KR101825637B1 - A pharmaceutical composition for prevention or treatment of cancer and use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, containing homoharringtonine and isolated natural killer (NK) cells. The present invention further relates to a use thereof. Homoharringtonine increases sensitivity in cancer cells against NK cells, bringing an increase in cytotoxicity in the NK cells against cancer cells. Thus, the composition of the present invention shows synergistic effects in remarkably increasing anticancer effects by combined treatment of NK cells and homoharringtonine.

Description

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

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising homoharringtonine and isolated natural killer (NK) cells, and uses thereof.

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, and as a result, the anticancer effect of the homoharring tonin and the isolated NK cell was significantly increased synergistically, 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 homoharringtonine and isolated natural killer (NK) cells.

Another object of the present invention is to provide a composition for enhancing sensitivity of cancer cells to NK cells, which comprises homoharring tonal.

It is still another object of the present invention to provide a composition for promoting the binding of cancer cells and NK cells, which comprises homoharringtonine.

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

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, which comprises homoharringtonine and isolated natural killer (NK) cells.

The term " Homoharringtonine "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 homoharringtonine 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 sensitivity of cancer cells to NK cells. Furthermore, the inventors of the present invention have for the first time concluded that the anticancer effect is significantly increased or decreased synergistically when the homoharring tonin and the isolated NK cell are used in combination. In particular, the pharmaceutical composition of the present invention not only exhibits anticancer effects due to the combination of the cancer cell killing ability of homoharringtonine and the cancer cell killing ability of NK cells, but also enhances the sensitivity of cancer cells to NK cells, And the cancerous effect was significantly increased synergistically.

The composition of the present invention may include a pharmaceutically acceptable salt of homoharring tonin in place of or in combination with a homoharring tonn.

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 homoharringtonine as well as possible solvates and hydrates thereof which may be prepared therefrom and may include all possible stereoisomers. The solvates, hydrates, and stereoisomers of the homoharring toners can also be prepared by 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, the homo-harringtonin and the isolated NK cells may be administered concurrently, 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 suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

Another embodiment of the present invention provides a composition for enhancing the sensitivity of cancer cells to NK cells, comprising homoharring tonal.

The homoharringtonine, 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, comprising homoharring tonal.

The homoharringtonine, 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 the homoharring tonon of the present invention increases the expression of the ligand for the NKG2D receptor of the NK cell in the cancer cell and can improve the binding of the NKG2D receptor with the cancer cell expressing the ligand.

Another aspect of the present invention provides a composition for enhancing cancer cell killing ability of NK cells, comprising homoharring tonal.

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

The composition comprising the homoharring tonon of the present invention can increase the expression of a ligand capable of binding to an activating receptor of NK cells and enhance the killing ability of NK cells even in cancer cells not killed by homoharringtonine.

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 kits of the present invention may be packaged in a form contained in a separate container, or in a single container divided into one or more compartments, wherein the homo-haring tonin and the separated NK cells are packaged in a separate container, But are not limited to, each packaged in unit dosage form in a single dose.

The homoharring tonnage and the separated 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, homoharringtonine 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, the pharmaceutical composition of the present invention can be used to co-administer the homoharring tonnens and the isolated NK cells to the 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.

Homoharringtonone 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 homoharringtonine and NK cells Synergy appears.

1 is a view showing the structure of Homoharringtonine.
FIG. 2 shows that cytotoxicity of hepatocarcinoma cells (HepG2) is greatly increased when NK cells and homoharringtonine are used in combination.
FIG. 3 shows that cytotoxicity of lung cancer cells (A549) and pancreatic cancer cells (PC1) is greatly increased when NK cells and homoharringtonine are co-administered.
FIG. 4 shows that the expression levels of ULBP1, ULBP2 and ULBP3, which are important factors in the activation and cytotoxicity of NK cells in liver cancer cells (HepG2) by homoharringtonine, are increased.
FIG. 5 is a graph showing the mechanism by which the anti-cancer activity is increased by the combination treatment of homoharringtonine and NK cells.
FIG. 6 is a graph showing that cytotoxicity against hepatocarcinoma cells (HepG2) is increased depending on the concentration alone by treatment with homoharringtonine 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 Homo-haring tonin  Anticancer effect analysis by combination treatment

To investigate the anticancer effect of NK cells and homoharringtonine, cytotoxicity of NK cells and homoharringtonine to cancer cells was analyzed. Specifically, 0.1 μM homo-harringtonin was treated with 5 × 10 ⁴ HepG2 hepatocarcinoma cells per well in a 96-well plate for 2 days. The number of cells was counted, and NK cells isolated from human blood in the surviving HepG2 liver cancer cells 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 liver cancer cells (cells not treated with homoharringtonine) 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 the hepatocarcinoma cell treated with homoharringtonine significantly increased the cytotoxicity of NK cell by about 150 ~ 200% compared to the hepatoma cell not treated with homoharringtonine (FIG. 2). In addition, it was confirmed that as the amount of NK cells treated with homoharringtonine increases, cytotoxicity against hepatocarcinoma cells increases (FIG. 2).

As a result, it was confirmed that NK cells and homoharringtonine-treated NK cells increased the cancer cell killing ability of NK cells when treated with NK cells alone or in combination with NK cells. It was found that the anticancer effect was more excellent than that of homo-treating alone.

Example  2. For various cancers NK  Cell and Homo-haring tonin  Analysis of concurrent treatment effect

To evaluate the anticancer effect of NK cells and homoharringtonine combination treatment on various cancers, cytotoxicity of NK cells and homoharringtonine in various cancers was analyzed. Specifically, 5 μM homo-harringtonin 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 for 2 days, And NK cells isolated from human blood were treated with cancer cells at 1: 1 and then further cultured in surviving cancer cells. As a control, NK cells were treated with the same number of cancer cells (cells not treated with homoharringtonine) 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 cytotoxicity of NK cells was significantly increased in lung cancer cells treated with homoharringtonine and pancreatic cancer cells than in lung cancer cells and pancreatic cancer cells not treated with homoharringtonine (FIG. 3).

As a result, it was confirmed that NK cells and homoharringtonine-treated NK cells increased the cancer cell killing ability of NK cells when treated with NK cells alone or in combination with NK cells. It was found that the anticancer effect was more excellent than that of homo-treating alone.

Example  3. Homo haring tonin and 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 homoharringtonine were measured using real time PCR method. The measurement method is as follows. First, HepG2 cells are treated with various concentrations of homoharring tonin (0, 0.05, 0.1, 0.25 μM) and cultured for 1 day or 2 days. Then, total RNA was extracted using Trizol, and cDNA was synthesized. Then, quantitative analysis using ULPB1, 2, and 3 specific PCR probes was performed and real time PCR was performed to determine the amounts of ULBP1, ULBP2 and ULBP3. The expression level of each of the measured amounts was relatively expressed based on the case where the homoharring tonin was not treated.

As a result, the expression of ULBP1, ULBP2 and ULBP3 in hepatocarcinoma cells was significantly increased according to the concentration of homo-harringtonin (Fig. 4).

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

Comparative Example  One. Homo Haringtonin  Anticancer effect analysis by single treatment

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

As a result, it was confirmed that cytotoxicity was increased in hepatocarcinoma cells according to the concentration only by homo-treatment of homoharringtonine (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)

A pharmaceutical composition for preventing or treating cancer, comprising homoharringtonine and isolated NK (Natural killer) 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 in combination with simultaneous, sequential, or reverse sequencing of homoharring toenine and isolated NK.
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 with simultaneous, sequential, or reverse sequencing of homoharring tolonine and isolated NK.

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