KR101458061B1 - Anticancer compositions - Google Patents

Abstract

 The present invention relates to (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) an anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient , And food compositions for the prevention or amelioration of cancer. The anticancer composition of the present invention is a synergistic combination of 5-hydroxymethylfurfural, metformin, phenformin, 2-deoxy-D-glucose and citric acid, Can inhibit the survival of cancer cells and can inhibit the cell cycle of cancer cells without inducing toxicity to normal cells and induce the death thereof. Therefore, it can be effectively used as an anticancer drug without side effects.

Description

Anticancer compositions

The present invention relates to (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) an anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient , And food compositions for the prevention or amelioration of cancer.

Among cancer deaths in recent years, one out of every four cancer deaths is on the rise. Thus, surgical treatment, radiation therapy, biotherapy, and chemotherapy are the treatment methods of cancer that account for most of the causes of death. Among them, chemotherapy with anticancer drugs is now widely used for cancer treatment and it is one of the well established treatment methods. Such anticancer agents interfere with the metabolism pathway of cancer cells and interfere with DNA replication, transcription, and translation by direct interaction with DNA, interfere with the synthesis of nucleic acid precursors, and inhibit cell division, thereby exhibiting toxicity to cells. Accordingly, the anticancer drug causes fatal damage to normal cells, resulting in cytopenia of leukocytes, platelets and red blood cells due to bone marrow destruction; Hair loss symptoms due to hair follicle cell destruction; Side effects of ovaries and testicles cause menstrual irregularities and male infertility; Side effects from digestive mucous membrane destruction include stomatitis, nausea and vomiting and food swallowing disorders and digestive disorders; Diarrhea symptoms; Renal toxicity by tubular necrosis; Peripheral neuritis and weakness caused by nervous system disorders; Vascular disorders such as vascular pain and rash; Skin and nail discoloration. Therefore, there is an urgent need for research to increase the therapeutic effect while minimizing side effects caused by anticancer drugs.

In addition, the main reason for the failure of chemotherapy is that the anticancer drug is initially effective, but the drug resistance is gradually developed and the immunity is extremely deteriorated. Therefore, there is a need for a method of improving cancer treatment efficacy without increasing the toxicity of the drug. Unfortunately, it is unexpected that combinations of drugs having anticancer effects can not be expected to all exhibit a synergistic effect, and it is very difficult to find combinations of drugs with synergistic effects It is difficult. Therefore, it is urgent to develop an anti-cancer combination preparation which can maximize the anticancer effect while minimizing the side effect of the anti-cancer drug.

The inventors of the present invention have made efforts to find an anticancer substance having no adverse effect on the human body while maximizing the therapeutic effect of the cancer while minimizing the use concentration. As a result, it has been found that a combination preparation The present inventors have completed the present invention by confirming that the combination preparation can inhibit the cell cycle even at a low concentration of the compound and activate AMPK (AMP-activated kinase) to effectively kill cancer cells.

It is an object of the present invention to provide an anticancer composition which can effectively treat cancer even in a small amount, exhibits a toxic effect specifically on cancer cells, and has reduced side effects.

It is another object of the present invention to provide a food composition for prevention or improvement of cancer.

In one aspect, the present invention provides a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) an anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient .

In another aspect, the present invention provides a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) prevention of cancer comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient Or improving food composition.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) an anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient .

(1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) a combination preparation comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient, Cancer drug that can effectively treat cancer with a small amount of side effects with a small amount.

The combination preparations of the present invention may use less of the individual compounds contained in the combination preparation than when treated with a single compound, which significantly reduces the risk and / or severity of side effects and significantly reduces the overall effect of the treatment There is an advantage to increase.

In the present invention, 5-hydroxymethylfurfural is a food additive added as a flavoring agent in fruit juice, food, and alcohol processing, and is a decomposition product easily produced from polysaccharides such as sugar at a food manufacturing process stage. It is often ingested. 5-Hydroxymethyl furfural has a structure represented by the following formula (1).

In the present invention, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof is used in combination with an acetylated compound (metformin or phenorphin) or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose or its pharmaceutical It has been confirmed that the combination with an acceptable salt and / or citric acid or a pharmaceutically acceptable salt thereof effectively induces the death of cancer cells. When 5-hydroxymethyl furfural was orally administered to rats, it was found that 5-hydroxymethyl furfural was an extremely safe compound with an LD ₅₀ of 2,500 mg / kg (National Technical Information Service OTS0544683).

In the present invention, metformin or phenformin, which is an acetylated compound, is known as a therapeutic agent for diabetes (Table 1) and is represented by the following formula (2).

Biguanide system
R ₁
R ₂
R ₃
R ₄
Metformin
H
H
CH ₃
CH ₃

Penformin

H

H

H

In the present invention, metformin or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a phenorphamine or a pharmaceutically acceptable salt thereof and 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof in combination with 5-hydroxymethyl It has been confirmed that the combination of furfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof shows a high anticancer effect even at a low concentration. Bigenanide drugs are known to activate the enzyme AMPK (AMP-activated kinase), which plays a pivotal role in the regulation of energy balance and nutrient metabolism in the cell. When metformin was orally administered to rats, it was found that metformin was a highly safe compound with an LD ₅₀ of 1,450 mg / kg (Gekkan Yakuji, Pharmaceuticals Monthly Vol. 9, Pg. 759, 1967) . However, penformin was developed in the late 1950s as an oral antidiabetic agent and was intended to be used for the treatment of non-insulin dependent diabetes mellitus (type 2 diabetes). However, due to the serious side effect of lactic acidosis, It was forbidden. Accordingly, the present invention provides a pharmaceutical composition comprising 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, / RTI > or a pharmaceutically acceptable salt thereof, wherein the composition comprises a phenorphin mono-agent or a combination of two compounds, a combination of a phenolamine and a 2-deoxy- It was confirmed that the anticancer effect was high even at a much lower concentration than the composition containing D-glucose, thereby effectively lowering the adverse effects of the penformin (Figs. 6, 13, 20, 27, 34, 41 48, 55, 62, 69).

In the present invention, 2-deoxy-D-glucose can be used as an inhibitor of the action, and it has the structure represented by the formula (3).

In the present invention, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof is added to 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof and a biguanide compound or a pharmaceutically acceptable salt thereof. The present inventors have confirmed that a high anticancer effect can be obtained even at a low concentration by combining with a salt capable and / or citric acid or a pharmaceutically acceptable salt thereof. 2-deoxy-D-glucose is a derivative of glucose, which prevents glycolysis during glucose metabolism and inhibits glycosylation of proteins in the endoplasmic reticulum, thereby inducing endoplasmic reticulum stress. 2-deoxy-D-glucose, an inhibitor of glucose degradation, was found not to kill cancer cells alone. However, it has shown remarkable results in anticancer therapy because cancer cells are sensitive to other drug treatments.

In the present invention, citric acid can be used as an inhibitor of the action, and it has the structure represented by the general formula (4).

In the present invention, citric acid or a pharmaceutically acceptable salt thereof, a biguanide compound or a pharmaceutically acceptable salt thereof and 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof and / or 5 -Hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, it has been confirmed that a high anticancer effect is obtained even at a low concentration. In addition, when citrate was orally administered to rats, it was found that LD50 was 1,548 mg / kg, and citric acid was a highly safe compound, respectively (Journal of Pharmacology and Experimental Therapeutics, Vol. 94, 1948.).

In the present invention, each of 5-hydroxymethyl furfural, an isocyanide compound, 2-deoxy-D-glucose and citric acid may be present in the form of a pharmaceutically acceptable salt. As salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. As used herein, "pharmaceutically acceptable salts" refers to those salts that are relatively non-toxic to a patient and have a beneficial effect that is innocuous, with side effects due to the salt being 5-hydroxymethyl furfural, - deoxy-D-glucose and any organic or inorganic addition salt that does not degrade the beneficial effects of citric acid.

The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. As organic acids, methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, Gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, But are not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble salt salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically acceptable to produce sodium, potassium or calcium salt, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts of each of 5-hydroxymethyl furfural, an acetylated compound (metformin or phenorphamine), 2-deoxy-D-glucose and citric acid, unless otherwise indicated, Methyl furfural, an acetyl-based compound (metformin or phenformin), 2-deoxy-D-glucose and citric acid, respectively. For example, pharmaceutically acceptable salts may include the sodium, calcium, and potassium salts of the hydroxy group, and the other pharmaceutically acceptable salts of amino groups include hydrobromides, sulfates, hydrogensulfates, phosphates, (Mesylate) and p-toluenesulfonate (tosylate) salts, which are known in the art, such as, for example, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate Can be prepared through a process for preparing a salt.

As the salt of an acetylenic compound (metformin or phenorphin) of the present invention, any pharmaceutically acceptable salt may be used as long as it is metformin or phenorphin salt exhibiting an anticancer effect equivalent to metformin or phenorphin, Metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride, metformin hydrochloride,

As the citrate salt of the present invention, any pharmaceutically acceptable salt may be used as long as it is an citrate salt exhibiting an anticancer effect equivalent to that of citric acid, preferably sodium citrate, potassium citrate, calcium citrate, ammonium citrate and magnesium citrate. However, the present invention is not limited thereto.

The 5-hydroxymethyl furfural, the biguanide compound, 2-deoxy-D-glucose, and citric acid of the present invention each include derivatives thereof. The "derivative" means a compound prepared by chemically changing a part of the compound, for example, introducing, substituting or eliminating a functional group to the extent that the anticancer activity of the compound does not change. .

Combining two or more kinds of drugs known to have anticancer effects can not be expected to show synergistic effects (synergistic effect) between the combined drugs. Rather, the function of the drug may be canceled by combination, Finding a combination of drugs is very difficult. In the present invention, an anticancer combination preparation capable of exhibiting the anticancer effect to the utmost while minimizing side effects by using the minimum concentration of the anticancer agent has been developed.

One embodiment of the preferred anti-cancer composition in the present invention is a composition comprising 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, A composition comprising a pharmaceutically acceptable salt thereof; A composition comprising 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, and an acetylenic compound or a pharmaceutically acceptable salt thereof; 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, Or a pharmaceutically acceptable salt thereof.

In the present invention, the above-mentioned biguanide compound may be metformin or a pharmaceutically acceptable salt thereof, or a phenorphamine or a pharmaceutically acceptable salt thereof.

As the acetylenic compound, a composition based on metformin may be used. The anticancer composition of the present invention may include 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof Acceptable salts, and metformin, or a pharmaceutically acceptable salt thereof; A composition comprising 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, and metformin or a pharmaceutically acceptable salt thereof; 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, and metformin or a pharmaceutical ≪ / RTI > acceptable salt.

As another example of a biguanide-based compound, a composition based on a penformin, the anticancer composition of the present invention may include 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or its A composition comprising a pharmaceutically acceptable salt, and a phenformin or a pharmaceutically acceptable salt thereof; A composition comprising 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, and a phenformin or a pharmaceutically acceptable salt thereof; 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, Or a pharmaceutically acceptable salt thereof. Most preferably, the anticancer composition of the present invention comprises 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof, citric acid or a pharmaceutically acceptable salt thereof, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof An acceptable salt, and an acetylenic compound or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, there is provided a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) a combination of at least three compounds consisting of 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof, It was confirmed that the preparation can inhibit cell proliferation by reducing the proliferation of cancer cells, suppressing the progression of G1 / S phase, G2 / M phase cell cycle, and inhibiting DNA synthesis of cancer cells (Example 2- 2).

(1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) a combination of at least three compounds consisting of 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof, When the preparation was used, synergistic anti-cancer effect was obtained due to the combination of compounds, and it was confirmed that the drug reduction index of each compound was remarkably reduced by decreasing the 50% cancer inhibitory concentration (Example 1).

That is, according to the present invention, each of 5-hydroxymethyl furfural, an isocyanide compound, 2-deoxy-D-glucose and citric acid is used as a single preparation, Was used as a combination preparation, it was confirmed that cancer cells can be effectively killed even in a small amount.

Particularly, a combination preparation of 5-hydroxymethyl furfural / metformin / 2-deoxy-D-glucose, a combination preparation of metformin / 2-deoxy-D-glucose / citric acid, 5- hydroxymethyl furfural / metformin / The combined preparation of 2-deoxy-D-glucose / citric acid is a combination of a single preparation of each compound, a combination formulation containing two compounds and a combination of 5-hydroxymethyl furfural / metformin / . In addition, a combination preparation of 5-hydroxymethylfurfural / phenformin / 2-deoxy-D-glucose, a combination preparation of penformin / 2-deoxy-D-glucose / citric acid, The combined preparation of fullerene / phenformin / 2-deoxy-D-glucose / citric acid may be a single formulation, a combination of two compounds and a combination of 5-hydroxymethylfurfural / phenformin / citric acid 50% reduction effect of cancer inhibition concentration was high.

Among the combination preparations, a combination preparation of 5-hydroxymethylfurfural / metformin / 2-deoxy-D-glucose / citric acid and 5-hydroxymethylfurfural / phenformin / 2-deoxy-D-glucose / citric acid Were found to have the highest reduction effect of 50% cancer inhibitory concentration and the drug reduction effect of each compound according to the combination.

In each combination preparation according to the present invention, the combination molar ratio of each compound is not particularly limited.

For example, 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof: metformin (or penformin) or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof Acceptable salt: citric acid or a pharmaceutically acceptable salt thereof may be in the range of 1: 2 (0.05 for phenol): 0.05: 0.3 to 1:30 (1.5 for phenol): 3: 5 , The combination molar ratio may vary depending on the type of cancer to be treated. (The values in parentheses are the molar ratios of penformin or a pharmaceutically acceptable salt thereof)

Preferably, 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof: metformin, penformin, or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof Possible salt: Combination molar ratio of citric acid or its pharmaceutically acceptable salt is 1: 3 (0.05): 0.1: 0.4 to 1:14 (0.5): 0.6: 2, preferably 1: 7 (0.2) (0.2): 0.5: 2.2 in cancer cell lines, 1: 4 (0.05): 0.2: 1 to 1:30 (0.8): 1: 4, preferably 1:16 (0.05): 0.1: 0.3 to 1:20 (0.05: 1) in a pancreatic cancer cell line (0.1: 0.5 to 1:20 (0.5): 0.8: 3, preferably 1:10 0.5: 0.7: 3, preferably 1: 7 (0.2): 0.2: 0.9 in colorectal cancer cell lines and 1: 3 (0.05): 0.4: 0.4 to 1:30 (0.5): 0.1: 5, preferably 1:10 (0.2): 0.3: 1 in cervical cancer cell line at 1:11 (0.2) , 1 in breast cancer cell line (0.1): 0.2: 1 to 1: 1 in a prostate cancer cell line, in a prostate cancer cell line of 2: 0.05 (0.05): 0.3: 0.3 to 1:18 (0.6) (0.05): 0.1: 0.2 to 1:30 (0.6): 0.8: 2.5 in ovarian cancer cell lines at a concentration of 1: 40 (0.1): 2.5: 5, preferably 1:13 (0.2): 0.2: 0.6 in a bladder cancer cell line, preferably 1: 2 (0.2): 0.2: 0.6: 0.8. At this time, the larger the combination ratio of metformin or phenformin, 2-deoxy-D-glucose and citric acid is, the greater the synergistic effect is with respect to the 5-hydroxymethylfurfural concentration, but the higher concentration of metformin or penformin, It is preferable to determine the combination index molar ratio in consideration of toxicity by -D-glucose and citric acid. Combination formulations of 5-hydroxymethyl furfural, metformin or phenformin, 2-deoxy-D-glucose and citric acid in the combined molar ratios exhibit a combination index (CI) corresponding to a synergistic effect, Low drug reduction index.

As used herein, the term "cancer" refers to a disease associated with cell death control, which is caused by excessive proliferation of cells when the normal apoptosis balance is broken. These abnormal hyperproliferative cells sometimes invade surrounding tissues and organs to form a mass, which destroys or transforms normal structures in the body. Such a condition is called cancer. In general, a tumor refers to a mass abnormally grown by autonomous overgrowth of the body tissue, and can be divided into benign tumor and malignant tumor. Malignant tumors have a much faster growth rate than benign tumors, and they invade the surrounding tissues, causing metastasis and endangering life. Such malignant tumors are commonly referred to as 'cancer'. The types of cancer include cerebrospinal fluid, brain cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, cervical cancer, colon cancer, liver cancer, pancreatic cancer, , Bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer. The anticancer composition of the present invention can be used without limitation in the kind of cancer but is preferably selected from the group consisting of liver cancer, lung cancer, gastric cancer, pancreatic cancer, colon cancer, cervical cancer, breast cancer, prostate cancer, ovarian cancer and bladder cancer for the purpose of the present invention. May be useful for preventing or treating cancer.

The term "preventive or therapeutic" as used herein refers to (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) an anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient Refers to all actions that inhibit or delay the onset of cancer by using the composition, and in particular, " treatment " means any action that improves or alters cancer by using the composition.

Accordingly, the scope of the present invention includes a method for preventing or treating cancer, which comprises administering the anti-cancer composition according to the present invention to a subject in need of prevention or treatment of cancer.

The anticancer composition of the present invention may further comprise a chemotherapeutic agent for cancer treatment, if necessary, in addition to the above-mentioned effective ingredient.

The anticancer composition of the present invention may further comprise a pharmaceutically acceptable carrier. The composition of the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations such as sterilized injection solutions, Suppositories, and diluents that can be included in such a composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Base materials for injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

The composition of the present invention may be administered orally, intravenously, subcutaneously, intradermally, intranasally, intraperitoneally, intramuscularly, transdermally, etc. The dosage may vary depending on the patient's age, sex, Can be readily determined by those skilled in the art. The dose of the composition according to the present invention may be increased or decreased according to the route of administration, degree of disease, sex, body weight, age, etc. Preferably, in the case of a combination of four compounds, 5-hydroxymethyl furfural Kg body weight per day when Metformin is used as an acetylated compound and between 0.1 and 40 mg / kg body weight per day when Penformin is used 2-deoxy-D-glucose is 0.1 to 40 mg / kg (body weight) per day, and citric acid is 1 to 200 mg / kg (body weight) per day. However, the scope of the present invention is not limited by the dosage.

In another aspect, the present invention provides a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) at least three compounds consisting of 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof, and / or citric acid or a pharmaceutically acceptable salt thereof, To a method for preventing or treating cancer.

In another aspect, the present invention provides a pharmaceutical composition comprising (1) a biguanide compound or a pharmaceutically acceptable salt thereof; (2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And (3) prevention of cancer comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and / or citric acid or a pharmaceutically acceptable salt thereof as an active ingredient Or improving food composition.

The above-mentioned biguanide compound may be metformin (hereinafter referred to as MET) or phenol (hereinafter referred to as PHE).

The composition may contain, in addition to the active ingredient, a pharmaceutically acceptable food-aid additive.

As used herein, the term "food-aid additive " refers to a component that can be added to foods in a supplementary manner, and is appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food aid additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. However, the types of the food-aid additives of the present invention are not limited by these examples.

A health functional food may be included in the food composition of the present invention. The term "health functional food " as used in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings using raw materials and components having useful functions in the human body. Here, 'functional' refers to the structure and function of the human body to obtain nutritional effects and obtain useful effects for health use such as physiological action. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, the formulations of the above health functional foods may also be manufactured without limitations as long as they are acceptable as health functional foods. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to enhance the effectiveness of anticancer drugs.

The anticancer composition of the present invention exhibits a synergistic anticancer effect through a combination of specific drugs which must be used in an excessive amount, thereby activating AMP-activated kinase (AMPK) in a small amount to inhibit cancer cell death and cell cycle, thereby effectively treating cancer . In addition, the anticancer composition of the present invention exhibits a toxic effect specifically on cancer cells without showing toxicity in normal cells, and can kill cancer cells without side effects, so that the composition can be effectively used as an anticancer agent.

FIG. 1 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours from treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the HepG2 cell line, which is a cancer cell derived from human liver, to be. The vertical bar of each point represents the standard error.
FIG. 2 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HMG, MET, 2DG, and CT with respect to the HepG2 cell line by volume. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 3 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, MET, 2DG and CT were combined for the HepG2 cell line (four combinations (HMF / MET / 2DG / (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 4 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration by HepG2 cell line. The vertical bar of each point represents the standard error.
FIG. 5 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HMG, PHE, 2DG, and CT with respect to the HepG2 cell line by the dose. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 6 shows how treatment of HMF, PHE, 2DG, and CT by dose for HepG2 cell lines can reduce the drug toxicity of PHE corresponding to 50% cancer inhibition rates in single and combination compositions by MTT assay after 48 hours FIG.
FIG. 7 is a Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT were combined for the HepG2 cell line (four combinations group (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 8 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours from the treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for each A549 cell line, which is a human lung- to be. The vertical bar of each point represents the standard error.
FIG. 9 is a graph showing the cell survival rate as a percentage by MTT assay after treatment with HMF, MET, 2DG, and CT by dose for A549 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 10 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, MET, 2DG and CT were combined for the A549 cell line (group of four species (HMF / MET / 2DG / (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 11 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration by A549 cell line. The vertical bar of each point represents the standard error.
FIG. 12 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HM5, PHE, 2DG, and CT by dose for A549 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 13 shows how treatment of HMF, PHE, 2DG, and CT by dose for A549 cell lines can reduce the drug toxicity of PHE corresponding to 50% cancer inhibition rates in single and combination compositions by MTT assay after 48 hours FIG.
FIG. 14 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT were combined for A549 cell line (4 groups (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 15 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the AGS cell line, which is a human cancer-derived cancer cell, . The vertical bar of each point represents the standard error.
FIG. 16 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, MET, 2DG, and CT with respect to the AGS cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
17 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, MET, 2DG and CT were combined for the AGS cell line (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 18 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG, and CT for each concentration by AGS cell line. The vertical bar of each point represents the standard error.
FIG. 19 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HMF, PHE, 2DG, and CT with respect to the AGS cell line by volume. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 20 shows how treatment of HMF, PHE, 2DG, and CT by dose for AGS cell lines can reduce the drug toxicity of PHE corresponding to 50% cancer inhibition rates in single and combination compositions by MTT assay after 48 hours FIG.
FIG. 21 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, PHE, 2DG and CT were combined for AGS cell line (four combinations group (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
22 is a graph showing the cell survival rate by MTT assay as a percentage after 48 hours after treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the Capan-2 cell line, which is a human pancreatic cancer- Fig. The vertical bar of each point represents the standard error.
23 is a graph showing the cell survival rate as a percentage by MTT assay after treating 48 hours after HMF, MET, 2DG and CT treatment of Capan-2 cell line by dose. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
24 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when combined with HMF, MET, 2DG and CT for the Capan-2 cell line ** p <0.01; ** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
25 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration by Capan-2 cell line. The vertical bar of each point represents the standard error.
26 is a graph showing the cell survival rate as a percentage by the MTT assay after 48 hours after treatment of HMF, PHE, 2DG, and CT with respect to the Capan-2 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 27 shows that treatment of HMF, PHE, 2DG, and CT by dose for Capan-2 cell line reduced the drug toxicity of PHE corresponding to 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours The graph shows a comparison of concentrations.
28 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when combined with HMF, PHE, 2DG and CT for the Capan-2 cell line 2DG and CTE): * p <0.05; ** p <0.01; *** p <0.001. Two groups (PHE / 2DG) and three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 29 is a graph showing the cell survival rate by MTT assay as a percentage after 48 hours from the treatment of each single preparation and combination preparation of HMF, MET, 2DG and CT for the DLD-1 cell line, which is a cancer cell derived from human colon, Fig. The vertical bar of each point represents the standard error.
30 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HMD, MET, 2DG, and CT with respect to the DLD-1 cell line after 48 hours. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
31 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, MET, 2DG and CT are combined for the DLD-1 cell line ** p <0.01; ** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
32 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration of DLD-1 cell line. The vertical bar of each point represents the standard error.
FIG. 33 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HMD, PHE, 2DG, and CT with respect to the DLD-1 cell line after 48 hours. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 34 shows that treatment of HMF, PHE, 2DG and CT by dose for the DLD-1 cell line reduced the drug toxicity of PHE corresponding to 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours The graph shows a comparison of concentrations.
35 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT are combined for the DLD-1 cell line (4 group combinations (HMF / PHE / 2DG and CTE): * p <0.05; ** p <0.01; *** p <0.001. Two groups (PHE / 2DG) and three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 36 shows the cell survival rate by MTT assay as a percentage after 48 hours after treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the HeLa cell line, which is a cancer cell derived from the human cervix, Graph. The vertical bar of each point represents the standard error.
FIG. 37 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HML, MET, 2DG, and CT with respect to the HeLa cell line by volume. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 38 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, MET, 2DG and CT were combined for HeLa cell line (group of four species (HMF / MET / 2DG / (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 39 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, PHE, 2DG, and CT for the HeLa cell line by concentration. The vertical bar of each point represents the standard error.
40 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of HML, PHE, 2DG, and CT with respect to the HeLa cell line by dose. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 41 shows how treatment of HMF, PHE, 2DG, and CT by dose for HeLa cell lines can reduce the drug toxicity of PHE corresponding to 50% cancer inhibition rates in single and combination compositions by MTT assay after 48 hours FIG.
FIG. 42 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT were combined for HeLa cell line (4 group combinations (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 43 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours from the treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the MCF7 cell line, which is a cancer cell derived from human breast, to be. The vertical bar of each point represents the standard error.
44 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, MET, 2DG and CT with respect to the MCF7 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
45 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, MET, 2DG and CT were combined for the MCF7 cell line (four combinations (HMF / MET / 2DG / (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
46 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for the MCF7 cell line by concentration. The vertical bar of each point represents the standard error.
47 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, PHE, 2DG, and CT with respect to the MCF7 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 48 shows how treatment of HMF, PHE, 2DG, and CT by dose with MCF7 cell line can reduce the drug toxicity of PHE corresponding to 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours FIG.
49 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, PHE, 2DG and CT were combined for the MCF7 cell line (4 group combinations (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
50 is a graph showing the cell survival rate by MTT assay as a percentage after 48 hours after treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the PC-3 cell line, which is a cancer cell derived from human prostate, Fig. The vertical bar of each point represents the standard error.
51 is a graph showing the cell survival rate as a percentage by MTT assay after treatment with HMF, MET, 2DG, and CT for the PC-3 cell line after 48 hours. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
52 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when combined with HMF, MET, 2DG and CT for the PC-3 cell line ** p <0.01; ** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 53 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, PHE, 2DG, and CT for the PC-3 cell line. The vertical bar of each point represents the standard error.
54 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, PHE, 2DG, and CT with respect to the PC-3 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 55 shows that treatment of HMF, PHE, 2DG, and CT by volume with PC-3 cell line resulted in lowering the drug toxicity of PHE corresponding to 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours The graph shows a comparison of concentrations.
56 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, PHE, 2DG and CT were combined for the PC-3 cell line 2DG and CTE): * p <0.05; ** p <0.01; *** p <0.001. Two groups (PHE / 2DG) and three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 57 shows the cell survival rate by MTT assay after 48 hours after treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the SK-OV-3 cell line, which is a cancer cell derived from human ovary, Lt; / RTI &gt; The vertical bar of each point represents the standard error.
FIG. 58 is a graph showing the cell survival rate as a percentage by MTT assay after treatment with HMF, MET, 2DG, and CT for the SK-OV-3 cell line after 48 hours. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
59 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, MET, 2DG and CT are combined for the SK-OV-3 cell line MET / 2DG / CT): * p <0.05; ** p <0.01; *** p <0.001. , T-test between MET / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 60 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration by SK-OV-3 cell line. The vertical bar of each point represents the standard error.
61 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, PHE, 2DG, and CT with respect to the SK-OV-3 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001)
Figure 62 shows the effect of treatment of HMF, PHE, 2DG and CT on the capacity of the SK-OV-3 cell line by dose to determine the drug toxicity of PHE corresponding to 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours This is a concentration comparison graph showing the ability to lower the temperature.
63 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT are combined for the SK-OV-3 cell line PHE / 2DG / CT): * p <0.05; ** p <0.01; *** p <0.001 The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG , PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 64 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours from the treatment of each single preparation and combination preparation of HMF, MET, 2DG, and CT for the T24 cell line derived from human bladder, to be. The vertical bar of each point represents the standard error.
FIG. 65 is a graph showing the cell survival rate as a percentage by MTT assay after treatment with HMF, MET, 2DG, and CT for each dose of T24 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
66 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when the HMF, MET, 2DG and CT were combined for the T24 cell line (the four species combination group (HMF / MET / 2DG / (P <0.05). ** p <0.01. *** p <0.001. The two groups (MET / 2DG) and the three groups (HMF / MET / 2DG, MET / / CT): † p <0.05; †† p <0.01; ††† p <0.001).
67 is a graph showing the cell survival rate as a percentage by MTT assay after treatment of each single preparation and combination preparation of HMF, PHE, 2DG and CT for each concentration by T24 cell line. The vertical bar of each point represents the standard error.
68 is a graph showing the cell survival rate as a percentage by MTT assay after 48 hours after treatment of HMF, PHE, 2DG, and CT with respect to the T24 cell line. (P <0.05; ** p <0.01; *** p <0.001). T-test among the combination groups: † p <0.05; †† p <0.01; ††† p <0.001).
Figure 69 shows how treatment of HMF, PHE, 2DG and CT by dose for the T24 cell line can reduce the drug toxicity of PHE corresponding to a 50% cancer inhibition rate in single and combination compositions by MTT assay after 48 hours FIG.
FIG. 70 is an Fa-CI graph showing the combination index (CI) for Fa (Fraction Affected) when HMF, PHE, 2DG and CT were combined for the T24 cell line (group of four species (HMF / PHE / 2DG / (P <0.05). ** p <0.01; *** p <0.001. The two groups (PHE / 2DG) and the three groups (HMF / PHE / 2DG, PHE / 2DG / CT): † p <0.05; †† p <0.01; ††† p <0.001).
FIG. 71 shows that when HepG2 cell lines were treated with HMF, MET, 2DG and CT at a concentration of 3.5: 25: 1: 3 mM for 24, 48 and 72 hours, the proliferation of HepG2 cells This is a graph showing the effect. The bar on each bar represents the standard error.
72 is a graph showing the effect of HMF, PHE, 2DG and CT on HepG2 cell proliferation for 24, 48 and 72 hours at a concentration of 3.5: 0.8: 1: 3 mM for each single agent and combination preparation This is a graph showing the effect. The bar on each bar represents the standard error.
73 is a graph showing cell cycle distribution by flow cytometry after culturing HepG2 cells to which HepG2 cell lines were added with HMF, MET, 2DG and CT combination agent for 24 hours, and control cells not added; The bar on each bar represents the standard error.
74 shows the effect of HMF, MET, 2DG, and CT on the single-agent and combination preparation of HepG2 cell line treated with the respective doses for 36 hours, followed by cell lysate to affect the protein will be.
FIG. 75 shows cell survival rates after 48 hours treatment of HepG2 cell line with normal hepatocyte CHANG and hepatoma cell HepG2 with 50% cancer inhibitory concentration of HMF, MET, 2DG, and CT combination preparation. The bar on each bar represents the standard error.
76 shows the cell survival rate after 48 hours treatment of normal hepatocyte CHANG and hepatoma cell HepG2 with 50% cancer inhibitory concentration of HMF, PHE, 2DG and CT combination preparation for HepG2 cell line. The bar on each bar represents the standard error.
FIG. 77 is a graph showing the degree of activation of AMPK after HMF, MET, 2DG, and CT were cultured for 36 hours in a single preparation and combination preparation for each dose of HepG2 cell line. The bar on each bar represents the standard error.
78 is a graph showing the degree of activation of AMPK after 36 hours of treatment of HMG, PHE, 2DG, and CT for each single preparation and combination preparation for HepG2 cell line by dose. The bar on each bar represents the standard error.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

In the following examples, sodium citrate, metformin hydrochloride, and phenorphine hydrochloride were used as representative examples of various pharmaceutically acceptable salts of metformin and phenorphamine as citric acid and an isocyanide compound. But is not limited thereto.

Used cell line

Human tumors such as hepatocellular carcinoma (HepG2), lung cancer (A549), gastric cancer (AGS), pancreatic cancer (Capan-2), colorectal cancer (DLD-1), cervical cancer (HeLa), breast cancer (MCF7) (Korean Cell Line Bank, Seoul, Korea), and the cell lines derived from the ovarian cancer (PC-3), ovarian cancer (SK-OV-3) and bladder cancer (T24) were cultured under established conditions. Respectively. Human normal hepatocytes (CHANG) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Cells were grown in monolayers in RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum, 2 mM glutamine, 100 units / ml penicillin, and 100 ug / ml streptomycin, or Dulbecco's modified Eagle's medium Respectively. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units / ml penicillin and 100 μg / ml streptomycin at 37 ° C, 5% carbon and 95% oxygen Lt; / RTI &gt; incubator. When the cells were about 80% of the plate, the cell monolayer was washed with phosphate buffer, followed by subculture with 0.25% trypsin-2.65 mM EDTA, and the medium was changed every 3 days.

Medication used

5-hydroxymethylfurfural (HMF), metformin hydrochloride (MET), phenformin hydrochloride (PHE), 2-deoxy-D-glucose -D-glucose (2DG) and sodium citrate (CT) were purchased from Sigma (St. Louis, USA). The table summarizing the results obtained through experiments in the present invention and all the drugs used in the drawings are abbreviated as abbreviations.

Reference Example  1: In vitro inhibition of cell growth assay

In order to measure the proliferation inhibitory effect of each cancer cell, yellow tetrazolium MTT (3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) analysis method was performed according to the method of Carmichael et al. MTT assay is a method for measuring the growth of living cells. The dehydrogenase in the mitochondria of living cells utilizes the principle of producing a purple formazan by MTT, a yellow water-soluble substance. The production of purple formazan is known to be almost proportional to the number of metabolically active living cells and can be used to measure cell growth and differentiation very effectively.

Each of the cultured cancer cells was added to a 96-well plate at a rate of 2 x 10 ⁴ cells / well per well and incubated for 24 hours at 37 ° C in a wet incubator supplied with 5% carbon and 95% oxygen, Each single agent and combination preparation was treated with cancer cells using a combination index representing the fraction affected for the concentration of drug corresponding to IC50, respectively. After incubation for 48 hours, 15 ㎕ of MTT (5 mg / ml) solution dissolved in phosphate buffered saline (PBS) was added to each well and incubated again for 4 hours. After confirming formation of formazan, the medium was completely removed and 100 μl of dimethylsulfoxide (DMSO) was added to dissolve the formazan formed in the bottom of the well. Then, the absorbance at 560 nm was measured using a microplate reader (Powerwave XS, Biotek), and relative cell proliferation inhibition rates were calculated when control cells were 100%.

Reference Example  2: Single formulation and combination test method

Cells were seeded in 96-well plates at 2 x 10 ⁴ cells / well and treated with HMF, MET or PHE, 2DG and CT, respectively, as single agent drugs to determine the concentration of drug corresponding to IC50.

The combination drug was treated with a concentration of the drug corresponding to IC50 of the combination preparation consisting of at least two compounds selected from the group consisting of HMF, MET or PHE, 2DG and CT. All cell lines were cultured for 48 hours at the concentration of single or combination preparation, and the growth inhibitory effect was measured by MTT assay.

The effect of concurrent exposures was determined using a combination index indicating the fraction affected for the concentration of drug corresponding to IC50. Combination index values of less than 0.9 are synergistic, 1.1 or more are antagonistic, and combination indexes of 0.9 to less than 1.1 are additive or additive.

Reference Example  3: Statistical analysis and measurement of synergistic activity

The effect of the drug combination was evaluated using the Chou and Talalay methods based on the intermediate effect principle [(Chou and Talalay, Adv. Enzyme Regul 22: 27-55, 1984) The dose-effect on the combination of ratios was calculated using the equationfa/fu = (D/Dm)^m. In the above formulaDIs the drug concentration,Dm Is the concentration required for a half-maximal effect (i. E., 50% inhibition of cell growth)fa The drug concentrationD (E.g., 0.9 when cell growth is inhibited by 90%),fu (1 -fa), And m is the slope coefficient of the sigmodicity of the concentration-effect curve. Based on the slope of the curve for each drug in the combination, one can determine whether the drug has a mutually non-exclusive effect (e.g., an independent or interactive mode of action). The combination index (CI) was determined here using the following equation.

In the equation, (D _{x) 1} is the concentration of drug required to produce an x% Efficacy 1 alone. 1, (D) ₁ is (D) in combination with a _second drug 1 required to produce the same x% effective Concentration. α is assumed to be α = 0 if there is a similarity between the actions of the drugs, and α = 1 if not. The CI value can be obtained by the above equation with respect to various values of fa . (Or synergistic) effect when the CI value indicating the cytotoxicity by the interaction of the two drugs is less than 0.9, and an addition (or adduct) action between 0.9 and 1.1 is shown by the interaction of the two drugs Indicating that they have no effect on each other. However, a value of CI greater than 1.1 indicates an antagonistic effect, which is offset by the interaction of the two drugs.

The Drug Reduction Index (DRI) is the degree to which the concentration of each single drug in the effect of a given drug is reduced by interaction of more than two drugs. (DRI) ₁ = (D _x ) ₁ / (D) ₁ and (DRI) ₂ = (D _x ) ₂ / (D) ₂ . The relationship between DRI and CI between two drugs can be expressed as CI = 1 / (DRI) ₁ + 1 / (DRI) ₂ .

Data were analyzed using concentration-effect analysis on Calcusyn software (BioSoft, Cambridge Bridge, England). Statistical analysis and graphing Instat and Prism software (graph pads from San Diego, USA) were used. The dose-effect relationship for the tested drugs alone or in pairs was treated with a mid-effect plot analysis to determine IC50, m and r in each cell line. As described above, IC50 and m values were used to calculate the synergistic and antagonistic effects, respectively, based on the CI equation. The results are shown as the mean ± standard deviation of two or more repeated experiments. In each experiment, cells were exposed to the paired combination for 48 hours as described above. All statistical procedures were performed using t-test using Minitab (version 16.0).

Reference Example  4: Cell cycle measurement

To investigate the effect of combination of HMF, MET, 2DG, and CT on the cell cycle progression of each cancer cell, the cells were diluted with a medium supplemented with 10% fetal bovine serum (FBS) and dispensed into a 24 well plate. After 24 hours, the cells were cultured in medium containing 10% fetal bovine serum (FBS) with medium supplemented with a combination of HMF, MET or PHE, 2DG and CT. Cells were cultured for 16 hours by adding HMF, MET or a combination of PHE, 2DG, and CT. Cells were treated with trypsin-EDTA to collect cells, which were then washed with phosphate buffered saline (PBS) , And the cells were fixed with 70% ethanol. Cells were fixed and stained with Guava cell cycle reagent containing propidium iodide. Cells stained with propidium iodide were measured by flow cytometry using a flow cytometer (Guava EasyCyte: Guava Technologies, Inc., Hayward, CA, USA) And analyzed using Guava CytoSoft version 2.5 software (Guava Technologies).

Reference Example  5: Western Blot  analysis( Western blot 분석 )

Each cancer cell was cultured for 8, 16, 24, and 40 hours in a 100 mM dish in the same manner as described above. Cell lysate was prepared by adding HMF, MET, 2DG, and CT in combination. Cell lysates were rinsed with cold phosphate buffered saline (PBS) and lysed in lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na ₃ VO ₄ ), and the mixture was stirred at 4 ° C for 40 minutes. The precipitate was removed by centrifugation at 12,000 xg for 30 minutes, and the supernatant was taken and used as a lysate.

Protein concentrations were measured using the BCA method (Pierce). 50 &lt; RTI ID = 0.0 &gt; pg &lt; / RTI &gt; of total protein was loaded into SDS-polyacrylamide gel containing Tris-glycine elution buffer. These proteins were electrophoresed using a Mini-Protein System (Bio-Rad) and electrophoresis was performed for 1 hour at 100 V using a nitrocellulose membrane (Bio-Rad) using an electrophoresis transfer cell : Bio-Rad). These nitrocellulose membranes were blocked with Tween 20 TBS (TBST) containing 5% skimmed milk for 30 minutes, added with antibodies to be measured in TBST containing 3% BSA, Or the mixture was stirred at room temperature for 2 hours. Horseradish peroxidase (Dako) was added and stirred for 1 hour. These membranes were then washed three times with TBST for five minutes each at room temperature, developed with an ECL Western blotting chemiluminescence system (Amersham / Pharmacia), and then transferred to an autoradiographic film (Hyperfilm ECL, Amersham ).

Reference Example  6: HMF , MET  or PHE , 2 DG   And CT Combination AMPK  Activation ability test

      AMPK is a phosphorylation enzyme that maintains energy and plays a pivotal role in the regulation of nutrient metabolism by increasing the activity of AMPK when the energy required for the survival of cancer cells becomes insufficient. In previous studies using Drosophila and cancer cells, it has been found that the activation of the AMPK gene reverts to abnormal normal cancer cell structure.

      AMPKα immunoassay kit (Invitrogen, catalog No. KHO0651) was used to investigate the effects of HMF, MET or PHE, 2DG and CT single and combination preparations on the AMPK activity of cancer cells. AMPKα (5'-AMP-activated protein kinase alpha) activation effect.

HepG2 cells derived from human liver were treated with HMF, MET or PHE, 2DG, and CT in the same manner as described above in the same manner as above, and the single preparation and combination preparation were cultured for 36 hours, The cell lysate was prepared by the method described in the description of the AMPKα immunoassay kit. After the cell lysate was obtained, the degree of phosphorylation of threonine 172 residue (Thr172) of AMPKa was confirmed from the cell lysate according to the method described in the instruction manual of the AMPKa immunoassay kit, and the results are shown (Figs. 77-78 ).

< Example  1> HMF , MET  Or 2 DG  And CT &Lt; / RTI &gt; &lt; RTI ID = 0.0 &gt;

Ten cell lines were used to compare cell proliferation inhibitory effects of HMF, MET or PHE, 2DG and CT combination.

Example  1-1: Liver cancer cell line ( HepG2 ) Survival inhibition effect

The cell proliferation inhibitory effect of HMG, an agonist (MET or PHE), a single agent of each of 2DG and CT, and a combination of two or more agents in HepG2 cell line derived from human liver was compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 1 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG and CT and a combination of two or more agents for 48 hours at a concentration according to the indicated dose in human HepG2 cell line derived from human liver, Effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 2 is a graph showing the effect of suppressing the survival rate of cancer cells after providing each single preparation of HMF, MET, 2DG and CT and two or more combinations of them in HepG2 cell line derived from human liver for 48 hours by a specific dose. It is. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 2, the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than the MET / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm ( mM ) m r HMF 2.99 1.60 0.98 MET 18.53 1.52 0.98 2 DG 1.16 1.95 0.97 CT 4.13 2.97 0.98 HMF / CT 1.63 / 1.40 1.75 1.00 MET /2 DG 9.03 / 0.36 1.50 1.00 HMF / MET /2 DG 0.78 / 5.58 / 0.22 1.52 1.00 HMF / MET / CT 0.87 / 6.21 / 0.75 1.55 1.00 MET /2 DG / CT 5.38 / 0.22 / 0.65 1.64 1.00 HMF / MET /2 DG / CT 0.44 / 3.16 / 0.13 / 0.38 1.64 1.00

Table 2 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.97 to 1.00, showing a high correlation between dose and effect. The m value ranged from 1.50 to 2.97, indicating that the dose - effect curves showed hyperbolic responses in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
3.5: 3 50 0.88 (synergistic effect) 1.83 - - 2.96 75 0.95 (additive action) 1.94 - - 2.29 90 1.05 (additive action) 2.06 - - 1.77
MET /2 DG
25: 1 50 0.80 (synergistic effect) - 2.05 3.22 - 75 0.86 (synergistic effect) - 2.03 2.72 - 90 0.93 (additive action) - 2.01 2.31 -
HMF / MET /2 DG
3.5: 25: 1 50 0.76 (synergistic effect) 3.82 3.32 5.21 - 75 0.80 (synergistic effect) 3.67 3.31 4.44 - 90 0.85 (synergistic effect) 3.53 3.30 3.79 -
HMF / MET / CT
3.5: 25: 3 50 0.81 (synergistic effect) 3.43 2.98 - 5.54 75 0.88 (synergistic effect) 3.35 3.02 - 3.95 90 0.99 (additive action) 3.27 3.05 - 2.81
MET /2 DG / CT
25: 1: 3 50 0.63 (synergistic effect) - 3.44 5.40 6.40 75 0.69 (synergistic effect) - 3.63 4.87 4.75 90 0.77 (synergistic effect) - 3.83 4.39 3.52
HMF / MET /2 DG / CT
3.5: 25: 1: 3 50 0.52 (synergistic effect) 6.75 5.86 9.20 10.89 75 0.54 (synergistic effect) 6.98 6.28 8.43 8.22 90 0.58 (synergistic effect) 7.21 6.73 7.73 6.20

Table 3 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75 and IC90 of the combination product containing two or more compounds according to the molar ratio of each drug in the HepG2 cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect in 50 ~ 90% cancer inhibition rate, HMF was 6.8 ~ 7.2 times, MET 5.9 ~ 6.7 times, 2DG 7.7 ~ 9.2 times, CT Showed 6.2 ~ 10.9 times of drug reduction effect, and the drug reduction index (DRI) was also the highest.

FIG. 3 is a graph of a fractionation effect-combination index (Fa-CI) graph of a HepG2 cell line treated with a combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.05, p < 0.01, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 4 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and a combination of two or more thereof for 48 hours at a concentration by the indicated dose in HepG2 cell line which is a cancer cell derived from human liver, &Lt; / RTI &gt; HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

FIG. 5 is a graph showing the effect of suppressing the survival rate of cancer cells after providing each single preparation of HMF, PHE, 2DG and CT and two or more combinations of them in a HepG2 cell line derived from human liver, It is. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 5, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm ( mM ) m r HMF 2.99 1.60 0.98 PHE 0.50 2.42 0.98 2 DG 1.16 1.95 0.97 CT 4.13 2.97 0.98 PHE /2 DG 0.24 / 0.30 1.95 1.00 HMF / PHE /2 DG 0.67 / 0.15 / 0.19 1.79 1.00 HMF / PHE / CT 0.76 / 0.17 / 0.65 1.96 1.00 PHE /2 DG / CT 0.15 / 0.19 / 0.56 2.01 1.00 HMF / PHE /2 DG / CT 0.28 / 0.07 / 0.08 / 0.24 1.93 1.00

Table 4 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a central-effect analysis using Calcusyn software for single and combination formulations of HMF, PHE, 2DG and CT. The r value ranged from 0.97 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.60 to 2.97, indicating that the dose - effect curves showed hyperbolic response in the compounds used alone or in combination.

The reduction of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / CTE / CT and HMF / PHE / 2DG / CT showed a lower inhibitory effect of 50% The highest inhibitory effect of 50% cancer inhibitory concentration was shown in the formulation.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF PHE 2 DG CT
PHE /2 DG
0.8: 1 50 0.74 (synergistic effect) - 2.09 3.87 - 75 0.79 (synergistic effect) - 1.87 3.87 - 90 0.86 (synergistic effect) - 1.68 3.87 -
HMF / PHE /2 DG
3.5: 0.8: 1 50 0.69 (synergistic effect) 4.46 3.28 6.08 - 75 0.74 (synergistic effect) 4.80 2.80 5.80 - 90 0.79 (synergistic effect) 5.16 2.40 5.53 -
HMF / PHE / CT
3.5: 0.8: 3 50 0.76 (synergistic effect) 3.93 2.89 - 6.34 75 0.80 (synergistic effect) 4.44 2.60 - 5.24 90 0.86 (synergistic effect) 5.03 2.34 - 4.33
PHE /2 DG / CT
0.8: 1: 3 50 0.59 (synergistic effect) - 3.38 6.26 7.41 75 0.64 (synergistic effect) - 3.08 6.36 6.20 90 0.70 (synergistic effect) - 2.80 6.47 5.19
HMF / PHE /2 DG / CT
3.5: 0.8: 1: 3 50 0.35 (synergistic effect) 10.48 7.71 14.28 16.92 75 0.37 (synergistic effect) 11.75 6.87 14.20 13.85 90 0.40 (synergistic effect) 13.17 6.12 14.12 11.33

Table 5 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75, and IC90 of combination products containing two or more compounds depending on the molar ratio of each drug in the HepG2 cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 10.5 ~ 13.2 times, PHE is 6.1 ~ 7.7 times, 2DG is 14.1 ~ 14.3 times, CT (11.3 ~ 16.9 times), and the drug reduction index (DRI) was the highest.

FIG. 6 shows the 50% cancer inhibitory concentration by PHE after 48 hours of a single and combination preparation consisting of HMF, PHE, 2DG, and CT in a HepG2 cell line derived from human liver, And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

FIG. 7 is an analysis of a HepG2 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 7 is a graph of a fractionation effect-combination index (Fa-CI) graph of a HepG2 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.05, p < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-2: Suppression of survival rate of lung cancer cell line (A549)

In the A549 cell line, which is a cancer cell derived from human lung, HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more Cell proliferation inhibitory effect was compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 8 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combinations thereof at a concentration of 48 hours according to the dose indicated in A549 cell line which is a cancer cell derived from a human lung, &Lt; / RTI &gt; The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 9 is a graph showing the effect of suppressing the survival rate of cancer cells after a single dose of each of HMF, MET, 2DG, and CT and a combination of two or more agents were administered to A549 cell lines derived from human lungs by a specific dose for 48 hours. It is. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 9, the combination of three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than MET / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 4.22 2.23 0.99 MET 34.26 2.20 0.97 2DG 1.83 1.98 0.90 CT 6.23 2.80 0.98 HMF / CT 1.64 / 3.61 2.04 1.00 MET / 2DG 18.08 / 0.54 1.92 1.00 HMF / MET / 2DG 0.57 / 9.18 / 0.28 1.84 1.00 HMF / MET / CT 0.71 / 11.32 / 1.56 1.91 1.00 MET /2 DG / CT 10.07 / 0.30 / 1.38 2.01 1.00 HMF / MET /2 DG / CT 0.27 / 4.31 / 0.13 / 0.59 1.99 1.00

Table 6 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET and 2DG. The r value ranged from 0.90 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.84 to 2.80, indicating that the dose - effect curve of the compounds used alone or in combination was almost hyperbolic.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
2.5: 5.5 50 0.97 (additive action) 2.57 - - 1.73 75 1.08 (additive action) 2.46 - - 1.49 90 1.20 (antagonism) 2.35 - - 1.29
MET /2 DG
40: 1.2 50 0.82 (synergistic effect) - 1.90 3.38 - 75 0.87 (synergistic effect) - 1.76 3.32 - 90 0.92 (additive action) - 1.64 3.26 -
HMF / MET /2 DG
2.5: 40: 1.2 50 0.55 (synergistic effect) 7.36 3.73 6.65 - 75 0.60 (synergistic effect) 6.63 3.39 6.38 - 90 0.66 (synergistic effect) 5.98 3.08 6.12 -
HMF / MET / CT
2.5: 40: 5.5 50 0.75 (synergistic effect) 5.97 3.03 - 4.00 75 0.84 (synergistic effect) 5.49 2.81 - 3.33 90 0.94 (additive action) 5.06 2.60 - 2.78
MET /2 DG / CT
0: 1.2: 5.5 50 0.68 (synergistic effect) - 3.40 6.06 4.50 75 0.73 (synergistic effect) - 3.25 6.11 3.86 90 0.79 (synergistic effect) - 3.10 6.16 3.30
HMF / MET /2 DG / CT
2.5: 40: 1.2: 5.5 50 0.35 (synergistic effect) 16.03 8.13 14.49 10.76 75 0.37 (synergistic effect) 15.11 7.72 14.54 9.17 90 0.40 (synergistic effect) 14.24 7.33 14.58 7.82

Table 7 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75, and IC90 of combination products containing two or more compounds according to the molar ratio of each drug in the A549 cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF was 14.2 ~ 16.0 times, MET was 7.3 ~ 8.1 times, 2DG was 14.5 ~ Was 7.8 ~ 10.8 times higher than the other drugs, and the drug reduction index (DRI) was also the highest.

FIG. 10 is an analysis of an A549 cell line treated with a combination preparation consisting of HMF, MET, 2DG, and CT for 48 hours in an Fa-CI graph. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

Fig. 11 is a graph showing the effect of the single agent and two or more combinations of HMF, PHE, 2DG and CT on the concentration by the indicated dose in the A549 cell line, which is a human lung-derived cancer cell, Time after the administration of the anticancer agent. HMF, PHE, 2DG, and CT, and combination of two or more combinations showed the highest inhibitory effect on cancer cell survival.

FIG. 12 is a graph showing the effect of suppressing the survival rate of cancer cells after a single dose of each of HMF, PHE, 2DG, and CT and a combination of two or more agents were administered to A549 cell lines derived from human lungs by a specific dose for 48 hours. It is. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 12, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm ( mM ) m r HMF 4.22 2.23 0.99 PHE 0.57 1.74 1.00 2 DG 1.83 1.98 0.90 CT 6.23 2.80 0.98 PHE /2 DG 0.27 / 0.54 1.61 1.00 HMF / PHE /2 DG 0.66 / 0.16 / 0.32 1.63 1.00 HMF / PHE / CT 0.74 / 0.18 / 1.64 1.96 1.00 PHE /2 DG / CT 0.13 / 0.27 / 1.22 1.70 1.00 HMF / PHE /2 DG / CT 0.22 / 0.05 / 0.11 / 0.48 1.81 1.00

Table 8 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a central-effect analysis using Calcusyn software for a single combination product of HMF, PHE, 2DG and CT. The r value ranged from 0.90 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.61 to 2.80, indicating that the dose-effect curve of the compounds used alone or in combination was almost hyperbolic.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF PHE 2 DG CT
PHE /2 DG
0.6: 1.2 50 0.77 (synergistic effect) - 2.11 3.40 - 75 0.83 (synergistic effect) - 2.00 2.99 - 90 0.91 (additive action) - 1.90 2.63 -
HMF / PHE /2 DG
2.5: 0.6: 1.2 50 0.61 (synergistic effect) 6.35 3.56 5.74 - 75 0.68 (synergistic effect) 5.31 3.41 5.11 - 90 0.75 (synergistic effect) 4.44 3.27 4.54 -
HMF / PHE / CT
2.5: 0.6: 5.5 50 0.75 (synergistic effect) 5.67 3.18 - 3.81 75 0.79 (synergistic effect) 5.31 3.41 - 3.22 90 0.84 (synergistic effect) 4.97 3.66 - 2.73
PHE /2 DG / CT
0.6: 1.2: 5.5 50 0.58 (synergistic effect) - 4.26 6.88 5.10 75 0.65 (synergistic effect) - 4.19 6.27 3.96 90 0.74 (synergistic effect) - 4.12 5.72 3.07
HMF / PHE /2 DG / CT
2.5: 0.6: 1.2: 5.5 50 0.28 (synergistic effect) 19.15 10.73 17.32 12.85 75 0.31 (synergistic effect) 17.12 11.01 16.47 10.39 90 0.34 (synergistic effect) 15.31 11.29 15.67 8.40

Table 9 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the A549 cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT, which is the combination of the four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 15.3 ~ 19.2 times, PHE is 10.7 ~ 11.3 times, 2DG is 15.7 ~ (8.4 ~ 12.9 times), and the drug reduction index (DRI) was the highest.

FIG. 13 shows the 50% cancer inhibitory concentration by PHE after 48 hours of the single and combination preparation consisting of HMF, PHE, 2DG, and CT was given to A549 cell line, which is a human lung-derived cancer cell, And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

14 is an analysis of A549 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 14 is a graph showing the Fraction-Effectiveness-Combination Index (Fa-CI) graph of the A549 cell line treated with the combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-3: Gastric cancer cell line ( AGS ) Survival inhibition effect

The cell proliferation inhibitory effect of a single preparation of HMF, an acetylated compound (MET or PHE), 2DG and CT, and a combination of two or more kinds of AGS cell lines derived from human cancer cells was compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 15 is a graph showing the results of inhibition of survival rate of cancer cells after providing each single preparation of HMF, MET, 2DG, and CT and two or more combinations of each agent by the indicated dose to the AGS cell line, FIG. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 16 shows the effect of suppressing the survival rate of cancer cells after 48 hours by providing a single preparation of HMF, MET, 2DG and CT and a combination of two or more kinds of AGS cell lines derived from human above, will be. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 16, the combination of three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than MET / 2DG combination exhibiting the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm ( mM ) m r HMF 2.98 1.83 0.97 MET 20.14 1.69 0.99 2 DG 0.68 1.65 0.99 CT 3.13 1.88 0.99 HMF / CT 1.14 / 1.50 1.62 1.00 MET /2 DG 9.21 / 0.25 1.55 1.00 HMF / MET /2 DG 0.53 / 5.64 / 0.15 1.55 1.00 HMF / MET / CT 0.62 / 6.60 / 0.81 1.57 1.00 MET /2 DG / CT 4.99 / 0.13 / 0.62 1.54 1.00 HMF / MET /2 DG / CT 0.27 / 2.90 / 0.08 / 0.37 1.58 1.00

Table 10 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a central-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.97 to 1.00, which showed a high correlation between dose and effect. The m value ranged from 1.54 to 1.88, indicating that the dose - effect curve of the compounds used alone or in combination was almost hyperbolic.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF MET 2DG CT
HMF / CT
2.8: 3.7 50 0.86 (synergistic effect) 2.63 - - 2.08 75 0.94 (additive action) 2.43 - - 1.90 90 1.02 (additive action) 2.25 - - 1.73
MET /2 DG
30: 0.8 50 0.82 (synergistic effect) - 2.19 2.78 - 75 0.86 (synergistic effect) - 2.06 2.66 - 90 0.91 (additive action) - 1.93 2.55 -
HMF / MET /2 DG
2.8: 30: 0.8 50 0.68 (synergistic effect) 5.66 3.57 4.53 - 75 0.73 (synergistic effect) 5.07 3.35 4.34 - 90 0.78 (synergistic effect) 4.54 3.15 4.16 -
HMF / MET / CT
2.8: 30: 3.7 50 0.79 (synergistic effect) 4.85 3.05 - 3.84 75 0.87 (synergistic effect) 4.38 2.90 - 3.43 90 0.94 (additive action) 3.97 2.76 - 3.05
MET /2 DG / CT
30: 0.8: 3.7 50 0.64 (synergistic effect) - 4.03 5.11 5.07 75 0.69 (synergistic effect) - 3.78 4.89 4.46 90 0.75 (synergistic effect) - 3.54 4.67 3.93
HMF / MET /2 DG / CT
2.8: 30: 0.8: 3.7 50 0.46 (synergistic effect) 11.01 6.94 8.80 8.73 75 0.50 (synergistic effect) 9.99 6.61 8.55 7.81 90 0.53 (synergistic effect) 9.07 6.30 8.31 6.98

Table 11 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75 and IC90 of the combination product containing two or more compounds according to the molar ratio of each drug in the AGS cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF was 9.1 ~ 11.0 times, MET was 6.3 ~ 6.9 times, 2DG was 8.3 ~ 8.8 times, CT Showed 7.0 ~ 8.7 times the drug reduction effect, and the drug reduction index (DRI) was also the highest.

FIG. 17 is an analysis of an AGS cell line treated with a combination preparation consisting of HMF, MET, 2DG, and CT for 48 hours in terms of a fractionation effect-combination index (Fa-CI) graph. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 18 is a graph showing the results of inhibition of survival rate of cancer cells after providing each single preparation of HMF, PHE, 2DG, and CT and two or more combinations of them by the concentration indicated in the AGS cell line, FIG. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

19 shows the effect of inhibiting the survival rate of cancer cells after 48 hours of providing a single preparation of HMF, PHE, 2DG and CT and a combination of two or more kinds of AGS cell lines derived from human above, will be. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 19, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 2.98 1.83 0.97 PHE 0.56 2.57 0.96 2DG 0.68 1.65 0.99 CT 3.13 1.88 0.99 PHE / 2DG 0.16 / 0.25 1.55 1.00 HMF / PHE / 2DG 0.47 / 0.08 / 0.13 1.49 1.00 HMF / PHE / CT 0.61 / 0.11 / 0.80 1.70 1.00 PHE /2 DG / CT 0.07 / 0.11 / 0.52 1.48 1.00 HMF / PHE /2 DG / CT 0.25 / 0.04 / 0.07 / 0.33 1.78 1.00

Table 12 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for HMF, PHE, 2DG and CT single and combination preparations. The r value ranged from 0.96 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.48 to 2.57, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF PHE 2 DG CT
PHE /2 DG
0.5: 0.8 50 0.65 (synergistic effect) - 3.54 2.70 - 75 0.76 (synergistic effect) - 2.67 2.60 - 90 0.90 (additive action) - 2.02 2.49 -
HMF / PHE /2 DG
2.8: 0.5: 0.8 50 0.50 (synergistic effect) 6.36 6.65 5.08 - 75 0.60 (synergistic effect) 5.55 4.89 4.75 - 90 0.71 (synergistic effect) 4.85 3.59 4.44 -
HMF / PHE / CT
2.8: 0.5: 3.7 50 0.66 (synergistic effect) 4.90 5.13 - 3.89 75 0.73 (synergistic effect) 4.68 4.12 - 3.66 90 0.82 (synergistic effect) 4.47 3.31 - 3.44
PHE /2 DG / CT
0.5: 0.8: 3.7 50 0.46 (synergistic effect) - 7.94 6.06 6.02 75 0.55 (synergistic effect) - 5.78 5.62 5.13 90 0.66 (synergistic effect) - 4.22 5.21 4.38
HMF / PHE /2 DG / CT
2.8: 0.5: 0.8: 3.7 50 0.37 (synergistic effect) 11.98 12.54 9.58 9.50 75 0.39 (synergistic effect) 11.76 10.36 10.06 9.19 90 0.41 (synergistic effect) 11.54 8.56 10.57 8.88

Table 13 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75, and IC90 of the combination product containing two or more compounds depending on the molar ratio of each drug in the AGS cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT, a combination of four compounds exhibiting the highest synergistic effect at 50-90% cancer inhibition rate, HMF was 11.5-12.0 times, PHE was 8.6-12.5 times, 2DG was 9.6-10.6 times, CT Showed a drug reduction effect of 8.9 to 9.5 times, and the drug reduction index (DRI) was also the highest.

FIG. 20 shows a 50% cancer inhibitory concentration by PHE after 48 hours of a single dose and a combination preparation consisting of HMF, PHE, 2DG, and CT were given to AGS cell line, which is a cancer cell from human, And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

21 is an analysis of an AGS cell line treated with HMF, PHE, 2DG, and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 21 is an analysis of an AGS cell line treated with a combination preparation consisting of HMF, PHE, 2DG, and CT for 48 hours in terms of a fractionation effect-combination index (Fa-CI) graph. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-4: Pancreatic cancer cell line ( Capan -2) to suppress the survival rate

The cell proliferation inhibitory effect of HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more compounds was compared in a Capan-2 cell line derived from a human pancreas.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

Figure 22 shows that each single formulation of HMF, MET, 2DG, and CT and two or more combinations are provided for 48 hours at a concentration indicated by Capan-2 cell line, which is a cancer cell derived from human pancreas, Showing the survival rate inhibiting effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

23 is a graph showing the results of inhibition of cancer cell viability after a single dose of each of HMF, MET, 2DG and CT and a combination of two or more agents were administered to a Capan-2 cell line derived from human pancreas, FIG. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

 In FIG. 23, the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than the MET / 2DG combination, which exhibited the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 5.97 3.04 0.98 MET 27.34 2.73 0.96 2DG 1.17 2.13 0.95 CT 4.77 2.85 0.99 HMF / CT 2.66 / 2.37 2.51 1.00 MET / 2DG 10.94 / 0.36 1.99 1.00 HMF / MET / 2DG 1.03 / 6.85 / 0.23 2.13 1.00 HMF / MET / CT 1.33 / 8.90 / 1.19 2.41 1.00 MET /2 DG / CT 6.55 / 0.22 / 0.87 2.11 1.00 HMF / MET /2 DG / CT 0.59 / 3.95 / 0.13 / 0.53 2.28 1.00

Table 14 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a central-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.95 to 1.00, showing a high correlation between dose and effect. The m value ranged from 1.99 to 3.04, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF MET 2DG CT
HMF / CT
4.5: 4 50 0.94 (additive action) 2.24 - - 2.01 75 1.00 (additive action) 2.08 - - 1.91 90 1.07 (additive action) 1.93 - - 1.82
MET /2 DG
30: 1 50 0.71 (synergistic effect) - 2.50 3.21 - 75 0.79 (synergistic effect) - 2.15 3.10 - 90 0.87 (synergistic effect) - 1.86 2.99 -
HMF / MET /2 DG
4.5: 30: 1 50 0.62 (synergistic effect) 5.81 3.99 5.13 - 75 0.68 (synergistic effect) 4.98 3.57 5.13 - 90 0.74 (synergistic effect) 4.28 3.19 5.13 -
HMF / MET / CT
4.5: 30: 4 50 0.80 (synergistic effect) 4.48 3.07 - 4.02 75 0.86 (synergistic effect) 4.07 2.91 - 3.75 90 0.92 (additive action) 3.71 2.76 - 3.49
MET /2 DG / CT
30: 1: 4 50 0.61 (synergistic effect) - 4.18 5.37 5.46 75 0.67 (synergistic effect) - 3.71 5.34 4.77 90 0.73 (synergistic effect) - 3.30 5.31 4.17
HMF / MET /2 DG / CT
4.5: 30: 1: 4 50 0.47 (synergistic effect) 10.08 6.92 8.90 9.05 75 0.50 (synergistic effect) 8.95 6.40 9.21 8.23 90 0.53 (synergistic effect) 7.94 5.92 9.53 7.48

Table 15 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the Capan-2 cell line .

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% inhibition of cancer, HMF is 7.9-10.1 times, MET is 5.9 ~ 6.9 times, 2DG is 8.9 ~ 9.5 times, CT Was 7.5 ~ 9.1 times more effective than other drugs (DRI).

FIG. 24 is a graph showing a Fraction-Effectiveness-Combination Index (Fa-CI) graph of Capan-2 cell line treated with combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 25 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products by the concentration indicated in the Capan-2 cell line, which is a cancer cell derived from human pancreas, Showing the survival rate inhibiting effect. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

26 is a graph showing the results of inhibition of cancer cell viability after a single dose of each of HMF, PHE, 2DG and CT and a combination of two or more agents were administered to a Capan-2 cell line derived from human pancreas, FIG. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 26, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 5.97 3.04 0.98 PHE 0.91 2.87 0.99 2DG 1.17 2.13 0.95 CT 4.77 2.85 0.99 PHE / 2DG 0.34 / 0.42 2.06 1.00 HMF / PHE / 2DG 1.05 / 0.19 / 0.23 2.11 1.00 HMF / PHE / CT 1.35 / 0.24 / 1.20 2.41 1.00 PHE / 2DG / CT 0.17 / 0.21 / 0.84 2.09 1.00 HMF / PHE /2 DG / CT 0.61 / 0.11 / 0.14 / 0.55 2.33 1.00

Table 16 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by the center-effect analysis using Calcusyn software for single and combination preparations of HMF, PHE, 2DG and CT. The r value ranged from 0.95 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 2.06 to 3.04, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
0.8: 1 50 0.72 (synergistic effect) - 2.72 2.80 - 75 0.79 (synergistic effect) - 2.34 2.75 - 90 0.87 (synergistic effect) - 2.01 2.70 -
HMF / PHE /2 DG
4.5: 0.8: 1 50 0.58 (synergistic effect) 5.67 4.86 5.01 - 75 0.64 (synergistic effect) 4.83 4.24 4.98 - 90 0.72 (synergistic effect) 4.12 3.69 4.95 -
HMF / PHE / CT
4.5: 0.8: 4 50 0.74 (synergistic effect) 4.42 3.79 - 3.97 75 0.80 (synergistic effect) 4.02 3.53 - 3.70 90 0.87 (synergistic effect) 3.67 3.28 - 3.46
PHE /2 DG / CT
0.8: 1: 4 50 0.54 (synergistic effect) - 5.42 5.58 5.67 75 0.60 (synergistic effect) - 4.71 5.53 4.94 90 0.66 (synergistic effect) - 4.09 5.47 4.30
HMF / PHE /2 DG / CT
4.5: 0.8: 1: 4 50 0.45 (synergistic effect) 9.74 8.36 8.60 8.75 75 0.48 (synergistic effect) 8.73 7.65 8.99 8.03 90 0.51 (synergistic effect) 7.82 7.01 9.38 7.37

Table 17 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the Capan-2 cell line .

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which have the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 7.8 ~ 9.7 times, PHE is 7.0 ~ 8.4 times, 2DG is 8.6 ~ 9.4 times, Was 7.4 ~ 8.8 times more effective than other drugs (DRI).

Figure 27 shows that a single and combination preparation consisting of HMF, PHE, 2DG, and CT was provided to Capan-2 cell line, which is a human pancreas-derived cancer cell, for 48 hours by a specific dose and then 50% This shows how much PHE can reduce drug toxicity. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

28 is an analysis of the Capan-2 cell line treated with HMF, PHE, 2DG, and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 28 is a graph showing a Fraction-effect-Combination Index (Fa-CI) graph of a Capan-2 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-5: Colorectal cancer cell line ( DLD -1) to suppress the survival rate

In the DLD-1 cell line derived from human colon, cell proliferation inhibitory effects of HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more compounds were compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 29 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combinations thereof at the concentration indicated by the DLD-1 cell line, which is a cancer cell derived from human colon, Showing the survival rate inhibiting effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 30 shows the results obtained after a single dose of each of HMF, MET, 2DG and CT and a combination of two or more thereof were given to a DLD-1 cell line derived from human colon cancer cells by a specific dose for 48 hours, FIG. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 30, the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than the MET / 2DG combination exhibiting the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm ( mM ) m r HMF 4.29 2.43 0.99 MET 29.25 1.64 0.99 2 DG 5.46 1.06 0.99 CT 4.59 2.64 0.99 HMF / CT 2.07 / 2.07 2.22 1.00 MET /2 DG 14.00 / 1.57 1.30 1.00 HMF / MET /2 DG 0.88 / 10.02 / 1.13 1.53 1.00 HMF / MET / CT 1.00 / 11.30 / 0.99 1.85 1.00 MET /2 DG / CT 8.76 / 0.99 / 0.77 1.52 1.00 HMF / MET /2 DG / CT 0.41 / 4.64 / 0.52 / 0.41 1.66 1.00

Table 18 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.99 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.06 to 2.64, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
3.5: 3.5 50 0.94 (additive action) 2.07 - - 2.22 75 0.99 (additive action) 1.98 - - 2.05 90 1.05 (additive action) 1.90 - - 1.90
MET /2 DG
40: 4.5 50 0.77 (synergistic effect) - 2.09 3.47 - 75 0.81 (synergistic effect) - 1.75 4.20 - 90 0.88 (synergistic effect) - 1.46 5.09 -
HMF / MET /2 DG
3.5: 40: 4.5 50 0.75 (synergistic effect) 4.89 2.92 4.84 - 75 0.78 (synergistic effect) 3.74 2.78 6.68 - 90 0.84 (synergistic effect) 2.86 2.65 9.21 -
HMF / MET / CT
3.5: 40: 3.5 50 0.83 (synergistic effect) 4.34 2.59 - 4.64 75 0.88 (synergistic effect) 3.77 2.80 - 3.90 90 0.94 (additive action) 3.27 3.02 - 3.27
MET /2 DG / CT
40: 4.5: 3.5 50 0.65 (synergistic effect) - 3.34 5.53 5.99 75 0.68 (synergistic effect) - 3.16 7.60 4.40 90 0.74 (synergistic effect) - 3.00 10.43 3.24
HMF / MET /2 DG / CT
3.5: 40: 4.5: 3.5 50 0.44 (synergistic effect) 10.56 6.31 10.45 11.31 75 0.45 (synergistic effect) 8.57 6.37 15.30 8.87 90 0.49 (synergistic effect) 6.96 6.43 22.38 6.95

Table 19 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combination form of two or more compounds depending on the molar ratio of each drug in the DLD-1 cell line .

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 7.0 ~ 10.6 times, MET is 6.3 ~ 6.4 times, 2DG is 10.5 ~ Was 7.0 ~ 11.3 times more effective in reducing the drug, and the drug reduction index (DRI) was also the highest.

FIG. 31 is a graph showing a fractional effect-combination index (Fa-CI) graph of a DLD-1 cell line treated with a combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.05, p < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 32 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products at the concentrations indicated in the DLD-1 cell line, which is a cancer cell derived from human colon, Showing the survival rate inhibiting effect. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

FIG. 33 is a graph showing the results of inhibition of cancer cell viability after a single dose of each of HMF, PHE, 2DG, and CT and a combination of two or more agents were administered to DLD-1 cell line derived from human colon, FIG. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 33, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 4.29 2.43 0.99 PHE 0.72 1.69 0.99 2DG 5.46 1.06 0.99 CT 4.59 2.64 0.99 PHE / 2DG 0.29 / 1.64 1.22 1.00 HMF / PHE / 2DG 0.71 / 0.16 / 0.91 1.45 1.00 HMF / PHE / CT 0.96 / 0.22 / 0.96 1.90 1.00 PHE / 2DG / CT 0.15 / 0.85 / 0.66 1.49 1.00 HMF / PHE / 2DG / CT 0.40 / 0.09 / 0.52 / 0.40 1.65 1.00

Table 20 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for HMF, PHE, 2DG and CT single and combination preparations. The r value ranged from 0.99 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.06 to 2.64, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
0.8: 4.5 50 0.70 (synergistic effect) - 2.49 3.33 - 75 0.78 (synergistic effect) - 1.93 3.82 - 90 0.90 (additive action) - 1.49 4.38 -
HMF / PHE /2 DG
3.5: 0.8: 4.5 50 0.55 (synergistic effect) 6.08 4.49 6.02 - 75 0.60 (synergistic effect) 4.48 4.03 7.99 - 90 0.67 (synergistic effect) 3.30 3.61 10.62 -
HMF / PHE / CT
3.5: 0.8: 3.5 50 0.74 (synergistic effect) 4.45 3.29 - 4.77 75 0.78 (synergistic effect) 3.93 3.53 - 4.06 90 0.84 (synergistic effect) 3.46 3.79 - 3.46
PHE /2 DG / CT
0.8: 4.5: 3.5 50 0.51 (synergistic effect) - 4.80 6.43 6.96 75 0.54 (synergistic effect) - 4.39 8.71 5.05 90 0.61 (synergistic effect) - 4.01 11.79 3.66
HMF / PHE /2 DG / CT
3.5: 0.8: 4.5: 3.5 50 0.41 (synergistic effect) 10.60 7.83 10.50 11.36 75 0.43 (synergistic effect) 8.56 7.69 15.27 8.85 90 0.47 (synergistic effect) 6.91 7.56 22.21 6.90

Table 21 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75 and IC90 of the combination product containing two or more compounds depending on the molar ratio of each drug in the DLD-1 cell line .

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which have the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 6.9 ~ 10.6 times, PHE is 7.6 ~ 7.8 times, 2DG is 10.5 ~ (6.9 ~ 11.4 times), and the drug reduction index (DRI) was the highest.

Figure 34 shows that a single and combination preparation consisting of HMF, PHE, 2DG, and CT was provided for 48 hours in a DLD-1 cell line, which is a cancer cell derived from human colon, by a specific dose, and then 50% This shows how much PHE can reduce drug toxicity. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

35 is an analysis of the DLD-1 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 35 is a graph of a fractional effect-combination index (Fa-CI) graph of a DLD-1 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P <0.01, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-6: Cervical cancer cell line ( HeLa ) Survival inhibition effect

The cell proliferation inhibitory effect of HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more kinds of compounds was compared in a HeLa cell line derived from a human cervix.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 36 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combinations thereof for 48 hours at a concentration by the indicated dose in a HeLa cell line derived from a human cervix, Inhibitory effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 37 shows the results of inhibiting cancer cell survival rate after 48 hours of providing a single dose of HMF, MET, 2DG and CT and a combination of two or more agents to a HeLa cell line derived from a human cervix, Respectively. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 37, the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than the MET / 2DG combination exhibiting the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 3.74 2.19 0.99 MET 30.91 2.51 0.95 2DG 1.45 1.92 0.93 CT 8.11 2.41 0.98 HMF / CT 1.67 / 3.89 2.04 1.00 MET / 2DG 14.30 / 0.48 2.02 1.00 HMF / MET / 2DG 0.84 / 8.44 / 0.28 1.93 1.00 HMF / MET / CT 0.91 / 9.10 / 2.12 2.04 1.00 MET / 2DG / CT 7.68 / 0.26 / 1.79 1.99 1.00 HMF / MET /2 DG / CT 0.38 / 3.78 / 0.13 / 0.88 1.99 1.00

Table 22 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.93 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.92 to 2.51, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT rather than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF MET 2DG CT
HMF / CT
3: 7 50 0.93 (additive action) 2.24 - - 2.09 75 0.98 (additive action) 2.16 - - 1.92 90 1.05 (additive action) 2.08 - - 1.77
MET /2 DG
30: 1 50 0.79 (synergistic effect) - 2.16 3.04 - 75 0.84 (synergistic effect) - 1.94 3.13 - 90 0.88 (synergistic effect) - 1.74 3.23 -
HMF / MET /2 DG
3: 30: 1 50 0.69 (synergistic effect) 4.43 3.66 5.15 - 75 0.75 (synergistic effect) 4.14 3.21 5.17 - 90 0.81 (synergistic effect) 3.86 2.81 5.19 -
HMF / MET / CT
3: 30: 7 50 0.80 (synergistic effect) 4.11 3.40 - 3.82 75 0.86 (synergistic effect) 3.96 3.07 - 3.52 90 0.93 (additive action) 3.82 2.77 - 3.24
MET /2 DG / CT
30: 1: 7 50 0.65 (synergistic effect) - 4.02 5.66 4.53 75 0.70 (synergistic effect) - 3.58 5.78 4.11 90 0.75 (synergistic effect) - 3.19 5.91 3.73
HMF / MET /2 DG / CT
3: 30: 1: 7 50 0.42 (synergistic effect) 9.91 8.19 11.52 9.21 75 0.45 (synergistic effect) 9.41 7.29 11.76 8.35 90 0.48 (synergistic effect) 8.93 6.49 12.01 7.57

Table 23 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, and IC90 of the combination product containing two or more compounds depending on the molar ratio of each drug in the HeLa cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of the four compounds showing the highest synergistic effect at 50-90% cancer inhibition rate, HMF was 8.9-9.9 times, MET was 6.5-8.2 times, 2DG was 11.5-12.0 times, CT Was 7.6 ~ 9.2 times more effective than other drugs, and the drug reduction index (DRI) was also the highest.

FIG. 38 is a graph showing a Fractional Effect-Combination Index (Fa-CI) graph of a HeLa cell line treated with a combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 39 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products for 48 hours according to the concentration indicated in the HeLa cell line, which is a cancer cell derived from a human cervix, Inhibitory effect. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

FIG. 40 shows the effect of inhibiting the survival rate of cancer cells after providing a single preparation of HMF, PHE, 2DG, and CT and two or more combinations of them in a HeLa cell line derived from a human cervix, Respectively. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 40, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 3.74 2.19 0.99 PHE 0.60 2.88 0.98 2DG 1.45 1.92 0.93 CT 8.11 2.41 0.98 PHE / 2DG 0.18 / 0.36 1.82 1.00 HMF / PHE / 2DG 0.64 / 0.11 / 0.21 1.82 1.00 HMF / PHE / CT 0.81 / 0.14 / 1.90 2.02 1.00 PHE / 2DG / CT 0.10 / 0.19 / 1.35 1.87 1.00 HMF / PHE / 2DG / CT 0.36 / 0.06 / 0.12 / 0.83 2.04 1.00

Table 24 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by the center-effect analysis using Calcusyn software for single and combination formulations of HMF, PHE, 2DG and CT. The r value ranged from 0.93 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.82 to 2.88, indicating that the dose - effect curve was almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
0.5: 1 50 0.55 (synergistic effect) - 3.29 4.00 - 75 0.64 (synergistic effect) - 2.63 3.87 - 90 0.74 (synergistic effect) - 2.10 3.75 -
HMF / PHE /2 DG
3: 0.5: 1 50 0.50 (synergistic effect) 5.83 5.58 6.77 - 75 0.57 (synergistic effect) 5.26 4.47 6.58 - 90 0.65 (synergistic effect) 4.75 3.58 6.39 -
HMF / PHE / CT
3: 0.5: 7 50 0.68 (synergistic effect) 4.60 4.40 - 4.27 75 0.75 (synergistic effect) 4.40 3.74 - 3.91 90 0.83 (synergistic effect) 4.21 3.17 - 3.57
PHE /2 DG / CT
0.5: 1: 7 50 0.46 (synergistic effect) - 6.18 7.50 6.00 75 0.52 (synergistic effect) - 5.03 7.41 5.26 90 0.60 (synergistic effect) - 4.10 7.32 4.62
HMF / PHE /2 DG / CT
3: 0.5: 1: 7 50 0.38 (synergistic effect) 10.53 10.08 12.24 9.79 75 0.40 (synergistic effect) 10.14 8.61 12.68 9.00 90 0.44 (synergistic effect) 9.77 7.36 13.13 8.28

Table 25 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75 and IC90 of a combination product containing two or more compounds according to the molar ratio of each drug in the HeLa cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which have the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 9.8 ~ 10.5 times, PHE is 7.4 ~ 10.1 times, 2DG is 12.2 ~ 13.1 times, CT Showed 8.3 ~ 9.8 times of drug reduction effect and the drug reduction index (DRI) was the highest.

41 is a graph showing the results of a single dose and a combination preparation consisting of HMF, PHE, 2DG, and CT in a HeLa cell line derived from a human cervix, 48 hours by a specific dose, And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

Figure 42 is an analysis of the HeLa cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 42 is a graph showing a Fraction-effect-Combination Index (Fa-CI) graph of a HeLa cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P <0.01, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-7: Breast cancer cell line ( MCF7 ) Survival inhibition effect

The cell proliferation inhibitory effect of HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more compounds in the MCF7 cell line derived from a human breast was compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

43 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combination products by the indicated dose in the MCF7 cell line, which is a cancer cell derived from human breast, for 48 hours by concentration, &Lt; / RTI &gt; The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

44 is a graph showing the effect of suppressing survival rate of cancer cells in MCF7 cell line, which is a cancer cell derived from human breast, for 48 hours by a single dose of HMF, MET, 2DG and CT, It is. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 44, the combination of three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than MET / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 4.39 2.47 0.99 MET 21.15 2.36 0.97 2DG 6.85 1.47 0.95 CT 4.66 2.67 0.99 HMF / CT 2.23 / 1.98 2.25 1.00 MET / 2DG 10.32 / 1.90 1.71 1.00 HMF / MET / 2DG 0.91 / 5.06 / 0.93 1.65 1.00 HMF / MET / CT 1.18 / 6.57 / 1.05 2.10 1.00 MET / 2DG / CT 5.54 / 1.02 / 0.89 1.73 1.00 HMF / MET / 2DG / CT 0.57 / 3.14 / 0.58 / 0.50 1.95 1.00

Table 26 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by the center-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.95 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 11.47 to 2.67, indicating that the dose - effect curves showed hyperbolic response in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
4.5: 4 50 0.93 (additive action) 1.97 - - 2.35 75 0.99 (additive action) 1.89 - - 2.18 90 1.05 (additive action) 1.81 - - 2.02
MET /2 DG
25: 4.6 50 0.77 (synergistic effect) - 2.05 3.61 - 75 0.83 (synergistic effect) - 1.72 4.01 - 90 0.92 (additive action) - 1.44 4.45 -
HMF / MET /2 DG
4.5: 25: 4.6 50 0.58 (synergistic effect) 4.83 4.18 7.36 - 75 0.68 (synergistic effect) 3.86 3.42 7.99 - 90 0.80 (synergistic effect) 3.09 2.80 8.67 -
HMF / MET / CT
4.5: 25: 4 50 0.81 (synergistic effect) 3.72 3.22 - 4.44 75 0.87 (synergistic effect) 3.44 3.04 - 3.98 90 0.94 (additive action) 3.18 2.88 - 3.56
MET /2 DG / CT
25: 4.6: 4 50 0.60 (synergistic effect) - 3.82 6.72 5.26 75 0.68 (synergistic effect) - 3.22 7.52 4.21 90 0.79 (synergistic effect) - 2.71 8.42 3.36
HMF / MET /2 DG / CT
4.5: 25: 4.6: 4 50 0.47 (synergistic effect) 7.76 6.73 11.84 9.27 75 0.51 (synergistic effect) 6.89 6.10 14.25 7.97 90 0.55 (synergistic effect) 6.12 5.53 17.16 6.85

Table 27 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75 and IC90 of the combination product containing two or more compounds depending on the molar ratio of each drug in the MCF7 cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF was 6.1-7.8 times, MET was 5.5 ~ 6.7 times, 2DG was 11.8 ~ 17.2 times, CT Was 6.9 ~ 9.3 times higher than the other drugs, and the drug reduction index (DRI) was also the highest.

FIG. 45 is an analysis of the MCF7 cell line treated with the combination preparation consisting of HMF, MET, 2DG and CT for 48 hours in terms of a fractionation effect-combination index (Fa-CI) graph. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

46 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products for 48 hours according to the concentration indicated in the MCF7 cell line, which is a cancer cell derived from human breast, for 48 hours, &Lt; / RTI &gt; HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

47 is a graph showing the effect of suppressing survival rate of cancer cells after providing a single preparation of HMF, PHE, 2DG, and CT and two or more combinations of them in a MCF7 cell line derived from a human breast for 48 hours, It is. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 47, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition rate among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 4.39 2.47 0.99 PHE 0.71 1.86 0.99 2 DG 6.85 1.47 0.95 CT 4.66 2.67 0.99 PHE / 2DG 0.40 / 2.06 1.58 1.00 HMF / PHE / 2DG 1.11 / 0.22 / 1.13 1.69 1.00 HMF / PHE / CT 1.13 / 0.23 / 1.00 2.01 1.00 PHE / 2DG / CT 0.24 / 1.25 / 1.09 1.73 1.00 HMF / PHE / 2DG / CT 0.63 / 0.13 / 0.64 / 0.56 1.93 1.00

Table 28 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by the center-effect analysis using Calcusyn software for single and combination formulations of HMF, PHE, 2DG and CT. The r values ranged from 0.95 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.47 to 2.67, indicating that the dose-effect curves were almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
0.9: 4.6 50 0.87 (synergistic effect) - 1.76 3.33 - 75 0.92 (additive action) - 1.59 3.51 - 90 0.97 (additive action) - 1.43 3.70 -
HMF / PHE /2 DG
4.5: 0.9: 4.6 50 0.73 (synergistic effect) 3.97 3.20 6.05 - 75 0.79 (synergistic effect) 3.22 3.01 6.67 - 90 0.87 (synergistic effect) 2.62 2.84 7.35 -
HMF / PHE / CT
4.5: 0.9: 4 50 0.79 (synergistic effect) 3.90 3.14 - 4.65 75 0.84 (synergistic effect) 3.51 3.28 - 4.06 90 0.89 (synergistic effect) 3.17 3.43 - 3.54
PHE /2 DG / CT
0.9: 4.6: 4 50 0.76 (synergistic effect) - 2.90 5.47 4.28 75 0.82 (synergistic effect) - 2.77 6.13 3.43 90 0.89 (synergistic effect) - 2.65 6.86 2.74
HMF / PHE /2 DG / CT
4.5: 0.9: 4.6: 4 50 0.53 (synergistic effect) 7.01 5.65 10.69 8.37 75 0.55 (synergistic effect) 6.19 5.79 12.80 7.16 90 0.58 (synergistic effect) 5.47 5.93 15.34 6.13

Table 29 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, and IC90 of the combination product containing two or more compounds according to the molar ratio of each drug in the MCF7 cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which have the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 5.5 ~ 7.0 times, PHE is 5.7 ~ 5.9 times, 2DG is 10.7 ~ 15.3 times, CT Showed 6.1 ~ 8.4 times of drug reduction effect and the drug reduction index (DRI) was the highest.

Figure 48 shows the 50% cancer inhibitory concentration by PHE after 48 hours of the single and combination preparation consisting of HMF, PHE, 2DG, and CT were given to MCF7 cell line, which is a cancer cell from human breast, by specific dose for 48 hours And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of quadruplets showed the highest effect among all combinations

49 is an analysis of an MCF7 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 49 is a graph showing a fractionation effect-combination index (Fa-CI) graph of an MCF7 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-8: Prostate cancer cell line ( PC -3) to suppress survival rate

The cell proliferation inhibitory effects of HMF, an acetylated compound (MET or PHE), a single preparation of 2DG and CT, and a combination of two or more compounds in the PC-3 cell line derived from a human prostate gland were compared.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

50 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combination products by the indicated dose in the PC-3 cell line which is a cancer cell derived from human prostate for 48 hours, Showing the survival rate inhibiting effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

51 is a graph showing the results of inhibition of survival rate of cancer cells after providing each single preparation of HMF, MET, 2DG and CT and two or more combinations of them in a PC-3 cell line derived from human prostate cancer cells by a specific dose for 48 hours, FIG. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 51, the combination of three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than MET / 2DG combination exhibiting the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 5.09 2.87 0.96 MET 31.24 1.65 0.99 2DG 4.59 1.55 0.97 CT 6.71 1.89 0.98 HMF / CT 1.90 / 3.75 1.98 1.00 MET / 2DG 19.88 / 1.06 1.49 1.00 HMF / MET / 2DG 0.95 / 12.98 / 0.69 1.71 1.00 HMF / MET / CT 0.93 / 12.74 / 1.84 1.78 1.00 MET / 2DG / CT 13.04 / 0.70 / 1.88 1.58 1.00 HMF / MET / 2DG / CT 0.54 / 7.37 / 0.39 / 1.07 1.78 1.00

Table 30 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r value ranged from 0.96 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.49 to 2.87, indicating that the dose - effect curve of the compounds used alone or in combination was almost hyperbolic.

The reduction of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
3.3: 6.5 50 0.93 (additive action) 2.68 - - 1.79 75 0.99 (additive action) 2.25 - - 1.84 90 1.06 (additive action) 1.89 - - 1.88
MET /2 DG
45: 2.4 50 0.87 (synergistic effect) - 1.57 4.33 - 75 0.92 (additive action) - 1.46 4.20 - 90 0.98 (additive action) - 1.36 4.07 -
HMF / MET /2 DG
3.3: 45: 2.4 50 0.75 (synergistic effect) 5.35 2.41 6.63 - 75 0.79 (synergistic effect) 4.12 2.46 7.08 - 90 0.84 (synergistic effect) 3.18 2.52 7.56 -
HMF / MET / CT
3.3: 45: 6.5 50 0.87 (synergistic effect) 5.45 2.45 - 3.64 75 0.90 (synergistic effect) 4.31 2.58 - 3.52 90 0.96 (additive action) 3.41 2.71 - 3.40
MET /2 DG / CT
45: 2.4: 6.5 50 0.85 (synergistic effect) - 2.40 6.60 3.56 75 0.90 (additive action) - 2.32 6.68 3.17 90 0.95 (additive action) - 2.25 6.76 2.82
HMF / MET /2 DG / CT
3.3: 45: 2.4: 6.5 50 0.59 (synergistic effect) 9.42 4.24 11.68 6.30 75 0.60 (synergistic effect) 7.46 4.46 12.81 6.08 90 0.62 (synergistic effect) 5.90 4.69 14.06 5.87

Table 31 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the PC-3 cell line .

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF was 5.9 ~ 9.4 times, MET was 4.2 ~ 4.7 times, 2DG was 11.7 ~ 14.1 times, CT Was 5.9 ~ 6.3 times higher than other drugs, and the drug reduction index (DRI) was also the highest.

FIG. 52 is a graph showing a Fraction-Effectiveness-Combination Index (Fa-CI) graph of a PC-3 cell line treated with the combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.01, p < 0.05, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 53 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products by the indicated dose in the PC-3 cell line, which is a cancer cell derived from human prostate, Showing the survival rate inhibiting effect. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

54 is a graph showing the results of inhibition of survival rate of cancer cells after providing a single preparation of HMF, PHE, 2DG, and CT and two or more combinations of them in a PC-3 cell line derived from a human prostate- FIG. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 54, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than the PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.01, p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 5.09 2.87 0.96 PHE 2.11 2.74 0.98 2DG 4.59 1.55 0.97 CT 6.71 1.89 0.98 PHE / 2DG 1.09 / 1.63 1.91 1.00 HMF / PHE / 2DG 1.35 / 0.66 / 0.98 2.07 1.00 HMF / PHE / CT 1.22 / 0.59 / 2.39 2.20 1.00 PHE / 2DG / CT 0.59 / 0.88 / 2.39 1.83 1.00 HMF / PHE / 2DG / CT 0.72 / 0.35 / 0.52 / 1.42 2.11 1.00

Table 32 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by a median-effect analysis using Calcusyn software for single and combination formulations of HMF, PHE, 2DG and CT. The r values ranged from 0.96 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.55 to 2.87, indicating that the dose - effect curves were almost hyperbolic in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
1.6: 2.4 50 0.87 (synergistic effect) - 1.95 2.82 - 75 0.92 (additive action) - 1.63 3.22 - 90 1.00 (additive action) - 1.37 3.67 -
HMF / PHE /2 DG
3.3: 1.6: 2.4 50 0.79 (synergistic effect) 3.76 3.22 4.66 - 75 0.84 (synergistic effect) 3.24 2.83 5.57 - 90 0.91 (additive action) 2.80 2.48 6.66 -
HMF / PHE / CT
3.3: 1.6: 6.5 50 0.88 (synergistic effect) 4.19 3.58 - 2.80 75 0.91 (additive action) 3.73 3.25 - 3.04 90 0.95 (additive action) 3.31 2.94 - 3.29
PHE /2 DG / CT
1.6: 2.4: 6.5 50 0.83 (synergistic effect) - 3.59 5.21 2.81 75 0.88 (synergistic effect) - 2.95 5.81 2.76 90 0.94 (additive action) - 2.42 6.48 2.71
HMF / PHE /2 DG / CT
3.3: 1.6: 2.4: 6.5 50 0.63 (synergistic effect) 7.08 6.05 8.77 4.73 75 0.64 (synergistic effect) 6.16 5.37 10.58 5.02 90 0.66 (synergistic effect) 5.36 4.76 12.76 5.33

Table 33 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the PC-3 cell line .

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which had the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF was 5.4 ~ 7.1 times, PHE 4.8 ~ 6.1 times, 2DG 8.8 ~ 12.8 times, CT Showed a 4.7 ~ 5.3 times decrease in drug, and the drug reduction index (DRI) was also the highest.

Figure 55 shows that a single and combination formulation consisting of HMF, PHE, 2DG, and CT was provided to a PC-3 cell line, which is a human prostate-derived cancer cell, for 48 hours by a specific dose, followed by 50% This shows how much PHE can reduce drug toxicity. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

56 is an analysis of the PC-3 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 56 is a graph of a fractionation effect-combination index (Fa-CI) graph of a PC-3 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-9: Ovarian cancer cell line ( SK - OV -3) to suppress survival rate

The cell proliferation inhibitory effect of HMF, a biguanide compound (MET or PHE), a single preparation of each of 2DG and CT, and a combination of two or more kinds was compared in the SK-OV-3 cell line derived from human ovary.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

57 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combinations thereof for 48 hours at a concentration by the indicated dose in a SK-OV-3 cell line which is a cancer cell derived from human ovary , Cancer cell survival rate inhibition effect. The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

FIG. 58 is a graph showing the results obtained by providing a single preparation of each of HMF, MET, 2DG and CT and two or more combinations thereof in a SK-OV-3 cell line which is a cancer cell derived from a human ovary for 48 hours, Inhibitory effect. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 58, the combination of three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than MET / 2DG combination exhibiting the highest tumor suppression rate among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 2.64 1.65 0.99 MET 30.00 1.76 0.98 2DG 1.21 1.25 0.97 CT 2.64 1.87 0.97 HMF / CT 1.52 / 0.95 1.58 1.00 MET / 2DG 15.28 / 0.38 1.39 1.00 HMF / MET / 2DG 0.82 / 8.25 / 0.21 1.49 1.00 HMF / MET / CT 0.89 / 8.89 / 0.56 1.59 1.00 MET / 2DG / CT 8.93 / 0.22 / 0.56 1.45 1.00 HMF / MET / 2DG / CT 0.45 / 4.52 / 0.11 / 0.28 1.54 1.00

Table 34 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r values ranged from 0.97 to 1.00, showing a high correlation between dose and effect. The m value ranged from 1.25 to 1.87, indicating that the dose - effect curves were almost hyperbolic in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
4: 2.5 50 0.94 (additive action) 1.73 - - 2.77 75 0.99 (additive action) 1.69 - - 2.50 90 1.06 (additive action) 1.64 - - 2.25
MET /2 DG
40: 1 50 0.83 (synergistic effect) - 1.96 3.17 - 75 0.89 (synergistic effect) - 1.66 3.47 - 90 0.97 (additive action) - 1.41 3.78 -
HMF / MET /2 DG
4: 40: 1 50 0.76 (synergistic effect) 3.20 3.64 5.88 - 75 0.79 (synergistic effect) 2.98 3.25 6.77 - 90 0.83 (synergistic effect) 2.78 2.91 7.80 -
HMF / MET / CT
4: 40: 2.5 50 0.84 (synergistic effect) 2.97 3.38 - 4.75 75 0.90 (synergistic effect) 2.89 3.15 - 4.28 90 0.96 (additive action) 2.81 2.93 - 3.86
MET /2 DG / CT
40: 1: 2.5 50 0.69 (synergistic effect) - 3.36 5.43 4.73 75 0.76 (synergistic effect) - 2.93 6.11 3.99 90 0.83 (synergistic effect) - 2.56 6.87 3.37
HMF / MET /2 DG / CT
4: 40: 1: 2.5 50 0.52 (synergistic effect) 5.84 6.65 10.74 9.35 75 0.55 (synergistic effect) 5.57 6.07 12.64 8.26 90 0.57 (synergistic effect) 5.31 5.55 14.88 7.29

Table 35 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75 and IC90 of the combination product containing two or more compounds according to the molar ratio of each drug in the SK-OV-3 cell line Respectively.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of the four compounds showing the highest synergistic effect in 50 ~ 90% cancer inhibition rate, HMF was 5.3 to 5.8 times, MET was 5.6 to 6.7 times, 2DG was 10.7 to 14.9 times, CT Showed 7.3 ~ 9.4 times of drug reduction effect and the drug reduction index (DRI) was the highest.

FIG. 59 shows the SK-OV-3 cell line treated with the combination preparation consisting of HMF, MET, 2DG, and CT for 48 hours in terms of a fractionation effect-combination index (Fa-CI) graph. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P < 0.05, p < 0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

(2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 60 shows the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and a combination of two or more agents by concentration at the indicated dose in the SK-OV-3 cell line, which is a cancer cell derived from human ovary, , Cancer cell survival rate inhibition effect. HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

FIG. 61 shows the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products for 48 hours in a SK-OV-3 cell line, which is a cancer cell derived from human ovary, Inhibitory effect. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 61, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 2.64 1.65 0.99 PHE 0.54 1.80 0.90 2DG 1.21 1.25 0.97 CT 2.64 1.87 0.97 PHE / 2DG 0.27 / 0.34 1.44 1.00 HMF / PHE / 2DG 0.73 / 0.15 / 0.18 1.45 1.00 HMF / PHE / CT 0.79 / 0.16 / 0.50 1.57 1.00 PHE / 2DG / CT 0.15 / 0.19 / 0.48 1.47 1.00 HMF / PHE / 2DG / CT 0.41 / 0.08 / 0.10 / 0.26 1.46 1.00

Table 36 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, PHE, 2DG and CT. The r value ranged from 0.90 to 1.00, showing a high correlation between dose and effect. The m value ranged from 1.25 to 1.87, indicating that the dose - effect curve of the compounds used alone or in combination was almost hyperbolic.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug Reduction Index (DRI) HMF PHE 2DG CT
PHE /2 DG
0.8: 1 50 0.79 (synergistic effect) - 1.96 3.54 - 75 0.85 (synergistic effect) - 1.68 3.95 - 90 0.92 (additive action) - 1.44 4.41 -
HMF / PHE /2 DG
4: 0.8: 1 50 0.70 (synergistic effect) 3.62 3.68 6.64 - 75 0.75 (synergistic effect) 3.29 3.17 7.47 - 90 0.82 (synergistic effect) 3.00 2.74 8.40 -
HMF / PHE / CT
4: 0.8: 2.5 50 0.78 (synergistic effect) 3.33 3.39 - 5.32 75 0.84 (synergistic effect) 3.22 3.10 - 4.77 90 0.91 (synergistic effect) 3.11 2.84 - 4.28
PHE /2 DG / CT
0.8: 1: 2.5 50 0.63 (synergistic effect) - 3.51 6.32 5.50 75 0.68 (synergistic effect) - 3.05 7.18 4.69 90 0.75 (synergistic effect) - 2.65 8.15 3.99
HMF / PHE /2 DG / CT
4: 0.8: 1: 2.5 50 0.49 (synergistic effect) 6.42 6.54 11.79 10.26 75 0.54 (synergistic effect) 5.87 5.66 13.32 8.70 90 0.59 (synergistic effect) 5.36 4.90 15.04 7.37

Table 37 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in IC50, IC75 and IC90 of a combination product containing two or more compounds according to the molar ratio of each drug in the SK-OV-3 cell line Respectively.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . In addition, combination effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination. The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT, a combination of four compounds exhibiting the highest synergistic effect at 50-90% cancer inhibition rate, HMF was 5.4 to 6.4 times, PHE was 4.9 to 6.5 times, 2DG was 11.8 to 15.0 times, CT Was 7.4 ~ 10.3 times more effective than other drugs (DRI).

Figure 62 shows that single and combination formulations consisting of HMF, PHE, 2DG, and CT were given to SK-OV-3 cell lines, which are cancer cells derived from human ovaries, for 48 hours by specific doses and then 50% Concentration, indicating how much the drug toxicity of PHE can be lowered. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

63 is an analysis of an SK-OV-3 cell line treated with HMF, PHE, 2DG and CT for 48 hours on an Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 63 is an analysis of the SK-OV-3 cell line treated with the combination preparation consisting of HMF, PHE, 2DG, and CT for 48 hours in terms of a fractionation effect-combination index (Fa-CI) graph. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

Example  1-10: Bladder cancer cell line T24 ) Survival inhibition effect

The cell proliferation inhibitory effect of HMF, an acetylated compound (MET or PHE), a single preparation of each of 2DG and CT, and a combination of two or more kinds was compared in a T24 cell line derived from a human bladder.

(One) HMF , MET , 2 DG , CT &Lt; / RTI &gt;

FIG. 64 is a graph showing the results obtained by providing each single preparation of HMF, MET, 2DG, and CT and two or more combination products at the concentrations indicated in the T24 cell line, which is a cancer cell derived from human bladder, &Lt; / RTI &gt; The higher the number of combination preparations than the single formulation of HMF, MET, 2DG and CT, the higher the effect of inhibiting cancer cell survival.

65 is a graph showing the effect of inhibiting cancer cell viability after a single dose of each of HMF, MET, 2DG, and CT and a combination of two or more agents were administered to a T24 cell line derived from a human bladder by a specific dose for 48 hours. It is. The combination of HMF, MET, 2DG, and CT showed a higher inhibitory effect on tumor cell survival than single agent.

In FIG. 65, the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT was more effective in inhibiting cancer cell survival than the MET / 2DG combination exhibiting the highest tumor suppression ratio among the two combinations p < 0.001). In addition, the combination of HMF / MET / 2DG / CT and HMF / MET / CT / CT showed a higher inhibitory effect on cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 3.52 2.16 0.98 MET 16.98 2.09 0.98 2DG 4.02 1.19 0.98 CT 3.11 2.22 1.00 HMF / CT 1.64 / 1.23 1.98 1.00 MET / 2DG 7.77 / 0.97 1.51 1.00 HMF / MET / 2DG 0.88 / 4.39 / 0.55 1.67 1.00 HMF / MET / CT 0.88 / 4.38 / 0.66 1.93 1.00 MET / 2DG / CT 4.27 / 0.53 / 0.64 1.57 1.00 HMF / MET / 2DG / CT 0.46 / 2.29 / 0.29 / 0.34 1.74 1.00

Table 38 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for single and combination formulations of HMF, MET, 2DG and CT. The r values ranged from 0.98 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.19 to 2.22, indicating that the dose - effect curves showed hyperbolic responses in the compounds used alone or in combination.

The reduction effect of 50% cancer inhibitory concentration was higher in the combination of the three combinations of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest 50% . In addition, the reduction effect of 50% cancer inhibitory concentration was higher in the combination of 4 combinations composed of HMF / MET / 2DG / CT than 3 combinations composed of HMF / MET / CT. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF MET 2 DG CT
HMF / CT
4: 3 50 0.86 (synergistic effect) 2.14 - - 2.53 75 0.91 (additive action) 2.05 - - 2.38 90 0.96 (additive action) 1.96 - - 2.24
MET /2 DG
20: 2.5 50 0.70 (synergistic effect) - 2.19 4.14 - 75 0.76 (synergistic effect) - 1.79 5.06 - 90 0.85 (synergistic effect) - 1.46 6.17 -
HMF / MET /2 DG
4: 20: 2.5 50 0.65 (synergistic effect) 4.01 3.87 7.33 - 75 0.69 (synergistic effect) 3.46 3.40 9.60 - 90 0.75 (synergistic effect) 2.99 2.98 12.58 -
MF / MET / CT
4: 20: 3 50 0.72 (synergistic effect) 4.02 3.88 - 4.74 75 0.76 (synergistic effect) 3.79 3.72 - 4.40 90 0.81 (synergistic effect) 3.57 3.56 - 4.08
MET /2 DG / CT
20: 2.5: 3 50 0.59 (synergistic effect) - 3.98 7.53 4.85 75 0.66 (synergistic effect) - 3.34 9.43 3.94 90 0.75 (synergistic effect) - 2.80 11.81 3.21
HMF / MET /2 DG / CT
4: 20: 2.5: 3 50 0.45 (synergistic effect) 7.70 7.43 14.07 9.06 75 0.48 (synergistic effect) 6.82 6.70 18.93 7.92 90 0.52 (synergistic effect) 6.05 6.04 25.46 6.91

Table 39 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75, IC90 combinations of two or more compounds depending on the molar ratio of each drug in the T24 cell line.

The combination index effect at 50-90% cancer inhibition rate was higher in the triple combinations consisting of HMF / MET / 2DG or MET / 2DG / CT than the MET / 2DG combination with the highest combination index among the two combinations . In addition, the combination index effect was higher at 50 ~ 90% cancer inhibition rate than the combination of HMF / MET / 2D / CT with HMF / MET / 2DG / CT. The combination index effect was the highest among all combinations.

In HMF / MET / 2DG / CT, which is the combination of four compounds showing the highest synergistic effect in 50 ~ 90% cancer inhibition rate, HMF is 6.1 ~ 7.7 times, MET is 6.0 ~ 7.4 times, 2DG is 14.1 ~ Showed 6.9 ~ 9.1 times of drug reduction effect and the drug reduction index (DRI) was the highest.

FIG. 66 is a graph of a fractionation effect-combination index (Fa-CI) graph of a T24 cell line treated with a combination preparation consisting of HMF, MET, 2DG and CT for 48 hours. The combination index effect was higher in the combination of the three combinations consisting of HMF / MET / 2DG or MET / 2DG / CT rather than the MET / 2DG combination with the highest combination index according to the fractional effect (P <0.01, p <0.001, respectively). The combined effect of HMF / MET / 2DG / CT and HMF / MET / 2DG / CT was higher than that of HMF / MET / CT (p <0.001) The combination index effect was the highest.

2) HMF , PHE , 2 DG , CT &Lt; / RTI &gt;

FIG. 67 is a graph showing the results obtained by providing each single preparation of HMF, PHE, 2DG, and CT and two or more combination products by the concentration indicated in the T24 cell line, which is a cancer cell derived from human bladder, by concentration for 48 hours, &Lt; / RTI &gt; HMF, PHE, 2DG, and CT, the effect of inhibiting the survival of cancer cells was increased as the number of combination preparations increased.

68 shows the effect of suppressing the survival rate of cancer cells after providing each single preparation of HMF, PHE, 2DG, and CT and two or more combinations of them in a T24 cell line derived from a human bladder by a specific dose for 48 hours. It is. HMF, PHE, 2DG, and CT were more effective in inhibiting the survival of cancer cells than the single agent.

In FIG. 68, the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT was more effective in inhibiting cancer cell survival than PHE / 2DG combination exhibiting the highest cancer inhibition ratio among the two combinations p < 0.001). In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / HMF / PHE / CT showed higher inhibition of cancer cell survival (p <0.001) Among all combinations, the highest cancer suppression rate was obtained.

Single and Combination Compounds Parameters Dm (mM) m r HMF 3.52 2.16 0.98 PHE 0.49 2.72 0.99 2DG 4.02 1.19 0.98 CT 3.11 2.22 1.00 PHE /2 DG 0.21 / 0.65 1.65 1.00 HMF / PHE / 2DG 0.61 / 0.12 / 0.38 1.75 1.00 HMF / PHE / CT 0.70 / 0.14 / 0.53 1.94 1.00 PHE / 2DG / CT 0.12 / 0.36 / 0.43 1.75 1.00 HMF / PHE / 2DG / CT 0.41 / 0.08 / 0.26 / 0.31 1.93 1.00

Table 40 shows Dm (or IC50), m (slope), and r (correlation coefficient) obtained by median-effect analysis using Calcusyn software for HMF, PHE, 2DG and CT single and combination preparations. The r value ranged from 0.98 to 1.00, indicating a high correlation between dose and effect. The m value ranged from 1.19 to 2.72, indicating that the dose - effect curves showed hyperbolic responses in the compounds used alone or in combination.

The reduction effect of the 50% cancer inhibitory concentration was higher in the combination of PHE / 2DG / PHE / 2DG or PHE / 2DG / CT than in the PHE / 2DG combination, which showed the highest 50% cancer inhibitory concentration among the two combinations . In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed lower reduction effect of 50% cancer inhibitory concentration. The highest inhibitory effect of 50% cancer inhibitory concentration was shown.

Combination compound
Molar ratio ( mM )
% Inhibition
Combination Index ( CI ) Drug reduction index ( DRI ) HMF PHE 2 DG CT
PHE /2 DG
0.8: 2.5 50 0.59 (synergistic effect) - 2.34 6.19 - 75 0.68 (synergistic effect) - 1.81 8.04 - 90 0.81 (synergistic effect) - 1.39 10.44 -
HMF / PHE /2 DG
4: 0.8: 2.5 50 0.52 (synergistic effect) 5.78 4.00 10.57 - 75 0.58 (synergistic effect) 5.15 3.21 14.27 - 90 0.66 (synergistic effect) 4.58 2.57 19.28 -
HMF / PHE / CT
4: 0.8: 3 50 0.66 (synergistic effect) 5.02 3.48 - 5.91 75 0.73 (synergistic effect) 4.74 2.96 - 5.50 90 0.82 (synergistic effect) 4.49 2.52 - 5.13
PHE /2 DG / CT
0.8: 2.5: 3 50 0.47 (synergistic effect) - 4.22 11.15 7.18 75 0.52 (synergistic effect) - 3.38 15.04 6.29 90 0.60 (synergistic effect) - 2.71 20.29 5.51
HMF / PHE /2 DG / CT
4: 0.8: 2.5: 3 50 0.45 (synergistic effect) 8.56 5.93 15.64 10.07 75 0.48 (synergistic effect) 8.05 5.02 22.34 9.34 90 0.51 (synergistic effect) 7.58 4.26 31.91 8.66

Table 41 shows the combination index (CI) and the drug reduction index (DRI) of each single agent in the IC50, IC75 and IC90 of the combination product containing two or more compounds according to the molar ratio of each drug in the T24 cell line.

The combination index effect at 50-90% cancer inhibition was higher in triple combination formulations consisting of HMF / PHE / 2DG or PHE / 2DG / CT than PHE / 2DG combinations with the highest combination index among the two combinations . The combination of HMF / PHE / 2DG / CT and HMF / PHE / CT / CT showed that the combined effects of 50% and 90% cancer suppression rate were higher than those of HMF / PHE / The combination index effect was the highest among all combinations.

In HMF / PHE / 2DG / CT preparations, which have the highest synergistic effect at 50 ~ 90% cancer inhibition rate, HMF is 7.6 ~ 8.6 times, PHE is 4.3 ~ 5.9 times, 2DG is 15.6 ~ 31.9 times, Showed a drug reduction effect of 8.7 ~ 10.1 times, and the drug reduction index (DRI) was also the highest.

69 shows the 50% cancer inhibitory concentration by PHE after 48 hours of the single and combination preparation consisting of HMF, PHE, 2DG, and CT were given to a T24 cell line derived from a human bladder by a specific dose for 48 hours And how it can lower the drug toxicity of PHE. PHE, PHE, 2DG, and CT, among the combination of PHE single agent and two or more combinations, showed the lowest drug toxicity.

The 50% cancer inhibitory concentration of PHE / PHE / 2DG combined with HME / PHE / 2DG or PHE / 2DG / CT was lower than that of PHE / 2DG combination. In addition, the combination of HMF / PHE / 2DG / CT and HMF / PHE / CT showed the lowest 50% inhibition concentration by PHE, The combination of four combinations showed the highest effect among all combinations.

Figure 70 is an analysis of the T24 cell line treated with HMF, PHE, 2DG and CT for 48 hours on a Fa-CI graph. Combination exponents according to Fraction Affected showed a synergistic effect both in low inhibition rate and high inhibition rate in combination of HMF, PHE, 2DG and CT.

FIG. 70 is a graph of a fractionation effect-combination index (Fa-CI) graph of a T24 cell line treated with a combination preparation consisting of HMF, PHE, 2DG and CT for 48 hours. The combination index effect was higher in the combination of three combinations of HMF / PHE / 2DG or PHE / 2DG / CT than the PHE / 2DG combination with the highest combination index according to the fractional effect (P < 0.001, respectively). The combined effect of HMF / PHE / 2DG / CT was higher than that of HMF / PHE / CT combination (p <0.001) The combination index effect was the highest.

< Example  2> HMF , MET  or PHE , 2 DG  And CT Effect of Combination Agents on Cell Cycle Progression and Cell Death

The effect of HMF, MET or PHE, 2DG and CT on the proliferation, cell cycle progression and apoptosis of HepG2 cell line derived from human liver was examined. Each drug used was not exceeding 40 mM.

Example  2-1: HMF , MET  or PHE , 2 DG  And CT Combination HepG2  Effect on cell proliferation

To investigate the effect of the combination of HMF, MET or PHE, 2DG and CT on the proliferation of HepG2 cells in human liver, HMF, MET or PHE, 2DG and CT were added 3.5: 25 (0.8 for PHE) The number of viable cells was measured by MTT assay after 24, 48 and 72 hours incubation in the control group with and without the combination preparation containing 1: 3 mM.

The combination of HMF, MET, 2DG, and CT at a concentration ratio of 3.5: 25: 1: 3 mM for 24, 48, and 72 hours resulted in significant cell proliferation (Fig. 71). When cultured for 24, 48, and 72 hours, the cell proliferation was decreased by 49.94%, 97.01%, and 99.62%, respectively, compared with the untreated control.

In addition, when HMF, PHE, 2DG, and CT were combined at a concentration ratio of 3.5: 0.8: 1: 3 mM for 24, 48, and 72 hours, (Fig. 72). When cultured for 24, 48, and 72 hours, the cell proliferation was decreased by 44.13%, 99.12%, and 99.85%, respectively, compared with the untreated control.

Example  2-2: HMF , MET , PHE , 2 DG  And CT Lt; / RTI &gt; HepG2  Effect of cell cycle progression and apoptosis

Delay in cell cycle progression is one of the ways to inhibit the proliferation of cancer cells. The effect of the combination of HMF, MET, 2DG, and CT on the cell cycle progression of HepG2 cells, a human liver cancer cell, was examined.

After the cells were cultured for 24 hours in the test group to which the combination preparation containing the combination of HMF, MET, 2DG, and CT was added at a concentration ratio of 1.7: 10: 0.5: 1.5 mM to the cell culture solution, Stained with propidium iodide (PI), and analyzed by flow cytometry.

Since the amount of DNA in the apoptotic cells is smaller than that in the G0 / G1 group, when stained with the PI solution, it appears on the left of the G0 / G1 group on the histogram, which is called Sub-G1. The amount of DNA was determined as% of cells. PI penetrates the cell membrane and stains DNA in the nucleus. As a result, not only early apoptotic cells but also late apoptotic cells and necrotic cells can be stained. Sub-G1 cells have increased over time. As a result, the number of cells staying in sub-G1 was significantly increased as the number of combination preparations was larger than the single preparation of HMF, MET, 2DG and CT in the cell culture (Fig. 73). The increase in Sub-G1 phase cell ratio indicates that apoptosis is increased under given experimental conditions. The number of cells in the S phase and G2 / M phase decreased significantly as the number of combination products increased. From the above results, it was found that the S group and G2 / M group of HepG2 cells inhibited cell proliferation by interfering with the highest DNA synthesis and mitosis process in the combination of the four species (Fig. 73).

Example  2-3: HMF , MET  or PHE , 2 DG  And CT Lt; / RTI &gt; HepG2  Effect on the protein expression of cells

In order to investigate the effects of the combined preparation inhibiting the cell cycle progression of HepG2 cells on a protein, a single formulation containing HMF, MET, 2DG and CT at a ratio of 4: 5: 0.5: 3 mM and two species The cells were treated with the above combination preparation, and the cells were cultured for 36 hours. Cell lysate was taken and subjected to Western blot analysis.

Hypophosphorylated Rb, a cell cycle regulator, is known to be a key substance to stop the cell cycle in G1 by inhibiting E2F function by binding to E2F, which plays an important role in regulating the transcription of genes necessary for S cycle entry. As a result, the expression level of low phosphorylated Rb increased in HMF, MET, 2DG and CT single agent and 4 combinations of two or more combinations. Increased hypophosphorylated Rb suggests that increased hypophosphorylated Rb is deeply involved in G1 arrest in inducing cell differentiation (Figure 74).

       In addition, the progression of the cell cycle is regulated by proteins in the cytoplasm. Many factors are required for this cell cycle regulation, and the most important role is the cyclin dependent kinase (CDK). As a result of the above experiment, the single formulation of HMF, MET, 2DG and CT and the combination of two or more agents markedly decreased the protein expression of CDK4, and the decrease of CDK4 protein expression was observed in the combination of the single agent and the combination of two or more kinds Showed a lot of decline.

Cyclin is also known to regulate the cell cycle by interacting with cell division cycle genes in the G1 and G2 phases of the cell cycle, and is reported to be overexpressed in cancer cells. Protein expression of Cyclin D1 was the most abundant in the combination of HMF, MET and CT single agent and two or more combinations. Proteins of p21 and p27, the major transcription factors for G1-phase cell cycle inhibition, inhibit the G1 to S transition from increased expression. As a result of the above experiment, the single HMF, MET, 2DG, and CT preparations and the combination of four or more of the combination of two or more kinds were the most increased (FIG. 74).

Example  2-4: HMF , MET  or PHE , 2 DG  And CT Effect of Combination Agent on Normal Cells at 50% Cancer Inhibitory Concentration

To investigate the effect of HMF, MET or PHE, 2DG and CT on the normal cells, the combined preparation was treated with hepatocyte CHANG and hepatoma cell line HepG2 at a 50% inhibitory concentration, and after 48 hours, MTT assay Toxicity was investigated. As a result of comparing the cell death of hepatocellular carcinoma cells with that of normal hepatocytes, HepG2 was found to be 1.90 times higher than that of normal cells when HMF, MET, 2DG and CT of 50% , 2DG and CT showed that HepG2 was 1.86 times more cytotoxic than normal cells and that the combined preparation was more toxic in liver cancer cells than normal hepatocytes. As a result, the combined preparation of HMF, MET or PHE, 2DG and CT was found to be more sensitive to cancer cells than normal hepatocytes, and showed the lowest toxicity to normal hepatocytes in the combination of four types (Fig. 75 -76).

Example  2-5: HMF , MET  or PHE , 2 DG   And CT Lt; / RTI &gt; HepG2  Effect on cell AMPK activity

AMPK is one of the enzymes that attach phosphate to proteins and regulates glucose and lipid metabolism and controls enzymes involved in energy production. If the function of AMPK is wrong, it can cause metabolic problems and diseases such as diabetes and cancer.

Single and Combination Compounds
density( mM ) pAMPK alpha
( Unit / mL ) Control correction value
( pAMPK α) Control group ( Control ) 0  5.15 ± 0.8 0 HMF 1.7  6.01 ± 0.9 0.86 MET 10.0 13.05 + 1.7 7.90 2 DG 0.5  7.17 ± 1.1 2.02 CT 1.5  6.52 ± 1.0 1.37 HMF / CT 1.7: 1.5 11.23 ± 1.4 6.08 MET /2 DG 10.0: 0.5 18.53 ± 2.2 13.38 HMF / MET /2 DG 1.7: 10.0: 0.5 19.87 ± 2.7 14.72 HMF / MET / CT 1.7: 10.0: 1.5 16.55 ± 2.0 11.40 MET /2 DG / CT 10.0: 0.5: 1.5 20.03 ± 3.0 14.88 HMF / MET /2 DG / CT 1.7: 10.0: 0.5: 1.5 29.45 ± 3.7 24.30

Table 42 shows the degree of phosphorylation of Threonine 172 residue (Thr172) of AMPKa. HMF / MET / 2DG / CT, HMF / MET / 2DG / CT, HMF / MET / 2DG / CT and HMF / MET / 2DG / 17.7 times higher than that of the control group.

Single and Combination Compounds
density( mM ) pAMPK alpha
( Unit / mL ) Control correction value
( pAMPK α) Control group ( Control ) 0  5.15 ± 0.8 0 HMF 1.7  6.01 ± 0.9 0.86 PHE 0.4 12.44 ± 1.6 7.29 2 DG 0.5  7.17 ± 1.1 2.02 CT 1.5  6.52 ± 1.0 1.37 HMF / CT 1.7: 1.5 11.23 ± 1.4 6.08 PHE /2 DG 0.4: 0.5 17.87 ± 2.1 12.72 HMF / PHE /2 DG 1.7: 0.4: 0.5 18.82 + - 2.5 13.67 HMF / PHE / CT 1.7: 0.4: 1.5 15.23 ± 1.9 10.08 PHE /2 DG / CT 0.4: 0.5: 1.5 19.93 + - 2.8 14.78 HMF / PHE /2 DG / CT 1.7: 0.4: 0.5: 1.5 27.31 ± 3.4 22.16

In addition, HMF / PHE / 2DG / CT, which is a combination of four compounds among HMF, PHE, 2DG and CT combinations and two or more combinations, has 25.8 times higher than HMF alone, 3.0 times higher than PHE, and 11.0 times higher , And CT increased 16.2-fold, indicating the highest AMPK activation level (Table 43).

As a result of the above experiment, it was confirmed that the anticancer action mechanism associated with AMPK activation shows a further enhanced anticancer activity effect due to the synergistic action of each drug in the combination treatment (FIG. 77-78).

Claims (8)

(1) metformin, phenformin, or a pharmaceutically acceptable salt thereof, among the biguanide compounds;
(2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And
(Iii) 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof, ii) citric acid or a pharmaceutically acceptable salt thereof, or iii) 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof. An anticancer composition comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and citric acid or a pharmaceutically acceptable salt thereof as an active ingredient.
7. The composition of claim 1 wherein the composition is selected from the group consisting of 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof, metformin, phenformin or a pharmaceutically acceptable salt thereof, Deoxy-D-glucose or a pharmaceutically acceptable salt thereof, and citric acid or a pharmaceutically acceptable salt thereof.
4. The compound according to claim 1, which is 5-hydroxymethyl furfural or a pharmaceutically acceptable salt thereof: metformin, phenformin or a pharmaceutically acceptable salt thereof in an acetonide compound: 2- Wherein the molar ratio of oxy-D-glucose or a pharmaceutically acceptable salt thereof: citric acid or a pharmaceutically acceptable salt thereof ranges from 1: 0.05: 0.2: 1 to 1: 30: 1: 4.
delete The composition according to claim 1, wherein the cancer is selected from the group consisting of liver cancer, lung cancer, gastric cancer, pancreatic cancer, colon cancer, cervical cancer, breast cancer, prostate cancer, ovarian cancer and bladder cancer.
The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
(1) metformin, phenformin, or a pharmaceutically acceptable salt thereof, among the biguanide compounds;
(2) 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof; And
(Iii) 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof, ii) citric acid or a pharmaceutically acceptable salt thereof, or iii) 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof. A food composition for preventing or ameliorating cancer comprising 5-hydroxymethylfurfural or a pharmaceutically acceptable salt thereof and citric acid or a pharmaceutically acceptable salt thereof as an active ingredient.


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