Pharmaceutical composition for blocking tumor blood vessels

Abstract

Exemplary embodiments of a pharmaceutical composition for tumor vascular occlusion may include a basic 5-fluorouracil and a nitric oxide synthase inhibitor. For example, the addition of alkaline 5-fluorouracil and a nitric oxide synthase inhibitor can induce apoptosis of tumor vascular endothelial cells and promote tumor vascular occlusion, thereby providing excellent anti-cancer efficacy.

Description

Technical Field

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

[0002] Fluorouracil (5-FU) is a chemotherapeutic agent widely used in the treatment of various cancers. 5-Fluorouracil is an analogue of uracil, which naturally exists in the human body and exerts its anti-cancer effect mainly by inhibiting DNA synthesis and repair by inhibiting thymidylate synthase and by interfering with RNA function.

[0003] 5-Fluorouracil is mainly administered by intravenous injection, which requires direct administration in a hospital environment and is associated with adverse reactions such as thrombosis. To solve these problems, oral prodrug formulations of 5-FU have been developed in recent years.

[0004] Similar to conventional anti-cancer agents, 5-fluorouracil exerts its anti-cancer effect by inhibiting intracellular nucleic acid synthesis or directly binding to nucleic acids, thereby impairing their functions. However, 5-fluorouracil not only has selective cytotoxicity to cancer cells but also damages normal cells, especially actively dividing tissue cells, resulting in frequent myelosuppression and gastrointestinal toxicity caused by phosphorylation in the gastrointestinal tract. In addition, although the incidence of cardiotoxicity is relatively low, severe and potentially life-threatening cases have been reported in 1.5% to 18% of patients.

[0005] Therefore, it is necessary to study anti-cancer agents that maintain the efficacy of 5-fluorouracil while reducing adverse reactions. For example, Korean Patent Publication No. 10-2013-0074325 discloses a pharmaceutical composition for cancer treatment, which contains 5-fluorouracil; however, this composition cannot simultaneously reduce adverse reactions and enhance anti-cancer efficacy. Summary of the Invention

[0006] An object of the present invention is to provide a pharmaceutical composition for blocking tumor blood vessels, which exerts excellent anti-cancer efficacy while reducing adverse reactions.

[0007] An exemplary embodiment of the pharmaceutical composition for blocking tumor blood vessels may include alkaline 5-fluorouracil and a nitric oxide synthase inhibitor.

[0008] In one embodiment, the nitric oxide synthase inhibitor may include one or more selected from L-NMMA (NG-Monomethyl-L-arginine), L-NAME (NG-Nitro-L-arginine methyl ester), L-NA (Nitroarginine), and 7NI (Nitroindazole).

[0009] In one embodiment, the nitric oxide synthase inhibitor may include L-NMMA (NG-monomethyl-L-arginine).

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

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

[0013] In one embodiment, alkaline 5-fluorouracil may increase the protein expression of thrombospondin-1 in tumor vascular endothelial cells, thereby inducing apoptosis of endothelial cells.

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

[0015] In one embodiment, the nitric oxide synthase inhibitor may cause tumor blood vessels to constrict and promote thrombosis in tumor blood vessels damaged by 5-fluorouracil, thereby blocking the blood vessels.

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

[0017] In one embodiment, the composition may further include one or more selected from cytotoxic anti-cancer agents, targeted anti-cancer agents, and immuno-oncology agents.

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

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

[0020] In one embodiment, the area around the tumor may be the submucosa where the tumor blood vessels are located.

[0021] Exemplary embodiments of the local preparation for administering to a solid tumor may include a pharmaceutical composition for tumor blood vessel blockade.

[0022] The pharmaceutical composition for tumor blood vessel blockade according to the present invention may include alkaline 5-fluorouracil and a nitric oxide synthase inhibitor.

[0023] For example, when alkaline 5-fluorouracil is injected into the bottom of a tumor, it can effectively induce apoptosis of vascular endothelial cells, thereby blocking the tumor. This therapeutic effect stems from the disruption of the blood vessels supplying the tumor rather than directly targeting the tumor itself, and thus can also be applied to other cancer types.

[0024] The pharmaceutical composition for tumor vascular occlusion according to the present invention, which comprises alkaline 5-fluorouracil and a nitric oxide synthase inhibitor, can induce apoptosis of tumor vascular endothelial cells, promote tumor vascular occlusion, and thus exhibit excellent anti-cancer efficacy.

[0025] In addition, the pharmaceutical composition for tumor vascular occlusion according to the present invention can also reduce the adverse reactions associated with anti-cancer agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 An example of local injection of the pharmaceutical composition for tumor vascular occlusion around a tumor according to an embodiment is shown.

[0027] Figure 2 The results of a Western blot experiment demonstrating the expression of TSP-1 protein in the HUVEC cell line are shown.

[0028] Figure 3 The results of TSP-1 expression in HUVEC cells according to the concentration and exposure time of 5-FU are shown.

[0029] Figure 4 The results of measuring LDH release in HUVEC cells according to the concentration and exposure time of 5-FU are shown.

[0030] Figure 5 The effect of the pH value of 5-FU on cell death in HUVEC cells is shown.

[0031] Figure 6 The CD31 staining results of a gastric cancer mass (right) after administration of alkaline 5-FU for one week compared with the control group (left) are shown.

[0032] Figure 7 The VEGF staining results of a gastric cancer mass (right) after administration of alkaline 5-FU for one week compared with the control group (left) are shown.

[0033] Figure 8 A photograph of the tumor size after administration of alkaline 5-FU after xenografting human gastric cancer cells into nude mice is shown.

[0034] Figure 9 A graph of the tumor size (volume) after administration of alkaline 5-FU after xenografting human gastric cancer cells into nude mice is shown.

[0035] Figure 10a andFigure 10b Shows the pathological changes observed in the surrounding tissues when alkaline 5-FU was injected around the tumor after xenotransplanting human gastric cancer cells into nude mice.

[0036] Figure 11 Shows the results of confirming the effect on cell death when L-NMMA and alkaline 5-FU were mixed.

[0037] Figure 12 Shows the photos of the tumor size (volume) after administering L-NMMA and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice.

[0038] Figure 13a and Figure 13b Shows the graph of the tumor size (volume) after administering L-NMMA and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice.

[0039] Figure 14 Shows the photos of the tumor size (volume) after administering bevacizumab after xenotransplanting human gastric cancer cells into nude mice.

[0040] Figure 15 Shows the photos of the tumor size (volume) after administering bevacizumab and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice.

[0041] Figure 16 Shows the graph of the tumor size (volume) after administering bevacizumab and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice.

[0042] Figure 17 Shows the graph of the viscosity of P407 changing according to temperature and concentration.

[0043] Figure 18 Shows the results of the 5-FU-sol-gel drug release test.

[0044] Figure 19 Shows the photos of the tumor size (volume) after administering 30% P407 and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice.

[0045] Figure 20 Shows the graph of the tumor size (volume) after administering 30% P407 and alkaline 5-FU after xenotransplanting human gastric cancer cells into nude mice. Detailed implementation mode

[0046] Exemplary embodiments of a pharmaceutical composition for tumor vascular occlusion (hereinafter referred to as "the composition") include alkaline 5-fluorouracil (5-FU) and nitric oxide synthase inhibitors. Thus, the composition can enhance apoptosis of tumor vascular endothelial cells and promote vascular occlusion, thereby providing excellent anti-cancer efficacy.

[0047] Hereinafter, the composition according to the exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings and embodiments. However, the drawings and embodiments are merely exemplary and should not be construed as limiting the scope of the present invention.

[0048] Exemplary embodiments of the composition may include alkaline 5-fluorouracil (5-FU).

[0049] In one embodiment, alkaline 5-fluorouracil (5-FU) can increase the expression of thrombospondin-1 (TSP-1) protein in tumor vascular endothelial cells, thereby inducing apoptosis of endothelial cells.

[0050] When fluorouracil is mixed with an alkaline solvent, a synergistic effect will be exhibited. For example, when mixed with an alkaline solvent, it can provide excellent anti-cancer effects compared to when mixed with a neutral solvent or an acidic solvent. For example, alkaline 5-fluorouracil can target vascular endothelial cells in tumor tissues and induce cell death. For example, when alkaline 5-fluorouracil is injected into the lower part of a tumor tissue, it will induce apoptosis of the vascular endothelial cells supplying blood to the tumor, resulting in vascular occlusion and tumor necrosis. In this case, the pH value of 5-fluorouracil is an important factor in inducing apoptosis of vascular endothelial cells. When the pH value is weakly alkaline, for example, in the range of 8.4 to 9.0, apoptosis of vascular endothelial cells will increase significantly compared to neutral 5-fluorouracil or acidic 5-fluorouracil.

[0051] In other words, alkaline 5-fluorouracil destroys the blood vessels near cancer cells, inhibits angiogenesis, blocks and inhibits the supply of nutrients or oxygen to cancer cells, thereby exerting anti-cancer effects. This effect is produced by destroying the blood vessels supplying blood to the tumor rather than targeting the tumor itself, so it may be applicable to other cancer types. In addition, the side effects of anti-cancer drugs can be reduced.

[0052] In one embodiment, alkaline 5-fluorouracil may be in a form in which 5-fluorouracil is dissolved in an alkaline solvent. The alkaline solvent may include, for example, alkaline water or physiological saline.

[0053] In one embodiment, the alkaline pH value may be in the range of 8.0 to 9.0. For example, the pH range may be 8.0 to 9.0, 8.2 to 9.0, 8.4 to 9.0, or 8.4 to 8.8. However, the pH value is not limited to these ranges.

[0054] In some embodiments, those skilled in the art can unrestrictedly select the concentration of alkaline 5-fluorouracil. For example, the concentration in the total composition can be in the range of 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. However, the concentration is not limited to these ranges.

[0055] Exemplary embodiments of the composition can include alkaline 5-fluorouracil and a nitric oxide synthase inhibitor.

[0056] The nitric oxide synthesis inhibitor can include, for example, L-NMMA (N G -methyl-L-arginine), L-NAME (N G -nitro-L-arginine methyl ester), L-NA (nitroarginine), or 7NI (nitroindazole).

[0057] Nitric oxide (NO) is synthesized and secreted by NO synthase in vascular endothelial cells, resulting in vasodilation. In addition, NO can protect cells by protecting vascular endothelial cells from apoptosis caused by external stimuli such as lipopolysaccharide (LPS), angiotensin II, caspase-3 overexpression, and TNF-α.

[0058] In some embodiments, the nitric oxide synthesis inhibitor can cause tumor blood vessels to constrict and promote thrombus formation within tumor blood vessels damaged by 5-FU. For example, alkaline 5-fluorouracil may cause damage or destruction to tumor vascular endothelial cells, resulting in thrombus formation within tumor blood vessels. The nitric oxide synthesis inhibitor can promote occlusion of such tumor blood vessels, thereby more effectively inducing tumor necrosis.

[0059] In some embodiments, the nitric oxide synthesis inhibitor is preferably L-NMMA (N G -monomethyl-L-arginine).

[0060] L-NMMA is a non-specific NO synthase inhibitor that can effectively increase blood pressure when administered to blood vessels. Under physiological conditions, inhibiting NO synthase alone with L-NMMA does not activate platelets in vivo. However, when endothelial cell damage occurs in tumor blood vessels, L-NMMA may induce platelet activation and thrombus formation. In other words, it can induce thrombus formation in damaged vascular endothelial cells. Therefore, the combination of L-NMMA and 5-fluorouracil can induce apoptosis of vascular endothelial cells and occlusion of tumor blood vessels, thereby producing excellent anti-cancer efficacy.

[0061] For example, the concentration range of the nitric oxide synthase inhibitor in the total composition can be 0.1 mM to 300 mM, 0.1 mM to 250 mM, 1 mM to 200 mM, or 10 mM to 200 mM, but the present invention is not limited thereto.

[0062] For example, based on the weight of the individual being treated, the content of L-NMMA can be 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. However, the present invention is not limited thereto.

[0063] In one embodiment, the composition may further comprise at least one selected from bevacizumab, capric acid or a pharmaceutically acceptable salt thereof, and poloxamer.

[0064] Bevacizumab is a recombinant humanized monoclonal antibody designed to inhibit vascular endothelial growth factor (VEGF) that promotes angiogenesis.

[0065] Bevacizumab is commonly used as an anticancer agent alone or in combination with other drugs. Conventionally, bevacizumab is mainly administered by intravenous injection. In the case of advanced gastric cancer, reports have shown that the combination of fluoropyrimidine-cisplatin treatment and bevacizumab improves progression-free survival and overall response rate.

[0066] However, in one embodiment, the composition may further comprise bevacizumab in a sustained-release form. By inhibiting tumor angiogenesis, this formulation can effectively eliminate the blood vessels distributed within the tumor.

[0067] Capric acid or a pharmaceutically acceptable salt thereof and poloxamer exhibit low toxicity, ensuring biocompatibility and excellent in vivo stability. For example, combining capric acid or a pharmaceutically acceptable salt thereof with poloxamer can increase viscosity and raise the sol-gel transition temperature. For example, the sol-gel transition temperature can be between room temperature and body temperature, such as 20°C to 40°C or 25°C to 37°C. Within this temperature range, the formulation remains in a sol state at room temperature, facilitating storage, transportation, and use. After administration, the transition to the gel state enhances diffusion within the tissue.

[0068] In some embodiments, the weight ratio of capric acid to poloxamer can be in the range of 0.25:30 to 3.4:30, 1:30 to 3.4:30, or 2:30 to 3:30. Specifically, the weight ratio of capric acid to poloxamer can be in the range of 2.75:30 to 3.3:30 or 2.8:30 to 3.2:30. Within these ranges, the sol-gel transition of the carrier may occur between 25°C and 36°C.

[0069] Capric acid is a compound represented by Chemical Formula 1 below and is a saturated fatty acid.

[0070] [Chemical Formula 1]

[0071]

[0072] Pharmaceutically acceptable salts can be salts prepared using capric acid and a relatively non-toxic acid or base. Pharmaceutically acceptable salts can include, for example, metal salts or acid addition salts.

[0073] Metal salts can be sodium salts, potassium salts or calcium salts. Bases can be used to prepare metal salts. For example, an alkali metal or alkaline earth metal salt can be obtained by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the resulting insoluble compound salt, and evaporating and / or drying the filtrate.

[0074] Acid addition salts can 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 from salts of non-toxic organic acids such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids or aliphatic sulfonic acids or aromatic sulfonic acids. These physiologically non-toxic salts can include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, caprates, octanoates, acrylates, formates, isobutyrates, caprates, heptanoates, acrylates, oxalates, malonates, succinates, octanedioates, decanedioates, fumarates, maleates, butene-1,4-dioates, hexane-1,6-dioates, benzoates, chlorobenzoates, toluobenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates or mandelates.

[0075] Poloxamer can be a triblock copolymer containing a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) structure. For example, poloxamer can be represented by Chemical Formula 2 below.

[0076] [Chemical Formula 2]

[0077]

[0078] The weight-average molecular weight of poloxamer can be, for example, in the range of 1000 to 100000, 10000 to 100000, 10000 to 20000, or 10000 to 15000. The molecular weight can be appropriately selected by those skilled in the art according to the substance to be delivered.

[0079] Poloxamers are generally represented using a numbering system that indicates the approximate molecular weight and percentage content of polyoxyethylene, also known as the trade name Pluronic.

[0080] For example, poloxamers 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, etc.

[0081] Poloxamer is a surfactant that can form micelles in an aqueous environment and is capable of loading substances. The hydrogel composed of poloxamer can form a matrix and can be used as a carrier for local and sustained release of substances. The poloxamer hydrogel can include crosslinking between poloxamer molecules. Poloxamers can be used in various molecular weights and ratios.

[0082] In one embodiment, the composition may further include a known anti-cancer agent. For example, the composition can include at least one selected from cytotoxic anti-cancer agents, targeted anti-cancer agents, and immuno-oncology agents.

[0083] Cytotoxic anti-cancer agents can interfere with the metabolic pathways of cancer cells to inhibit DNA or RNA synthesis and division, or can induce cell death by directly damaging DNA. Cytotoxic anti-cancer agents can refer to chemotherapeutic agents or chemical drug-based anti-cancer agents. For example, cytotoxic anti-cancer agents can be alkylating agents, platinum-based compounds, antimetabolites, or alkaloids of plant origin. However, the present invention is not limited thereto, as long as an anti-cancer effect is provided by a chemical agent.

[0084] For example, an alkylating agent refers to a substance capable of introducing an alkyl group (R-CH 2 ) into another compound. Examples of alkylating agents include cyclophosphamide, ifosfamide, and bendamustine.

[0085] For example, a platinum-based compound contains platinum and refers to a substance that forms an oxide, a chloride, or a coordination complex. Examples of platinum-based compounds include cisplatin, carboplatin, and oxaliplatin.

[0086] For example, an antimetabolite refers to a substance that inhibits cell growth and proliferation by antagonizing essential metabolites required for the metabolism or growth of tumor cells. Examples of antimetabolites include methotrexate, cladribine, fludarabine, pemetrexed, and mercaptopurine.

[0087] For example, an alkaloid of plant origin refers to a compound containing basic nitrogen that exhibits strong physiological activity in animals and is derived from plant extracts. Examples of alkaloids of plant origin include docetaxel, cabazitaxel, paclitaxel, vincristine, and vinblastine.

[0088] Targeted anticancer agents can exert anticancer effects by interfering with the molecular activities involved in cancer growth and development, targeting specifically altered proteins or genes in cancer cells or cancer tissues. For example, targeted anticancer agents can be tyrosine kinase inhibitors, PARP (poly ADP-ribose polymerase) inhibitors, CDK4 / 6 (cyclin-dependent kinase 4 / 6) inhibitors, or antibody-drug conjugates.

[0089] Different from conventional anticancer agents that directly attack cancer cells, immuno-oncology agents can be therapeutic agents that stimulate the immune system by introducing artificial immune proteins into the body, enabling immune cells to selectively attack only cancer cells. For example, immuno-oncology reagents can include immune checkpoint inhibitors, immune cell therapies, or therapeutic antibodies for passive immunotherapy, as well as cancer treatment vaccines or immunomodulators for active immunotherapy. However, the present invention is not limited thereto.

[0090] The additional components can also include other carriers and can be formulated together with the carrier. Those skilled in the art can appropriately select the additional components considering the type of bioactive substance and the administration route of the carrier.

[0091] In some embodiments, the composition can be a sustained-release formulation.

[0092] The longer the exposure time to vascular endothelial cells, the greater the vascular endothelial cell death effect. To provide a composition as a sustained-release formulation, the composition can also contain capric acid or a pharmaceutically acceptable salt thereof and poloxamer.

[0093] In one embodiment, bevacizumab can be added to the composition and provided in a sustained-release form. By inhibiting angiogenesis in cancer cells, the blood vessels distributed within the tumor can be effectively eliminated.

[0094] Compositions according to some embodiments may include one or more active ingredients that exhibit the same or similar functions in tumor treatment.

[0095] Compositions according to some embodiments may also include compounds that maintain or enhance the solubility and / or absorbability of the active ingredients.

[0096] In one embodiment, the tumor may include solid tumors.

[0097] In some embodiments, the tumor may include, for example, gastric cancer, liver cancer, pancreatic cancer, osteosarcoma, skin cancer, lung cancer, neuroblastoma, uterine cancer, kidney cancer, prostate cancer, breast cancer, colorectal cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, brain tumor, cervical cancer, prostate cancer, testicular cancer, penile cancer, urogenital cancer, seminoma, esophageal cancer, laryngeal cancer, gastrointestinal 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, cholangiocarcinoma, kidney cancer, bone cancer, myeloid disorders, hairy cell leukemia, oral and oropharyngeal cancer, lip cancer, tongue cancer, oral cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, colon cancer, rectal cancer, vulvar cancer, thyroid cancer, endometrial cancer, central nervous system cancer, peritoneal cancer, hepatocellular carcinoma, head and neck cancer. However, the present invention is not limited thereto.

[0098] The formulation of the composition can be prepared as an oral formulation or a parenteral formulation. For example, the formulation may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration. Alternatively, the formulation may be suitable for administration by inhalation or insufflation.

[0099] The formulation of the composition can be an injectable formulation. The formulation does not form a precipitate in a biological environment such as blood and allows administration through a fine injection needle.

[0100] The formulation of the composition is preferably an injectable formulation.

[0101] In one embodiment, the composition can be administered locally around the tumor by injection.

[0102] For example, with reference to Figure 1 , an endoscope can be used to deliver the composition to the basal region of the tumor through a syringe. Specifically, the composition can be injected into the submucosal region of the normal gastric wall adjacent to the tumor, targeting the basal layer of the tumor. A drug delivered to the vascular endothelial cells of the blood vessels supplying the tumor tissue can induce endothelial cell apoptosis or inhibit neovascularization.

[0103] Compared with conventional intravenous administration of anticancer agents, local injection can deliver not only more drugs to the submucosa around the tumor, but also to the surrounding lymph nodes. In addition, compared with systemic administration, local injection can reduce the side effects of anticancer agents.

[0104] The compositions according to the exemplary embodiments can be administered in a pharmaceutically effective amount. The effective dose level can be determined according to the type and severity of the patient's disease, the drug activity, the sensitivity to the drug, the administration time, the administration route, the elimination rate, the treatment duration, the combined use of drugs, and other factors well-known in the medical field.

[0105] The local formulations for the administration of solid tumors according to the exemplary embodiments can include the compositions. Specific examples of solid tumors are the same as those described above.

[0106] In some embodiments, the compositions can be administered as an independent therapeutic agent or in combination with other therapeutic agents. The components included in the combined anticancer formulations can be administered sequentially or simultaneously, and can also be administered as a single dose or multiple doses. Considering all these factors, it is important to administer the minimum effective amount that maximizes the therapeutic effect and minimizes the side effects, which can be easily determined by those skilled in the art.

[0107] For example, the dose of the composition can vary according to factors such as the patient's weight, age, gender, health status, diet, administration time, administration method, elimination rate, and disease severity. The appropriate dose can also vary according to the amount of drug accumulated in the patient's body and / or the specific efficacy of the delivery system used in the present invention. For example, the dose range is from 0.01 μg to 1 g per kilogram of body weight, and can be administered once or multiple times per unit time (such as daily, weekly, monthly, or annually). Alternatively, the composition can be continuously administered using an infusion pump over an extended period of time. The frequency of repeated administration can be determined according to factors such as the residence time of the drug in the body and the drug concentration in the blood. Even after the treatment is completed, the composition can be administered according to the course of the disease treatment to prevent recurrence.

[0108] The following examples are provided to describe the present invention in detail.

[0109] <Example>

[0110] Example 1: Verification of the destruction mechanism of gastric cancer tissue by 5-FU (cytotoxicity of endothelial cells in tumor neovascularization) 1) Upregulation of thrombospondin-1 (TSP-1) expression in endothelial cells by 5-FU

[0111] Figure 2

[0112] Thrombospondin (TSP) is a macromolecule with various functions. Among its isomers, TSP-1 acts as an angiogenesis inhibitor. The following experiments were conducted to detect the effect of different concentrations of 5-FU on the expression of TSP-1.

[0113] Western blotting was performed to confirm the expression of TSP-1 protein in the HUVEC (human umbilical vein endothelial cell) cell line.

[0114] HUVEC cultured in a dish was lysed using 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 1× protease inhibitor mixture. The lysate was incubated on ice for 30 minutes and then centrifuged at 14,000 g for 10 minutes to collect the supernatant.

[0115] Protein quantification was performed using a bicinchoninic acid (BCA) protein assay kit (Pierce Chemical, Rockford, IL, USA). A total of 30 μg of protein was electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked with blocking milk for 1 hour and then incubated with the same TSP-1 antibody for immunostaining at room temperature for 1 hour. After washing three times at 15-minute intervals with Tris buffer containing 0.1% Tween 20, the membrane was incubated with a secondary antibody specific for the primary antibody at room temperature for 1 hour. Detection was performed using a chemiluminescent substrate (Amersham Life Science, Arlington Heights, IL, USA). The results are as Figure 3 shown.

[0116] The protein extracted from HUVEC was used as a positive control. The results of TSP-1 expression in HUVEC according to the 5-FU concentration and exposure time are as Figure 3 shown. As 2) Determination of LDH release in HUVEC induced by 5-FU shown, long-term exposure to 5-FU and high drug concentrations led to an increase in the expression level of TSP-1.

[0117] Figure 4

[0118] It has been generally reported that increased expression of thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) induces endothelial cell apoptosis. To investigate whether the administration of 5-fluorouracil (5-FU) would enhance endothelial cell apoptosis, a LDH release assay was performed in human umbilical vein endothelial cells (HUVEC). Cytotoxicity was evaluated based on the amount of lactate dehydrogenase (LDH) released due to cell membrane damage. According to the manufacturer's protocol, the released LDH was quantified using an LDH assay kit (EZ-LDH1000; DoGenBio, Seoul, Korea).

[0119] HUVEC (5×10 4Cells / well) were seeded in 96-well polystyrene plates and incubated at 37 °C for 24 h. LDH substrate was added to each well and then incubated at 37 °C for an additional 10 min. The plates were centrifuged at 600 g for 5 min. A 10-μL aliquot of the supernatant was transferred from the culture plate wells to the assay plate. Subsequently, 100 μL of the LDH reaction mixture reagent was added to the supernatant in each well of the assay plate. After 30 min of reaction, the absorbance was measured at 450 nm using a microplate reader (model 680; Bio-Rad). The cytotoxicity of 5-FU against HUVEC was determined according to the amount of LDH released. According to the 5-FU concentration and exposure time, the results of LDH release in HUVEC are as Figure 4 shown.

[0120] As Figure 5 shown, the amount of LDH released increased in a time-dependent manner after treatment with 1 mM, 5 mM, and 10 mM 5-FU.

[0121] 3) Effect of pH of 5-FU on the cytotoxicity against HUVEC

[0122] To evaluate the cytotoxic effect of 5-fluorouracil (5-FU) against human umbilical vein endothelial cells (HUVEC) under different pH conditions, an MTS cell proliferation assay was performed. The cytotoxicity of 5 μM 5-FU against HUVEC at different pH levels was evaluated using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA).

[0123] HUVEC were seeded at 5 × 10 3The cells were seeded at a density of cells / well in a 96-well plate and incubated for 24 hours. Subsequently, the cells were cultured for an additional 24 hours in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5-FU under different pH conditions (pH 6.4, 7.4, 8.4, and 9.0). After incubation, the cells were washed twice with phosphate-buffered saline (PBS), then 100 μL of fresh DMEM growth medium was added, and the cells were incubated for an additional 48 hours. Then, the medium was replaced with 100 μL of fresh DMEM growth medium, and 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (Promega Corp., Madison, WI, USA) was added. After an additional 2-hour incubation, the absorbance was measured at 490 nm using a microplate reader (Model 680, BIO-RAD; Spark, TECAN, CA, USA). The results of the MTS cell proliferation assay for 5-FU cytotoxicity in HUVEC according to the pH values are as Figure 5 shown.

[0124] As 4) CD31 and VEGF staining of gastric tumor tissue after local injection of 5 mg 5-FU into nude mice shown, the cytotoxic effect of 5-FU increased with the increasing alkalinity of the drug, and showed significantly higher toxicity under alkaline conditions compared with neutral 5-FU or acidic 5-FU.

[0125] Figure 6

[0126] To observe the changes in vascular tissue within the gastric tumor mass after local injection of 5-FU, immunohistochemical staining for CD31 and VEGF was performed.

[0127] For the control group, gastric tumor tissues were implanted into the dorsal region of nude mice and excised and used when the tumors reached a size of 1 cm × 1 cm. For the 5-FU injection group, when the implanted tumors reached a size of 1 cm × 1 cm, 5 mg of 5-FU was injected at the tumor base. One week after injection, the gastric tumor masses were excised and immunohistochemical staining was performed. The immunohistochemical staining method was as follows.

[0128] Tissue microarray (TMA) blocks were prepared from formalin-fixed, paraffin-embedded gastric tumor tissues. The blocks were cut into 3.5-μm thick sections, mounted on slides in a uniform orientation, and dried. To enhance immunoreactivity, the slides were immersed in 10 mM / L citrate buffer (pH 6.0) and microwaved for 15 minutes. Then the slides were washed with triple-distilled water (3’DW) for 5 minutes. To reduce the endogenous peroxidase activity present in blood cells within the tissue, the slides were treated with 3% H 2 O 2 dissolved in methanol for 15 minutes at room temperature. After washing twice with 3’DW, 3 minutes each time, a blocking antibody containing normal serum albumin was applied for 30 minutes at room temperature to prevent non-specific binding. The excess blocking antibody was removed, and the slides were incubated with VEGF antibody (1:100 dilution, sc-7269, Santa Cruz Biotechnology) for 60 minutes at room temperature. The slides were washed twice with TBST (Tris-buffered saline containing 0.1% Tween 20) for 3 minutes each time, then incubated with detection kit reagent 1 (HRP polymer anti-mouse / rabbit IgG) for 15 minutes at room temperature. After washing twice more with TBST, 3 minutes each time, the slides were incubated with the detection kit reagent 2 mixture for 1 minute at room temperature, then washed with 3’DW (triple-distilled water) for 1 to 2 minutes. Counterstaining was performed using Harris hematoxylin. In each staining step, smooth muscle endothelial cells were used as a positive control.

[0129] To evaluate angiogenesis, an antibody specific for CD31 (PECOM-1, sc-376764, Santa Cruz Biotechnology) (a marker for vascular endothelial cells) was applied using the same method as for VEGF.

[0130] For result analysis, the staining areas for CD31-positive blood vessels and VEGF-positive cancer cells were calculated as a proportion of the total tissue area and compared between the control group and the 5-FU local injection group. Table 1 summarizes the results.

[0131] Figure 7 Immunostaining results of CD31 in gastric tumor blocks one week after injection in the control group (left) and 5-FU injection group (right) are shown. Immunopositive area (%) Immunostaining results of VEGF in gastric tumor masses one week after injection in the control group (left) and 5-FU injection group (right) are shown.

[0132] [Table 1]

[0133] <![CDATA[Total area [μm 2 > <![CDATA[Immunopositive area [μm 2 > Control group (CD31) 5-FU injection group (CD31) 29556494.76 257505.634 0.87 Control group (VEGF) 47304976.18 257079.525 0.54 5-FU injection group (VEGF) 29556494.76 113422.564 0.38 Figure 6 47304976.18 46077.917 0.10

[0134] Reference Figure 7 , immunohistochemical staining results showed that CD31 stained the cytoplasm of vascular endothelial cells brown. In the tumor masses of the 5-FU injection group, compared with the control group, the distribution of necrotic tissue increased, and the proportion of the stained area of CD31 decreased.

[0135] Reference 5) In vivo experiment to evaluate the response to anticancer agents after transplantation of human gastric cancer cells into the back of nude mice , VEGF was mainly stained as granular deposits located in the cytoplasm of cancer cells. Compared with the control group, the stained area of the 5-FU injection group was smaller. Therefore, compared with the control group, the blood vessel distribution in the tumor masses of the 5-FU injection group decreased, and the VEGF secretion level also decreased. These findings suggest that 5-FU injection induced damage to vascular endothelial cells, resulting in reduced blood vessel distribution and inhibition of neovascular regeneration, ultimately leading to tumor necrosis and reduction in tumor size.

[0136] Referring to Table 1, compared with the control group, the stained areas of CD31 and VEGF indicating the vascular area were smaller in the 5-FU injection group.

[0137] Figure 8

[0138] The animals used in the experiment were six male nude mice (Crj: BALB / c-nu / nu mice, male), five weeks old, weighing approximately 30 g, purchased from Orient. Before use, the mice underwent a one-week adaptation period in the laboratory. 5×10 6 human gastric cancer cells suspended in 100 μL PBS were subcutaneously transplanted into the subcutaneous layer of the back of each nude mouse. The tumor size was measured regularly, and when the tumor diameter reached 1 cm, the drug administration experiment was conducted.

[0139] The experimental animals were divided into two groups. One group served as the control group and received only normal saline, while the other group was administered 5 mg of 5-FU with a pH of 8.4. 0.2 cc of the drug was injected three times near the tumor every other week. One month later, the major axis and minor axis of the tumors growing on the dorsal epidermis were measured. Using the formula: average tumor volume = (major axis × minor axis 2 ) / 2 (mm 3 ), the tumor volumes before and after drug administration were calculated and compared and analyzed. The results are as Figure 9 and Figure 8 shown.

[0140] In Figure 9 , after 4 weeks of drug treatment, the tumor size decreased compared with that before injection (P < 0.05).

[0141] In 6) Observation of pathological changes in the subcutaneous muscle layer after subcutaneous injection of 5-FUAmong them, compared with the control group treated with PBS, the tumor size of the group administered with alkaline 5-FU decreased (P < 0.05).

[0142] Figure 10a

[0143] To observe the pathological changes in the surrounding tissues after injecting 5 mg of 5-FU at pH 8.4 near the tumor, the drug was locally administered to nude mice. After 24 hours, tissues including the muscle layer were collected and stained with H&E to examine degeneration, necrosis, inflammation, and fibrosis of the muscle layer. The degree of each pathological change was scored as follows: 0 = none, 1 = mild, 2 = moderate, 3 = severe, and the results are as Figure 10b and Figure 10a shown. Figure 10b The observation results at 40-fold magnification are shown, Figure 10a and the observation results at 100-fold magnification are shown.

[0144] As Figure 10b and Example 2: Verification of the necrosis mechanism of L-NMMA-induced gastric cancer tissue shown, no pathological changes were observed in the subcutaneous muscle layer at the 5-FU injection site (score = 0).

[0145] Figure 11

[0146] 1) Effect of L-NMMA concentration on the cytotoxicity of HUVEC cells

[0147] To evaluate the effects of 5-FU and L-NMMA concentrations on HUVEC apoptosis, an MTS cell proliferation assay was performed.

[0148] The cytotoxicity of 5 μM 5-FU on HUVEC at different concentrations of L-NMMA was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. HUVEC were seeded in 96-well plates at a density of 5 × 10 3 cells / well and incubated for 24 hours. Subsequently, the cells were incubated in DMEM medium containing different concentrations of L-NMMA for 24 hours. Then the cells were washed twice with PBS, followed by the addition of 100 μL of fresh DMEM growth medium and incubation for another 48 hours. The medium was replaced with 100 μL of fresh DMEM growth medium, and then 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added. After incubation for another 2 hours, the absorbance was measured at 490 nm using a Bio-Rad Model 680 microplate reader (Spark, Tecan, CA, USA).

[0149] The results are as Figure 11 shown. Refer toIn vivo experiment to observe the response to anticancer agents after transplantation of human gastric cancer cells into the back of nude mice As shown, a decrease in cell viability was observed after administration of L-NMMA alone, and an additional apoptotic effect was confirmed when administered in combination with 5-FU.

[0150] 2) Figure 12

[0151] The animals used in the experiment were two male nude mice (Crj: BALB / c-nu / nu, male), approximately 5 weeks old, with an average body weight of 30 grams, produced by Orient. The mice underwent a one-week adaptation period in the laboratory before use. 5×10 6 cells / 100 μL of human gastric cancer cells (PBS) were subcutaneously implanted into the dorsal region of each nude mouse. The tumor size was measured regularly, and when the tumor diameter reached 1 cm, the drug administration experiment was conducted. The experimental animals were divided into two groups. One group served as the control group and received only normal saline, while the other group was administered 5 mg of 5-FU with a pH of 8.4. These drugs were injected at a dose of 0.2 cc near the tumor, and administered three times every other week. After one month, the length and width of the tumors growing on the dorsal skin were measured. Using the formula: average tumor volume = (length × width 2 ) / 2 (mm 3 ), the tumor volumes before and after drug administration were calculated and compared and analyzed. The results are as Figure 13a , Figure 13b and Figure 12 shown.

[0152] Refer to Figure 13a , Figure 13b and Example 3: Verification of the antitumor effect of bevacizumab on gastric cancer tissue , after administration of 5-FU and L-NMMA, the tumor size decreased significantly. Compared with administration of 5-FU alone, the reduction in tumor size was greater.

[0153] 1) In vivo experiment to observe the response to the anticancer agent (bevacizumab) after xenotransplantation of human gastric cancer cells into the back of nude mice

[0154] Figure 14 Figure 14

[0155] The animals used in the experiment were three male nude mice (Crj: BALB / c-nu / nu, male), each weighing approximately 30 grams, 5 weeks old, produced by Orient. The mice underwent a one-week adaptation period in the laboratory before use. Human gastric cancer cells (5×10 6 cells / 100 μL in PBS) were xenografted into the dorsal subcutaneous fat layer of each nude mouse. The tumor size was measured regularly, and drug administration began when the tumor diameter reached 1 cm.

[0156] The experimental animals were administered 1 mg of bevacizumab by injecting 0.2 cc three times every other week near the tumor. One month later, the length and width of the tumors growing on the back skin were measured. The tumor volume before and after administration was calculated using the following formula: average tumor volume = (length × width 2 ) / 2 (mm 3 ) for comparative analysis. The results are as 2) In vivo experiment to evaluate the response to the anticancer agents (alkaline 5-FU and bevacizumab) after xenotransplantation of human gastric cancer cells into the back of nude mice shown.

[0157] As Figure 15 shown, compared with before injection, the tumor size remained almost unchanged four weeks after administration (P > 0.05). The tumor size did not change in the following two months and began to increase in the third month.

[0158] Figure 16 Figure 15

[0159] The experimental animals used in this study were three five-week-old male nude mice (Crj: BALB / c-nu / nu mice, male), each weighing about 30 g, purchased from Orient Bio. Before use, the animals were acclimated in the research facility for one week. Human gastric cancer cells (5 × 10 6 cells / 100 μL in PBS) were implanted into the subcutaneous fat layer of the back of each nude mouse. The tumor size was measured regularly, and when the tumor diameter reached 1 cm, the drug administration experiment was carried out.

[0160] The experimental animals were administered 0.2 mg of bevacizumab and 5 mg of 5-FU with a pH of 8.4 by injecting 0.2 cc three times every other week near the tumor. One month later, the length and width of the tumors growing on the back skin were measured. The tumor volume before and after administration was calculated using the following formula: average tumor volume = (length × width2) / 2 (mm3) for comparative analysis. The results are as Figure 16 and Example 4: Treatment of gastric cancer using sustained-release 5-FU sol-gel by local injection shown.

[0161] Referring to Figure 17 and Figure 17 , compared with before injection, the tumor size decreased four weeks after administration (P < 0.05). An obvious pattern of tumor regression was observed. The overall tumor size initially remained stable, and then necrosis started from the center and gradually spread outwards.

[0162] 2) Drug release test of 5-FU-sol-gel (UV-VIS spectrophotometer)

[0163] 1) Determination of the concentration of Pluronic F-127 (P407) during the preparation of 5-FU-sol-gel

[0164] To evaluate the viscosity of P407 according to temperature and concentration, a Brookfield viscometer was used to measure the viscosity while increasing the temperature from approximately 24 °C to 38 °C. The concentrations of P407 were set at 10%, 20%, and 30% respectively. The results are as Figure 18 shown.

[0165] Refer to Figure 18 , it was confirmed that a concentration of 30% of P407 is required for gelation at 37 °C. In addition, when the concentration exceeds 30%, the viscosity becomes too high, making local injection of the drug difficult. Therefore, 30% was determined to be the most suitable concentration.

[0166] Figure 19

[0167] A drug release experiment was conducted using 5 mL of PBS and 0.76 mL of gel (2 mm thick, 30% P407, 4.3 mg 5-FU). Changes were observed at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, and 24 hours in a 37 °C environment. The results are as Figure 20 shown.

[0168] Refer to Figure 19 , it was confirmed that the drug was completely released after 12 hours.

[0169] 3) In vivo experiment to evaluate the anti-cancer agent response after xenografting human gastric cancer cells onto the back of nude mice

[0170] The experimental animals used were five male nude mice (Crj: BALB / c-nu / nu, male), each approximately 5 weeks old and weighing approximately 30 g, purchased from Orient. The animals were acclimated to the laboratory for one week before use. 5 × 10 6 human gastric cancer cells / 100 μL (PBS) were subcutaneously implanted into the subcutaneous fat layer on the back of each nude mouse. The tumor size was measured regularly, and when the tumor diameter reached 1 cm, a drug administration experiment was conducted. A solution containing 30% P407 and 2.5% 5-FU with a pH of 8.4 (5 mg 5-FU) was injected three times near the tumor every other week (0.2 cc). One month later, the length and width of the tumors growing on the back skin were measured. The following formula was used to calculate the tumor volume before and after drug administration: average tumor volume = (length × width 2 ) / 2 (mm 3 ) for comparative analysis. The results are as Figure 20 and ​ shown.

[0171] Refer to ​ and ​, the drug was injected below the gastric tumor, resulting in almost complete necrosis of the malignant tumor. In addition, the comparison of tumor volume before and after drug administration confirmed a significant reduction in tumor size (P<0.05).

Claims

1. A pharmaceutical composition for tumor vascular occlusion, which comprises alkaline 5-fluorouracil and a nitric oxide synthase inhibitor.

2. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the nitric oxide synthase inhibitor comprises one or more selected from L-NMMA (NG-methyl-L-arginine), L-NAME (NG-nitro-L-arginine methyl ester), L-NA (nitroarginine), and 7NI (nitroindazole).

3. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the nitric oxide synthase inhibitor comprises L-NMMA (NG-methyl-L-arginine).

4. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the alkalinity is in the range of pH 8 to 9.

5. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the alkaline 5-fluorouracil is in the form in which 5-fluorouracil is dissolved in an alkaline solvent.

6. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the concentration of the alkaline 5-fluorouracil in the total composition ranges from 0.1 mM to 600 mM.

7. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the alkaline 5-fluorouracil increases the protein expression of thrombospondin-1 in tumor vascular endothelial cells, thereby inducing apoptosis of endothelial cells.

8. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the concentration of the nitric oxide synthase inhibitor in the total composition ranges from 0.1 mM to 300 mM.

9. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the nitric oxide synthase inhibitor causes tumor blood vessels to contract and promotes thrombosis formation in tumor blood vessels damaged by 5-fluorouracil, thereby occluding the blood vessels.

10. The pharmaceutical composition for tumor vascular occlusion according to claim 1, which further comprises one or more selected from bevacizumab, capric acid or a pharmaceutically acceptable salt thereof, and poloxamer.

11. The pharmaceutical composition for tumor vascular occlusion according to claim 1, which further comprises one or more selected from cytotoxic anticancer agents, targeted anticancer agents, and immuno-oncology agents.

12. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the tumor comprises solid tumors.

13. The pharmaceutical composition for tumor vascular occlusion according to claim 1, wherein the composition is administered locally by injection around the tumor.

14. The pharmaceutical composition for tumor vascular occlusion according to claim 13, wherein the area around the tumor is the submucosa where tumor blood vessels are located.

15. A topical preparation for administering to solid tumors, which comprises the pharmaceutical composition for tumor vascular occlusion according to claim 1.

Images