KR102150040B1 - A composition for preventing or treating liver cancer comprising 4-methylpyrazole - Google Patents

Abstract

The present invention relates to a composition for preventing or treating liver cancer comprising 4-methylpyrazole, specifically for preventing or treating liver cancer comprising 4-methylpyrazole or a pharmaceutically acceptable salt thereof It relates to a pharmaceutical composition, a method for preventing or treating liver cancer comprising administering the composition to an individual other than humans, and a health functional food for preventing or improving liver cancer comprising 4-methylpyrazole or a pharmaceutically acceptable salt thereof. . 4-methylpyrazole according to the present invention inhibits retinol metabolism of hepatic stellate cells, reduces the activity of myeloid-derived inhibitory cells, which helps the survival of cancer cells, and increases the activity of CD8+ T cells and natural killer cells to form liver cancer. Since it can inhibit the liver cancer can be usefully used as a composition for preventing or treating.

Description

Composition for preventing or treating liver cancer comprising 4-methylpyrazole {A composition for preventing or treating liver cancer comprising 4-methylpyrazole}

The present invention relates to a composition for preventing or treating liver cancer comprising 4-methylpyrazole, specifically for preventing or treating liver cancer comprising 4-methylpyrazole or a pharmaceutically acceptable salt thereof It relates to a pharmaceutical composition, a method for preventing or treating liver cancer comprising administering the composition to an individual other than humans, and a health functional food for preventing or improving liver cancer comprising 4-methylpyrazole or a pharmaceutically acceptable salt thereof. .

Cancer is a disease with the highest mortality rate in Korea. According to data from the National Statistical Office in 2009, cancer, cerebrovascular disease, and heart disease accounted for about 50% of all deaths. It is a serious problem socially. In addition, among OECD countries, Korea ranked first in the incidence rate of liver cancer, with 27.8% of the total deaths in 2011, of which 22.2% died of lung cancer, and liver cancer ranked second with 15.3%. Despite the remarkable progress in recent treatments for liver cancer (liver resection, liver transplantation, and chemotherapy), the 5-year survival rate of liver cancer is reported to be the third lowest after pancreatic cancer and lung cancer. Although it shows an increase of 26.7%, the recurrence rate within 5 years after surgery is reported to be over 70%, so it is classified as a refractory cancer.

In the case of early liver cancer, resection is used as the best treatment, and in the case of liver cancer (less than 3 nodules or less than 3 cm), hepatectomy, radiofrequency therapy, and liver transplant are initially performed depending on the stage, and in the middle stage, transarterial chemoembolization is performed. , TACE) is being used, and at the end, systemic anticancer therapy is being used as a treatment for liver cancer.

In the case of advanced liver cancer, systemic anticancer therapy is used. Currently, Sorafenib (Nexaba) is the only one used. Sorafenib is a multikinase inhibitor, and when administered orally, it is known that it can prolong life for only about 3 months compared to the placebo group. The main side effects are diarrhea, fatigue, decreased body pressure, and limb syndrome. Sorafenib showed a partial response in less than about 5% of 137 patients in phase 2 clinical trials, and recently, patients with resistance to sorafenib were also reported, as an aid to replace sorafenib or suppress sorafenib resistance in the treatment of liver cancer. There is a desperate need for effective new drugs that can be used in various ways in the form of drugs or liver cancer.

Accordingly, the present inventors have made intensive research efforts to develop a composition for treating liver cancer, as a result of which 4-methylpyrazole (4-MP) reduces the activity of myeloid-derived suppressor cells, which helps cancer cells survive, and CD8+ By confirming that it is possible to inhibit the formation of liver cancer by increasing the activity of T cells and natural killer cells, the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer, including 4-methylpyrazole or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a health functional food for preventing or improving liver cancer, including 4-methylpyrazole or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for preventing or treating liver cancer comprising administering 4-methylpyrazole or a pharmaceutically acceptable salt thereof to an individual other than humans.

One aspect of the present invention for achieving the above object is to provide a pharmaceutical composition for preventing or treating liver cancer, including 4-methylpyrazole or a pharmaceutically acceptable salt thereof.

In the present invention, the term “4-methylpyrazole (4-MP)” is composed of the following formula (1), is also referred to as pomepizole, and converts methanol and ethylene glycol into toxic decomposition products. It has the function of blocking alcohol dehydrogenase, and is known as a drug mainly used in the treatment of methanol and ethylene glycol poisoning. However, the therapeutic effect of 4-MP on liver cancer was first identified by the present inventors.

[Formula 1]

The method of obtaining the 4-MP is not particularly limited, and may be chemically synthesized using a method known in the art, or a commercially available material may be used.

In addition, the compound may exist in a solvated or unsolvated form, and may exist in a crystalline or amorphous form, and all such physical forms are included in the scope of the present invention.

In the present invention, the term "hepatocellular carcinoma" refers to a malignant tumor originating from liver cells that occupy most of the liver. In a broad sense, it includes all types of malignant tumors in the liver (e.g., intrahepatic bile duct cancer) and metastatic liver cancer caused by metastasis to the liver of other organs, but hepatocellular carcinoma (HCC) is the most common among liver cancers. May mean only hepatocellular carcinoma arising from liver cells.

In the present invention, the term "prevention" refers to any action of suppressing or delaying the symptoms of liver cancer by administration of a composition comprising 4-MP according to the present invention.

In the present invention, the term "treatment" refers to any action in which symptoms of liver cancer are improved or advantageously changed by administration of a composition containing 4-MP according to the present invention.

The 4-MP liver cancer prevention or treatment may be achieved by increasing the activity of NK, NKT, and CD8+ T cells that induce apoptosis of liver cancer cells (FIGS. 2 and 4).

Specifically, it is widely known that NKT and CD8T cells secrete substances such as Interferon-gamma when liver cancer is generated to suppress proliferating hepatocellular carcinoma cells (NKT and CD8 lymphocytes mediate suppression of hepatocellular carcinoma growth via tumor antigen-pulsed dendritic). cells.Int J Cancer. 2003 Aug 20;106(2):236-43; Natural Killer Cells in Liver Disease and Hepatocellular Carcinoma and the NK Cell-Based Immunotherapy.J Immunol Res. 2018 Sep 4;2018:1206737; Inflammation- induced IgA+ cells dismantle anti-liver cancer immunity.Nature. 2017 Nov 16;551(7680):340-345), the present inventors found that 4-MP is an activity of CD8+ T cells that induces cell death of NK, NKT, and liver cancer cells. By confirming that it increases, it was confirmed that 4-MP can be effectively used for preventing or treating liver cancer.

In a specific embodiment of the present invention, in the process of co-culturing hepatic stellate cells and myeloid-derived suppressor cells, 4-MP was treated and the cell proliferation response to CD8+ T cells was confirmed. It was confirmed that the proliferation of T cells increased again when 4-MP was administered (FIG. 4).

In addition, the liver cancer prevention or treatment may be achieved by reducing bone marrow-derived suppressor cells.

In a specific embodiment of the present invention, as a result of confirming the distribution of Ly6C+ myeloid-derived inhibitory cells through immunostaining in the liver of mice in which liver cancer was induced with diethylnitrosamine (DEN), hepatic astrocytes in the 4-MP non-administration group The accumulation of Ly6C+ bone marrow-derived suppressor cells was confirmed in the vicinity, but the accumulation of Ly6C+ bone marrow-derived suppressor cells was decreased in the 4-MP administration group (FIG. 1).

The Ly6C+ bone marrow-derived suppressor cells are known to help the survival of cancer cells by inhibiting CD8+ T cells or natural killer cells, etc., which are involved in the death of cancer cells (Higher frequencies of GARP(+)CTLA-4(+)Foxp3( +) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.Cancer Res. 2013 Apr 15;73(8):2435-44; Myeloid derived suppressor cells inhibit natural killer cells in patients with hepatocellular carcinoma via the NKp30 receptor. Hepatology. 2009 Sep;50(3):799-807.

Therefore, it can be determined that the therapeutic effect of liver cancer of the present invention is achieved by reducing bone marrow-derived suppressor cells.

The reduction of the bone marrow-derived inhibitory cells may be achieved by inhibiting retinol metabolism.

In a specific embodiment of the present invention, in the process of co-culturing hepatic stellate cells and bone marrow-derived suppressor cells, 4-MP was treated and the expression of genes related to retinol metabolism was analyzed. As a result, compared to the 4-MP non-administered group, 4- It was confirmed that the expression of various genes (ADH3, Raldh1, CRABP1, CX3CL1, RARα, RARβ, RXRβ) affecting retinol metabolism and signal transduction in astrocytes in the MP administration group was reduced (FIG. 3 ).

The term "pharmaceutical composition" used in the present invention means that it is prepared for the purpose of preventing or treating diseases, and may be formulated and used in various forms according to conventional methods. For example, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, and can be formulated in the form of external preparations, suppositories, and sterile injectable solutions. Specifically, it may be formulated and used in a form suitable for eye drop administration, for example, eye drops, creams, ointments, gels or lotions.

The term "pharmaceutically acceptable salt" of the present invention refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bound by electrostatic attraction, and generally, metal salts, organic bases and Salts, 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.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt, and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine May be salts with the like; Salts with inorganic acids may be salts of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids may be salts of 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; Salts with acidic amino acids may be salts with aspartic acid and glutamic acid.

The pharmaceutical composition of the present invention may contain 0.01 to 80%, specifically 0.01 to 70%, more specifically 0.01 to 60% by weight of 4-MP or a pharmaceutically acceptable salt thereof based on the total weight of the composition, but liver cancer As long as it shows a prophylactic or therapeutic effect, it is not limited thereto.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for preventing or treating liver cancer further comprising an appropriate carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, the carrier being a non-natural carrier ( non-naturally occuring carrier).

In the present invention, the carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the upper leaf extract and its fractions. , Sucrose (sucrose) or lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include water and liquid paraffin, which are simple diluents commonly used for suspensions, liquid solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, etc. according to a desired method, and the active ingredient of the pharmaceutical composition is the age of the subject to be administered. , Sex, weight, pathological condition and severity, route of administration, or the judgment of the prescriber. Determination of the application amount based on these factors is within the level of those skilled in the art, and its daily dose is, for example, 0.1 mg/g/day to 100 mg/g/day, more specifically 5 mg/g/day to 50 mg/day. It may be g/day, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage does not in any way limit the scope of the present invention.

Another aspect of the present invention is to provide a health functional food for preventing or improving liver cancer, including 4-methylpyrazole or a pharmaceutically acceptable salt thereof.

In the present invention, the terms “4-methylpyrazole” and “liver cancer” are as described above.

Functional food is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects that have been processed to efficiently display bioregulatory functions in addition to nutritional supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in inhibiting cancer metastasis.

The health functional food of the present invention may further include a food pharmaceutically acceptable carrier.

There is no particular limitation on the type of food to which the composition containing 4-MP of the present invention can be added, and examples include various beverages, gums, teas, vitamin complexes, and health supplement foods. Other ingredients that do not interfere with the liver cancer treatment effect may be added to the food composition, and the type is not particularly limited. For example, it may contain various herbal extracts, food additives, or natural carbohydrates as an additional component, as in ordinary foods.

The food supplementary additives are added to prepare health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. For example, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. are included, but the types are not limited by the above examples.

At this time, the content of the extract contained in the food is not particularly limited thereto, but may be included in 0.01 to 100% by weight, more preferably 1 to 80% by weight based on the total weight of the food composition.

When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g, based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve smell, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleached and highly bleached, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoleuene ( BHT), etc.), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweeteners (dulsin, cyclamate, saccharin, sodium, etc.) , Flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen stannate, etc.), reinforcing agents, emulsifiers, thickeners (thickening agents), coating agents, gum base agents, foam inhibitors, solvents, and food additives such as improving agents. ) Can be added. The additive is selected according to the type of food and used in an appropriate amount.

The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of the production, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and it can be excellent in portability.

Another aspect of the present invention is to provide a method for preventing or treating liver cancer, comprising administering 4-methylpyrazole or a pharmaceutically acceptable salt thereof to an individual other than a human.

In the present invention, the terms “4-methylpyrazole” and “liver cancer” are as described above.

The term "individual" used in the present invention may mean all animals including humans who have or are likely to develop liver cancer. The animals may be mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. in need of treatment for symptoms similar to humans, but are not limited thereto.

The prevention or treatment method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to an individual who has developed or is at risk of developing liver cancer.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

4-methylpyrazole according to the present invention inhibits retinol metabolism of hepatic stellate cells, reduces the activity of myeloid-derived inhibitory cells, which helps the survival of cancer cells, and increases the activity of CD8+ T cells and natural killer cells to form liver cancer. Since it can inhibit the liver cancer can be usefully used as a composition for preventing or treating.

1 is a graph confirming the effect of reducing the incidence of liver cancer induced by diethylnitrosamine (DEN) by administration of 4-methylpyrazole (4-MP); A) It is a diagram explaining the overall experimental method, and it specifies the formation of DEN-induced liver cancer and the administration method of 4-MP. B) The result of induction of liver cancer due to DEN visually and DEN-induced liver cancer by 4-MP C) The maximum tumor size and the number of tumors in mice induced with liver cancer by DEN were reduced by 4-MP administration, and ALT (alanine), an indicator of liver damage in the serum. aminotransferase) and AST (aspartate aminotransferase) also showed decreased results.D and E) inhibited Ly6C-positive bone marrow in the vicinity of α-smooth muscle actin (α-SMA) positive hepatic stellate cells in mice induced liver cancer with DEN. Cells are accumulated, and the accumulation of Ly6C-positive myeloid-derived suppressor cells is suppressed by 4-MP.
2 is a graph confirming the decrease of myeloid-derived suppressor cells and the increase of anti-tumor killer cells by 4-MP administration in the process of DEN-induced liver cancer development; A) In the DEN-induced liver cancer model, there was no significant change in blood by 4-MP administration, but among the immune cells infiltrated into the liver, inflammatory macrophages (CD11b+F4/80+) and bone marrow-derived inhibitory cells (CD11b+Ly6G-Ly6Chigh or MHC-II-Ly6G-Ly6Chigh) was reduced. B) In the DEN-induced liver cancer model, there was no similarly significant change in blood by 4-MP administration, but natural killer cells among the infiltrated immune cells in the liver ( NK cells, CD3-NK1.1+) and natural killer T cells (NKT cells, CD3+NK1.1+) were increased in proportion. C) In the DEN-induced liver cancer model, by 4-MP administration The expression of Arg1, iNOS, CX3CR1, and CCR2, which contribute to increasing liver cancer, decreases, and IFN-γ, Granzyme B, Perforin, and TRAIL, which play an important role in inhibiting liver cancer, show an increase in expression.
3 is a graph confirming the effect of 4-MP on genes related to retinol metabolism and liver cancer formation; A) The result of treatment with 4-MP on the 4th day of in vitro culture of mouse hepatic stellate cells showed an increase in the content of lipid droplets in hepatic stellate cells compared to the control. B) 4 days of mouse hepatic stellate culture Fourth, the expression of ADH3, Raldh1, LRAT, CRABP1, CX3CR1, RARα, RARβ, RXRβ, which is dependent on retinol metabolism and metabolite retinoic acid, decreased by 4-MP treatment, C) mouse hepatic stellate cell culture 4 days Second, when Ly6C-positive was co-cultured with bone marrow cells, the expression of Arg1 in bone marrow cells decreased by 4-MP treatment. D) When co-cultured with hepatic stellate cells on the 4th day of culture, 4-MP was observed. The effect of inhibition of retinol metabolism on the expression of Arg1 is shown.E) In a DEN-induced liver cancer model, the expression of α-SMA, Col1α1, ADH3, Raldh1, and CX3CR1 in the liver tissue is reduced by 4-MP administration. F) In the DEN-induced liver cancer model, CX3CR1 decreased in blood and liver tissue by 4-MP administration.
4 is a graph confirming the T cell proliferation inhibitory effect of hepatic stellate cell mediated Ly6C+ cells reduced by 4-MP treatment; A) Ly6C-positive bone marrow cells co-cultured with hepatic stellate cells after separating Ly6C-positive cells from bone marrow and co-cultured with hepatic stellate cells on day 4 of culture, and CD8+ T cells were isolated from the spleen and stimulated with α-CD3 and CD28. It is a diagram schematically explaining the experimental method that confirmed the effect on cell proliferation through co-culture with B) when co-cultured with hepatic stellate cells on the fourth day of culture, the expression of Arg1 in Ly6C-positive bone marrow cells significantly increased, but 4 -MP treatment showed the result of decreasing again, C and D) CD8+ T cell proliferation was inhibited by Ly6C-positive bone marrow cells co-cultured with hepatic stellate cells, but when co-cultured with hepatic stellate cells and Ly6C-positive bone marrow cells 4 -MP treatment shows that the proliferation of CD8+ T cells is again increased.

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

Example 1.4- In methylpyrazole (4-methylpyrazole, 4-MP) by Diethylnitrosamine (diethylnitrosamine, DEN)-induced liver cancer formation inhibitory effect

Liver cancer was induced by intraperitoneal injection of DEN to 14-day male mice at 20 mg/kg, and vehicle or 4-MP (10 mg/kg) was treated three times a week from 30 weeks of age and sacrificed after 10 weeks. Proceeded. In the overall examples, statistics were expressed as mean±SEM, and compared with the corresponding control group, *P<0.05, **P<0.01.

1-1. 4- MP Liver cancer formation inhibitory effect

After opening the abdominal cavity of the mouse and extracting the liver, the level of liver cancer formation was confirmed. As a result, as shown in Fig. 1B, it was confirmed that the size and number of cancer tissues were reduced in the liver of the mice treated with 4-MP. In addition, it was confirmed that the maximum size and number of tumors were reduced by 4-MP treatment as shown in FIG. 1C.

1-2. ALT in serum, AST Content measurement

Blood was collected from the abdominal aorta of the mouse, centrifuged at 8,000 rpm for 10 minutes with a centrifuge, and then only the serum was separated, and the ALT and AST contents in the serum were determined using a kit from the IDEXX Laboratories (USA) company. It was measured by the vet test (VetTEST, IDEXX, USA) according to the experimental method provided by the manufacturer.

As a result, as shown in Fig. 1C, it was confirmed that ALT and AST levels indicating damage to hepatocytes were reduced by 4-MP.

1-3. 4- MP On by Ly6C + Of myeloid-derived inhibitory cells Reduced infiltration

According to the existing literature, Ly6C+ bone marrow-derived suppressor cells are known to help the survival of cancer cells by inhibiting CD8+ T cells or natural killer cells, which are involved in the death of cancer cells. Immunostaining and flow cytometric analysis (LSRII, BD bioscience) were performed to observe changes in Ly6C+ myeloid-derived suppressor cells between the mice.

As a result, as shown in Fig. 1D, the accumulation of Ly6C+ myeloid-derived inhibitory cells occurs in the vicinity of α-SMA-positive hepatic stellate cells during DEN treatment as a result of immunostaining. It was confirmed that the accumulation was reduced. As shown in FIG. 2A, the same result was confirmed even during flow cytometric analysis.

1-4. 4- MP Of immune cells and increased anticancer activity

In the case of immune cells and anticancer related genes, it was confirmed that natural killer cells and natural killer T cells were increased among immune cells in the liver by 4-MP as shown in FIG. 2B. In addition, as shown in Figure 2C, the expression of Arg1, iNOS, CX3CR1, and CCR2 showing carcinogenic effects decreased, and the expression of IFN-γ, Granzyme B, Perforin, and TRAIL showing anticancer activity increased.

Therefore, it was confirmed that the 4-MP of the present invention inhibits the formation of DEN-induced liver cancer and decreases Ly6C+ myeloid-derived suppressor cells that help the survival of cancer cells.

Example 2. 4- MP On by Hepatic stellate cells And Ly6C + Bone marrow-derived inhibitory cells Liver interaction inhibitory effect

In vitro and in vivo experiments were conducted to confirm the specific mechanism of the inhibitory effect on the occurrence of liver cancer by 4-MP identified in Example 1. 4-MP inhibits alcohol dehydrogenase, which is involved in the primary metabolism of retinol, thereby causing a disorder in retinol metabolism. Therefore, it was hypothesized that 4-MP treatment would lead to changes in various immune responses by inhibiting retinol metabolism in hepatic stellate cells, which mainly store retinol, and experiments such as co-culture were conducted to confirm this.

2-1. 4- MP end Hepatic stellate cells And Ly6C + Bone marrow-derived suppressor cells Effect

Taking advantage of the characteristic that primary hepatic stellate cells directly collected from mice are activated only by in vitro culture, hepatic stellate cells in an activated state after culture until the 4th day were used for the experiment. The functional changes of myeloid-derived suppressor cells by 4-MP treatment were evaluated through co-culture with activated hepatic stellate cells and Ly6C+ myeloid-derived suppressor cells.

As a result, as shown in FIG. 3A, it was confirmed that the content of lipid droplets in hepatic stellate cells increased during 4-MP treatment when observed with a phase contrast microscope. As shown in FIG. 3B, the duration of 4-MP treatment It was confirmed that the expression of various genes (ADH3, Raldh1, CRABP1, CX3CL1, RARα, RARβ, RXRβ) affecting retinol metabolism and signaling in astrocytes was reduced. In addition, hepatic stellate cells are cells containing retinol and may be involved in retinol signal transduction in other cells by delivering retinol to other cells. When co-cultured with hepatic stellate cells in Ly6C+ myeloid-derived suppressor cells, the expression of Arg1, which is involved in the inhibition of CD8+ T cells and natural killer cells, was remarkably increased, but it was confirmed that the result was reduced by 4-MP. In addition, as shown in Fig. 3D, it was confirmed that the expression of Arg1, which was increased in Ly6C+ myeloid-derived suppressor cells by retinol treatment, was decreased by 4-MP.

As shown in Fig. 3E, in vivo as shown in the above results, expression of α-SMA and Col1α1 representing the degree of hepatic fibrosis decreased by 4-MP treatment, and expression of ADH3 and Raldh1 involved in retinol metabolism decreased. It was confirmed that the expression of CX3CL1, a chemokine involved in the recruitment of Ly6C+ bone marrow-derived suppressor cells, decreased. Enzymatic immunoassay was performed to measure the level of CX3CL1, and as shown in FIG. 3F, it was confirmed that CX3CL1 decreased by 4-MP in serum and liver tissue.

2-2. 4- MP end CD8 + Effects on T cells

In order to confirm the change of CD8+ T cells involved in the inhibition of liver cancer, the cell proliferation response to CD8+ T cells was confirmed using Ly6C+ bone marrow-derived suppressor cells co-cultured with hepatic stellate cells as shown in FIG. 4A. Cell proliferation response was measured according to the experimental method provided by the manufacturer using EZ-Cytox (Daeil Lab Service, Korea).

As a result, as shown in Fig. 4B, the expression of Arg1, which is involved in the inhibition of proliferation of CD8+ T cells in Ly6C+ bone marrow-derived suppressor cells, significantly increased when co-cultured with hepatic stellate cells, as in the result of Example 2-1. -It was confirmed that it decreased again with MP treatment. As a result of this, as shown in Figs. 4C and 4D, the proliferation of CD8+ T cells is remarkably suppressed by Ly6C+ myeloid-derived inhibitory cells co-cultured with hepatic stellate cells, but the inhibitory effect is reduced when co-cultured with 4-MP treatment. It was confirmed that the proliferation of T cells increased again.

Therefore, the mechanism of inhibiting the formation of DEN-induced liver cancer of 4-MP of the present invention reduces the activity of myeloid-derived inhibitory cells increased by retinol by inhibiting the metabolism of retinol in hepatic stellate cells, thereby killing and inhibiting cancer cells. It was confirmed that it was due to an increase in cell activity.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

Claims (6)

A pharmaceutical composition for preventing or treating non-alcoholic liver cancer, comprising as an active ingredient 4-methylpyrazole of Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

The pharmaceutical composition of claim 1, wherein the composition increases the activity of CD8+ T cells.
The pharmaceutical composition of claim 1, wherein the composition reduces bone marrow-derived suppressor cells.
The pharmaceutical composition of claim 3, wherein the reduction of myeloid-derived suppressor cells is achieved by inhibiting retinol metabolism.
A health functional food for preventing or improving non-alcoholic liver cancer, including 4-methylpyrazole of the following formula (1) or a pharmaceutically acceptable salt thereof.
[Formula 1]

A method for preventing or treating non-alcoholic liver cancer comprising administering 4-methylpyrazole of Formula 1 or a pharmaceutically acceptable salt thereof to an individual other than a human.
[Formula 1]

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