KR102714321B1 - Anticancer composition comprising cancer metabolism regulator - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol. The composition according to the invention exhibits a synergistic anticancer effect by appropriately combining specific drugs that have the problem of having to be used in excessive amounts, thereby killing cancer cells even with a small amount and effectively treating cancer. Furthermore, the composition of the present invention exhibits a toxic effect specifically on cancer cells without exhibiting toxicity on normal cells, thereby killing only cancer cells without side effects, and thus can be usefully used as an anticancer agent and a composition for preventing or improving cancer.

Description

{Anticancer composition comprising cancer metabolism regulator}

The present invention relates to a composition for preventing or treating cancer, comprising a glutaminase inhibitor; a biguanide compound; 2-deoxy-D-glucose; and an inositol compound, and optionally comprising an immune checkpoint inhibitor, a method of treatment using the same, and uses thereof.

When considered as a single entity, cancer is the second leading cause of death worldwide. The cost of cancer treatment is skyrocketing as demands on the health budgets of all countries increase. Cancer cells have a very different metabolism from normal cells, which allows them to proliferate at a higher rate and resist some cell death signals.

The importance of cellular metabolism in cancer development was initially observed when Warburg discovered that tumor cells exhibit enhanced glycolytic activity compared to normal cells. This phenomenon, now known as the “Warburg effect,” has recently become the focus of intensive efforts in the discovery of new therapeutic targets and new cancer drugs. However, cancer cells do not simply enhance their glycolytic activity in metabolism by upregulating the expression of enzymes in the pathway to promote many reactions in the pathway.

They were observed to produce energy primarily by lactic acid fermentation in the cytoplasm after a high rate of glycolysis, rather than by oxidation of pyruvate following a relatively low rate of glycolysis in the mitochondria as in most normal cells. The former is anaerobic, not using oxygen, while the latter is aerobic, using oxygen.

This shift in cancer cell metabolism occurs even in oxygen-rich environments. It was discovered that this shift in cancer cell metabolism occurs because the conversion of phosphoenolpyruvate to pyruvate catalyzed by the enzyme pyruvate kinase is not accelerated but rather attenuated in cancer cells (Christofk et al., Nature 2008;452:230-233).

Under aerobic conditions, cancer cells tend to favor glycolysis over the much more efficient oxidative phosphorylation pathway favored by most other cells in the body (Alfarouk et al., Oncoscience 2014;12:777-802). In tumor cells, the end product of glycolysis, pyruvate, is converted to lactate (Alfarouk et al., J ENZYM INHIB MED CH. 2016;31:859-866).

Recently, there has been much interest in developing anticancer treatments that target cell signaling pathways important for cancer cell metabolism and growth. Representative drugs involved in cancer cell metabolism include biguanide compounds such as metformin and 2-deoxy-D-glucose (Wokoun et al., Oncol Rep. 2017;37:2418-2424).

As an antihyperglycemic agent, metformin has been used as the first-line treatment for type 2 diabetes for decades. Despite the widespread use of metformin as an antidiabetic agent, its potential anticancer effects in mammals were first reported in 2001. Furthermore, the first report on a reduced cancer risk in type 2 diabetes patients treated with metformin was published only 10 years ago. Since then, numerous studies have shown consistent antiproliferative effects of metformin in several cancer cell lines, including ovarian cancer, and in xenograft or transgenic mice. With regard to metabolism, metformin has been identified as a new class of complex I and ATP synthase inhibitors, which act directly on mitochondria to restrict respiration, making it energy inefficient, and reducing glucose metabolism via the citric acid cycle (Andrzejewski et al., Cancer Metab. 2014;2:12-25).

2-Deoxy-D-glucose has been considered as a potential anticancer agent because of the dependence of tumor cells on this process. 2-Deoxy-D-glucose is a glucose analog that can be readily taken up by glucose transporters and acts as a competitive inhibitor of this process, decreasing ATP production and inducing apoptosis through the activation of caspase-3 in solid tumors (Zhang et al., Cancer Lett. 2014;355:176-183).

Commonly used doses of metformin and 2-deoxy-D-glucose are insufficient to sufficiently treat cancer, and have limitations in that high-dose treatment can cause adverse reactions (Raez et al., Cancer Chemother Pharmacol. 2013;71:523-530). Combination therapy of metformin and 2-deoxy-D-glucose studied by Cheong et al. showed that it was effective against breast cancer cell lines, but the concentration was higher than the tolerable concentration in the human body or the concentration that can be administered in plasma (Cheong et al., Mol Cancer Ther. 2011;10:2350-2362). In another paper, the combination of metformin and 2-deoxy-D-glucose was successfully tested in prostate cancer cells using a metformin concentration higher than the concentration available in human plasma (Ben Sahra et al., Cancer Res. 2010;70:2465-2475). This suggests that it would be desirable to lower the therapeutic concentrations of metformin and 2-deoxy-D-glucose, both clinically effective anticancer agents, to a range reasonably achievable in vivo. This suggests the need to develop combination therapies as well as strategies for tumor-selective delivery (He et al., J CONTROL RELEASE. 2015;219:224-236).

Meanwhile, pyruvate generated through a unique enzymatic mechanism is mainly converted to lactate rather than acetyl-CoA for citrate synthesis, which normally enters the citric acid cycle. This process is enhanced in cancer cells, but is no longer a source of biosynthetic precursors. To accommodate the alteration of this pathway, cancer cells shift to an increased rate of glutamine metabolism to maintain the citric acid cycle, especially given the loss of input from pyruvate (Erickson et al., Oncotarget. 2010;1:734-740). This demonstrates an important role for glutamine in energy production, biosynthesis, and cellular homeostasis in cancer cells, in addition to glucose (Altman et al., Nat Rev Cancer. 2016;16:619-634).

The shift toward increased glutamine metabolism occurs through accelerated conversion of cytosolic glutamine to glutamate in the mitochondria, catalyzed by the mitochondrial membrane enzyme glutaminase. Glutamate is subsequently converted to α-ketoglutarate by glutamate dehydrogenase or glutamate oxaloacetate aminotransferase (Emadi et al., Exp. Hematol. 2014;42:247-251).

A consequence of increased glutamine metabolism in cancer cells is increased production of α-ketoglutarate for the citric acid cycle. This outcome is of great importance to cancer cells because it provides carbon for the citric acid cycle, which produces glutathione, fatty acids, and nucleotides, and contributes nitrogen to produce hexosamines, nucleotides, and many nonessential amino acids (Xiang et al., J. Clin. Invest. 2015;125:2293-2306).

Cancer cells are dependent on glutamine metabolism, and this dependency has been shown to make cancer cells highly sensitive to exogenous levels of glutamine (Abdel-Magid et al., ACS Med. Chem. Lett. 2016;7:207-208).

Thus, the most direct approach to targeting glutaminolysis is inhibition of glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. Inhibition of glutaminase can starve cancer cells by blocking glutamate synthesis and thus preventing α-ketoglutarate from supplying the TCA cycle.

As glutaminase inhibitors, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), CB-839, Compound 968, JHU083, which are structurally related to BPTES, have been demonstrated to inhibit cancer cell growth in vitro and slow tumor growth in vivo (Zhang et al., Oxidative Medicine and Cellular Longevity, 2016;2016:1616781; Robinson et al., Biochemical Journal, 2007;406(3):407-414; Elgadi et al., Physiological Genomics, 1999;1(2):51-62).

The only glutaminase inhibitor that has entered clinical trials is CB-839, commercially known as Telaglenastat. CB-839 completed a phase 1 trial in advanced RCC (NCT02071862) in March 2019 and a phase 2 trial in non-small cell lung cancer (NCT04265534) in 2020. Phase 1 trials in hematological malignancies (NCT02071888) and leukemia (NCT02071927) have been completed, although the clinical data have not been released.

Although simultaneous targeting of glucose and glutamine metabolism in this way can reduce tumor growth to some extent, it has the disadvantage of having limitations in suppressing angiogenesis and metastasis.

Meanwhile, inositol hexaphosphate and inositol are natural organic compounds that are abundant in most grains, seeds, and legumes and also exist in mammalian cells, and exist together with the phosphate form with low phosphate (IP1-5). Inositol hexaphosphate plays an important role in regulating important cellular functions such as signal transduction, cell proliferation, and differentiation of various cells, and is recognized as a natural antioxidant (Shamsuddin et al., J Nutr. 2003;133:3778S-3784S).

Recently, inositol hexaphosphate has been reported to have some anticancer effects and to inhibit the growth, progression, and metastasis of experimental tumors (Vucenik et al., Nutr Cancer. 2006;55:109-125).

Preliminary clinical studies have reported that co-administration of inositol hexaphosphate and inositol with chemotherapy reduces the side effects of chemotherapy and improves the quality of life of patients with breast cancer or colon cancer with liver metastases. However, it is still difficult to effectively inhibit cancer cell growth with only inositol hexaphosphate or a combination of inositol hexaphosphate and inositol.

Meanwhile, suppression of anticancer immune responses has emerged as an important mechanism of tumor resistance to treatment, and it has become possible to stimulate and/or expand the patient's endogenous anticancer immune responses through the development of monoclonal antibodies that block the immune checkpoint receptors CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) and PD-1 (programmed death-1) or their ligand PD-L1 (programmed death-ligand 1).

These pathways may play distinct roles in immune regulation, with CTLA-4 primarily regulating T cell proliferation in lymph nodes, while PD-1 primarily suppresses T cells in the tumor microenvironment. These immune checkpoint inhibitors have recently demonstrated remarkable efficacy against a variety of tumor types, particularly melanoma and non-small cell lung cancer, and have quickly become standard of care for patients.

However, immune checkpoint inhibitors generally have immune-related side effects that harm the gastrointestinal tract, endocrine glands, skin, and liver. These side effects are known to be mostly associated with abnormal immune system responses resulting from activated T lymphocytes.

In addition, immune checkpoint inhibitors are only effective in a small subset of patients. In most advanced cancers, the response rate of anti-PD-1/PD-L1 monotherapy is approximately 20%, and that of anti-CTLA-4 is approximately 12%, indicating a need for improvement (Borghaei et al., N Engl J Med. 2015; 373:1627-1639). This low efficacy may be due to the lack of preexisting tumor-specific T cell immunity (Elizabeth et al., Am J Clin Oncol. 2016 Feb; 39(1): 98-106).

In order to maximize the therapeutic effects of these immune checkpoint inhibitors, clinical trials are being conducted in combination with other anticancer drugs. For example, two immunotherapy drugs (nivolumab and ipilimumab combination therapy) for the treatment of advanced melanoma showed a greater survival improvement effect than either monotherapy. However, the side effects related to the treatment were so severe that 4 out of 10 patients stopped treatment. In this way, combining two or more treatments when cancer is induced has become the cornerstone of cancer treatment today, but in the case of immune checkpoint inhibitors, they show a high response rate and a relatively high treatment discontinuation rate.

Therefore, research is needed to increase therapeutic effects while minimizing side effects caused by combinations of anticancer drugs.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, a food composition, a method for treating cancer using the same, and uses thereof, which can effectively kill cancer even with a low concentration of a drug and exhibit specific toxicity to cancer cells, thereby reducing side effects.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of 1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

In addition, the present invention provides a combination for preventing or treating cancer, comprising at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

The present invention also provides a combination for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

The above pharmaceutical composition and/or combination can effectively kill cancer cells even with a low concentration combination of each compound, and is expected to be greatly utilized in the field of cancer treatment in the future, along with ensuring human safety. In particular, it can serve as a pharmaceutical composition for preventing or treating cancer with reduced side effects by exhibiting a toxic effect specifically on cancer cells.

In addition, the present invention provides a food composition for preventing or improving cancer, comprising the above-mentioned ingredients.

Furthermore, the present invention provides a method for treating cancer, comprising a step of administering the above-mentioned components to a subject in need thereof.

The present invention also provides a composition and/or combination comprising the above-mentioned ingredients for the treatment of cancer.

The present invention also provides the use of a composition and/or combination comprising the above-mentioned ingredients in the manufacture of a medicament for the treatment of cancer.

As one embodiment to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

In addition, the pharmaceutical composition for preventing or treating cancer may further include (5) an immune checkpoint inhibitor as an additional effective ingredient.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising: 1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

The pharmaceutical composition according to the present invention has an excellent effect of effectively treating cancer while having fewer side effects by including individual small amounts of effective ingredients. The pharmaceutical composition of the present invention can use a smaller amount of individual compounds included in the complex preparation than when treating with a single compound, and thus has the advantage of significantly increasing the overall effect of the treatment while significantly reducing the risk and/or severity of side effects.

In particular, according to the present invention, immune checkpoint inhibitors that block the immune checkpoint receptors CTLA-4 and PD-1 or their ligand PD-L1 have the advantage of exhibiting excellent anticancer effects even at low concentrations when included in the pharmaceutical composition according to the present invention and used.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

In the present invention, the pharmaceutical composition may include (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof.

Additionally, the pharmaceutical composition may include (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol.

Additionally, the pharmaceutical composition may include (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate and inositol.

Glutamine plays an important role as a transporter of nitrogen, carbon, and energy. Glutamine is used in urea synthesis in the liver, ammoniagenesis in the kidney, glucogenesis, and as a respiratory fuel in many cells. The conversion of glutamine to glutamate is initiated by the mitochondrial enzyme glutaminase (GLS). There are two major forms of the enzyme, the K-form (GLS1) and the L-form (GLS2). In addition, an alternative splice form of GLS1, referred to as glutaminase C or "GAC," has been recently identified and has activity properties similar to GLS1.

In certain embodiments, a glutaminase inhibitor according to the present invention can selectively inhibit GLS1, GLS2 and GAC.

In the present invention, the glutaminase inhibitor may be preferably at least one selected from the group consisting of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof, a compound represented by the following chemical formula 3 or a pharmaceutically acceptable salt thereof, and a compound represented by the following chemical formula 4 or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

The chemical formula 1 above is named Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, abbreviated as BPTES. BPTES is an allosteric and selective glutaminase inhibitor.

[Chemical formula 2]

The chemical formula 2 above is called Telaglenastat, 2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide, and its abbreviation is CB-839. CB-839 is known as a reversible orally active glutaminase 1 (GLS1) inhibitor. CB-839 can selectively inhibit GLS1 splice variants KGA (elongated glutaminase) and GAC (glutaminase C).

[Chemical Formula 3]

The chemical formula 3 above is named 5-[3-bromo-4-(dimethylamino)phenyl]-2,3,5,6-tetrahydro-2,2-dimethyl-benzo[a]phenanthridin-4(1H)-one, also abbreviated as Compound 968. Compound 968 is a cell-permeable, reversible mitochondrial glutaminase inhibitor that has the properties of inhibiting growth and invasive activity in glutaminase-upregulated fibroblasts and tumor cells.

[Chemical Formula 4]

The above chemical formula 4 is a prodrug of 6-diazo-5-oxo-L-norleucine, named ethyl 2-(2-amino-4-methylpentanamido)-DON, and abbreviated as JHU-083. JHU-083 is an orally active and selective glutaminase antagonist that has the property of blocking glutaminase activity in brain CD11b.

In the present invention, the glutaminase inhibitor is a substance capable of inhibiting the metabolism of cancer cells. According to the present invention, when the glutaminase inhibitor is applied in combination with metformin, 2-deoxy-D-glucose, and an inositol compound, not only can the level of inhibition of glucose and glutamine metabolism in cancer cells be lowered, but also the problems of angiogenesis and metastasis inhibition can be solved.

Glutamine is one of the most abundant non-essential amino acids in the body, but it is essential for cancer cells. Glutamine metabolism is very important for energy metabolism for tumor cell growth and proliferation. Tumor cells have been shown to consume glutamine more than other amino acids, and they take up glutamine through a variety of glutamine transporters. Glutamine starvation can interfere with tumor metabolism and inhibit tumor growth and survival. Glutamine is important for the synthesis of TCA cycle metabolites, nucleotides, spleen acid, antioxidants, and ATP energy.

The composition according to the present invention can effectively block the metabolic process of cancer by using a combination of a biguanide compound, such as metformin, which blocks glucose decomposition in a tumor and reduces energy utilization; 2-deoxy-D-glucose; an inositol compound; and a glutaminase inhibitor, which inhibits glutamine absorption, and also exhibits a synergistic effect on anticancer through combined treatment. That is, the composition according to the present invention is preferable because the anticancer effect exhibited by the combination of a biguanide compound, such as metformin, 2-deoxy-D-glucose, an inositol compound, and a glutaminase inhibitor is not simply additive, but exhibits a significantly enhanced effect.

In the present invention, the biguanide compound may be metformin or phenformin, and preferably metformin. Specifically, metformin has the structural formula of the following chemical formula 5.

[Chemical Formula 5]

Specifically, phenformin has the structural formula of the following chemical formula 6.

[Chemical formula 6]

The types of biguanide compounds are not limited to these.

Metformin, a biguanide compound, can exhibit anticancer effects through a mechanism of action that activates an enzyme called AMPK (AMP-activated kinase), which plays a central role in regulating intracellular energy balance and nutrient metabolism. When metformin was orally administered to rats, the LD50 was 1,450 mg/kg, indicating that metformin is a very safe compound, but there is still the problem that it must be used in high doses.

In the present invention, the combined preparation of the present invention exhibits a high anticancer effect even at a much lower concentration than each single preparation or a composition comprising a combination of two compounds, while improving the problems of high-dose administration or side effects of metformin or phenformin.

In the present invention, 2-deoxy-D-glucose has a structure represented by the following chemical formula 7.

[Chemical formula 7]

The compound of the above chemical formula 7 has an effect as an inhibitor of the corresponding action.

2-deoxy-D-glucose is a derivative of glucose, and has the effect of inhibiting glycolysis in the glucose metabolism process and inducing endoplasmic reticulum stress by inhibiting glycosylation of proteins in the endoplasmic reticulum. Thus, 2-deoxy-D-glucose, a glucose degradation inhibitor, was found to not kill cancer cells alone, but when formed into a complex preparation of the present invention, it exhibits excellent anticancer effects.

In the present invention, inositol hexaphosphate and/or inositol can regulate various important pathways in cancer cells, and specifically, inositol hexaphosphate has a structure represented by the following chemical formula 8.

[Chemical formula 8]

Additionally, in the present invention, inositol may include D-chiroinositol, L-chiro-inositol, myo-inositol, scyllo-inositol, etc., and preferably may be myo-inositol.

In the present invention, it was confirmed that a high anticancer effect was exhibited even at a low concentration by combining inositol hexaphosphate or a pharmaceutically acceptable salt thereof or inositol with a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, and 2-deoxy-D-glucose.

In the present invention, the biguanide compound, such as metformin or phenformin, preferably metformin, may be present in the form of a pharmaceutically acceptable salt. In addition, in the present invention, inositol hexaphosphate may be present in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As used herein, the term "food-acceptable salt" or "pharmaceutically acceptable salt" means any organic or inorganic addition salt having a relatively non-toxic and harmless effective effect on a patient, and the side effects due to the salt do not reduce the beneficial effects of the biguanide compound and inositol hexaphosphate.

At this time, as an additional salt, inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used, and as organic acids, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used, but are not limited thereto.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. An alkali metal salt or an alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium, calcium, magnesium salt or a mixed salt thereof as a metal salt, but is not limited thereto.

Pharmaceutically acceptable salts of each of the biguanide compound (e.g., metformin or phenformin) and inositol hexaphosphate, unless otherwise indicated, include salts of acidic or basic groups which may be present in each of the biguanide compound (e.g., metformin or phenformin) and inositol hexaphosphate. For example, pharmaceutically acceptable salts may include sodium, potassium, calcium or magnesium salts of a hydroxy group, and other pharmaceutically acceptable salts of an amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, and can be prepared by methods for preparing salts known in the art.

As a salt of the biguanide compound of the present invention (e.g., metformin or phenformin), any metformin salt or phenformin salt that is a pharmaceutically acceptable salt and exhibits an anticancer effect equivalent to that of metformin or phenformin may be used, preferably, metformin hydrochloride, metformin succinate, metformin citrate, or phenformin hydrochloride, phenformin succinate, phenformin citrate, etc., but is not limited thereto.

The inositol hexaphosphate salt of the present invention may be any inositol hexaphosphate salt that is a pharmaceutically acceptable salt and exhibits an anticancer effect equivalent to that of inositol hexaphosphate, and preferably includes, but is not limited to, sodium inositol hexaphosphate, potassium inositol hexaphosphate, calcium inositol hexaphosphate, ammonium inositol hexaphosphate, magnesium inositol hexaphosphate, and calcium magnesium inositol hexaphosphate.

The biguanide compound, 2-deoxy-D-glucose, and inositol hexaphosphate of the present invention each also include derivatives thereof. The term "derivative" refers to a compound prepared by chemically changing a portion of the compound, for example, introducing, substituting, or deleting a functional group, within the range where the anticancer activity of the compound is not changed, and may be included in the present invention without limitation.

In the present invention, inositol may exist in the form of various isomers. Isomers include both enantiomers and diastereomers. Any inositol that exhibits pharmaceutically anticancer effects may be used, and preferably, at least one selected from the group consisting of D-chiro-inositol, L-chiro-inositol, myo-inositol, and scyllo-inositol may be used, but is not limited thereto.

When combining two or more drugs, each of which is known to have anticancer effects, it is not expected that the combined drugs will exhibit a synergistic effect. Rather, there are cases where the functions of the drugs are offset by the combination, so it is very difficult to find a combination of drugs that has a synergistic effect. In the present invention, an anticancer combination use that can minimize side effects while maximizing anticancer effects by using the minimum concentration of the anticancer agent has been developed.

The pharmaceutical composition of the present invention may further include (5) an immune checkpoint inhibitor as an additional effective ingredient.

Immune checkpoint inhibitors can treat cancer by blocking immune checkpoints that block the progression of immune responses in cancers with high immunosuppression, thereby suppressing cancer immune evasion. Immune checkpoint inhibitors are new tumor treatment agents developed as a result of the advancement of immunology and the resulting understanding of the human immune system, and are widely used in anticancer strategies. Exemplary mechanisms for demonstrating anticancer effects using immune checkpoint inhibitors include the CTLA-4-mediated T lymphocyte suppression mechanism and the PD-1/PD-L1 mechanism that suppresses activated T lymphocytes. However, treatment using only immune checkpoint inhibitors has been reported to have limitations such as low therapeutic efficiency and minimal effectiveness.

However, the pharmaceutical composition of the present invention can improve the prevention and treatment of cancer due to the synergistic effect by co-administering at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol, and (5) an immune checkpoint inhibitor.

The immune checkpoint inhibitor according to the present invention may be an antibody, a fusion protein, an aptamer or an immune checkpoint protein-binding fragment thereof. For example, the immune checkpoint inhibitor is an anti-immune checkpoint protein antibody or an antigen-binding fragment thereof.

The immune checkpoint inhibitor of the present invention may be at least one selected from the group consisting of an anti-CTLA-4 antibody, a derivative thereof or an antigen-binding fragment thereof; an anti-PD-L1 antibody, a derivative thereof or an antigen-binding fragment thereof; an anti-LAG-3 antibody, a derivative thereof or an antigen-binding fragment thereof; an anti-OX40 antibody, a derivative thereof or an antigen-binding fragment thereof; an anti-TIM3 antibody, a derivative thereof or an antigen-binding fragment thereof; and an anti-PD-1 antibody, a derivative thereof or an antigen-binding fragment thereof, and preferably at least one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-PD-L1. The antibody may be purchased and used, for example, from a conventional antibody manufacturer, or may be manufactured and used according to a known antibody manufacturing method.

For example, the immune checkpoint inhibitor is Ipilimumab, a derivative thereof or an antigen-binding fragment thereof; Tremelimumab, a derivative thereof or an antigen-binding fragment thereof; Nivolumab, a derivative thereof or an antigen-binding fragment thereof; Pembrolizumab, a derivative thereof or an antigen-binding fragment thereof; Pidilizumab, a derivative thereof or an antigen-binding fragment thereof; Atezolizumab, a derivative thereof or an antigen-binding fragment thereof; Durvalumab, a derivative thereof or an antigen-binding fragment thereof; Avelumab, a derivative thereof or an antigen-binding fragment thereof; BMS-936559, a derivative thereof or an antigen-binding fragment thereof; BMS-986016, a derivative thereof or an antigen-binding fragment thereof; GSK3174998, a derivative thereof or an antigen-binding fragment thereof; TSR-022, a derivative thereof or an antigen-binding fragment thereof; MBG453, a derivative thereof or an antigen-binding fragment thereof; LY3321367, a derivative thereof or an antigen-binding fragment thereof; and IMP321 recombinant fusion protein may be at least one selected from the group consisting of an antibody or other form of an immune checkpoint inhibitor that can be used as an immune checkpoint inhibitor, without limitation.

The above-mentioned immune checkpoint inhibitor may be a small molecule compound that has an effect as an immune checkpoint inhibitor mentioned above or is involved in the inhibitory mechanism thereof. Such small molecule compounds may be, for example, small molecule compounds that bind to an immune checkpoint protein or are involved in the mechanism involved in immune checkpoint inhibition.

Specifically, the small molecule compounds may be BMS-202 (Source: BMS), BMS-8 (Source: BMS), CA170 (Source: Curis/Aurigene), CA327 (Source: Curis/Aurigene), Epacadostat, GDC-0919, BMS-986205, etc. Any small molecule compound that is used as an immune checkpoint inhibitor or has a related effect may be used without limitation.

In the present invention, a preferred embodiment of a pharmaceutical composition for preventing or treating cancer is:

A composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, and inositol hexaphosphate or a pharmaceutically acceptable salt thereof;

A composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, and inositol;

A composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, inositol hexaphosphate or a pharmaceutically acceptable salt thereof, and inositol; or

A composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, inositol hexaphosphate or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor;

A composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, inositol and an immune checkpoint inhibitor; and

It may be a composition comprising a glutaminase inhibitor, a biguanide compound or a pharmaceutically acceptable salt thereof, 2-deoxy-D-glucose, inositol hexaphosphate or a pharmaceutically acceptable salt thereof, inositol, and an immune checkpoint inhibitor.

In the present invention, the glutaminase inhibitor may be at least one selected from the group consisting of compounds such as BPTES, CB-839, Compound 968, and JHU083.

For example, as one embodiment for achieving the above purpose, let us look at a composition centered on CB-839 as a glutaminase inhibitor.

The composition for preventing or treating cancer of the present invention comprises a combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate.

Combination of CB-839/Metformin/2-Deoxy-D-Glucose/Inositol

Combination of CB-839/Metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-CTLA-4 antibody,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-1 antibody, or

It may be a combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-L1 antibody, wherein the metformin and inositol hexaphosphate may be in the form of a pharmaceutically acceptable salt.

For example, as one embodiment for achieving the above purpose, let us look at a composition centered on BPTES as a glutaminase inhibitor.

The composition for preventing or treating cancer of the present invention comprises a combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate.

Combination of BPTES/metformin/2-deoxy-D-glucose/inositol,

Combination of BPTES/Metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol

Combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-CTLA-4 antibody,

A combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-1 antibody, or

It may be a combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-L1 antibody, wherein the metformin and inositol hexaphosphate may be in the form of a pharmaceutically acceptable salt.

For example, as one embodiment for achieving the above purpose, let us look at a composition centered on Compound 968 as a glutaminase inhibitor.

The composition for preventing or treating cancer of the present invention comprises a combination of Compound 968/metformin/2-deoxy-D-glucose/inositol hexaphosphate.

A combination of Compound 968/Metformin/2-deoxy-D-glucose/inositol,

A combination of Compound 968/Metformin/2-deoxy-D-glucose/Inositol hexaphosphate/Inositol,

Combination of Compound 968/Metformin/2-deoxy-D-glucose/Inositol hexaphosphate/Inositol/Anti-CTLA-4 antibody,

A combination of Compound 968/Metformin/2-deoxy-D-glucose/Inositol hexaphosphate/Inositol/Anti-PD-1 antibody, or

It may be a combination of Compound 968/Metformin/2-deoxy-D-glucose/Inositol hexaphosphate/Inositol/Anti-PD-L1 antibody, and the metformin and inositol hexaphosphate may be in the form of a pharmaceutically acceptable salt.

For example, as one embodiment for achieving the above purpose, let us look at a composition centered on JHU083 as a glutaminase inhibitor.

The composition for preventing or treating cancer of the present invention comprises a combination of JHU083/metformin/2-deoxy-D-glucose/inositol hexaphosphate.

Combination of JHU083/Metformin/2-deoxy-D-glucose/inositol,

Combination of JHU083/Metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol,

Combination of JHU083/Metformin/2-deoxy-D-glucose/Inositol hexaphosphate/Inositol/Anti-CTLA-4 antibody,

Combination of JHU083/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-1 antibody, or

It may be a combination of JHU083/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-L1 antibody, wherein the metformin and inositol hexaphosphate may be in the form of a pharmaceutically acceptable salt.

As one aspect to achieve the above purpose,

Combination of CB-839/Metformin/2-deoxy-D-glucose/inositol hexaphosphate,

A combination of CB-839/metformin/2-deoxy-D-glucose/inositol, or

The combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two or three compounds.

As one aspect to achieve the above purpose,

Combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate,

A combination of BPTES/metformin/2-deoxy-D-glucose/inositol, or

The combination of BPTES/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two or three compounds.

As one aspect to achieve the above purpose,

Combination of Compound 968/Metformin/2-deoxy-D-glucose/inositol hexaphosphate,

A combination of Compound 968/Metformin/2-deoxy-D-glucose/inositol, or

The combination of Compound 968/Metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two or three compounds.

As one aspect to achieve the above purpose,

Combination of JHU083/Metformin/2-deoxy-D-glucose/inositol hexaphosphate,

JHU083/Metformin/2-deoxy-D-glucose/inositol combination, or

The combination of JHU083/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol was shown to be significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two or three compounds.

As another aspect for achieving the above purpose,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/anti-CTLA-4 antibody,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol/anti-CTLA-4 antibody, or

The combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-CTLA-4 antibody was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two to four compounds.

As another aspect for achieving the above purpose,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/anti-PD-1 antibody,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol/anti-PD-1 antibody, or

The combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-1 antibody was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two to four compounds.

As another aspect for achieving the above purpose,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/anti-PD-L1 antibody,

Combination of CB-839/metformin/2-deoxy-D-glucose/inositol/anti-PD-L1 antibody, or

The combination of CB-839/metformin/2-deoxy-D-glucose/inositol hexaphosphate/inositol/anti-PD-L1 antibody was significantly more effective in reducing tumor size than single agents of each compound or combination agents containing two to four compounds.

In one embodiment of the present invention, it was confirmed that the pharmaceutical composition according to the present invention can suppress cancer with a remarkably excellent effect in a cancer animal model. That is, according to the present invention, a glutaminase inhibitor; a biguanide compound; 2-deoxy-D-glucose; inositol hexaphosphate; inositol; and an immune checkpoint inhibitor; each has a low anticancer effect when used alone and must be used in an excessive amount, but when the compounds are used as a combined preparation, it was confirmed that cancer cells can be effectively killed even in a small amount.

In each pharmaceutical composition according to the present invention, the weight ratio of the combination of each compound is not particularly limited.

For example, any one or more selected from the group consisting of BPTES, CB-839, Compound 968 and JHU083 as glutaminase inhibitors can be used in a single dose range of 1-200 mg/kg.

As another example, one or more monoclonal antibodies selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies and anti-PD-L1 antibodies as immune checkpoint inhibitors can be used in a single dose range of 0.01 to 25 mg/kg.

Excluding glutaminase inhibitors and immune checkpoint inhibitors, ingredients can be used within the following ranges depending on the type of cancer being treated.

For example, the weight ratio of metformin or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose can range from 1:0.2 to 1:5, and the relative amounts of the combination can vary depending on the type of cancer being treated.

As another example, the weight ratio of metformin or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose: inositol hexaphosphate or a pharmaceutically acceptable salt thereof may be in the range of 1:0.2:0.5 to 1:5:20, and the relative amounts of the combination may vary depending on the type of cancer to be treated.

As another example, the weight ratio of metformin or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose: inositol can range from 1:0.2:0.5 to 1:5:20, and the relative amounts of the combination can vary depending on the type of cancer being treated.

As another example, the weight ratio of metformin or a pharmaceutically acceptable salt thereof: 2-deoxy-D-glucose: inositol hexaphosphate or a pharmaceutically acceptable salt thereof: inositol can be in the range of 1:0.2:0.5:0.5 to 1:5:20:20, and the combined relative amounts can vary depending on the type of cancer to be treated.

The present invention includes a combination of the above-mentioned components. That is, a combination for preventing or treating cancer is provided, comprising at least one selected from the group consisting of 1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

Also provided is a pharmaceutical combination for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

In the present invention, the combination may mean a combination in which the above-mentioned components are individually or two or more are combined together.

The term "cancer" used in the present invention refers to a disease related to the regulation of cell death, and refers to a disease caused by excessive cell proliferation when the normal apoptosis balance is disrupted. These abnormally excessively proliferating cells, in some cases, invade surrounding tissues and organs to form a tumor, and destroy or deform the normal structure of the body, and this condition is called cancer. In general, a tumor refers to a lump that has grown abnormally due to the autonomous excessive growth of body tissue, and can be divided into benign tumors and malignant tumors. Malignant tumors grow much faster than benign tumors, and metastasize by infiltrating surrounding tissues, threatening life. Such malignant tumors are commonly called 'cancer', and the types of cancer include cerebrospinal tumors, brain cancer, head and neck cancer, lung cancer, breast cancer such as triple-negative breast cancer, thymoma, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, leukemia such as acute leukemia or chronic leukemia, osteosarcoma, multiple myeloma, sarcoma, melanoma, malignant melanoma, skin cancer, etc. The anticancer composition of the present invention can be used without limitation on the type of cancer, but for the purpose of the present invention, it can be usefully used for the prevention or treatment of any one or more cancers selected from the group consisting of liver cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, cervical cancer, breast cancer, prostate cancer, ovarian cancer, brain cancer, osteosarcoma, bladder cancer, head and neck cancer, kidney cancer, melanoma, leukemia, and lymphoma.

The term "prevention or treatment" used in the present invention refers to any act of inhibiting or delaying the onset of cancer by using the pharmaceutical composition or combination according to the present invention, and in particular, 'treatment' refers to any act of improving or beneficially altering cancer by using the composition.

Accordingly, the present invention provides a method for treating cancer, comprising administering to a subject in need of prevention or treatment of cancer at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

The method may further comprise administering (5) a therapeutically effective amount of an immune checkpoint inhibitor.

Accordingly, the present invention provides a method for treating cancer, comprising administering to a subject in need of treatment of cancer at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

The pharmaceutical composition for preventing or treating cancer of the present invention may additionally contain a chemotherapeutic agent for treating cancer, as needed, in addition to the above-mentioned effective ingredients.

In addition, the pharmaceutical composition for preventing or treating cancer of the present invention may additionally contain a pharmaceutically acceptable carrier. The composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, in the form of sterile injectable solutions, in external preparations such as ointments, and in suppositories, according to the respective intended uses, according to conventional methods, and carriers, excipients, and diluents that can be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing the above composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The base of the injection may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

The composition of the present invention can be administered in various ways, such as orally, intravenously, subcutaneously, intradermally, intranasally, intraperitoneally, intramuscularly, and transdermally, and the dosage can vary depending on the patient's age, sex, and weight and can be easily determined by a person skilled in the art. The dosage of the composition according to the present invention may increase or decrease depending on the administration route, type of cancer, degree of disease, gender, body weight, age, etc., for example, in the case of the combination of each compound, a single dosage of the glutaminase inhibitor is 1 to 200 mg/kg (body weight), a biguanide compound is 1 to 50 mg/kg (body weight) per day, 2-deoxy-D-glucose is 1 to 100 mg/kg (body weight) per day, at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol is 1 to 200 mg/kg (body weight) per day, and a single dosage of the immune checkpoint inhibitor is 0.01 to 10 mg/kg (body weight). However, the scope of the present invention is not limited by the above dosage.

In one embodiment, for example, in the case of a combination of six compounds, when CB-839 is used as a glutaminase inhibitor, a single dose is 1 to 200 mg/kg (body weight), when metformin is used, 1 to 50 mg/kg (body weight) per day, 2-deoxy-D-glucose is 1 to 100 mg/kg (body weight) per day, inositol hexaphosphate is 1 to 200 mg/kg (body weight) per day, and inositol is 1 to 200 mg/kg (body weight) per day, and when anti-PD-1, a monoclonal antibody, is used as an immune checkpoint inhibitor, a single dose is 0.01 to 10 mg/kg (body weight). However, the scope of the present invention is not limited by the above dosages.

In another aspect, the present invention relates to a method for treating cancer by administering to a subject at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

In another aspect, the present invention relates to a method for treating cancer by administering to a subject at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

In another aspect, the present invention relates to a composition for use in the prevention or treatment of cancer, comprising at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

In another aspect, the present invention relates to a composition for use in the prevention or treatment of cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

In another aspect, the present invention relates to the use of at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol.

In another aspect, the present invention relates to the use of (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) at least one selected from the group consisting of 2-deoxy-D-glucose; (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

In another aspect, the present invention relates to a food composition for preventing or improving cancer, comprising at least one selected from the group consisting of (1) a glutaminase inhibitor; (2) a biguanide compound or a food-related acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a food-related acceptable salt thereof and inositol.

In another aspect, the present invention relates to a food composition for preventing or improving cancer, further comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a food-related acceptable salt thereof; (3) 2-deoxy-D-glucose; (4) at least one selected from the group consisting of inositol hexaphosphate or a food-related acceptable salt thereof and inositol; and (5) an immune checkpoint inhibitor.

The term "improvement" as used in the present invention means any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms.

The above composition may contain, in addition to the active ingredient, a food additive acceptable from a food science perspective.

The term "food supplement additive" used in the present invention means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like, but the types of food supplement additives of the present invention are not limited by the above examples.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time using food as a raw material, and is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effect of an anticancer agent.

The composition according to the present invention exhibits a wide and improved indication for various cancers by combining the inhibitor with limited indications and the metabolic anticancer agent as a glutaminase inhibitor. Therefore, compared to the use of a single drug, it can effectively kill cancer with a much smaller amount of the drug, thereby resolving the problem of having to use an excessive amount, and selectively treating cancer while minimizing side effects. In addition, the composition according to the present invention can exhibit a more remarkable synergistic effect by additional combination with an immune checkpoint inhibitor, and can expand the indications to various cancers, so it can be usefully used as an anticancer agent for various cancers and as a combination preparation for preventing or improving cancer.

Figure 1 is a graph showing the percentage of cell viability by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in HepG2 cell line, a human liver-derived cancer cell line. The drugs were treated at low concentrations usable in human plasma, and the MTT assay was performed after 48 hours. The vertical bar of each bar represents the standard deviation. Statistical analysis was performed by one-way ANOVA test with Tukey's multiple comparison post-hoc analysis using GraphPad Prism 6.0 software. **** p<0.0001.
Figure 2 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the A549 cell line, a human lung cancer cell line. **** p<0.0001.
Figure 3 is a graph showing the cell survival rate as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the AGS cell line, a human gastric cancer cell line. **** p<0.0001.
Figure 4 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the PANC-1 cell line, a cancer cell line derived from human pancreas. **** p<0.0001.
Figure 5 is a graph showing the cell survival rate as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the DLD-1 cell line, a cancer cell line derived from human colon. **** p<0.0001.
Figure 6 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in HeLa cell line, a cancer cell line derived from human cervix. **** p<0.0001.
Figure 7 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the MDA-MB-231 cell line, a human breast cancer cell line. **** p<0.0001.
Figure 8 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in PC-3 cell line, a cancer cell line derived from human prostate. **** p<0.0001.
Figure 9 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the SK-OV-3 cell line, a human ovarian cancer cell line. **** p<0.0001.
Figure 10 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the T24 cell line, a cancer cell line derived from human bladder. **** p<0.0001.
Figure 11 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in a human cancer cell line, glioblastoma (U-87 MG). **** p<0.0001.
Figure 12 is a graph showing the cell viability as a percentage by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in the Saos-2 cell line, a cancer cell line derived from human osteosarcoma. **** p<0.0001.
Figure 13 is a graph showing the percentage of cell survival by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), CB-839, and inositol hexaphosphate (IP6) in triple-negative breast cancer 4T1 cell line in an animal model. **** p<0.0001.
Figure 14 is a graph showing the percentage of cell viability by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), BPTES, and inositol hexaphosphate (IP6) in triple-negative breast cancer 4T1 cell line in an animal model. **** p<0.0001.
Figure 15 is a graph showing the percentage of cell survival by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), Compound 968, and inositol hexaphosphate (IP6) in triple-negative breast cancer 4T1 cell line in an animal model. **** p<0.0001.
Figure 16 is a graph showing the percentage of cell survival by single and combined treatment with metformin (MET), 2-deoxy-D-glucose (2DG), JHU083, and inositol hexaphosphate (IP6) in triple-negative breast cancer 4T1 cell line in an animal model. **** p<0.0001.
Figure 17 is a graph showing the change in tumor volume measured at 3-day intervals according to the administration of single and combined preparations of MET, 2DG, CB-839, IP6+Ins, and anti-PD-1 antibody in an animal model. Statistical analysis was performed by two-way ANOVA test with Tukey's multiple comparison post hoc analysis using GraphPad Prism 6.0 software. ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 18 is a graph showing the change in tumor volume measured at 3-day intervals according to the administration of single and combined formulations of MET, 2DG, CB-839, IP6+Ins, and anti-PD-L1 antibody in a water model. Statistical analysis was performed by two-way ANOVA test with Tukey's multiple comparison post hoc analysis using GraphPad Prism 6.0 software. ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 19 is a graph showing the change in tumor volume measured at 3-day intervals according to the administration of single and combined preparations of MET, 2DG, CB-839, IP6+Ins, and anti-CTLA-4 antibody in an animal model. Statistical analysis was performed by two-way ANOVA test with Tukey's multiple comparison post hoc analysis using GraphPad Prism 6.0 software. ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 20 is a graph showing the expression level of VEGF-A protein related to angiogenesis according to single and combined administration of MET, 2DG, CB-839, IP6+Ins, and anti-PD-1 antibody to triple-negative breast cancer 4T1 tumor tissue lysate in an animal model. *** p<0.001, **** p<0.0001.
Figure 21 is a graph showing the level of Angiopoietin-2 protein expression related to angiogenesis according to the administration of single and combined preparations of MET, 2DG, CB-839, IP6+Ins, and anti-PD-1 antibody to triple-negative breast cancer 4T1 tumor tissue lysate in an animal model. ** p<0.01, **** p<0.0001.

The advantages and features of the present invention, and the method for achieving them, will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

In the following examples, CB-839, BPTES, 968, and JHU083 were used as glutaminase inhibitors. In addition, metformin HCl was used among several pharmaceutically acceptable salt forms of metformin. The form of these salts is not limited by the examples. In addition, inositol hexaphosphate (Phytic acid) was used among several pharmaceutically acceptable salt forms of inositol hexaphosphate. The form of these is not limited by the examples. In addition, myo-inositol was used among inositol isomers. The isomers of these isomers are not limited by the examples. Monoclonal antibodies anti-mouse PD-1 mAb (RMP1-14), anti-mouse PD-L1 mAb (10F. 9G2), and anti-mouse CTLA-4 mAb (UC10-4F10-11) were used as immune checkpoint inhibitors.

Cell culture

The cells used in this study were tumor cells, including liver cancer (HepG2), lung cancer (A549), gastric cancer (AGS), pancreatic cancer (PANC-1), colon cancer (DLD-1), cervical cancer (HeLa), breast cancer (MDA-MB-231), prostate cancer (PC-3), ovarian cancer (SK-OV-3), bladder cancer (T24), glioblastoma (U-87 MG), osteosarcoma (Saos-2), and mouse-derived breast cancer (4T1). Non-tumor cells included prostate (PZ-HPV-7), colon (CCD-18Co), and lung (MRC5) cell lines. All cell lines were purchased from the Korean Cell Line Bank or the American Type Culture Collection (ATCC, Rockville, MD).

The above cells were cultured in a cell culture medium containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Hyclone) added to Roswell Park Memorial Institute 1640 Medium (RPMI1640, Hyclone, Logan, UT, USA), and maintained and cultured in a 37°C incubator (5% CO2/95% air). When the cells filled approximately 80% of the culture dish, the cell monolayer was washed with phosphate-buffered saline (PBS, Hyclone) and subcultured by treating with 0.25% trypsin-2.65 mM EDTA (Hyclone). The medium was changed every 2 days.

Drugs used

Glutaminase inhibitors CB-839, BPTES, 968, and JHU083 were purchased from Selleck Chemicals and stored and handled according to the manufacturer's instructions. Metformin HCl (MET), 2-deoxy-D-glucose (2DG), inositol hexaphosphate (IP6), and myoinositol (Ins) were purchased from Sigma (St. Louis, USA). Anti-mouse PD-1 mAb (RMP1-14), anti-mouse PD-L1 mAb (10F.9G2), and anti-mouse CTLA-4 mAb (UC10-4F10-11) monoclonal antibodies were purchased from BioXcell and stored and handled according to the manufacturer's instructions.

All drugs used in the tables and drawings summarizing the results obtained through experiments in the present invention are indicated by abbreviations.

Reference Example 1: In vitro cell growth inhibition assay

The cytotoxicity of glutaminase inhibitors (CB-839, BPTES, 968, JHU083), MET, 2DG, and IP6 was confirmed by MTT assay [3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide assay]. The cells (3–4 × 105 cells/well) were dispensed into a 96-well culture plate and stabilized for more than 12 hours. The medium from each well was removed, and each cell was treated with glutaminase inhibitors (CB-839, BPTES, 968, JHU083), MET, 2DG, and IP6 at various concentrations or as a mixture together with serum-free medium. For the control cells, PBS was added to the medium. After incubation at 37°C with _CO2 for 48 h, the medium including the control and mixture was cleanly removed and incubated with MTT (Sigma Aldrich, St. Louis, MO, USA) reagent (0.5 mg/ml) at 37°C for 4 h. Thereafter, the medium containing the MTT reagent was cleanly removed, and the MTT formazan crystals formed by living cells were dissolved by adding DMSO (Sigma) and leaving it at room temperature for more than 16 minutes. The absorbance was measured at a wavelength of 560 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Reference Example 2: Single and Combination Administration Experimental Methods

The above cells (3-4 × 10 ⁵ cells/well) were seeded in a 96-well plate and treated with glutaminase inhibitors (CB-839, BPTES, 968, JHU083), MET, 2DG, and IP6 as single agent drugs at different concentrations to confirm the cell proliferation inhibition rate.

As a combination drug, the cells were treated with a concentration of a drug corresponding to the IC ₅₀ of combination administration consisting of two or more compounds selected from the group consisting of glutaminase inhibitors (CB-839, BPTES, 968, JHU083), MET, 2DG, and IP6. All cell lines were cultured for 48 hours at the concentration of single or combination administration, and the growth inhibition effect was measured using the MTT assay.

Reference Example 3: Experimental Animals

Five-week-old, specific pathogen-free, female BALB/c mice were purchased from Dooyeol Biotech Co., Ltd. After a one-week quarantine and acclimatization period, healthy animals without weight loss were selected and used in the experiment.

The experimental animals were raised in a breeding environment set at temperature 23 ± 3℃, relative humidity 50 ± 10%, ventilation rate 10-15 times/hour, lighting time 12 hours (08:00 - 20:00), and illuminance 150 - 300 Lux. Throughout the test period, the experimental animals were allowed free access to solid animal feed (Cargill Agri Purina Co., Ltd.) and water.

Reference Example 4: Tumor cell transplantation and test substance administration

After a one-week adaptation period, BALB/c mice were injected with breast cancer cells, 4T1 cells (1× ¹⁰⁵ cells/mouse), into the left mammary fat tissue of the experimental animals, and tumor tissue development was observed with the naked eye. When the tumor tissue of the experimental animals reached a size of approximately 50 mm ³ , 10 test groups (control group, MET group (MET 500 mg/kg), 2DG group (2DG 1000 mg/kg), IP6(+Ins) group (IP6 600 mg/kg + Ins 600 mg/kg), CB-839 group (CB-839 200 mg/kg), mAb group (150 μg/mouse as monoclonal antibody (anti-PD1 antibody or anti-PD-L1 antibody or anti-CTLA-4 antibody)), MET+2DG+IP6(+Ins) group (MET 500 mg/kg + 2DG 1000 mg/kg, IP6 600 mg/kg + Ins 600 mg/kg), CB-839+mAb group (CB-839 200 mg/kg, 150 μg/mouse as monoclonal antibody (anti-PD1 antibody or anti-PD-L1 antibody or anti-CTLA-4 antibody), CB-839+MET+2DG+IP6(+Ins) group (CB-839 200 mg/kg, MET 500 mg/kg + 2DG 1000 mg/kg + IP6 600 mg/kg + Ins 600 mg/kg), CB-839+MET+2DG+IP6(+Ins)+mAb group (CB-839 200 mg/kg, MET 500 mg/kg + 2DG 1000 mg/kg + IP6 600 mg/kg + Ins 600 mg/kg + 150 μg/mouse as monoclonal antibody (anti-PD1 antibody or anti-PD-L1 antibody or anti-CTLA-4 antibody).

Five experimental animals were used for each test group. The test substance monoclonal antibody was administered intraperitoneally in 4-day cycles, starting from the test group separation time of day 1, and the test substances CB-839, MET, 2DG, and IP6(+Ins) were dissolved in distilled water and orally administered at regular intervals for 3 weeks until the end of the test, starting from the test group separation time of day 1.

Reference Example 5: Measurement of body weight and tumor volume of experimental animals

During the test period, the body weight of the experimental animals was measured once a week at a fixed time from the day of test substance administration. The tumor volume was calculated by measuring the length and width of the tumor using a digital caliper at three-day intervals and substituting the tumor volume into the following formula.

Tumor volume (mm ³ ) = (width ² × length) / 2

Reference Example 6: Statistical Processing

All analysis values are expressed as mean ± SD. To compare the differences between the control group and the test substance administration group, significance was verified by one-way ANOVA or two-way ANOVA test with Tukey's multiple comparison post hoc analysis using GraphPad Prism 6.0 software. Statistically significant was determined only when p < 0.05 or higher.

Example 1: Cell viability after single and combined administration of CB-839, MET, 2DG, and IP6

The cytotoxic effects of single and combined administration of CB-839, MET, 2DG, and IP6 were compared using 12 cancer cell lines.

Figures 1 to 12 show the results of evaluating the cell viability (% cell viability) after single and combined administration of MET, 2DG, and IP6, focusing on human cancer cell lines including liver cancer (HepG2), lung cancer (A549), gastric cancer (AGS), pancreatic cancer (PANC-1), colon cancer (DLD-1), cervical cancer (HeLa), breast cancer (MDA-MB-231), prostate cancer (PC-3), ovarian cancer (SK-OV-3), bladder cancer (T24), glioblastoma (U-87 MG), and osteosarcoma (Saos-2), respectively.

Figure 1 shows the survival rate in hepatocellular carcinoma (HepG2) cells treated alone and in combination with 4 mM MET, 1 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 2 shows the survival rate in lung cancer (A549) cell lines treated alone and in combination with 4 mM MET, 1 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 3 shows the viability in gastric cancer (AGS) cell lines treated alone and in combination with 2 mM MET, 0.7 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001).

Figure 4 shows the survival rate in pancreatic cancer (PANC-1) cell lines treated alone and in combination with 5 mM MET, 0.7 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 5 shows the survival rate in colorectal cancer (DLD-1) cell lines treated alone and in combination with 5 mM MET, 0.4 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 6 shows the viability in cervical cancer (HeLa) cell lines treated alone and in combination with 6 mM MET, 0.5 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001).

Figure 7 shows the survival rate in breast cancer (MDA-MB-231) cell lines treated alone and in combination with 6 mM MET, 1 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 8 shows the survival rate in prostate cancer (PC-3) cell lines treated alone and in combination with 5 mM MET, 1 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 9 shows the survival rate in ovarian cancer (SK-OV-3) cell lines treated alone and in combination with 5 mM MET, 0.5 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 10 shows the survival rate in bladder cancer (T24) cell lines treated alone and in combination with 4 mM MET, 1 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001).

Figure 11 shows the viability in glioblastoma (U-87 MG) cells treated alone and in combination with 5 mM MET, 0.4 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001).

Figure 12 shows the viability in osteosarcoma (Saos-2) cell lines treated alone and in combination with 5 mM MET, 0.7 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001).

Example 2: Cell viability after single and combined administration of MET, 2DG and IP6 with glutaminase inhibitors (CB-839, BPTES, Compound 968, JHU083) in triple-negative breast cancer cell lines

Figure 13 shows the survival rate in breast cancer (4T1) cell lines treated alone and in combination with 5 mM MET, 2 mM 2DG, 0.8 μM CB-839, and 1 mM IP6. The triple combination (MET + 2DG + CB-839) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + CB-839 + IP6) group showed a significant decrease in survival compared to the single, double, and triple combination treatments including the control group (P <0.0001).

Figure 14 shows the viability in breast cancer (4T1) cell lines treated alone and in combination with 5 mM MET, 2 mM 2DG, 2 μM BPTES, and 1 mM IP6. The triple combination (MET + 2DG + BPTES) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + BPTES + IP6) group showed a significant decrease in viability compared to the single, double, and triple combination treatments including the control group (P <0.0001).

Figure 15 shows the survival rate in breast cancer (4T1) cell lines treated alone and in combination with 5 mM MET, 2 mM 2DG, 10 μM Compound 968, and 1 mM IP6. The triple combination (MET + 2DG + Compound 968) group showed a significant decrease in survival compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + Compound 968 + IP6) group showed a significant decrease in survival compared to the single, double, and triple combination treatments including the control group (P <0.0001).

Figure 16 shows the viability in breast cancer (4T1) cell lines treated alone and in combination with 5 mM MET, 2 mM 2DG, 10 μM JHU083, and 1 mM IP6. The triple combination (MET + 2DG + JHU083) group showed a significant decrease in viability compared to the single and double combination treatments including the control group (P <0.0001). The quadruple combination (MET + 2DG + JHU083 + IP6) group showed a significant decrease in viability compared to the single, double, and triple combination treatments including the control group (P <0.0001).

Example 3. Effect of adding immune checkpoint inhibitors on tumor volume changes

Example 3.1. Effect of single and combined administration of CB-839, MET, 2DG, IP6, Ins, and anti-PD-1 on tumor volume changes

When the tumor tissue size was approximately 50 mm ³ , the test groups were classified and test substance administration was started, and the tumor volume was measured at 3-day intervals from the start of test substance administration. As shown in Fig. 17, the tumor volume of the single and combination treatment groups tended to decrease compared to the control group from the 3rd day of test substance administration.

On the 21st day of test substance administration, the combination treatment (CB-839 + MET + 2DG + IP6(+Ins)) group and the (CB-839 + MET + 2DG + IP6(+Ins) + anti-PD-1) group showed a high significant difference compared to the single and double combination treatment groups including the control group (P <0.0001). In terms of tumor volume, the (CB-839 + MET + 2DG + IP6(+Ins) + anti-PD-1) group showed the highest reduction among all groups, indicating a high tumor growth inhibition effect.

Example 3.2. Effect of single and combined administration of CB-839, MET, 2DG, IP6, Ins, and anti-PD-L1 on tumor volume changes

When the tumor tissue size was approximately 50 mm ³ , the test groups were classified and test substance administration was started, and the tumor volume was measured at 3-day intervals from the start of test substance administration. As shown in Fig. 18, the tumor volume of the single and combination treatment groups tended to decrease compared to the control group from the 3rd day of test substance administration.

On the 21st day of test substance administration, the combination treatment (CB-839 + MET + 2DG + IP6(+Ins)) group and the (CB-839 + MET + 2DG + IP6(+Ins) + anti-PD-L1) group showed a high significant difference compared to the single and double combination treatment groups including the control group (P <0.0001). In terms of tumor volume, the (CB-839 + MET + 2DG + IP6(+Ins) + anti-PD-L1) group showed the highest reduction among all groups, indicating a high tumor growth inhibition effect.

Example 3.3. Effect of single and combined administration of CB-839, MET, 2DG, IP6, Ins, and anti-CTLA-4 on tumor volume changes

When the tumor tissue size was approximately 50 mm ³ , the test group was classified and test substance administration was started, and the tumor volume was measured at 3-day intervals from the start of test substance administration. As shown in Fig. 19, the tumor volume of the single and combination treatment groups tended to decrease compared to the control group from the 3rd day of test substance administration.

On the 21st day of test substance administration, the combined treatment (CB-839 + MET + 2DG + IP6(+Ins)) group and the (CB-839 + MET + 2DG + IP6(+Ins) + anti-CTLA-4) group showed a high significant difference compared to the single and double combination treatment groups including the control group (P <0.0001). In terms of tumor volume, the (CB-839 + MET + 2DG + IP6(+Ins) + anti-CTLA-4) group showed the highest reduction among all groups, indicating a high tumor growth inhibition effect.

Example 4: Effects of CB-839, MET, 2DG, IP6, Ins and anti-PD-1 on lung metastasis after single and combined administration

To observe cancer metastasis to the liver and lungs in this tumor animal model, the liver and lungs were extracted, fixed with Bouin’s solution, and the number of metastatic tumor nodules was measured. While no metastasis to the liver tissue was observed in any animal, tumor nodules were observed in the lungs of all animals. The number of tumor nodules metastasized to the lungs was significantly reduced in the G6, G7, and G8 groups compared to 26.5 ± 4.6 in the control group (G1). Among all the groups, the (CB-839 + MET + 2DG + IP6(+Ins) + anti-PD-1) group showed the highest decrease in the number of tumor nodules, indicating a high metastasis inhibition effect.

[Table 1] Number of metastatic tumor nodules to the lungs in animal models

*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to control group)

Example 5: Inhibition of angiogenesis after single and combined administration of CB-839, MET, 2DG, IP6, Ins, and anti-PD-1

Currently, combination therapy with anti-angiogenic drugs and other treatments is being attempted as one of several strategies to improve the response rate and treatment period of immunotherapy. To investigate the effect of test substances on the changes in various metastasis-related proteins related to angiogenesis in tumor tissue, Proteome Profiler Mouse XL Cytokine Array was performed using tumor tissue lysate. As a result of analysis with the Proteome Profiler Mouse XL Cytokine Array kit, the expression of VEGF-A and Angiopoietin-2 proteins, which are representative proteins affecting angiogenesis and metastasis, was investigated.

In Figure 20, the (CB-839+MET+2DG+IP6(+Ins)) group and the (CB-839+MET+2DG+IP6(+Ins)+anti-PD-1) group showed a highly significant difference in VEGF-A protein expression compared to the control group and the single and double to triple combination preparation groups (P <0.0001).

In Figure 21, the (CB-839+MET+2DG+IP6(+Ins)) group and the (CB-839+MET+2DG+IP6(+Ins)+anti-PD-1) group showed a highly significant difference in angiopoietin-2 protein expression compared to the control group and the single and double to triple combination preparation groups (P <0.0001).

Claims (14)

(1) Glutaminase inhibitor;
(2) Biguanide compound or a pharmaceutically acceptable salt thereof;
(3) 2-deoxy-D-glucose; and
(4) A pharmaceutical composition for preventing or treating cancer, comprising inositol hexaphosphate or a pharmaceutically acceptable salt thereof, or inositol hexaphosphate or a pharmaceutically acceptable salt thereof and inositol;
The above (1) glutaminase inhibitor is at least one selected from the group consisting of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof, a compound represented by the following chemical formula 3 or a pharmaceutically acceptable salt thereof, and a compound represented by the following chemical formula 4 or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]
,
[Chemical formula 2]
,
[Chemical Formula 3]
,
[Chemical Formula 4]
,
The above (2) biguanide compound is a pharmaceutical composition for preventing or treating cancer, which is metformin. A pharmaceutical composition for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate or a pharmaceutically acceptable salt thereof, in claim 1. delete A pharmaceutical composition for preventing or treating cancer, comprising: (1) a glutaminase inhibitor; (2) a biguanide compound or a pharmaceutically acceptable salt thereof; (3) 2-deoxy-D-glucose; and (4) inositol hexaphosphate and inositol. A pharmaceutical composition for preventing or treating cancer, wherein in claim 1, the composition further comprises (5) at least one immune checkpoint inhibitor selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody. delete delete A pharmaceutical composition for preventing or treating cancer, wherein in the first paragraph, (4) inositol is at least one selected from the group consisting of D-chiroinositol, L-chiro-inositol, myo-inositol, and scyllo-inositol. delete delete A pharmaceutical composition for preventing or treating cancer, wherein in claim 1, the cancer is selected from the group consisting of liver cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, cervical cancer, breast cancer, prostate cancer, ovarian cancer, brain cancer, osteosarcoma, bladder cancer, head and neck cancer, kidney cancer, melanoma, leukemia, and lymphoma. (1) Glutaminase inhibitor;
(2) Biguanide compound or a food-acceptable salt thereof;
(3) 2-deoxy-D-glucose; and
(4) A food composition for preventing or improving cancer, comprising inositol hexaphosphate or a food-based acceptable salt thereof, or inositol hexaphosphate or a food-based acceptable salt thereof and inositol;
The above (1) glutaminase inhibitor is at least one selected from the group consisting of a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof, a compound represented by the following chemical formula 2 or a food-based acceptable salt thereof, a compound represented by the following chemical formula 3 or a food-based acceptable salt thereof, and a compound represented by the following chemical formula 4 or a food-based acceptable salt thereof.
[Chemical Formula 1]
,
[Chemical formula 2]
,
[Chemical Formula 3]
,
[Chemical Formula 4]
,
The above (2) biguanide compound is a food composition for preventing or improving cancer, which is metformin. In claim 12, the composition further comprises (5) an immune checkpoint inhibitor, which is at least one selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody. A food composition for preventing or improving cancer. A food composition for preventing or improving cancer, wherein in claim 12, the cancer is selected from the group consisting of liver cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, cervical cancer, breast cancer, prostate cancer, ovarian cancer, brain cancer, osteosarcoma, bladder cancer, head and neck cancer, kidney cancer, melanoma, leukemia, and lymphoma.