KR101828997B1 - A pharmaceutical composition for prevention or treatment of cancer and use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical compositions for the prevention or treatment of cancer including anisomycin and isolated natural killer (NK) cells, and a use thereof. Anisomycin increases the susceptibility of cancer cells to NK cells and increases the cytotoxicity of NK cells to cancer cells. As a result, the composition of the present invention significantly increases the anticancer effect by the combination treatment of anisomycin and NK cells and provides a synergetic effect.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention or treatment of cancer,

The present invention relates to pharmaceutical compositions for the prevention or treatment of cancer, and uses thereof, including anisomycin and isolated NK (Natural killer) cells.

Cancer is one of the world's most deadly diseases, with the cancer age gradually decreasing, while the average life expectancy is increasing, and cancer incidence is expected to increase further.

Cancer chemotherapy is a typical cancer treatment method. However, it can damage not only cancer cells but also cells that rapidly proliferate among normal cells, so that it has low target of cancer cells and decrease of anemia, leukocyte and platelet count after chemotherapy , Vomiting, nausea, and diarrhea. Accordingly, there is a need for a chemotherapeutic agent having an excellent anticancer effect in a smaller amount (Korean Patent Publication No. 10-2015-0135796).

On the other hand, NK (natural killer) cells are nonspecifically able to kill cancer cells, but many studies have been made, but the treatment effect of solid cancer is insufficient.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a therapeutic agent having superior anticancer effect. As a result, the anticancer effect was synergistically greatly increased when the anismaicin and the isolated NK cells were treated in combination, The present inventors have completed the present invention by confirming that the expression of a ligand for NKG2D, which is an important receptor for anticancer activity of cells, is increased and the sensitivity of cancer cells to NK cells is increased.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises anisomycin and isolated NK (Natural killer) cells.

Another object of the present invention is to provide a composition for enhancing the sensitivity of cancer cells to NK cells, including an isomicin.

Another object of the present invention is to provide a composition for promoting the binding of cancer cells and NK cells, including an isomicin.

It is still another object of the present invention to provide a composition for enhancing killing ability of NK cells, which comprises an isomicin.

It is still another object of the present invention to provide a kit for preventing or treating cancer, which comprises the above pharmaceutical composition.

Another object of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition to a subject.

To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising anisomycin and isolated natural killer (NK) cells.

The term " Anisomycin "as used in the present invention is a compound having the formula shown in Fig. 1 and is used as an anticancer agent. However, the inventors of the present invention for the first time confirmed that anismaicin increases the expression of a ligand for NKG2D, which is an important receptor for the anticancer activity of NK cells in cancer cells, and increases the susceptibility of cancer cells to NK cells. In addition, the inventors of the present invention have for the first time concluded that the anticancer effect is significantly increased or decreased synergistically when the anismaicin and the isolated NK cells are used in combination. In particular, the pharmaceutical composition of the present invention not only exhibits an anti-cancer effect due to the combination of the cancer cell killing ability of anismaicin and the cancer cell killing ability of NK cells, but also enhances the sensitivity of NK cells to an isomycin, And the cancerous effect was significantly increased synergistically.

The composition of the present invention may include a pharmaceutically acceptable salt of an isomycin in place of, or in combination with, an isomicin.

The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition.

The term "pharmaceutically acceptable salt " of the present invention means salts in which the cation and the anion are pharmaceutically usable among the salts in which they are bound by electrostatic attraction. Usually, the salt is a salt with a metal salt, an organic base Salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt and the like; Examples of salts with organic bases include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, And the like; The salt with inorganic acid may be a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid and the like.

The compositions of the present invention include both pharmaceutically acceptable salts of anismaicin as well as possible solvates and hydrates which may be prepared therefrom, and may include all possible stereoisomers. The solvates, hydrates and stereoisomers of the anismaic acid can also be prepared using conventional methods.

As used herein, the term "NK (natural killer) cell" refers to a cytotoxic lymphocyte that plays an important role in the body's immune system. For purposes of the present invention, the NK cell may be an endogenous NK cell of an individual and / or an exogenous NK cell, and may be, but is not limited to, a separate NK cell administered externally. In addition, the NK cell can be obtained by a conventional method such as purchasing a commercially available product, isolating it from an individual, or culturing it.

The pharmaceutical composition of the present invention can increase the expression of a ligand for an activating receptor of NK cells in cancer cells. The activation receptor is a receptor associated with activation and cytotoxicity of NK cells, and may be, but is not limited to, NKG2D. The ligand is not particularly limited as long as it can bind to an activating receptor of an NK cell, but specifically it may be ULBP (UL16 binding protein) 1, ULBP2 or ULBP3.

For the purposes of the present invention, NK cells isolated from anismajine can be administered simultaneously, sequentially, or in reverse order.

The term "prophylactic " as used in the present invention means any action that inhibits or delays cancer by administration of the pharmaceutical composition.

The term "treatment" as used in the present invention means all the actions of improving or ameliorating the symptoms of cancer by the administration of the pharmaceutical composition.

The cancer includes both solid cancer and blood cancer and specifically includes liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, adenocarcinoma, adenocarcinoma, adenocarcinoma, adenocarcinoma, vulvar carcinoma, vulvar carcinoma, gastric cancer, Renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system (CNS) tumors, cancer of the uterine cervix, Nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and the like, and more specifically, may be solid cancer in the cancer, but is not limited thereto.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent conventionally used in the production of a pharmaceutical composition, and the carrier may include a non-naturally occuring carrier .

Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical 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 and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. 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 or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Another aspect of the present invention provides a composition for enhancing sensitivity of cancer cells to NK cells, including an isomicin.

The anismaicin, NK cell, and cancer are as described above.

As used herein, the term "enhancing sensitivity to NK cells" means enhancing the sensitivity of cancer cells. Specifically, "enhancing sensitivity to NK cells" means that the expression of a ligand for an activating receptor (for example, NKG2D) important for the anticancer activity of NK cells is activated in cancer cells so as to activate NK cell cytotoxicity against cancer cells , But is not limited thereto.

The ligand is not particularly limited as long as it can bind to an activating receptor of an NK cell, but specifically it may be ULBP (UL16 binding protein) 1, ULBP2 or ULBP3.

In the present invention, "sensitivity" is mixed with "susceptibility ".

Another aspect of the present invention provides a composition for promoting the binding of cancer cells and NK cells, including an isomicin.

The anismaicin, NK cell and cancer are as described above.

As used herein, the term "enhancing the binding of cancer cells and NK cells" means enhancing the binding of NK cells to cancer cells so that NK cells can exhibit cytotoxicity against the cancer cells.

The composition comprising an isomicin of the present invention increases the expression of a ligand for an NKG2D receptor of NK cells in cancer cells, thereby enhancing the binding of NKG2D receptor-bearing NK cells to cancer cells expressing the ligand.

Another aspect of the present invention provides a kit for preventing or treating cancer, which comprises the above pharmaceutical composition.

The pharmaceutical composition, cancer, prevention and treatment are as described above.

The type of the kit of the present invention is not particularly limited, and a kit of a type commonly used in the art can be used.

The kit of the present invention can be packaged in a form contained in an individual vessel or in a single container divided into one or more compartments, wherein the anismaicin and the separated NK cells and the NK cells in the body are respectively contained in an individual vessel, The isolated NK cells may be packaged in the form of a single dosage unit dose, but are not limited thereto.

The anismaicin and the isolated NK cells in the kit can be administered separately at appropriate times according to the health condition of the subject to be administered and the like. In addition, anismaicin and isolated NK cells may be co-administered to an individual in a concurrent, sequential, or reverse order.

Another aspect of the present invention provides a method of preventing or treating cancer, comprising the step of administering the pharmaceutical composition to a subject.

The pharmaceutical composition, cancer, prevention and treatment are as described above.

The term "individual" of the present invention means all animals such as rats, mice, livestock, etc., including humans who have developed or can develop cancer. But are not limited to, mammals including humans. In addition, in the present invention, an individual may be excluded from human, but is not limited thereto.

The term "administering" of the present invention means introducing a given substance into a subject in an appropriate manner. Specifically, using the pharmaceutical composition of the present invention, anismaicin and isolated NK cells can be administered to an individual simultaneously, sequentially, or in reverse order.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dose level is determined by the sex, age And other medical fields, including drugs used in combination or concurrently, with respect to body weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, administration time, route of administration, Can be readily determined by those skilled in the art according to well known factors. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The route of administration and the mode of administration for administering the composition of the present invention are not particularly limited, and any route of administration and administration mode may be used as long as the composition containing the composition can reach the desired site. Specifically, the composition may be administered orally or parenterally through various routes. Non-limiting examples of routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, Intramuscularly or through inhalation or the like.

In addition, the cancer prevention or treatment method of the present invention can be used in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using a biological response modifier.

An isomicin increases the susceptibility of cancer cells to NK cells, and the cytotoxicity of NK cells to cancer cells is increased. As a result, the composition of the present invention shows that the anticancer effect is greatly increased by the combination treatment of anismaicin and NK cells Synergy appears.

Figure 1 shows the structure of Anisomycin.
FIG. 2 shows that the cytotoxicity of hepatocellular carcinoma cells (HepG2) is greatly increased when NK cells and anisoma are treated together.
FIG. 3 shows that cytotoxicity of lung cancer cells (A549) and pancreatic cancer cells (PC1) is greatly increased when NK cells and an isomycin are used in combination.
FIG. 4 shows that the expression levels of ULBP1, ULBP2 and ULBP3, which are important factors for NK cell activation and cytotoxicity in liver cancer cells (HepG2), are increased by anismaicin.
FIG. 5 shows a mechanism of increasing anticancer activity by the combination treatment of anismaicin and NK cells.
FIG. 6 is a graph showing that cytotoxicity against liver cancer cells (HepG2) is increased according to concentration only by treatment with anismaicin alone.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example  One. NK  Cell and Anomalous  Anticancer effect analysis by combination treatment

In order to confirm the anticancer effect of NK cells and Anisomycin in combination, cytotoxicity of NK cells and anismacein to cancer cells was analyzed. Specifically, 5 μM anismaicin was treated with 5 × 10 ⁴ HepG2 hepatocarcinoma cells per well of a 96-well plate for 2 days, and NK cells isolated from human blood after surviving HepG2 hepatoma cells were counted to 0.5 : 1 and 1: 1 (NK cell number: HepG2 cell number), followed by further incubation for 4 hours. As a control, NK cells were treated with the same number of HepG2 hepatocarcinoma cells (anismaicin-untreated cells) as the surviving HepG2 hepatoma cells treated with NK cells. Subsequently, MTT assay was performed on liver cancer cells to determine cytotoxicity.

As a result, it was confirmed that NK cell-induced cytotoxicity was remarkably increased to about 150 ~ 200% in case of hepatocarcinoma cells treated with anismacein (Fig. 2). In addition, it was confirmed that as the amount of NK cells treated with anismaicin increases, the cytotoxicity against liver cancer cells increases (FIG. 2).

As a result, it was confirmed that NK cells and an isomycin-treated NK cells increased the cancer cell killing ability of NK cells compared with the case where NK cells were treated alone. When NK cells and anismaicin were treated in combination, NK cells or It was found that the anticancer effect was superior to that of the single treatment with an isomicin.

Example  2. For various cancers NK  Cell and Anomalous  Analysis of concurrent treatment effect

To examine the anticancer effect of NK cells and anismaximin on various cancers, the cytotoxicity of NK cells and an isomycin in various cancers was analyzed. Specifically, 5 μM ansomycin was treated with 5 × 10 ⁴ lung cancer cell lines (A549) per 96 well plate wells or 5 × 10 ⁴ pancreatic cancer cell lines (PC1) per 96 well plate wells. Cells were cultured for 2 days, , And NK cells isolated from human blood were treated with cancer cells at a ratio of 1: 1 to the surviving cancer cells, followed by further incubation. In addition, as a control, NK cells were treated with the same number of cancer cells (cells not treated with anismaicin) as the surviving cancer cells treated with NK cells as described above. Then, cytotoxicity was measured by MTT assay for each cancer cell.

As a result, it was confirmed that NK cell-induced cytotoxicity was significantly increased in lung cancer cells treated with anismacein and pancreatic cancer cells compared with lung cancer cells and pancreatic cancer cells not treated with somaicin (FIG. 3).

As a result, it was confirmed that NK cells and an isomycin-treated NK cells increased the cancer cell killing ability of NK cells compared with the case where NK cells were treated alone. When NK cells and anismaicin were treated in combination, NK cells or It was found that the anticancer effect was superior to that of the single treatment with an isomicin.

Example  3. No Soma Isin NK  Increased anticancer activity by co-treatment of cells Mechanism  Confirm

The expression levels of ULBP1, ULBP2 and ULBP3, which are important factors in the activation and cytotoxicity of NK cells in hepatocarcinoma cells after treatment with an isomycin in HepG2 liver cancer cells, were measured using real time PCR method. The measurement method is as follows. First, HepG2 cells were treated with various concentrations of anismaicin (0, 0.05, 0.1, 0.25 μM) and cultured for 1 day or 2 days. Total RNA was extracted with Trizol and cDNA was synthesized. Then, ULPB1,2 And ULBP1, ULBP2, and ULBP3 were quantitatively analyzed by real-time PCR using a PCR probe specific for each strain. The amount of each of the measured amounts was expressed relative to the case in which no anisoma was treated.

As a result, it was confirmed that the expression of ULBP1, ULBP2 and ULBP3 was significantly increased in hepatoma cells according to the concentration of an isomycin (Fig. 4).

As a result of the increase of the expression of ligands (ULBP1, ULBP2, ULBP3) to NKG2D, an activating receptor important for the anticancer activity of NK cells in cancer cells, anomasicin induced the sensitivity of NK cells to cancer cells due to the increased ligand (Fig. 5). That is, as an isomicin increases the sensitivity of cancer cells to NK cells, the cytotoxicity of NK cells to cancer cells increases, and the anticancer effect is synergistically increased or decreased by the combination treatment of an isomycin and NK cells .

Comparative Example  One. No Somaiyin  Anticancer effect analysis by single treatment

In order to confirm the anticancer effect of anisomycin alone, the cytotoxicity of an isomycin to cancer cells was analyzed. Specifically, an isomicin was treated with liver cancer cell line (HepG2) in a concentration-dependent manner, and the liver cancer cell line was cultured for 2 days. Subsequently, MTT assay was performed on liver cancer cell lines to determine cytotoxicity.

As a result, it was confirmed that the cytotoxicity of liver cancer cells was increased according to the concentration only by treatment with an isomicin alone (Fig. 6).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (10)

Anisomycin and isolated natural killer (NK) cells.
2. The composition of claim 1, wherein the composition increases the expression of a ligand for the NKG2D receptor of NK cells in cancer cells.
2. The composition of claim 1, wherein the composition is administered concomitantly with anismaicin and isolated NK cells simultaneously, sequentially, or in reverse order.
The composition of claim 1, wherein the composition enhances the sensitivity of cancer cells to NK cells.
delete 2. The composition of claim 1, wherein the composition enhances the binding of cancer cells and NK cells.
2. The composition of claim 1, wherein the composition enhances killing ability of NK cells.
A kit for preventing or treating cancer, comprising the composition of any one of claims 1 to 4, 6, and 7.
A method of preventing or treating cancer in an animal other than a human, comprising administering to the individual a composition according to any one of claims 1 to 4, 6 and 7.
10. The method of claim 9, wherein said administering is co-administered sequentially, sequential, or in reverse order with anismaicin and isolated NK cells.

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