, vincristine, vinblastine, etc.

[0067] Targeted anticancer drugs exert their anticancer effects by targeting proteins or genes that are specifically altered in cancer cells or tissues and interfering with molecular activities involved in the growth and development of cancer. Examples include tyrosine kinase inhibitors, PARP inhibitors (poly-ADP ribose polymerase inhibitors), CDK4 / 6 inhibitors (cyclin-dependent kinases 4 / 6 inhibitors), and antibody-drug conjugates.

[0068] Unlike existing anticancer drugs that attack cancer itself, immunological anticancer drugs can be therapeutic agents that stimulate the immune system by injecting artificial immune proteins into the body, thereby inducing immune cells to selectively attack only cancer cells. Examples of such immunological anticancer drugs include immune checkpoint inhibitors, immune cell therapy agents, and therapeutic antibodies that are used in passive immunotherapy, as well as cancer treatment vaccines and immunomodulators that are used in active immunotherapy. However, they are not limited to these.

[0069] The additional components may further include other carriers, etc., or may be formulated together with a carrier, and may be selected appropriately by a person skilled in the art taking into consideration the type of physiologically active substance, the route by which the delivery agent is administered, etc.

[0070] In some embodiments, the composition may be a sustained release formulation.

[0071] The longer the exposure time to vascular endothelial cells, the greater the effect of killing vascular endothelial cells. To provide the composition as a sustained-release formulation, the composition may further comprise capric acid or a physiologically acceptable salt thereof, and poloxamer.

[0072] In one embodiment, the composition may contain bevacizumab, which may be provided in a sustained release form, thereby suppressing the formation of new blood vessels by cancer cells and effectively eliminating blood vessels within tumors.

[0073] Compositions according to some embodiments may contain one or more active ingredients that exhibit the same or similar functions with respect to tumor treatment.

[0074] Compositions according to some embodiments may further comprise compounds that maintain or increase the solubility and / or absorption of the active ingredient.

[0075] In one embodiment, the tumor may comprise a solid tumor.

[0076] In some embodiments, the tumor may be, for example, stomach 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, urogenital tract cancer, testicular tumor, esophageal cancer, laryngeal cancer, gastrointestinal tract cancer, keratoacanthoma, follicular carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, thyroid cancer, papillary carcinoma, bile duct cancer, kidney, bone tumor, bone marrow disease, hair follicle cancer, oral and pharyngeal cancer, lip cancer, tongue cancer, oral cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, colon cancer, rectal cancer, vulvar cancer, endometrial cancer, central nervous system tumor, peritoneal cancer, hepatocellular carcinoma, head tumor, neck tumor, etc., but is not limited to these.

[0077] The composition may be prepared as an oral or parenteral dosage form. For example, the composition may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration. Alternatively, the composition may be suitable for administration by inhalation or injection.

[0078] The composition may be in the form of an injection, which does not form precipitates in the body environment or blood, and can be administered using a thin syringe.

[0079] The dosage form of the composition is preferably an injection dosage form.

[0080] In one embodiment, the composition may be administered by local injection around the tumor.

[0081] Referring to Figure 1, for example, an endoscope can be used to deliver the composition to the base of the cancer via an injector. Specifically, the composition can be injected toward the basal layer of the cancer and into the submucosal layer of the normal stomach wall immediately adjacent to the cancer. The drug delivered to endothelial cells of the blood vessels supplying blood to the cancer tissue can have the effect of killing endothelial cells or inhibiting the formation of new blood vessels.

[0082] Compared with intravenous administration of typical anticancer drugs, the local injection described above can deliver large amounts of drug not only to the submucosal layer surrounding the tumor but also to the surrounding lymph nodes, thereby reducing the side effects of anticancer drugs compared with systemic administration.

[0083] The compositions according to exemplary embodiments may be administered in a pharmaceutically effective amount, which may depend on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical arts.

[0084] According to an exemplary embodiment, a topical administration agent for a solid cancer may contain the above-described composition. Specific examples of solid cancers are as described above.

[0085] In some embodiments, the compositions may be administered as individual therapeutic agents or in combination with other therapeutic agents. The components contained in the formulations used in combination with anticancer drugs may be administered sequentially or simultaneously, and may be administered once or multiple times. Taking all of the above factors into consideration, it is important to administer the minimum amount that will produce the maximum effect without causing side effects, which can be easily determined by those skilled in the art.

[0086] For example, the dosage of the composition varies widely 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 depending on, for example, 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, the composition may be administered at 0.01 μg to 1 g per kg of body weight, daily, weekly, monthly, or yearly, in one or several divided doses per unit period, or continuously over a long period using an infusion pump. The number of repeated doses is determined based on factors such as the drug's retention time in the body and the drug concentration in the body. The composition may also be administered after treatment to prevent recurrence.

[0087] The present invention will be described in detail below with reference to examples. [Example]

[0088] Example 1: Confirmation of the mechanism of action of 5-FU in gastric cancer tissue (endothelial cell toxicity of newly formed blood vessels in tumor tissue) 1) 5-FU increases the expression of thrombospondin-1 (TSP-1) in endothelial cells TSP is a polymer with various functions, and one of its subtypes, TPS-1, functions as an anti-angiogenic factor. Below, we examine the changes in TSP-1 expression in response to 5-FU concentration.

[0089] Western blot was performed to confirm the expression of TSP-1 protein in the human umbilical vein endothelial cell (HUVEC) cell line.

[0090] HUVEC cells cultured 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 cooled on ice for 30 minutes and centrifuged at 14,000 g for 10 minutes, after which the supernatant was separated.

[0091] Protein was quantified using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). 30 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, followed by incubation with the same TSP-1 antibody used for immunostaining at room temperature for 1 hour. After three 15-minute washes with Tris buffer containing 0.1% Tween 20, the membrane was incubated with the primary antibody-specific secondary antibody at room temperature for 1 hour. Detection was performed using a chemiluminescent reagent (Amersham Life Science, Arlington Heights, IL, USA). The results are shown in Figure 2.

[0092] Protein extracted from HUVEC was used as a positive control. The results of TSP-1 expression in HUVEC cells according to the concentration and duration of 5-FU administration are shown in Figure 3. Figure 3 confirms that the amount of TSP-1 expressed increases with the duration of 5-FU administration and the drug concentration of 5-FU.

[0093] 2) Study on LDH release from HUVECs induced by 5-FU Increased expression of TSP-1 and TSP-2 is generally known to induce endothelial cell apoptosis. To confirm whether 5-FU treatment increases endothelial cell apoptosis, an LDH release assay was performed on HUVECs. Cytotoxicity was measured by the amount of LDH released by cell membrane damage. Released LDH was quantified using an LDH assay kit (EZ-LDH1000, DoGenBio, Seoul, Korea) according to the manufacturer's protocol.

[0094] HUVEC 5×10 4 Cells per well were seeded into a 96-well polystyrene plate and cultured at 37°C for 24 hours. LDH substrate was added to each well, followed by an additional 10 minutes of incubation at 37°C. The plate was centrifuged at 600g for 5 minutes. The supernatant (10 μl) was transferred from the culture plate wells to the assay plate. LDH reaction mixture reagent (100 μl) was added sequentially to the supernatant in each well of the assay plate. After 30 minutes of reaction, absorbance was measured at 450 nm using a microplate reader (Model 680, Bio-Rad). The toxicity of 5-FU to HUVEC cells was determined by the amount of LDH released. The results of measuring the amount of LDH released in HUVECs as a function of 5-FU concentration and exposure time are shown in Figure 4.

[0095] As shown in Figure 4, the amount of released LDH increased after treatment with 1 mM, 5 mM, and 10 mM 5-FU in proportion to the treatment time (P<0.05).

[0096] 3) Changes in the toxicity of 5-FU to HUVECs depending on pH To examine the cell-killing effect of 5-FU on HUVECs depending on their pH, an MTS cell proliferation assay was performed. The cytotoxicity of 5 μM 5-FU at various pH levels on HUVECs was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA).

[0097] HUVEC cells were plated in a 96-well plate at 5 × 10 3Cells were seeded at a density of 1000 cells / well and cultured for 24 hours. The cells were then cultured for 24 hours with DMEM medium containing 5-FU at various pH levels (6.4, 7.4, 8.4, and 9.0). The cells were then rinsed twice with PBS, 100 μl of fresh growth DMEM medium was added, and the cells were further cultured for 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) (Promega Corp., Madison, WI, USA) was added. After a further 2 hours of incubation, the absorbance was measured at 490 nm using a BIO-RAD Model 680 microplate reader (Spark, TECAN, CA, USA). The results of the MTS cell proliferation assay in response to 5-FU and pH in HUVECs are shown in Figure 5.

[0098] As shown in Figure 5, the more alkaline the drug, the more pronounced the cytotoxicity was observed compared to 5-FU in acidic or neutral states.

[0099] 4) CD31 and VEGF staining in gastric cancer tumors after injection of 5mg of 5-FU into standard nude mice To observe changes in vascular tissue within gastric cancer tumors after local injection of 5-FU, CD31 and VEGF immunohistochemistry for vascular staining was performed.

[0100] For the control group, gastric cancer tissue was transplanted into the dorsal side of nude mice, and then excised when it reached a size of 1cm x 1cm. For the 5-FU-injected gastric cancer tumors, gastric cancer tissue was transplanted into the dorsal side of nude mice, and when it reached a size of 1cm x 1cm, 5mg of 5-FU was injected into the base of the tumor. One week later, the gastric cancer tumors were excised and subjected to immunohistochemical staining. The immunohistochemical test method was as follows.

[0101] Tissue microarray (TMA) blocks were prepared from preserved formalin-fixed, paraffin-embedded gastric cancer tissue. They were then sectioned at 3.5 μm thickness, oriented, and dried on slides. To enhance immunoreactivity, slides were immersed in 10 mM citrate buffer (pH 6.0) and microwaved for 15 minutes. After washing for 5 minutes with 3'DW (triple distilled water), the slides were incubated with a 3% HO / MeOH solution at room temperature for 15 minutes to reduce endogenous peroxidase activity present in blood cells within the tissue. After two 3-minute washes with 3'DW, the slides were incubated with a blocking antibody containing bovine serum albumin for 30 minutes at room temperature to prevent nonspecific binding. After removing excess blocking antibody, the slides were incubated with a VEGF antibody (sc-7269, Santa Cruz Biotechnology) diluted 1:100 at room temperature for 60 minutes. After washing twice for 3 minutes with TBST (Tris-buffered saline containing 0.1% Tween® 20), the sections were incubated with Detection Kit Reagent 1 (HRP Polymer-anti-mouse / rabbit IgG) at room temperature for 15 minutes and then washed twice for 3 minutes with TBST. The sections were incubated with Detection Kit Reagent 2 mixture at room temperature for 1 minute and then washed with 3'DW for 1-2 minutes. Harris hematoxylin was used as a control staining, and smooth muscle vascular endothelial cells were used as a positive control for staining.

[0102] To evaluate angiogenesis, we used an antibody against CD31 (PECOM-1, sc-376764, Santa Cruz Biotechnology), a marker for vascular endothelial cells, in the same manner as for VEGF.

[0103] For analysis of the results, the percentage of the area of ​​cancer cells stained for CD31 vascular staining and VEGF staining relative to the total tissue area was calculated based on the immunostaining results, and the results were compared between the control group and the 5-FU local injection group. The results are shown in Table 1.

[0104] Figure 6 shows the results of CD31 staining of gastric cancer tumors one week after injection of the control group (left) and 5-FU (right). Figure 7 shows the results of VEGF staining of gastric cancer tumors one week after injection of the control group (left) and 5-FU (right).

[0105] [Table 1]

[0106] As shown in Figure 6, immunochemical staining revealed that the cytoplasm of vascular endothelial cells was stained brown with CD31. Compared to the control group, the distribution of necrotic tissue within the tumor mass in the 5-FU-injected group was increased, and the area stained with CD31 was also lower.

[0107] As shown in Figure 7, VEGF staining was mainly localized in the cytoplasm of cancer cells in a granular form, and the staining area was smaller in the 5-FU-injected group compared to the control group. This indicates that the vascularity and level of VEGF secretion inside the 5-FU-injected cancer tumors were reduced compared to the control group. This suggests that 5-FU injection induced damage to vascular endothelial cells, reducing vascularity and inhibiting the regeneration of new blood vessels, thereby inducing necrosis of the cancer tumors and resulting in a reduction in tumor size.

[0108] Referring to Table 1, the areas stained with CD31 and VEGF, which indicate blood vessels, were smaller after administration of 5-FU compared to the control group.

[0109] 5) In vivo experiments in which human gastric cancer cells were xenografted into the dorsal side of nude mice and the response to anti-cancer drugs was observed. The animals used in the experiment were six male nude mice (Crj:BALB / c-nu / nu mice, male) manufactured by Orient Co., Ltd., 5 weeks old and weighing an average of approximately 30 grams. They were used after a one-week inspection period at the laboratory. Each nude mouse was inoculated with 5 x 10 human gastric cancer cells. 6Cells / 100 μl (PBS) were implanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were performed when the tumor reached a diameter of 1 cm.

[0110] The experimental animals were divided into two groups. One group served as a control and received only saline, while the other group received 0.2 cc of 5 mg of 5-FU (pH 8.4) injected immediately adjacent to the tumor, three times weekly. One month later, the long and short diameters of the tumors growing in the dorsal epidermis were measured, and the size before and after drug administration was calculated as the average tumor volume = (long diameter x short diameter). 2 ) / 2(mm 3 ) and comparative analysis was performed. The results are shown in Figures 8 and 9.

[0111] As shown in Figure 8, the tumor size was reduced 4 weeks after drug treatment compared with that before injection (P<0.05).

[0112] As shown in FIG. 9, tumor size was reduced when alkaline 5-FU was administered compared to the control group administered PBS (p<0.05).

[0113] 6) Observation of pathological phenomena occurring in the subcutaneous muscle layer after injecting 5-FU into the subcutaneous tissue To observe the pathological changes occurring in the surrounding tissues after 5 mg of 5-FU (pH 8.4) was injected into nude mice, tissue samples were collected 24 hours after local injection, including muscle tissue samples. H&E staining was performed and the occurrence of degeneration, necrosis, inflammation, fibrosis, and other changes in the muscle layer was observed. The severity of each pathological change was quantified as 0 = absent, 1 = mild, 2 = moderate, and 3 = severe, and is shown in Figure 10A and Figure 10B. Figure 10A is a 40x magnification, and Figure 10B is a 100x magnification.

[0114] As shown in Figures 10A and 10B, the pathological changes in the subcutaneous muscle layer at the site where 5-FU was injected were observed and no pathological changes were observed (score = 0).

[0115] Example 2: Confirmation of the mechanism by which L-NMMA promotes gastric cancer tissue death 1) Changes in toxicity of HUVECs depending on L-NMMA concentration To examine the cell-killing effects of 5-FU and L-NMMA on HUVECs depending on their concentrations, an MTS cell proliferation assay was performed.

[0116] The cytotoxicity of 5 μM 5-FU at various L-NMMA concentrations against HUVECs was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC cells were plated in 96-well plates at 5 × 10 3 Cells were seeded at a density of 1000 cells / well and cultured for 24 hours. The cells were then cultured with DMEM medium containing various L-NMMA concentrations for 24 hours. The cells were then rinsed twice with PBS, 100 μl of fresh growth DMEM medium was added, and the cells were further cultured for 48 hours. The medium was replaced with 100 μl of fresh growth DMEM medium, followed by the addition of 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). After a further 2 hours of incubation, the absorbance was measured at 490 nm using a BIO-RAD Model 680 microplate reader (Spark, TECAN, CA, USA).

[0117] The results are shown in Figure 11. Referring to Figure 11, when L-NMMA was administered alone, a decrease in cell viability was observed, and when it was administered in combination with 5-FU, a further cell killing effect was confirmed.

[0118] 2) In vivo experiments in which human gastric cancer cells were xenografted into the dorsal side of nude mice and the response to anti-cancer drugs was observed. The animals used in the experiment were two male nude mice (Crj:BALB / c-nu / nu mice, male) manufactured by Orient Co., Ltd., 5 weeks old and weighing an average of approximately 30 g. They were used after a one-week inspection period at the laboratory. Each nude mouse was inoculated with 5 x 10 human gastric cancer cells. 6Cells / 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. The experimental animals were divided into two groups. One group served as the control group and received only saline, while the other group received 0.2 cc of 5 mg of 5-FU (pH 8.4) and 0.1 mg of L-NMMA injected immediately adjacent to the tumor, three times weekly. One month later, the long and short diameters of the tumor growing in the dorsal epidermis were measured, and the size before and after drug administration was calculated as the average tumor volume = (long diameter x short diameter). 2 ) / 2(mm 3 ) and comparative analysis was performed. The results are shown in Figures 12, 13A, and 13B.

[0119] 12, 13A and 13B, the tumor size after injection of 5-FU and L-NMMA was significantly reduced, more significantly than when only 5-FU was injected.

[0120] Example 3: Confirmation of the killing effect of bevacizumab on gastric cancer tissue 1) In vivo experiments in which human gastric cancer cells were xenografted into the dorsal side of nude mice and the response to an anti-cancer drug (bevacizumab) was observed. The animals used in the experiment were three male nude mice (Crj:BALB / c-nu / nu mice, male) manufactured by Orient Co., Ltd., 5 weeks old and weighing an average of approximately 30 g. They were used after a one-week inspection period at the laboratory. Each nude mouse was inoculated with 5 x 10 human gastric cancer cells. 6 Cells / 100 μl (PBS) were implanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were performed when the tumor reached a diameter of 1 cm.

[0121] Experimental animals were injected with 1 mg of bevacizumab in 0.2 cc doses immediately adjacent to the tumor, three times weekly. One month later, the long and short diameters of the tumors growing in the dorsal epidermis were measured, and the size before and after drug administration was calculated as the average tumor volume = (long diameter x short diameter). 2 ) / 2(mm 3 ) and comparative analysis was performed. The results are shown in Figure 14.

[0122] As shown in Figure 14, the tumor size did not change significantly after 4 weeks of drug treatment compared to before injection (P>0.05). The tumor size remained unchanged for the next 2 months, but only began to increase in size at the 3rd month.

[0123] 2) In vivo experiments in which human gastric cancer cells were xenografted into the dorsal side of nude mice and the response to anticancer drugs (alkaline 5-FU and bevacizumab) was observed. The animals used in the experiment were three male nude mice (Crj:BALB / c-nu / nu mice, male) manufactured by Orient Co., Ltd., 5 weeks old and weighing an average of approximately 30 g. They were used after a one-week inspection period at the laboratory. Each nude mouse was inoculated with 5 x 10 human gastric cancer cells. 6 Cells / 100 μl (PBS) were implanted into the dorsal subcutaneous fat layer. Tumor size was measured periodically, and drug administration experiments were performed when the tumor reached a diameter of 1 cm.

[0124] Experimental animals were injected with 0.2 mg of bevacizumab and 5 mg of 5-FU (pH 8.4) in 0.2 cc doses immediately adjacent to the tumor, three times weekly. One month later, the long and short diameters of the tumor growing in the dorsal epidermis were measured, and the size before and after drug administration was calculated as the average tumor volume = (long diameter x short diameter). 2 ) / 2(mm 3 ) and comparative analysis was performed. The results are shown in Figures 15 and 16.

[0125] As shown in Figures 15 and 16, tumor size decreased 4 weeks after drug treatment compared to pre-injection (P<0.05). The pattern of tumor disappearance was unusual. While the overall tumor size remained the same, necrosis progressed from the center and spread to the periphery.

[0126] Example 4: Treatment of gastric cancer by local injection of sustained-release 5-FU sol-gel 1) Determination of Pluronic F-127 (P407) concentration for the preparation of 5-FU sol-gel To confirm the viscosity of P407 as a function of temperature and viscosity, the P407 concentrations were set to 10%, 20%, and 30%, and the viscosity was measured using a Brookfield viscometer while the temperature was raised from approximately 24°C to 38°C. The results are shown in Figure 17.

[0127] 17, it was confirmed that the P407 concentration must be 30% in order to gel at 37° C. It was also confirmed that a concentration of 30% or more results in too high viscosity, making local injection of the drug difficult, and therefore 30% is the most appropriate concentration.

[0128] 2) Drug release experiment of 5-FU sol-gel (UV-VIS spectrophotometer) A drug release experiment was conducted using 5 ml of PBS and 0.76 ml of gel (2 mM thickness, 30% P407, 4.3 mg of 5-FU). The changes were observed at 1, 2, 3, 4, 5, 6, 9, 12, and 24 hours after incubation at 37°C. The results are shown in Figure 18.

[0129] Referring to Figure 18, it can be seen that the drug was completely released after 12 hours.

[0130] 3) In vivo experiments in which human gastric cancer cells were xenografted into the dorsal side of nude mice and the response to anti-cancer drugs was observed. The animals used in the experiment were five male nude mice (Crj:BALB / c-nu / nu mice, male) manufactured by Orient Co., Ltd., 5 weeks old and weighing an average of approximately 30 g. They were used after a one-week inspection period at the laboratory. Each nude mouse was inoculated with 5 x 10 human gastric cancer cells. 6 Cells / 100 μl (PBS) were transplanted into the dorsal subcutaneous fat layer. The size of the tumor was measured periodically, and when the diameter reached 1 cm, drug administration experiments were carried out. 0.2 cc (5 mg of 5-FU) of 30% P407 and 2.5% 5-FU (pH 8.4) was injected immediately adjacent to the tumor, three times weekly. One month later, the long and short diameters of the tumor growing in the dorsal epidermis were measured, and the size before and after drug administration was calculated as the average tumor volume = (long diameter × short diameter). 2 ) / 2(mm 3 ) and comparative analysis. The results are shown in Figures 19 and 20.

[0131] 19 and 20, it was confirmed that administration of the drug to the base of gastric cancer tumors almost completely killed the malignant tumors. Furthermore, by comparing the tumor volume before and after drug administration, it was confirmed that the tumor size was reduced (p<0.05).

Claims

1. A pharmaceutical composition for disrupting tumor blood vessels, comprising alkaline 5-fluorouracil and an inhibitor of nitric oxide production.

2. The nitric oxide production inhibitor is L-NMMA (N Ｇ -Monomethyl-L-arginine), L-NAME (N Ｇ 2. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, comprising one or more selected from L-nitro-L-arginine methyl ester), L-NA (nitroarginine), and 7NI (nitroindazole).

3. The nitric oxide production inhibitor is L-NMMA (N Ｇ 2. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, comprising:

4. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, wherein the alkaline state is pH 8 to 9.

5. 2. The pharmaceutical composition for disrupting 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.

6. 2. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, wherein the alkaline 5-fluorouracil has a concentration of 0.1 to 600 mM based on the total composition.

7. 2. The 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.

8. 2. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, wherein the concentration of the nitric oxide production inhibitor is 0.1 to 300 mM based on the total composition.

9. The pharmaceutical composition for destroying tumor blood vessels according to claim 1, wherein the nitric oxide production inhibitor constricts tumor blood vessels and promotes the onset of thrombosis in tumor blood vessels damaged by 5-fluorouracil, thereby closing the blood vessels.

10. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, further comprising one or more selected from bevacizumab, capric acid or a physiologically acceptable salt thereof, and poloxamer.

11. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, further comprising one or more selected from the group consisting of cytotoxic anticancer agents, targeted anticancer agents, and immune anticancer agents.

12. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1 , wherein the tumor comprises a solid cancer.

13. The pharmaceutical composition for disrupting tumor blood vessels according to claim 1, which is administered by local injection around the tumor.

14. The pharmaceutical composition for disrupting tumor blood vessels according to claim 13, wherein the area surrounding the tumor is the submucosal layer in which tumor blood vessels are located.

15. A topical administration agent for solid cancer, comprising the pharmaceutical composition for disrupting tumor blood vessels according to claim 1.

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