KR20210096019A - Composition for preventing or treating cancer comprising polyP as active ingredient - Google Patents

Abstract

The present invention pertains to a pharmaceutical composition for preventing or treating cancer, containing polyP as an active component. The pharmaceutical composition has a remarkable effect in terms of killing only cancer cells without damaging normal cells, thereby being expected to be widely used in the fields of medicine and health.

Description

A composition for preventing or treating cancer comprising polyP as an active ingredient

The present invention relates to a pharmaceutical composition capable of effectively preventing or treating cancer.

Cancer is one of the incurable diseases to be solved by mankind, and huge capital is being invested in development to cure it worldwide. diagnosed, and more than 60,000 people have died.

Examples of carcinogens that cause cancer include smoking, ultraviolet rays, chemicals, food, and other environmental factors. Substances currently used as therapeutic agents have considerable toxicity and cannot selectively remove only cancer cells, so there is an urgent need to develop an effective anticancer agent with less toxicity to prevent cancer as well as treatment after the onset of cancer. . Although the diagnosis and treatment of cancer has made rapid progress in the past decade, the mortality rate due to cancer is still high. Looking at the research trends of anticancer drugs so far, the first generation can be divided into chemical anticancer drugs, second generation targeted anticancer drugs, and third generation immune anticancer drugs, and recently, fourth generation anticancer drugs are emerging.

First-generation chemical anticancer drugs have severe side effects because they damage not only cancer cells but also normal cells. That is, in order to kill cancer cells, it attacks even normal cells, destroys the patient's immune system, and due to its strong toxicity, various side effects such as hair loss, vomiting, loss of appetite, fatigue, and extreme physical strength occur. In contrast, second-generation targeted anticancer drugs have the advantage of identifying and attacking only cancer cells, but they can only be used for patients with genetic mutations, making it impossible to treat various cancers, and there are disadvantages in that treatment becomes impossible due to resistance. The third-generation immune anticancer drug has a new mechanism to kill cancer cells by activating the immune cells of the body, which have been suppressed, and has the advantage that it can be widely used in various carcinomas without specific gene mutations. Because there is a risk, the fourth-generation metabolizing anticancer drug that kills only cancer cells by starvation is attracting more attention. Accordingly, in recent years, active research has been conducted around the world paying attention to cancer metabolism that occurs only in cancer cells.

The present invention relates to a pharmaceutical composition for cancer treatment as a metabolic anticancer agent containing a new active ingredient, and since it is possible to more effectively treat cancer by selecting only cancer cells without damaging normal cells, it is a new alternative to anticancer treatment in medicine and health. It is expected to be of great use.

An object of the present invention is to provide a method for effectively treating cancer by selecting only cancer cells without damaging normal cells.

One object of the present invention is to provide a composition that can effectively prevent or treat cancer for cancer patients.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

According to one embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating cancer, comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the invention, "polyphosphate (polyP)" is a linear polymer of orthophosphate residues linked by ATP-like phosphoanhydride bonds. PolyP is found in a wide range from bacteria to mammalian cells and is widely known as a blood coagulation factor. In particular, short-chain polyP is found in human platelets and mast cells, and long-chain polyP is found in some microorganisms and mammals. PolyP has been mainly studied in bacteria and yeast with a focus on polyP metabolism, and metabolic processes in eukaryotic cells and mammals are currently being studied. In addition, recently mentioning the action related to blood coagulation such as thrombosis (Front Med (Lausanne). 2019 Apr12;6:76.), polyP is used as an agent to inhibit cell proliferation and cell migration, so it may be used for cancer treatment. (Biochemistry (Mosc). 2018 Aug;83(8):961-968.).

In the present invention, the polyphosphate is divided by the length of the phosphates connected, for example, P60 refers to a linear polymer in which 60 phosphates are connected, and P200 refers to a linear polymer in which 200 phosphates are connected. As an active ingredient for cancer treatment of the present invention, the polyphosphate chain may have a length of P20 to P200, preferably P30 to P120, and more preferably P40 to P101, but is not limited thereto.

The pharmaceutically acceptable salts may include addition salts of acids or bases, and stereochemical isomers thereof. For example, the compound may be in the form of an addition salt of an organic or inorganic acid. Salts have a desirable effect in a patient when administered to a patient, and include, but are not particularly limited to, any salts that retain the activity of their parent compound. These salts include inorganic and organic salts such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, Benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, ecylline Acid, pamoic acid, gluconic acid, methyl nitric acid, malonic acid, hydrochloric acid, hydroiodic acid, hydroxynaphtolic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, nafsilic acid, muconic acid, p -nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilic acid, salts of methanesulfonic acid, and the like. Addition salts of bases include salts of alkali metals or alkaline earth metals, such as salts of ammonium, lithium, sodium, potassium, magnesium, calcium and the like; salts with organic bases, such as salts of benzathine, N-methyl-D-glucamine, hydrabamine and the like; and salts with amino acids, such as arginine, lysine, and the like. In addition, these salts can be converted to their free form by treatment with an appropriate base or acid.

The pharmaceutical composition of the present invention is characterized in that it is remarkably excellent in preventing or treating cancer cells without damaging normal cells.

According to another embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating cancer, further comprising a biguanide-based compound in a composition comprising polyphosphate (polyP) as an active ingredient.

In one embodiment of the present invention, “biguanide-based compound” refers to a role of lowering mitochondrial oxidative phosphorylation by activating AMP-activated protein kinase (AMPK), a key enzyme that physiologically regulates carbohydrate metabolism and lipid metabolism. compounds known to do It is mainly used as a treatment for diabetes to help control blood sugar. Meanwhile, it has been recently proven that biguanide-based drugs have anticancer effects. However, there is a study result showing that the apoptosis effect of cancer cells is relatively low in the case of single use, so research on combination-administered drugs that have the potential to show a more improved anticancer effect is being actively conducted.

The biguanide-based compound of the present invention may be metformin, phenformin, buformine, etc. If it is a biguanide-based compound, it is not limited thereto.

In one embodiment of the present invention, the "culture medium" is a liquid or gel nutrient source designed to grow microorganisms, cells, or small plants such as moss. Different types of media are used depending on the type of cells to be cultured. The growth medium is largely divided into two types: a cell medium and a microbial medium: A cell medium is a type of medium for culturing specific cells derived from plants or animals, and a microbial medium is used for propagating microorganisms such as bacteria and yeast. It is the medium used.Some organisms require a special environment for propagation, and in some cases it is difficult to propagate.For example, a virus must parasitize a host cell to proliferate, so it is like adding a live host cell to the medium. Various substances can be mixed with the nutrients required by the culture as the main component for culturing the tissues of microorganisms or animals and plants.The types of medium include DMEM, RPMI1640, IMDM, M199, alpha-MEM, CMRL1066 medium, Fisher's medium. , Waymouth's medium, McCoy's 5A medium, Ham's F-10, BME, etc. exist, and since the components of each medium are various, it can be selectively used depending on the cell line.

In one embodiment of the present invention, the pharmaceutical composition of the present invention is a component contained more abundantly in the RPMI-1640 medium compared to the DMEM medium in polyP as an active ingredient, for example, glutathione, arginine, asparagine, aspartic acid, glutamic acid, histidine. , proline, inositol, or vitamin B group, etc. may be further included, and an improved cancer treatment effect can be expected therefrom. In addition, the pharmaceutical composition of the present invention contains polyP as an active ingredient and a substance that inhibits the component contained more abundantly in the DMEM medium compared to the RPMI-1640 medium or inhibits cellular uptake, for example, calcium, iron, magnesium, glucose, Substances that inhibit pyruvic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, tyrosine, folic acid, nicotinamide, pyridoxine, or thiamine, or substances that inhibit intracellular absorption and improved cancer treatment effect can be expected from this.

As a disease to be prevented or treated in the pharmaceutical composition of the present invention, it may be cancer that has occurred or is likely to develop in an individual.

In the present invention, the "individual" refers to mammals including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats. It may be selected from, and preferably may be a human, but is not limited thereto.

In the present invention, the term "cancer" as a disease to be prevented or treated refers to or refers to a physiological condition characterized by unregulated cell growth in mammals typically. The cancer is thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervoussystem tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma If the cancer progression, such as differentiation and/or proliferation, is a type of cancer dependent on cancer cells and/or cancer stem cells described in the present invention, the present invention is not limited thereto.

In one embodiment of the present invention, "cancer stem cell (CSC)" refers to a cancer cell in a comprehensive sense that has a self-renewal ability or differentiation ability, which is a unique ability of a stem cell, and a cancer stem cell in a cancer tissue Cells are estimated to be present between 0.1 and 5%. The cancer stem cells are under normal tumor growth conditions (the term "normal tumor growth conditions" refers to a state in which nutrients (glucose) necessary for cell growth are sufficient and the growth conditions of the tumor microenvironment are abundant and there is no cellular stress.) It has the characteristic of slowly proliferating differently from normal cancer cells. In addition, it may have resistance to anticancer drugs by maintaining a dormant state, and unlike normal tumor cells, the expression of transcriptional regulators such as PGC-1α is regulated, so the functions of major metabolic regulators are lower than those of normal cancer cells. There are different characteristics. Through these different metabolic regulation and regulation of cell signaling systems linked thereto, it refers to a cell with infiltrating and/or metastatic potential by acquiring resistance to apoptosis in a nutrient-deficient state. However, if it is a cell capable of differentiating into a general cancer cell, it is not limited thereto.

In one embodiment of the present invention, "prevention" may include, without limitation, any action that blocks cancer symptoms or suppresses or delays cancer symptoms using the pharmaceutical composition of the present invention.

In one embodiment of the present invention, "treatment" refers to a series of activities performed for alleviation or/and amelioration of a desired disease. For the purposes of the present invention, treatment may include, without limitation, any action in which cancer symptoms are improved or beneficial by examining the pharmaceutical composition of the present invention.

In one embodiment of the present invention, "pharmaceutical composition" refers to a composition administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention is to prevent or treat cancer, and may include a compound involved therein and a pharmaceutically acceptable carrier, excipient or diluent. In addition, the pharmaceutical composition according to the present invention comprises 0.1 to 50% by weight of the active ingredient of the present invention with respect to the total weight of the composition.

In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical composition of the present invention is not limited thereto, but each can be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents A topical agent, solubilizer, isotonic agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

The route of administration of the pharmaceutical composition according to the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Oral or parenteral administration is preferred.

In one embodiment of the present invention, "administration" means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention is through any general route as long as it can reach the target tissue. may be administered. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, intrathecal administration may be made, but is not limited thereto does not In the present invention, the effective amount refers to the type of disease, the severity of the disease, the type and content of active ingredients and other components contained in the composition, the type of formulation and the age, weight, general health status, sex and diet, administration time, administration route of the patient. And it can be adjusted according to various factors including the secretion rate of the composition, the duration of treatment, and drugs used simultaneously. For adults, the therapeutic pharmaceutical composition can be administered into the body in an amount of 50ml to 500ml at a time, 0.1ng/kg-10mg/kg in the case of a compound, 0.1ng/kg-10mg in the case of a monoclonal antibody It can be administered at a dose of /kg. The administration interval may be 1 to 12 times a day, and if administered 12 times a day, it may be administered once every 2 hours. In addition, the pharmaceutical composition of the present invention may be administered alone or with other therapies known in the art, for example, chemotherapeutic agents, radiation and surgery for the treatment of desired cancer stem cells. In addition, the pharmaceutical composition of the present invention may be administered in admixture with other treatments designed to enhance the immune response, for example, adjuvants or cytokines (or nucleic acids encoding cytokines) as well known in the art. Other standard delivery methods may be used, such as biolistic delivery or ex vivo treatment. In ex vivo treatment, for example, antigen-presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells can be obtained from a patient or an appropriate donor and activated in vitro with the present pharmaceutical composition and then administered to the patient. there is.

As used herein, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day per day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

According to another embodiment of the present invention, it relates to a food composition for preventing or improving cancer comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient. The food composition may further include a biguanide-based compound.

In one embodiment of the present invention, "food composition" is used in various ways for the prevention or improvement of indications for the purpose of the present invention. For example, it may be prepared in the form of beverage, gum, tea, vitamin complex, powder, granule, tablet, capsule, confectionery, rice cake, bread, and the like. Since the food composition of the present invention is improved from the existing food intake with little toxicity and side effects, it can be safely used even when taken for a long period of time for the purpose of prevention. When the composition of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, and common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). Others of the present invention of various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, It may contain stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverage, etc. These components can be used independently or in combination.The proportion of these additives is usually per 100 parts by weight of the composition of the present invention. It is generally selected in the range of 0.1 to 100 parts by weight, but is not limited thereto.

In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the polyphosphate is 20 to 200 phosphate salts. It provides a pharmaceutical composition for preventing or treating cancer that is linked, wherein the polyphosphate provides a pharmaceutical composition for preventing or treating cancer in which 30 to 120 phosphates are linked, and the polyphosphate is 40 to 101 phosphates It provides a pharmaceutical composition for the prevention or treatment of cancer that is linked, wherein the pharmaceutical composition provides a pharmaceutical composition for preventing or treating cancer further comprising a biguanide-based compound, wherein the biguanide-based compound comprises: Provided is a pharmaceutical composition for the prevention or treatment of any one or more cancers selected from the group consisting of metformin, phenformin, and buformine, wherein the cancer comprises cancer stem cells It provides a pharmaceutical composition for the prevention or treatment of cancer.

In another embodiment of the present invention, there is provided a pharmaceutical composition for inhibiting metastasis of cancer comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the pharmaceutical composition is a biguanide series It provides a pharmaceutical composition for inhibiting metastasis of cancer further comprising a compound, and provides a pharmaceutical composition for inhibiting metastasis of cancer, wherein the cancer includes cancer stem cells.

In another embodiment of the present invention, there is provided a food composition for the prevention or improvement of cancer comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the food composition is biguanide (biguanide). ) provides a food composition for preventing or improving cancer further comprising a series compound, wherein the cancer provides a food composition for preventing or ameliorating cancer comprising cancer stem cells.

Hereinafter, the present invention will be described in detail step by step.

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, and since the pharmaceutical composition of the present invention has a remarkable effect in selectively killing only cancer cells without damaging normal cells, it is expected to be widely used in medicine and health. It is expected.

1A and 1B show cell viability in normal lung cells (L-132) and lung cancer cells (A549) according to the dose of glucose and P67 (polyphosphate67, polyP67) treatment for 24 hours, according to an embodiment of the present invention. viability).
Figure 1c is, according to an embodiment of the present invention, using a cell counting kit (cell counting kit) after P67 treatment for 24 hours to observe the cell viability in normal lung cells (L-132) and lung cancer cells (A549) The result is shown as a linear graph.
2a and 2b show the length of each polyphosphate (polyP) chain (P65, P45, P41, P25, and P13) in breast cancer cells (MCF7) and lung cancer cells (A549) according to an embodiment of the present invention; It is the result of observing the degree of cell death according to the molar concentration (μmol/L).
3a and 3b show the concentration of each polyphosphate (polyP) chain length (P65, P101, P157, and P199) in breast cancer cells (MCF7) and lung cancer cells (A549) according to an embodiment of the present invention; mg/mL) is the result of observing the degree of cell death according to the change.
4 is a result of observing the degree of cell death according to the polyP64 concentration (mg/mL) change in brain cancer cells (U87) using a cell counting kit according to an embodiment of the present invention.
Figure 5a is a result of confirming the degree of cell death according to the polyP64 concentration (mg / mL) change in lung cancer cells (A549) according to an embodiment of the present invention through flow cytometry (Fluorescence Activated Cell Sorting; FACS).
Figure 5b is a graph showing the ratio of apoptosis (apoptosis) and necrosis (necrosis) from the FACS result of Figure 5a, according to an embodiment of the present invention.
6A and 6B show the chain length of polyP in breast cancer cells (MDA-MB-231 parental cell, p cell) and breast cancer stem cells (MDA-MB-231 stem cell, s cell) according to an embodiment of the present invention; (P65, P101, P157, and P199) is the result of observation of apoptosis according to the concentration (mg / mL) change.
7A and 7B are results of confirming the therapeutic effect according to the polyP67 concentration (mg/mL) in lung cancer cells (A549) in RPMI medium or DMEM medium, respectively, according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Preparation for experiment

The inventors of the present invention obtained from the American Type Culture Collection (ATCC), a human breast cancer cell line MDA-MB-231 (a parental cell line and a stem cell line), another breast cancer cell line MCF7, a lung cancer cell line A549, and a brain cancer cell line U87. , and L-132, a normal lung cell line, were obtained and used in the experiment.

In addition, the cancer cells were cultured and tested using RPMI-1640 medium or DMEM medium supplemented with FBS 10%, and the components of the medium are shown in Table 1 below.

RPMI-1640 DMEM COMPONENTS, [mg/L]) INORGANIC SALTS Ca(NO ₃ ) _2· 4H ₂ O 100.00 - CaCl ₂ (anhydrous) - 200.00 Fe(NO ₃ ) ₃ 9H ₂ O - 0.10 KCl 400.00 400.00 MgSO ₄ (anhydrous) 48.84 97.67 NaCl 6000.00 6400.00 NaHCO ₃ 2000.00 3700.00 Na ₂ HPO ₄ (anhydrous) 800.00 - NaH ₂ PO ₄ H ₂ O - 125.00 OTHER COMPONENTS D-Glucose 2000.00 4500.00 Glutathione (reduced) 1.00 HEPES - - Phenol Red 5.00 15.00 Sodium Pyruvate - 110.00 Amino Acids L-Arginine 200.00 - L-Arginine HCl - 84.00 L-Asparagine (free base) 50.00 - L-Aspartic acid 20.00 - L-Cystine·2HCl 65.00 63.00 L-Glutamic acid 20.00 - L-Glutamine 300.00 584.00 Glycine 10.00 30.00 L-Histidine (free base) 15.00 - L-Histidine HCl H ₂ O - 42.00 L-Hydroxyproline 20.00 - L-Isoleucine 50.00 105.00 L-Leucine 50.00 105.00 L-Lysine HCl 40.00 146.00 L-Methionine 15.00 30.00 L-Phenylalanine 15.00 66.00 L-Proline 20.00 - L-Serine 30.00 42.00 L-Threonine 20.00 95.00 L-Tryptophan 5.00 16.00 _{L-Tyrosine · 2Na · 2H 2} O 29.00 104.00 L-valine 20.00 94.00 Vitamins Biotin 0.20 - D-Ca Pantothenate 0.25 4.00 Choline chloride 3.00 4.00 folic acid 1.00 4.00 i-Inositol 35.00 7.20 Niacinamide 1.00 4.00 Para-aminobenzoic acid 1.00 - Pyridoxine HCl 1.00 4.00 Riboflavin 0.20 0.40 Thiamin HCl 1.00 4.00 Vitamin B12 0.005 - Add (ADD) L-Glutamine - - L-Methionine - - SPECIFICATIONS pH at RT (without NaHCO ₃ ) N/A N/A pH at RT (with NaHCO ₃ ) 7.3±0.3 7.3±0.3 Osmolality-mOsm/kg H2O(without NaHCO ₃ ) N/A N/A Osmolality-mOsm/kg H2O(with NaHCO ₃ ) 278±5% 330±5%

Example 2: Confirmation of selective therapeutic effect of polyP (polyphosphate) drug

The present inventors tried to measure cell viability after P67 (polyP67) treatment in L-132, a normal lung cell line, and A549, a lung cancer cell line, in order to confirm the effect of polyP (polyphosphate).

To this end, L-132 cells and A549 cells were aliquoted in a 96-well plate at a ^{concentration of 5×10 3} cells/well, and after overnight culture, each was dissolved in DMSO (Dimethyl Sulfoxide) to obtain polyP67 at 0 to 2 mg/well. It was treated by concentration within the mL range, and further cultured for 24 hours. At this time, each cell was divided into an environment without glucose, an environment with 1 g/L glucose, and an environment with 4 g/L glucose, and experiments were performed. For each glucose environment, nothing was treated in the negative control group, and as the positive control group, the polyP67 drug was used as a solute concentration rather than a molar concentration (mol/L). 0.01 mg/mL, 0.1 mg/mL, 1 mg/mL, 1.25 mg/mL , 1.5 mg/mL, 1.75 mg/mL or 2 mg/mL. After that, 50 μl of the cell culture solution diluted with DPBS (Dulbecco's Phosphate-Buffered Saline) and 50 μl of the assay reaction buffer (reaction buffer, Promega, Madison, WI, USA) were mixed, put in a 96-well plate, and reacted at room temperature for 1 hour. After incubation, luminescence was measured with a spectrophotometer (Synergy HTX Multi-Reader, BioTek). At the same time, the number of cells in each sample was measured with a cell viability analysis kit (Cell Counting Kit-8), and the measured value was calculated so that the same number of cells in each sample could be compared with the number of cells in the negative control group. All experiments were repeated three times, and the average is shown in FIGS. 1A and 1B. As a result of the experiment, it was confirmed that polyP had an apoptosis effect selectively only on cancer cells regardless of the influence of the glucose environment. The above results were compared in L-132, a normal cell, and A549, a cancer cell, to show the effect of polyP67 treatment in a graph (see FIG. 1c ).

Example 3: Confirmation of cancer treatment effect according to polyP chain length

The present inventors used P13, P25, P41, P45, or P65 drugs to confirm the effect according to the chain length of polyP (polyphosphate). The degree of cell death was observed by treating the drug with a molar concentration (mol/L, M) of 0.1 μM, 1 μM, 10 μM, 100 μM, 200 μM, 400 μM, or 500 μM. At this time, an experiment was performed in the same manner as in Example 2 using MCF7, a breast cancer cell line, and A549, a lung cancer cell line.

The number of cells in each sample was measured with a cell viability analysis kit (Cell Counting Kit-8), and the measured value was calculated so that the same number of cells in each sample could be compared with the number of cells in the negative control group. All experiments were repeated three times, and are shown in FIGS. 2A and 2B, and the results of this experiment are shown as the average value of repeated experiments at weekly intervals.

As a result of the experiment, the longer the polyP chain length in the range of P13 to P65, the more effective the cancer treatment was. More specifically, it was confirmed that the apoptosis effect increased with the increase in chain length and concentration. In the P41 to P65 groups, polyP treatment at a high concentration had about 80% apoptosis effect on both breast and lung cancer cells compared to the control group, and it was also confirmed that the effect of the treatment was different depending on the carcinoma. On the other hand, in the case of the P65 group, the drug reactivity was good regardless of the carcinoma, and the killing effect was also high (see FIGS. 2a and 2b).

The present inventors performed an experiment in the same manner as in Example 2 by additionally treating P65, P101, P157, or P199 drugs to determine whether the cancer treatment effect is proportionally improved as the polyP chain length increases. As a result, it was confirmed that the cancer treatment effect did not increase in proportion to the chain length, and it was confirmed that the breast cancer cell line (MCF7) and the lung cancer cell line (A549) showed a similar tendency. There was a cancer treatment effect at a certain concentration or more, and when the polyP chain length was longer than P101, there was a tendency that the treatment effect was not significantly changed (see FIGS. 3A and 3B ). Accordingly, in order to check whether a drug having a chain length close to P65, which is judged to have good overall effect, has the same effect on other carcinomas, an experiment was performed using the U87 cell line, a brain cancer cell. As a result of observing the degree of apoptosis after treatment with polyP64 drug by solute concentration, it showed the same tendency as in breast cancer and lung cancer cell lines (FIG. 4). More specifically, the degree of cell death according to the polyP64 drug concentration (mg / mL) change in lung cancer cells (A549) was confirmed through flow cytometry (Fluorescence Activated Cell Sorting; FACS) (see Fig. 5a), and apoptosis ( The ratio of apoptosis) and necrosis was confirmed for each concentration of the corresponding drug (see FIG. 5b ). Through the above experiments, the cancer treatment effect of polyP64 to polyP67 length was confirmed.

Example 4: Confirmation of the effect of co-administration of polyP (polyphosphate) and phenformin (Phenformin)

The present inventors P65, P101, P157 to confirm the cancer treatment effect of stem cells (s cells; s cells) and parental cells (p cells) when phenformin and a drug according to the polyP chain length are co-administered. , or P199 drug was additionally treated and the experiment was performed in the same manner as in Example 2. In this case, MDA-MB-231 p cells and MDA-MB-231 s cells, which are breast cancer cell lines, were used. As a result of the experiment, it was confirmed that the degree of cell death was improved compared to the negative control when administered in combination with phenformin. Furthermore, it was confirmed that when the drug was co-administered, even when treated at a low concentration, it was more effective in killing cancer stem cells, which are difficult to treat compared to relatively general cancer cells (see FIGS. 6A and 6B ). In particular, the increase in the therapeutic effect within the low concentration range was markedly.

Example 5: Confirmation of different effects of polyP67 treatment according to the type of medium

The inventors of the present invention dispensed lung cancer cell line A549 in a 96-well plate at a ^{concentration of 5×10 3} cell/well, cultured overnight, and tested RPMI-1640 medium containing 10% FBS and 10% FBS Cell viability was observed after 24 hours or 48 hours of treatment using DMEM medium. As a result of the experiment, it was confirmed that when the RPMI-1640 medium was used, the drug sensitivity was increased compared to the case where the DMEM medium was used.

In addition, when the concentration of the polyP67 drug was 2.5 mg/mL or more, it was confirmed that the cancer treatment effect was shown in both media, and in the case of the RPMI-1640 medium, the therapeutic effect was higher even at a relatively low concentration such as 1.25 mg/mL. (See Figures 7a and 7b).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A pharmaceutical composition for preventing or treating cancer, comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 1,
The polyphosphate is a pharmaceutical composition for preventing or treating cancer, wherein 20 to 200 phosphates are linked. 3. The method of claim 2,
The polyphosphate is a pharmaceutical composition for preventing or treating cancer, wherein 30 to 120 phosphates are linked. 4. The method of claim 3,
The polyphosphate is a pharmaceutical composition for preventing or treating cancer, wherein 40 to 101 phosphates are linked. The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating cancer, further comprising a biguanide-based compound. The method of claim 1,
The cancer is a pharmaceutical composition for the prevention or treatment of cancer, including cancer stem cells. A pharmaceutical composition for inhibiting metastasis of cancer, comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient. 8. The method of claim 7,
The pharmaceutical composition further comprises a biguanide-based compound, a pharmaceutical composition for inhibiting metastasis of cancer. 8. The method of claim 7,
The cancer comprising cancer stem cells, a pharmaceutical composition for inhibiting metastasis of cancer. A food composition for preventing or improving cancer, comprising polyphosphate (polyP) or a pharmaceutically acceptable salt thereof as an active ingredient. 11. The method of claim 10,
The food composition is a food composition for preventing or improving cancer, further comprising a biguanide-based compound. 11. The method of claim 10,
The cancer is a food composition for the prevention or improvement of cancer, including cancer stem cells.

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