KR20240038394A - Pharmaceutical composition for the prevention or treatment of cancer comprising a glycolysis inhibitor and a mitochondrial function inhibitor - Google Patents

Abstract

One aspect relates to a pharmaceutical composition for preventing or treating cancer comprising a glycolysis inhibitor and a mitochondrial function inhibitor. According to one aspect, when a glycolysis inhibitor and a mitochondrial function inhibitor are administered in combination, the growth of cancer cells is inhibited, ATP energy production is blocked in cancer cells, and PI3K/Akt/Akt/AKT in cancer cells are inhibited compared to the single administration of the glycolysis inhibitor or mitochondrial function inhibitor. It was confirmed that the effect of inhibiting mTORC1 signaling, inducing the expression of REDD1 (regulated in development and DNA damage responses 1) in cancer cells, and suppressing the expression of proteins related to cell proliferation was significantly superior. In addition, when glycolysis inhibitors and mitochondrial function inhibitors are loaded onto extracellular vesicles and administered simultaneously, superior cancer prevention, improvement, or treatment effects were confirmed, which can contribute to the cancer treatment market/industry.

Description

Pharmaceutical composition for the prevention or treatment of cancer comprising a glycolysis inhibitor and a mitochondrial function inhibitor}

It relates to a pharmaceutical composition for preventing or treating cancer containing a glycolysis inhibitor and a mitochondrial function inhibitor.

In the early stages of hepatocellular carcinoma (HCC), various treatment methods exist, such as radiofrequency ablation, resection, radiation therapy, and transplantation. However, targeted treatment is recommended for advanced hepatocellular carcinoma.

Recently, it has been emphasized that magnetic resonance imaging (MRI) can be helpful in early detection of hepatocellular carcinoma. However, there is a problem that the medical insurance issue for high-cost MRI has not been resolved, making it difficult to easily perform MRI examinations. In fact, 30-35% of hepatocellular carcinomas are discovered in advanced stages, and the average survival time of hepatocellular carcinoma patients in advanced stages is 7-9 months, making the limitations of the current standard treatment for hepatocellular carcinoma clear. In addition, there are the following limitations in clinically treating HCC patients: (1) lack of available drugs after failure of tyrosine kinase inhibitors. (2) the need for targeted therapy increases in patients with tumors that do not respond to transarterial chemoembolization after transarterial chemoembolization, which is a local treatment, and (3) there is a significant risk of hepatocellular carcinoma recurrence even after 5 years in patients who have undergone radical resection. and (4) no drugs are available for targeted therapy in patients with decompensated cirrhosis. Therefore, a new strategy is needed as an alternative to the drugs currently used in patients with advanced hepatocellular carcinoma.

Accordingly, the present inventors confirmed that when a glycolysis inhibitor and a mitochondrial function inhibitor are administered in combination, the cancer prevention or treatment effect is excellent, and thus developed the present invention.

Korean Patent Publication No. 10-2005-0098244

One aspect is to provide a pharmaceutical composition for preventing or treating cancer comprising a glycolysis inhibitor and a mitochondrial function inhibitor.

Another aspect is to provide health functional foods for preventing or improving cancer containing glycolysis inhibitors and mitochondrial function inhibitors.

Another aspect is to provide a method for preventing or treating cancer comprising administering a glycolysis inhibitor and a mitochondrial function inhibitor to a subject in need thereof.

Another aspect is to provide a use of a glycolysis inhibitor and a mitochondrial function inhibitor for the manufacture of a medicament for the prevention or treatment of cancer.

One aspect provides a pharmaceutical composition for preventing or treating cancer comprising a glycolysis inhibitor and a mitochondrial function inhibitor.

The term “glycolysis” refers to a metabolic pathway that converts glucose (C ₆ H ₁₂ O ₆ ) into pyruvic acid (CH ₃ COCOO ^- ), and in one aspect, the glycolysis inhibitor is an inhibitor of the glycolysis process. It means inhibiting conversion enzymes that act at various stages.

The glycolysis inhibitors include dichloroacetic acid (DCA), FX-11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic acid), 2-DG (2 -Deoxy-d-glucose), iodoacetate, and 3-bromopyruvate, and may be one or more selected from the group consisting of, specifically, dichloroacetic acid (DCA: Dichloroacetic acid), FX. -11(7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic Acid), 2-DG(2-Deoxy-d-glucose) and 3-bromopyruvate (3 -bromopyruvate), and more specifically, dichloroacetic acid (DCA: Dichloroacetic acid) or FX-11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl- naphthalene-1-carboxylic Acid).

The term “Dichloroacetic acid (DCA)” is a type of organic chlorine acid and refers to a compound having the chemical formula Cl ₂ CHCO ₂ H.

The term "FX-11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic Acid)" refers to an inhibitor of lactate dehydrogenase A. FX-11 is known to lower ATP levels, induce oxidative stress, reactive oxygen species (ROS) generation, and cell death, and exhibit antitumor activity in lymphoma and pancreatic cancer xenografts.

In one aspect, the glycolysis inhibitor is administered in an amount of 1 to 200 mg/kg, 1 to 170 mg/kg, 1 to 140 mg/kg, 1.3 to 200 mg/kg, 1.3 to 170 mg/kg, 1.3 to 140 mg/kg. kg, 1.6 to 200 mg/kg, 1.6 to 170 mg/kg, or 1.6 to 140 mg/kg. Specifically, when the glycolysis inhibitor is FX-11, the glycolysis inhibitor is 1 to 3 mg/kg, 1 to 2.7 mg/kg, 1 to 2.4 mg/kg, 1.3 to 3 mg/kg, 1.3 to 2.7 mg/kg. It may be administered at mg/kg, 1.3 to 2.4 mg/kg, 1.6 to 3 mg/kg, 1.6 to 2.7 mg/kg, or 1.6 to 2.4 mg/kg, and when the glycolysis inhibitor is DCA, the glycolysis The inhibitor is 10 to 200 mg/kg, 10 to 170 mg/kg, 10 to 140, 40 to 200 mg/kg, 40 to 170 mg/kg, 40 to 140 mg/kg, 70 to 200 mg/kg, 70 to 70 mg/kg. It may be administered at 170 mg/kg or 70 to 140 mg/kg.

In one aspect, when the glycolysis inhibitor is administered at less than 1 mg/kg, the effect of the glycolysis inhibitor may not be effectively shown, and when the glycolysis inhibitor is administered at more than 200 mg/kg, the administration target It may cause toxicity to normal cells other than cancer cells.

The term "mitochondria" refers to an important organelle in a cell where the process of oxygen respiration occurs in eukaryotes, and the mitochondria convert energy stored in various organic substances into ATP required for life activities through the oxidative phosphorylation process. It has the function of converting into form. In one aspect, the "mitochondrial function inhibitor" refers to inhibiting the function of the mitochondrial respiratory complex (mitochondiral respiratory complex) in the mitochondria, and the subtypes of the mitochondrial respiratory complex (mitochondiral respiratory complex) are from type I to type V. exist.

In one aspect, the mitochondrial function inhibitor is metformin, IACS-010759 (4-(methylsulfonyl)-1-[3-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]- 1,2,4-oxadiazol-5-yl]-1H-1,2,4-triazol-1-yl]methyl]phenyl]-piperidine), rotenone, Piericidin A, It may be one or more selected from the group consisting of Streptozotocin and Antimycin A, specifically metformin, IACS-010759 (4-(methylsulfonyl)-1-[3-[[ 5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-1,2,4-triazol-1-yl]methyl]phenyl]- It may be one or more selected from the group consisting of piperidine and rotenone, and more specifically, metformin or IACS-010759 (4-(methylsulfonyl)-1-[3-[[5-methyl-3 -[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-1,2,4-triazol-1-yl]methyl]phenyl]-piperidine) .

The term "metformin ( N,N- Dimethylimidodicarbonimidic diamide)" is a compound with the chemical formula of C ₄ H ₁₁ N ₅ , has a molecular weight of 129.16, and induces gluconeogenesis by activating AMP-activated protein kinase (AMPK) in hepatocytes. It refers to a drug that inhibits synthesis.

The term "IACS-010759(4-(methylsulfonyl)-1-[3-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl] -1H-1,2,4-triazol-1-yl]methyl]phenyl]-piperidine)" refers to an oxidative phosphorylation inhibitor that blocks cellular respiration by inhibiting mitochondrial respiratory complex I.

In one aspect, the mitochondrial function inhibitor is administered in an amount of 1 to 300 mg/kg, 1 to 270 mg/kg, 1 to 240 mg/kg, 1.3 to 300 mg/kg, 1.3 to 270 mg/kg, 1.3 to 240 mg/kg. kg, 1.6 to 300 mg/kg, 1.6 to 270 mg/kg, or 1.6 to 240 mg/kg. Specifically, when the mitochondrial function inhibitor is IACS-010759, the mitochondrial function inhibitor is 1 to 3 mg/kg, 1 to 2.7 mg/kg, 1 to 2.4 mg/kg, 1.3 to 3 mg/kg, 1.3 to 2.7. It may be administered at mg/kg, 1.3 to 2.4 mg/kg, 1.6 to 3 mg/kg, 1.6 to 2.7 mg/kg, or 1.6 to 2.4 mg/kg, and when the mitochondrial function inhibitor is metformin, the mitochondrial function The inhibitor is 100 to 300 mg/kg, 100 to 270 mg/kg, 100 to 240, 130 to 300 mg/kg, 130 to 270 mg/kg, 130 to 240 mg/kg, 160 to 300 mg/kg, 160 to 300 mg/kg. It may be administered at 270 mg/kg or 160 to 240 mg/kg.

In one aspect, when the mitochondrial function inhibitor is administered at less than 1 mg/kg, the effect of the mitochondrial function inhibitor may not be effective, and when the mitochondrial function inhibitor is administered at more than 300 mg/kg, the administration target It may cause toxicity to normal cells other than cancer cells.

In one example, an in vivo subcutaneous It was confirmed that MH-134 significantly inhibited tumor growth compared to the control group at 13, 14, and 15 days after tumor budding (see Example 1.(7) and Example 2.(1)5).

In another example, when 2 mg/kg of FX-11 and 2 mg/kg of IACS-010759 were subcutaneously administered to Balb-c nude mice for 3 weeks, FX-11 loaded on exosomes and IACS-loaded on exosomes It was confirmed that the 010759 administration group showed the strongest tumor suppression effect, and the exosome-loaded FX-11 and exosome-loaded IACS-010759 administration group showed a stronger tumor growth inhibition effect than the FX-11 and IACS-010759 administration groups. This was confirmed (see Example 2.(2)7).

In one aspect, the glycolysis inhibitor and mitochondrial function inhibitor are 60:1 to 1:5, 60:1 to 1:3, 60:1 to 1:2, 3:2 to 1:5, 3:2 to It may be administered at a mass ratio of 1.3, 3:2 to 1:2, 1:1 to 1:5, 1:1 to 1:3, or 1:1 to 1:2. Specifically, when the glycolysis inhibitor is DCA and the mitochondrial function inhibitor is metformin, the glycolysis inhibitor and the mitochondrial function inhibitor are 8:5 to 1:5, 8:5 to 1:2, and 4:5 to It may be administered at a mass ratio of 1:5 or 4:5 to 1:2, and specifically, when the glycolysis inhibitor is FX-11 and the mitochondrial function inhibitor is IACS-010759, the glycolysis inhibitor and the The mitochondrial function inhibitor may be administered at a mass ratio of 60:1 to 3:10, 60:1 to 4:7, 3:2 to 3:10, or 3:2 to 4:7.

In one aspect, when the glycolysis inhibitor and the mitochondrial function inhibitor are administered in a mass ratio other than 60:1 to 1:5, the synergistic effect of the glycolysis inhibitor and the mitochondrial function inhibitor, that is, the glycolysis inhibitor alone or the mitochondrial function inhibitor The cancer prevention or treatment effect may not be as effective compared to the drug alone.

In one aspect, the cancer is glioblastoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, and ureteral cancer. , bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer. Specifically, breast cancer, lung cancer, liver cancer, pancreatic cancer, uterine cancer, ovarian cancer, It may be one or more selected from the group consisting of stomach cancer, colon cancer, and skin cancer, and more specifically, it may be breast cancer, liver cancer, or pancreatic cancer.

The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that grow uncontrollably and have the ability to invade and destroy normal body tissues.

The term “prevention” may refer to any act of suppressing or delaying the onset of cancer in an individual by administering a pharmaceutical composition according to one aspect.

The term “treatment” may refer to any action in which an individual's cancer symptoms are improved or beneficially changed by administration of a pharmaceutical composition according to one aspect.

The term “administration” means introducing a predetermined substance into an individual in an appropriate manner, and “individual” refers to all living organisms such as rats, mice, and livestock, including humans, that can harbor cancer. As a specific example, it may be a mammal, including humans.

In one aspect, the intracellular or extracellular environment of the cancer may be a glucose-deficient environment. The extracellular environment refers to the environment surrounding the cancer cells, specifically, the environment outside the cell membrane of the cancer cells, as well as the environment between cancer cells, the environment between cancer cells and normal cells, or normal cells near cancer cells. It can also include my environment.

In one example, when a liver cancer cell line was treated in combination with FX-11 and IACS-010759 according to the glucose concentration around the liver cancer cells, changes in the apoptosis effect were confirmed, and as a result, low glucose around the tumor cells was observed. ) It was confirmed that cell death occurs more easily under environmental conditions (see Example 2.(2)2).

In one aspect, the composition may inhibit ATP production in cancer cells. Specifically, the composition may inhibit survival signal transmission within cancer cells by inhibiting ATP production within cancer cells.

In one example, it was confirmed whether the combined treatment of metformin and DCA inhibits ATP production in liver cancer cells. As a result, it was confirmed that the combined treatment of metformin and DCA significantly inhibited intracellular ATP production in liver cancer cells compared to the control group (see Example 2.(1)2).

In another example, the degree of inhibition of ATP production in tumor tissue was compared between the control group and the exosome-loaded FX-11 and exosome-loaded IACS-010759 administration groups. As a result, it was confirmed that the group administered FX-11 loaded on exosomes and IACS-010759 loaded on exosomes significantly suppressed ATP production in tumor tissue compared to the control group (see Example 2.(2)8). ).

In one aspect, the composition may inhibit PI3K/Akt/mTORC1 signaling in cancer cells.

Additionally, in one aspect, the composition may enhance the expression of REDD1 (regulated in development and DNA damage responses 1) protein in cancer cells.

In one example, it was evaluated whether the combined treatment of metformin and DCA effectively inhibits PI3K/Akt/mTORC1 signaling. As a result, it was confirmed that treatment with metformin or DCA inhibits the expression of PI3K/Akt/mTORC1 signaling in liver cancer cells. In addition, it was confirmed that the combined treatment of metformin and DCA significantly inhibited PI3K/Akt/mTORC1 signaling compared to the control group (see Example 2.(1)3).

In another example, it was evaluated whether energetic stress induced by the combined treatment of metformin and DCA affects REDD1 expression. As a result, we found a strong anticancer effect of metformin and DCA on the induction of REDD1 expression, but the induction of REDD1 expression by metformin and DCA was not affected by compound C, and REDD1 induction by metformin and DCA was AMPK independent. It was confirmed that it contributes to the inhibition of mTORC1 activity (see Example 2.(1)3).

Additionally, in one aspect, the composition is a group consisting of cyclin D1, Ki-67, p-Akt, mTOR, proliferating cell nuclear antigen, and mitochondrial respiratory complex I in cancer cells. It may inhibit the expression of one or more proteins selected from, specifically, cyclin D1, Ki-67, p-Akt, mTOR, proliferating cell nuclear antigen, or mitochondrial respiratory complex I ( It may inhibit the expression of mitochondrial respiratory complex I) protein, and more specifically, cyclin D1, Ki-67, p-Akt, mTOR, proliferating cell nuclear antigen, and mitochondrial respiratory complex I in cancer cells. It may be suppressing the expression of (mitochondrial respiratory complex I) protein.

In one example, an in vivo Subcutaneous In the group treated with , it was confirmed that the expression of cyclin D1, Ki-67 protein, and proliferating cell nuclear antigen was decreased in tumor tissue (see Example 2.(1)5).

In another example, in order to confirm the correlation between glycolysis and energy metabolism through mitochondria in liver cancer cell lines (HepG2 cells, Huh-7 cells), FX, a glycolysis inhibitor, was added to liver cancer cell lines (HepG2 cells, Huh-7 cells). -11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic Acid, C22H22O4, CAS Number: 213971-34-7) is treated, and IACS-, a mitochondrial function inhibitor, is treated. 010759(4-(methylsulfonyl)-1-[3-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-1, 2,4-triazol-1-yl]methyl]phenyl]-piperidine, C25H25F3N6O4S, CAS Number: 1570496-34-2). As a result, when treating liver cancer cell lines (HepG2 cell, Huh-7 cell) with IACS-010759, the change in expression of mitochondrial respiratory complex I, the target protein of IACS-010759, decreased on Western blot. It was confirmed that the expression of mitochondrial respiratory complex I decreased even when treated with FX-11 (see Example 2.(2)1).

In another example, after administering a drug to a liver cancer model formed by injecting a liver cancer cell line (HepG2 cell) into a Balb/c nude mouse xenograft animal model, Ki-67, p-Ak, and mTOR are related to tumor growth inhibition. Immunostaining for proteins was performed to compare and evaluate the control group and the group administered simultaneously with FX-11 and IACS-010759. As a result, it was confirmed that the group administered simultaneously with FX-11 and IACS-010759 had an increased incidence of apoptosis compared to the control group, and the expression of proteins related to tumor growth (Ki-67, p-Akt, and mTOR) was decreased ( (see Example 2.(2)4)).

In one aspect, the glycolysis inhibitor and the mitochondrial function inhibitor may be loaded into extracellular endoplasmic reticulum.

The term “extracellular vesicle” refers to a small sphere surrounded by a membrane derived from cells.

In one aspect, the extracellular vesicle may be one or more selected from the group consisting of exosomes, microvesicles, ectosomes, and apoptotic bodies, and specifically, It may be one or more selected from the group consisting of exosomes, microvesicles, and ectosomes, and more specifically, it may be exosomes.

The term “exosome” refers to a small membrane vesicle of endosome origin that is released into the extracellular environment following fusion of the plasma membrane and multivesicles.

In one aspect, the exosome may be an exosome derived from one or more selected from the group consisting of immune cells, macrophages, cancer cells, stem cells, plant cells, blood, body fluids, microbiome, lactic acid bacteria, and milk, , Specifically, it may be an exosome derived from one or more selected from the group consisting of immune cells, macrophages, cancer cells, and stem cells, and more specifically, it may be an exosome derived from human fetal kidney cells.

The term “microvesicle” refers to extracellular vesicles having a diameter of 100 to 1000 nm.

The term “ectosome” refers to vesicles of various sizes (e.g., 0.1-1 mm in diameter) that sprout directly from the plasma membrane.

The term “apoptotic body” refers to extracellular vesicles that are remnants of cells that have undergone apoptosis or programmed cell death.

In one aspect, the glycolysis inhibitor and the mitochondrial function inhibitor may each be loaded into separate extracellular vesicles or simultaneously loaded into one extracellular vesicle.

In one aspect, when the glycolysis inhibitor and the mitochondrial function inhibitor are each loaded into separate extracellular vesicles or simultaneously loaded into one extracellular vesicle, the glycolysis inhibitor and the mitochondrial function inhibitor are loaded into the extracellular vesicle. The effect of preventing or treating cancer is better than if it does not work.

In one example, in a liver cancer model formed by injecting a liver cancer cell line (HepG2 cell) into a Balb/c nude mouse The inhibitory effect on liver cancer growth was compared and evaluated when administered and when FX-11 and IACS-010759 were each loaded onto exosomes and administered intraperitoneally simultaneously. As a result, liver cancer compared to the control group in both cases of intraperitoneal simultaneous administration of FX-11 and IACS-010759 not loaded on exosomes and intraperitoneal simultaneous administration of FX-11 and IACS-010759 loaded on exosomes, respectively. It was confirmed that the growth was significantly inhibited (see Example 2.(2)3).

In another example, FX-11 and IACS-010759 were each loaded into exosomes in a breast cancer cell line (MCF-7) and treated alone or in combination. As a result, simultaneous intraperitoneal treatment of IACS-010759-loaded exosomes and FX-11-loaded exosomes showed a higher anticancer effect compared to intraperitoneal treatment of FX-11 and IACS-010759 alone. It was confirmed that the cancer cell death rate was 80%. In addition, it was confirmed that loading the drug into exosomes showed higher anticancer efficiency even at a lower drug concentration than when not loading the drug into exosomes (see Example 2.(2)5).

In another example, a pancreatic cancer cell line (MIA PaCa-2) was simultaneously treated intraperitoneally with exosomes loaded with FX-11 and exosomes loaded with IACS-010759. As a result, the simultaneous intraperitoneal treatment of exosomes loaded with FX-11 and exosomes loaded with IACS-010759 was more effective than the simultaneous intraperitoneal treatment of exosomes loaded with FX-11 or exosomes loaded with IACS-010759. It was confirmed that the anticancer effect increased compared to the treatment case (see Example 2.(2)6).

In another example, a liver cancer cell line (Huh-7 sorafenib resistant [SR] cell) that is resistant to sorafenib, the current standard treatment for hepatocellular carcinoma, was subcutaneously transplanted into a Balb-c nude mouse and loaded into exosomes into the animal's abdominal cavity. As a result of comparing the degree of inhibition of ATP production in tumor tissue in animal groups administered with FX-11 and IACS-010759 loaded on exosomes, the group administered with FX-11 loaded on exosomes and IACS-010759 loaded on exosomes It was confirmed that ATP production in tumor tissue was significantly suppressed compared to the control group. In addition, as a result of comparing the degree of inhibition of lactate production in tumor tissue between the control group and the group administered with FX-11 loaded on exosomes and IACS-010759 loaded on exosomes, FX-11 loaded on exosomes and IACS-010759 loaded on exosomes were compared. It was confirmed that lactate production in tumor tissue was significantly suppressed in the IACS-010759 administration group compared to the control group (see Example 2.(2)8).

The pharmaceutical composition may contain the active ingredient alone or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable excipients or diluents.

When the pharmaceutical composition is formulated, it may be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. You can. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may contain at least one excipient, such as starch, calcium carbonate, or sucrose. ) or it can be prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the 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 may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used, and when manufacturing in the form of eye drops, known diluents or excipients can be used. there is.

In one aspect, the pharmaceutical composition may further include a pharmaceutical composition for preventing or treating cancer other than the glycolysis inhibitor or the mitochondrial function inhibitor.

The pharmaceutical composition may be provided by mixing with a pharmaceutical composition for the prevention or treatment of other cancers, and the pharmaceutical composition for the prevention or treatment of other cancers may be a conventionally known pharmaceutical composition for the prevention or treatment of cancer or It may be a newly developed pharmaceutical composition for preventing or treating cancer.

When the pharmaceutical composition further includes a pharmaceutical composition for the prevention or treatment of other cancers or is provided in combination with a pharmaceutical composition for the prevention or treatment of other cancers, the maximum effect can be obtained with the minimum amount without side effects. It is important that the amounts are mixed, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition is not mixed with other pharmaceutical compositions for preventing or treating cancer other than the glycolysis inhibitor or the mitochondrial function inhibitor, and may be administered in parallel, simultaneously, separately, or sequentially, and may be administered as a single Or it can be administered in multiple doses. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, it can be administered externally to the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, or intracardiac injection. You can choose the injection method: injection, intrathecal injection, transdermal injection, intranasal injection, enteral injection, local injection, sublingual injection, or intrathoracic injection.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the drug's effect. It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

In one aspect, the pharmaceutical composition may be administered once a day or may be administered several times. For example, it may be administered every other day, or it may be administered once a week. Specifically, the pharmaceutical composition may be administered at 0.001 to 1000 mg/kg/day, more specifically at 0.1 to 100 mg/kg/day. The administration may be administered once a day, or may be administered several times.

Another aspect provides a health functional food for preventing or improving cancer containing a glycolysis inhibitor and a mitochondrial function inhibitor.

The terms “inhibitor of glycolysis,” “inhibitor of mitochondrial function,” “cancer,” “prevention,” etc. may be within the above-mentioned range.

The term “improvement” may refer to any action that results in at least a reduction in the severity of a parameter associated with the condition being treated, such as symptoms. At this time, the health functional food can be used simultaneously or separately with drugs for treatment before or after the onset of the disease in order to prevent or improve cancer.

In the health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, when manufacturing a food or beverage, the health functional food may be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The health functional food may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of diluents, excipients, and additives. Foods to which compounds according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

In addition to containing the effective ingredients, the health functional food may contain other ingredients as essential ingredients without any particular restrictions. For example, like regular beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. You can. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, health functional foods according to one aspect include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

In one aspect, the health functional food may further include a health functional food for preventing or improving cancer other than the glycolysis inhibitor or the mitochondrial function inhibitor.

The health functional food may be provided by mixing with health functional food for preventing or improving other cancers, and the health functional food for preventing or improving other cancer is a health functional food for preventing or improving cancer known in the art or It may be a newly developed health functional food for preventing or improving cancer.

If the health functional food further contains the health functional food for preventing or improving other cancers, or is mixed with the health functional food for preventing or improving other cancers, the maximum effect can be obtained with the minimum amount without side effects. It is important that the amounts are mixed, and this can be easily determined by a person skilled in the art.

In addition, the health functional food may be consumed in parallel with other health functional foods for preventing or improving cancer other than the glycolysis inhibitor or the mitochondrial function inhibitor, and may be consumed simultaneously, separately, or sequentially, singly or in multiple forms. It can be ingested. Considering all of the above factors, it is important to consume an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect provides a method of preventing or treating cancer comprising administering a glycolysis inhibitor and a mitochondrial function inhibitor to a subject in need thereof.

The terms “inhibitor of glycolysis,” “inhibitor of mitochondrial function,” “individual,” “administration,” “cancer,” “prevention,” “treatment,” etc. may be within the scope described above.

In the method, pharmaceutical compositions for preventing or treating cancer other than the glycolysis inhibitor or the mitochondrial function inhibitor may be administered in parallel, and may be administered simultaneously, separately, or sequentially, and may be administered singly or multiple times. You can. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect provides the use of glycolysis inhibitors and mitochondrial function inhibitors for the manufacture of medicaments for the prevention or treatment of cancer.

The terms “cancer,” “prevention,” “treatment,” “glycolysis inhibitor,” “mitochondrial function inhibitor,” etc. may be within the above-mentioned range.

According to one aspect, when a glycolysis inhibitor and a mitochondrial function inhibitor are administered in combination, the growth of cancer cells is inhibited, ATP energy production is blocked in cancer cells, and PI3K/Akt/Akt/AKT in cancer cells are inhibited compared to the single administration of the glycolysis inhibitor or mitochondrial function inhibitor. It was confirmed that the effect of inhibiting mTORC1 signaling, inducing the expression of REDD1 (regulated in development and DNA damage responses 1) in cancer cells, and suppressing the expression of proteins related to cell proliferation was significantly superior. In addition, when glycolysis inhibitors and mitochondrial function inhibitors are loaded onto extracellular vesicles and administered simultaneously, superior cancer prevention, improvement, or treatment effects were confirmed, which can contribute to the cancer treatment market/industry.

Figure 1 is a diagram showing the results of confirming apoptosis of metformin and dichloroacetic acid (DCA) in liver cancer cells. Figure 1A is a diagram showing the results of confirming the cytotoxicity of metformin on liver cancer cells. Figure 1B is a diagram showing the results of confirming the cytotoxicity of DCA to liver cancer cells, Figure 1C is a diagram showing the results of confirming the cytotoxicity of metformin and DCA to liver cancer cells, and Figure 1D is a diagram showing the results of confirming the cytotoxicity of metformin and DCA to liver cancer cells. This diagram shows the results of confirming PARP expression when treated alone (* p <0.05; ** p <0.01; and *** p < 0.001).
Figure 2 is a diagram showing the results of apoptosis of HepG2 and PLC/PRF5 cells stained with annexin V-FITC after combined treatment with metformin and DCA (***P < 0.001).
Figure 3 is a diagram showing the results of confirming cytotoxicity of normal hepatocytes after combined treatment of metformin and DCA. Figure 3A is a diagram showing the results of confirming the cytotoxicity of DCA on normal hepatocytes, and Figure 3B is a diagram showing the results of confirming the cytotoxicity of DCA on normal hepatocytes. A diagram showing the results of confirming the cytotoxicity of , and Figure 3C is a diagram showing the results of confirming the cytotoxicity of the combined treatment of metformin and DCA in normal hepatocytes (* p < 0.05, *** p < 0.001).
Figure 4 is a diagram showing the results of ATP production by metformin and DCA in HepG2 and PLC/PRF5 cells, Figure 4A is a diagram showing the results of confirming the lactate level in liver cancer cells, and Figure 4B is a diagram showing the results of confirming the ATP level in liver cancer cells. A diagram showing the results, and Figure 4C is a diagram showing the results of confirming the function of PDK (pyruvate dehydrogenase kinase), the target protein of DCA, in liver cancer cells through phosphorylation of PDH (pyruvate dehydrogenase) (* p <0.05; ** p < 0.01 and ***p < 0.001).
Figure 5 shows the results of treating liver cancer cells with metformin, DCA, or metformin and DCA for 24 hours. Figure 5A shows PI3K/Akt/mTORC1 signaling and mTORC1 effectors S6K1 and 4EBP after combined treatment with metformin and DCA. A diagram showing the results of confirming the expression level of , Figure 5B is a diagram showing the results of confirming the expression level of p-ACC, an AMPK effector, after combined treatment with metformin and DCA, and Figure 5C shows REDD1 expression after combined treatment with metformin and DCA. This figure shows the results of confirming the level (* p <0.05; ** p <0.01; and *** p < 0.001).
Figure 6 shows the results of Western blotting of PI3K/Akt/mTORC1 signaling after combined treatment with metformin and DCA, and Figure 6A shows the results confirming that PI3K/Akt/mTORC1 signaling is inhibited after combined treatment with metformin and DCA. Figure 6B shows the results confirming that the expression of p-ACC, an AMPK effector, is stimulated after the combined administration of metformin and DCA, and Figure 6C shows the results confirming that the combined administration of metformin and DCA effectively induces REDD1 expression. It is a degree that represents .
Figure 7 shows the results of confirming the level of reactive oxygen species (ROS) after treating liver cancer cells with metformin, DCA, or metformin and DCA for 1 hour. Figure 7A shows liver cancer cells after combined administration of metformin and DCA. This diagram shows the results confirming that ROS production in cells significantly increases, and Figure 7B shows the cytotoxic effect of treatment with the antioxidant N-acetylcysteine (NAC: N-acetylcysteine) on liver cancer cells after combined treatment with metformin and DCA. This figure shows the results confirming the significant reduction (* p <0.05; ** p <0.01; and *** p < 0.001.)
Figure 8 shows the results of confirming changes in mitochondrial reactive oxygen species in HepG2 cells treated with metformin, DCA, or metformin and DCA for 1 hour. Figure 8A shows mitochondrial reactive oxygen species (ROS) in HepG2 cells after combined administration of metformin and DCA. ) This figure shows the results confirming that the production was significantly increased, and Figure 8B shows the results confirming that 4-hydroxynonenal (4-HNE: 4-hydroxynonenal) was highly expressed in the metformin and DCA treated groups in the tumor tissue. Doida (x100).
Figure 9 shows the results of confirming the anticancer effect of metformin and DCA combination treatment in a mouse model containing MH-134 cells, and Figure 9A shows the tumor volume of the combination treatment group (metformin and DCA) for 15 days after the start of drug treatment. This is a diagram showing the results of confirmation, and Figure 9B is a diagram showing the results of TUNEL-positive staining (400x magnification) in tumor tissue in the combination treatment group (metformin and DCA) (*p < 0.05).
Figure 10 shows the results of confirming the expression of Cyclin D1, Ki-67 protein, and proliferating cell nuclear antigen (PCNA) after treating tumor tissue with metformin and DCA. Figure 10A shows the expression of Cyclin D1 protein in the control group. A diagram showing the results, Figure 10B is a diagram showing the results confirming the expression of cyclin D1 protein in the metformin and DCA treated group, Figure 10C is a diagram showing the results confirming the expression of Ki-67 protein in the control group, and Figure 10D is a diagram showing the results confirming the expression of the Ki-67 protein in the control group. A diagram showing the results of confirming the expression of Ki-67 protein in the metformin and DCA treatment group, Figure 10E is a diagram showing the results of confirming the expression of PCNA in the control group, and Figure 10F is a diagram showing the results of confirming the expression of PCNA in the metformin and DCA treatment group. It is a diagram that shows the result.
Figure 11 is a diagram showing the results of confirming changes in tumor volume in each individual in the combination treatment group treated with metformin and DCA over time.
Figure 12 is a diagram showing the results confirming that intracellular lactate production is suppressed when the concentration of FX-11, a glycolysis inhibitor, is increased in liver cancer cell lines (HepG2 cell, Huh-7 cell).
Figure 13 shows that when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with IACS-010759, a mitochondrial function inhibitor, intracellular lactate production is increased, and FX-11, a glycolysis inhibitor, and IACS-010759, a mitochondrial function inhibitor, are increased. This diagram shows the results confirming that intracellular lactate production is suppressed when treated simultaneously.
Figure 14 is a diagram showing the results confirming that ATP production, an energy source within cancer cells, is reduced when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with FX-11 and IACS-010759.
Figure 15 shows changes in the expression of lactate dehydrogenase A (LDHA: lactate dehydrogenase A), the target protein of FX-11, when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with FX-11 and IACS-010759. This diagram shows the results confirmed on Western blot.
Figure 16 shows the change in expression of mitochondrial respiratory complex I, the target protein of IACS-010759, on Western blot when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with FX-11 and IACS-010759. This diagram shows the confirmed results.
Figure 17 is a diagram showing the results of confirming changes in expression of mitochondrial respiratory complex I when liver cancer cell lines (HepG2 cell, Huh-7 cell) were treated with FX-11 and IACS-010759.
Figure 18 is a diagram showing the results of confirming the synergistic effect of apoptosis through MTT assay when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated in combination with FX-11 and IACS-010759.
Figure 19 is a diagram showing the results confirming that apoptosis occurs as a cell death mechanism when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated in combination with FX-11 and IACS-010759.
Figure 20 shows the results of confirming the expression levels of S6K1 and 4EBP, which are mTORC1 effectors, as one of the mechanisms of apoptosis when liver cancer cell lines (HepG2 cell, Huh-7 cell) were treated in combination with FX-11 and IACS-010759. It is a degree that represents.
Figure 21 is a diagram showing the results of confirming changes in the apoptosis effect when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with FX-11 and IACS-010759 in combination according to the glucose concentration around the liver cancer cells.
Figure 22 shows the simultaneous administration of FX-11 and IACS-010759 as a free drug not loaded on exosomes in a liver cancer model formed by injecting a liver cancer cell line (HepG2 cell) into a Balb/c nude mouse xenograft animal model and the case of simultaneous administration to exosomes. This diagram shows the results of a comparative evaluation of the inhibitory effect on liver cancer growth when combined with FX-11 and IACS-010759.
Figure 23 shows TUNEL (apoptosis), Ki-67 (proliferation of cancer cells), p-Akt (energy generation of cancer cells) and mTOR ( This diagram shows the results of comparing changes in cancer cell proliferation.
Figure 24 is a diagram showing the results of confirming the cell death effect through MTT assay when FX-11 and IACS-010759 were loaded onto exosomes in a breast cancer cell line (MCF-7) and treated alone or in combination.
Figure 25 is a diagram showing the results of confirming the apoptosis effect of FX-11 and IACS-010759 when pancreatic cancer cells were loaded with exosomes and treated alone or in combination.
Figure 26 shows the transplantation of Huh-7 SR cells, which are resistant to sorafenib, the current standard treatment for liver cancer, into Balb-c nude mice, followed by loading FX-11 and IACS-010759 into exosomes and processing them alone or in combination. This diagram shows the results of confirming the tumor growth inhibition effect during treatment and treatment with sorafenib.
Figure 27 shows the transplantation of Huh-7 SR cells, which are resistant to sorafenib, the current standard treatment for liver cancer, into Balb-c nude mice, followed by loading FX-11 and IACS-010759 into exosomes and processing them alone or in combination. This diagram shows the results of confirming the inhibitory effect on ATP production during treatment and treatment with sorafenib.
Figure 28 shows the transplantation of Huh-7 SR cells, which are resistant to sorafenib, the current standard treatment for liver cancer, into Balb-c nude mice, followed by loading FX-11 and IACS-010759 into exosomes and processing them alone or in combination. This diagram shows the results of confirming the effect of inhibiting lactate production during treatment and treatment with sorafenib.
Figure 29 shows the transplantation of Huh-7 SR cells, which are resistant to sorafenib, the current standard treatment for liver cancer, into Balb-c nude mice, followed by loading FX-11 and IACS-010759 into exosomes and processing them alone or in combination. This figure shows the results of confirming the change in body weight in each treatment group over time during treatment and treatment with sorafenib.

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

Example

1. Experimental materials and methods

(1) Acquisition of cell lines and chemicals

HepG2 and PLC/PRF5 human liver cancer cell lines for in vitro studies and MH-134 murine liver cancer cell lines for in vivo studies were purchased from the Korean Cell Line Bank. Both cell lines were incubated with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin G, and 100 g/mL streptomycin at 37°C in a 5% CO ₂ incubator. The cells were maintained in Dulbecco's modified Eagle's medium containing streptomycin. Human normal hepatocytes were purchased from Nexel (Hepatosight®-S H-002, Seoul, Korea), plated on collagen 1 (Sigma Aldrich)-coated 12-well plates, and incubated in hepatocyte culture medium provided by the manufacturer. maintained (Hepatosight®-S H-002, Seoul, Korea). Compound C, an AMPK inhibitor (CC: compound C; 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine , C ₂₄ H ₂₅ N ₅ O, CAS number: 866405-64-3) from Sigma-Aldrich (St. Louis, MO, USA, Cat. No. 171260), dissolved in DMSO, 20 for each specific study. M concentration was used. When treating HepG2 and PLC/PRF5 cells with metformin, Compound C was pretreated with 20 M Compound C for 1 hour and then treated with 10 mM metformin.

(2) Cell viability assay

Cell viability was measured using MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS (0.5 mg/mL). On the day of measurement, the medium was replaced with MTT medium and incubated for 4 hours at 37°C in the dark. After removing the incubation medium, formazan crystals were dissolved in DMSO for 1 hour. Untreated cells were considered control (100% cell survival).

(3) Western Blot analysis

Cells were lysed using protein extraction buffer (iNTRON Biotechnology, Seongnam-Si, Gyeonggi-do, Korea), and the protein concentration of the cell lysate was assessed by BCA protein assay (Thermo Scientific, Pittsburgh, PA, USA). Aliquots of 20-30 g proteins from whole cell lysates were separated by 8-12% sodium dodecyl sulfatepolyacrylamidegel electrophoresis and transferred to poly(vinylidene fluoride) (PVDF) membranes. . After blocking with 5% skim milk for 1 hour, the membrane was incubated with poly-ADP-ribose polymerase (PARP), cleaved PARP, β-actin, and pyruvate dehydrogenase (PDH). : pyruvate dehydrogenase)-E1, p-PDH-El, p-phosphatidylinositol-3-kinase (PI3K: p-phosphatidylinositol-3-kinase), anti-PI3 K, serine/threonine kinase (Akt) ), p-Akt, mammalian target of rapamycin complex 1 (mTORC1), p-mTORC1, p-AMPKα (Thr172), primary antibody against AMPKα (1:1000 dilution), development and DNA damage response 1 (REDD1: regulated in development and DNA damage responses 1), p-70 S6 Kinase (S6K1) (Th389), S6K1, 4E-binding protein 1 (4EBP1), p-4EBP1, Acetyl-CoA carboxylase (p-ACC) and p-ACC were probed overnight at 4°C (Cell Signaling Technology, Danvers, MA, USA).

Then, it was incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) at room temperature for 2 hours. Antibody-reactive bands were detected by chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA).

(4) Flow cytometry using Annexin V-FITC staining

Apoptotic analyzes were performed by flow cytometry using a FACScalibur system (Becton Dickinson Biosciences, San Diego, CA, USA). Cells were treated in combination with metformin and dichloroacetic acid (DCA) for 24 hours. After treatment, cells were collected using trypsin, washed twice with cold PBS, and resuspended in 0.5 mL of Annexin V-FITC solution for 15 min in the dark at room temperature. After staining, cells were injected through flow cytometry and FITC fluorescence signals were analyzed. Apoptotic cells were analyzed by quadrant statistics for propidium iodide-negative and annexin V-positive cells. Mean ± SD was calculated from cell populations of three replicate data.

(5) Extracellular Lactate and Intracellular ATP Analysis

Lactate production was measured using the EZ-Lactate Assay Kit (DoGenBio, Seoul, Korea). Cells were treated with metformin and DCA for 24 hours, cell culture medium was collected, and lactate levels were analyzed using colorimetric detection at 450 nm according to the manufacturer's instructions. ATP production level was measured using the EZ-ATP Assay Kit. After 24 hours of treatment, HepG2 and PLC/PRF5 cells were collected and analyzed using an enzyme-linked immunosorbent assay (ELISA) spectrophotometer with colorimetric detection at 570 nm. Absorbance was normalized to protein concentration.

(6) Evaluation of intracellular and mitochondrial reactive oxygen species production

Dihydroethidium (DHE) is converted to red fluorescent ethidium (Sigma-Aldrich, St. Louis, MO, USA) by superoxide. Intracellular reactive oxygen species (ROS) levels were detected using 5 μM DHE under dark conditions in a 37°C incubator for 30 minutes. Afterwards, cells were obtained after 1 hour incubation with 0.1 N NaOH. The intensity of DHE cell fluorescence was measured at 590 nm excitation and 650 nm emission using a Spectra Max M2 ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Experiments were performed in triplicate and final values were normalized to the total number of cells.

Additionally, mitochondrial reactive oxygen species (Mitochondrial Reactive Oxygen Species) were detected using a MitoSOX-based assay (Invitrogen, Waltham, MA, USA). A flow cytometry protocol involving the use of 5 μM MitoSOX detected mitochondrial ROS in HepG2 cells with treatment of metformin and/or DCA. Measurement of MitoSOX-derived fluorescence intensity reflected the level of total mitochondrial ROS. After treatment, cells were stained with dye at 37°C for 10 min according to the manufacturer's instructions, and mitochondrial ROS activity was analyzed using flow cytometry.

(7) Production of in vivo subcutaneous xenograft model

MH-134 cells ( ⁵ Tumor volume was measured using a Vernier caliper and calculated ^as [length Once the tumor volume reached a size of approximately 200 mm ³ , mice were treated daily with saline or metformin (200 mg/kg) or a combination of metformin (200 mg/kg) and DCA (100 mg/kg) orally. ) was done. The length and width of each nodule were measured daily for 15 days. To confirm tumor cell death, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed. Tumor specimens were fixed in 4% formaldehyde, embedded in paraffin, and cut into 4 m sections for immunohistochemical (IHC) analysis. IHC staining was performed with anti-mouse Ki-67 antibody (Abcam, Cambridge, UK) at 1:3000 dilution, anti-cyclin D1 antibody (Cell Signaling Technology, Beverly, MA, USA) at 1:50 dilution, and anti-mouse Ki-67 antibody (Abcam, Cambridge, UK) at 1:3000 dilution. -Mouse proliferating cell nuclear antigen antibody (Thermo Scientific, Pittsburgh, PA, USA) at 1:5000 dilution, 4-hydroxynonenal (HNE) antibody (LSBio, Seattle, WA, USB) was performed using a 1:1000 dilution. All protocols for animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (No. IACUC200170, Ethics Approval Date: November 2019, 2020). All animal procedures followed the “Guide for the Care and Use of Laboratory Animals” published by the US National Research Council, Institute of Laboratory Animal Resources Commission on Life Science.

(8) Statistical analysis

All results were expressed as means ± standard deviations (SD). A paired Student's-test was applied to detect differences between the two groups. A p value <0.05 was considered significant. All statistical analyzes were performed using SPSS version 18.0 (IBM, Armonk, NY, USA).

2. Experimental results

(1) Confirmation of liver cancer treatment efficacy through combined administration of metformin and dichloroacetic acid (DCA)

1) Apoptosis induced by metformin and dichloroacetic acid

To investigate whether an anticancer effect exists between metformin, a mitochondrial function inhibitor, and DCA, a glycolysis inhibitor, in inhibiting the growth of liver cancer cells, HepG2 and PLC/PRF5 cells were treated with metformin, DCA, or a combination of metformin and DCA (Figures 1A and Figure 1B). Combination treatment of metformin and DCA significantly inhibited the growth of hepatoma cells compared to metformin or DCA alone (p < 0.001 in HepG2 and p < 0.01 in PLC/PRF5, respectively; Figure 1C), and 10 mM metformin and 8 It was confirmed that the combined treatment with mM DCA inhibited cell viability (IC ₅₀ value) by 50% compared to the control group.

The cytotoxic effect of combined metformin and DCA treatment on HepG2 and PLC/PRF5 cells was evaluated using the combination index (CI) using Chou and Talalay: CI = (dA/DA) + (dB/DB) ), where dA and dB are the IC ₅₀ values of compounds A and B combined, and DA and DB are the IC ₅₀ values of single compounds A or B, respectively. CI values <1, 1, and >1 indicate synergism, additivity, and antagonism in the combined agonist action, respectively. Since the CI values for metformin and DCA treated in combination were 0.63 and 0.58 for HepG2 and PLC/PRF5 cells, respectively, it can be confirmed that metformin and DCA have a strong anticancer effect on inhibition of liver cancer cell growth, and that metformin and DCA have a strong anticancer effect on inhibition of liver cancer cell growth. was confirmed to effectively induce apoptosis in liver cancer cells. It was confirmed that cleaved PARP (marker of apoptosis) expression was enhanced in liver cancer cells treated in combination with metformin and DCA compared to those treated with metformin or DCA alone (Figure 1D). Annexin V-FITC staining showed that when HepG2 and PLC/PRF5 cells were treated in combination with 10 mM metformin and 8 mM DCA for 24 hours, the proportion of apoptotic cells was significantly higher than that of the control group (all p <0.001; Figure 2).

On the other hand, there was no significant cytotoxicity against normal hepatocytes at the respective IC ₅₀ values of 8mM DCA (Figure 3A) or 10mM metformin (Figure 3B) against hepatoma cells. It was confirmed that the combined treatment of metformin (10mM) and DCA (8mM) did not show cytotoxicity to normal hepatocytes (Figure 3C). However, it was confirmed that metformin is cytotoxic to normal hepatocytes at high concentrations (80 and 120 mM).

2) Confirmation of the inhibitory effects of metformin and DCA on lactate production and ATP production in liver cancer cells

Metformin is known to inhibit respiration complex I in mitochondria. Inhibition of mitochondrial respiratory complex I induces energy stress that promotes the activation of AMPK and subsequent catabolic metabolism that restores energy homeostasis. To determine whether inhibition of complex I using metformin induces glycolysis and depletes intracellular ATP in liver cancer cells, extracellular lactate levels and intracellular ATP levels were measured. Metformin increased lactate acid production in liver cancer cells in a dose-dependent manner (p = 0.007 in HepG2, p = 0.01 in PLC/PRF5 for 10 mM metformin, p < 0.01 in HepG2 for 20 mM metformin). 0.001, p = 0.002 in PLC/PRF5; Figure 4A), and it was confirmed that the intracellular ATP level was reduced in liver cancer cells (p = 0.02 for HepG2, p = 0.03 in PLC/pRF5, Figure 4B).

Because phosphorylation of the PDH complex is related to the Warburg effect, we sought to determine whether DCA treatment alters lactate production in liver cancer cells. 24-hour incubation with 8 mM DCA resulted in a significant decrease in lactate compared to the control, suggesting a shift toward glucose oxidation (p = 0.07 in HepG2, PLC/PRF5 with 8 mM DCA) p = 0.02 in, p = 0.008 in HepG2, p = 0.005 in PLC/PRF5 in DCA 16 mM; Figure 4A). DCA treatment reduced the phosphorylation of the E1 subunit of the PDH complex in a dose-dependent manner after 4 hours of incubation, and it was confirmed that DCA inhibited lactate production in hepatoma cells (Figure 4C). Additionally, DCA was confirmed to reduce intracellular ATP levels in liver cancer cells (p = 0.11 in PLC/PRF5 for 8 mM DCA; Figure 4B).

Combination treatment of metformin and DCA significantly inhibited lactate acid production and intracellular ATP production in liver cancer cells compared to the control group (p=0.005 in HepG2, p=0.02 in PLC/PRF5 for inhibition of lactate acid production) , and p<0.001 in HepG 2 and p=0.01 in PLC/PRF5 for inhibition of ATP production) (Figures 4A and 4B).

3) Confirmation of the inhibitory effect of metformin and DCA on mTORC1 expression in liver cancer cells

It is well known that mTORC1 is one of the key proteins involved in the carcinogenesis process of liver cancer. The present inventors evaluated whether the combined treatment of metformin and DCA effectively inhibits PI3K/Akt/mTORC1 signaling. Treatment with metformin or DCA inhibited the expression of PI3K/Akt/mTORC1 signaling in liver cancer cells. It was confirmed that the combined treatment of metformin and DCA significantly inhibited PI3K/Akt/mTORC1 signaling compared to the control group (p<0.001 for p-PI3K/PI3k, p=0.02 for p-AKt/Akt, HepG2 p = 0.005 for p-mTOR/mTOR and p < 0.001 for p-PI3K/PI3K, p < 0.001 for p-Akt/Akt, p < 0.001 for p-mTOR/mTOR in PLC/PRF5 cells. (respectively; Figure 5A). Regarding mTORC1 effectors such as S6K1 and 4EBP1, it was confirmed that the combined treatment of metformin and DCA significantly inhibited S6k1 and 4EBPl signaling compared to the control group (p<0.001 in HepG2 cells and PLC/PRF5 cells; Figure 5A and Figure 6).

Considering that metformin inhibits mTORC1 activity in cancer cells through energy stress to activate AMPK, we evaluated whether the combined treatment of metformin and DCA affects mTORC1 expression by activating AMPK expression.

The combined treatment of metformin and DCA significantly enhanced AMPK expression compared to the control group: The combined treatment of metformin and DCA increased the expression of p-ACC as an effector of AMPK signaling compared to the control group (p=0.03 in HepG2) , p=0.01 in PLC/PRF5; Figure 5B). However, Compound C, a known AMPK inhibitor (20 M), inhibited the expression of p-ACC when treated with metformin and DCA in both cell lines; AICAR as an activator of AMPK (2 mM) potentiated the expression of p-ACC compared to control (p = 0.006 in HepG2, p = 0.004 in PLC/PRF5 for compound C, p = 0.04 in HepG2, p = 0.004 for ACAR) p < 0.0001 in PLC/PRF5; Figures 5B and 6).

Although combined treatment with metformin and DCA activated AMPK signaling, inhibition of mTORC1 expression was mediated by REDD1 expression, not AMPK expression. Considering that energetic stress induces REDD1 expression in cancer, we evaluated whether energetic stress induced by the combined treatment of metformin and DCA affects REDD1 expression. REDD1 is known to interact with TSC2 and increase the inhibitory effect of TSC1/TSC2 heterodimer on mTORC1 activity. We found a strong anticancer effect of metformin and DCA on the induction of REDD1 expression (Figures 5C and 6), but the induction of REDD1 expression by metformin and DCA was not affected by compound C, but was affected by metformin and DCA. It was confirmed that REDD1 induction contributes to the inhibition of mTORC1 activity in an AMPK-independent manner.

4) Confirmation of ROS regulation important for the anticancer effect of metformin and DCA

Since glycolysis and oxidative metabolism are intrinsically involved in the generation of ROS, we assessed whether activation of pyruvate dehydrogenase by DCA alters ROS generation in liver cancer cells. did. In response to DCA treatment, the intracellular ROS level in liver cancer cells was confirmed to significantly increase after 1 hour of DCA treatment compared to the control group (p<0.001 for HepG2 and p=0.07 for PLC/PRF5, Figure 7A). , it was confirmed that metformin significantly increased compared to the control group (p=0.02 for HeptG2 and P<0.001 for PLC/PRF5). Additionally, when adding metformin, it was confirmed that ROS generation was enhanced in the presence of DCA compared to metformin or DCA alone (p = 0.03 metformin + DCA vs. DCA in HepG2 cells, and p = 0.02 vs. metformin + DCA in PLC/PRF5). DCA, p = 0.04 metformin + DCA vs. metformin; Figure 7A).

To clarify the relationship between increased ROS and cell death following DCA and/or metformin treatment, the anticancer effect of DCA and/or metformin was evaluated when ROS levels were adjusted in liver cancer cells. It was confirmed that the antioxidant N-acetylcysteine significantly attenuated the cytotoxicity of combined treatment with metformin and DCA (p = 0.03 in HepG2, p = 0.02 in PLC/PRF5; Figure 7B). The above results suggest that oxidative stress increased by the combined treatment of metformin and DCA plays an important role in relation to the strong anticancer effect in liver cancer cells.

In response to metformin and DCA treatment, the mitochondrial ROS level in HepG2 cells was confirmed to significantly increase after 1 hour of DCA treatment compared to the control group (p < 0.001, Figure 8). Measurement of ROS in tumor tissue Liou, G.Y.; Storz, P. Detecting reactive oxygen species by immunohistochemistry. Methods Mol. Biol. 2015, 1292, 97-104. Serum 4-hydroxynonenal (HNE) antibody (LSBio, Seattle, USA) against an antigen that can act as a susceptibility marker of increased oxidative stress. WA, USA) was used. It was confirmed that 4-HNE was highly expressed in the tumor tissue in the group treated with metformin and DCA (Figure 8).

5) Confirmation of anticancer effect of combined treatment of metformin and DCA in murine models

The anticancer effect of combined treatment with metformin and DCA was investigated using an in vivo xenograft model. Specifically, treatment of mice began after tumor budding, and the size of the tumor was 200 mm ³ or more. It took 6 to 8 days after transplanting ⁵ The time from implantation to tumor budding did not differ between the three groups. It was confirmed that the combined treatment of metformin and DCA significantly inhibited tumor growth compared to the control group at 13, 14, and 15 days after MH-134 tumor budding (Figure 9A; all, p < 0.05). There was a tendency for the metformin-only treatment group to have a lower tumor growth rate than the control group, but there was no significant difference in tumor growth rate between the metformin-only treatment group and the control group. In the combination treatment group of metformin and DCA, apoptotic cells were TUNEL-positive (Figure 9B). Compared to the control group, the expression of cyclin D1, Ki-67 protein, and proliferating cell nuclear antigen was confirmed to be decreased in tumor tissues in the group treated with metformin and DCA (FIG. 10). Individual changes in tumor volume in the combination treatment group of metformin and DCA over time are shown in Figure 11.

(2) Confirmation of liver cancer treatment efficacy through combined administration of FX-11 and IACS-010759

1) Confirmation of correlation between glycolysis and energy metabolism through mitochondria

In order to confirm the correlation between glycolysis and energy metabolism through mitochondria, the liver cancer cell lines (HepG2 cell, Huh-7 cell) distributed from the Liver Research Institute of Seoul National University Hospital were used. Process inhibitor FX-11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic Acid, C ₂₂ H ₂₂ O ₄ , Sigma-Aldrich CAS Number: 213971-34-7) was processed. When the concentration of FX-11 increases (HepG2 cell: 0 μM, 60 μM, 120 μM, 150 μM, Huh-7 cell: 0 μM, 60 μM, 120 μM, 150 μM), intracellular lactate production is significant. It was confirmed that the mitochondrial function inhibitor, IACS-010759 (4-(methylsulfonyl)-1-[3-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2, 4-oxadiazol-5-yl]-1H-1,2,4-triazol-1-yl]methyl]phenyl]-piperidine, C ₂₅ H ₂₅ F ₃ N ₆ O ₄ S, Cayman chemicals, CAS Number: 157049 6 When treated with -34-2) (HepG2 cells and Huh-7 cells: 0 nM, 50 nM, 100 nM, 150 nM), it was confirmed that intracellular lactate production was significantly increased (FIGS. 12 and 13) . In addition, FX-11 (HepG2 cell and Huh-7 cell: 0 μM, 60 μM, 120 μM, 150 μM) and IACS-010759 (HepG2 cell and Huh-7 cell) were added to liver cancer cell lines (HepG2 cell, Huh-7 cell). : 0 nM, 50 nM, 100 nM, 150 nM), it was confirmed that ATP production, an energy source in cancer cells, was significantly reduced (FIG. 14).

In addition, liver cancer cell lines (HepG2 cell, Huh-7 cell) were treated with FX-11 (HepG2 cell: 0 μM, 10 μM, 30 μM, 60 μM, 120 μM, Huh-7 cell: 0 μM, 10 μM, 30 μM) μM, 60 μM, 120 μM), there was no change in the expression of lactate dehydrogenase A (LDHA: lactate dehydrogenase A), the target protein of FX-11, on Western blot, and treatment with IACS-010759 (HepG2 cells and Even in the case of Huh-7 cells: 0 nM, 100 nM), it was confirmed that there was no change in the expression of LDHA on Western blot (FIG. 15). Therefore, it was determined that the decrease in lactate production when FX-11 was administered to liver cancer cells was more likely to be due to functional inhibition rather than a change in the expression level of LDHA.

In addition, when liver cancer cell lines (HepG2 cell, Huh-7 cell) are treated with IACS-010759 (HepG2 cell and Huh-7 cell: 0 nM, 100 nM), mitochondrial respiratory complex I (mitochondrial complex I), the target protein of IACS-010759, The change in expression of respiratory complex I) showed a decrease in Western blot, and even when treated with FX-11 (HepG2 cells and Huh-7 cells: 0 μM, 120 μM, 150 μM), mitochondrial respiratory complex I (mitochondrial complex I) showed a decrease. It was confirmed that the expression of respiratory complex I) was decreased (FIGS. 16 and 17).

2) Confirmation of inhibition of proliferation of FX-11 and IACS-010759 in liver cancer cell lines

To confirm the inhibition of liver cancer cell proliferation when administering FX-11 and IACS-010759 to liver cancer cell lines (HepG2 cell, Huh-7 cell), FX-11 (HepG2 cell and Huh-7 cell: 0 μM) , 30 μM, 60 μM, 120 μM) and IACS-010759 (HepG2 cell and Huh-7 cell: 0 nM, 10 nM, 50 nM, 100 nM, 150 nM), the synergistic effect of apoptosis was observed using MTT. This was confirmed through assay (Figure 18). In addition, FX-11 (HepG2 cell and Huh-7 cell: 0 μM, 120 μM, 150 μM) and IACS-010759 (HepG2 cell and Huh-7 cell: 0 nM, 100 nM, 150 nM) were used in combination with liver cancer cell lines. When treated, it was confirmed that apoptosis occurs as a cell death mechanism (Figure 19), and it was confirmed that mTOR, a major target in the carcinogenesis of liver cancer, is inhibited as one of the cell death mechanisms, and inhibition of mTOR was confirmed. It was confirmed that the proliferation of liver cancer cells was suppressed (FIG. 20).

Furthermore, the low glucose environment around tumor cells causes metabolism within cancer cells to depend on energy production through mitochondrial respiration rather than glycolysis using glucose, and the high glucose environment around tumor cells causes metabolism within cancer cells to depend on energy production through mitochondrial respiration. Because it relies primarily on glycolysis, inhibiting these two energy processes may be important in blocking energy production in cancer cells. FX-11 (HepG2 cell and Huh-7 cell: 0 μM, 120 μM, 150 μM) and IACS-010759 (HepG2 cell and Huh-7 cell: 0 μM) according to the concentration of glucose around the liver cancer cell line. nM, 100 nM), changes in the apoptosis effect were confirmed, and it was confirmed that apoptosis occurs more easily under low glucose environmental conditions around tumor cells (FIG. 21).

3) Confirmation of proliferation inhibition in liver cancer model of FX-11 and IACS-010759 loaded on exosomes

In a liver cancer model formed by injecting a liver cancer cell line (HepG2 cell) into a Balb/c nude mouse FX-11 and IACS-010759 were each loaded into exosomes and the inhibitory effect on liver cancer growth when administered intraperitoneally was compared and evaluated. Specifically, exosomes derived from human embryonic kidney (HEK293) cells, which have a very low immune response and tumor formation potential, were used. To isolate exosomes from cultured cells, cell debris (debris) in the medium was used. After removal, exosomes were isolated using the ExoQuick-TC kit (System Biosciences, Palo Alto, USA). The isolated exosomes were used after confirming their size and concentration using Nanoparticle Tracking Analysis (NTA). For loading of FX-11 and IACS-010759 into ^exosomes , exosomes (5 It was mixed with -010759 and stirred in the dark for 2 hours (4° C.) Afterwards, the sample was purified using a PD G-25 desalting column.

Liver cancer growth when FX-11 and IACS-010759 were simultaneously administered intraperitoneally as free drugs not loaded on exosomes, and when FX-11 and IACS-010759 were respectively loaded on exosomes and administered simultaneously intraperitoneally. As a result of comparing and evaluating the inhibitory effect of , the control group was found to have In comparison, it was confirmed that the growth of liver cancer was significantly inhibited (FIG. 22).

4) Confirmation of tumor tissue changes in FX-11 and IACS-010759 loaded on exosomes

As in Example 2.(2)3), after intraperitoneally administering the drug to a liver cancer model formed by injecting a liver cancer cell line (HepG2 cell) into a Balb/c nude mouse xenograft animal model, immunity is generated in the tumor tissue of the animal. Changes in tumor tissue according to drug administration were compared using chemical staining. The occurrence of tumor apoptosis was assessed using TUNEL staining, and in relation to tumor growth inhibition, immunostaining for Ki-67, p-Akt, and mTOR proteins was performed in the control group and the group administered simultaneously with FX-11 and IACS-010759. Comparison and evaluation were conducted across groups. As a result, it was confirmed that the group administered simultaneously with FX-11 and IACS-010759 had an increased incidence of apoptosis compared to the control group, and the expression of proteins related to tumor growth (Ki-67, p-Akt, and mTOR) was decreased ( Figure 23).

5) Confirmation of cell death effect of FX-11 and IACS-010759 loaded in exosomes on breast cancer cell line (MCF-7)

When FX-11 (1 μM, 5 μM, 10 μM) and IACS-010759 (1 μM, 5 μM, 10 μM) were treated alone or in combination with the breast cancer cell line (MCF-7) distributed from the Korea Cell Line Bank, the cells The killing effect was confirmed through MTT assay. Specifically, the cell survival rate was evaluated after the cells were treated with the drug for 24 hours. As a result, it was confirmed that when 10 μM of IACS-010759 and 10 μM of FX-11 were combined, the cell survival rate was 22% (Figure 24A). In addition, FX-11 (7 μM) and IACS-010759 (4 μM) were loaded into exosomes and treated alone or in combination with breast cancer cell lines. As a result, the case of simultaneous intraperitoneal treatment of exosomes loaded with 4 μM of IACS-010759 and the exosomes loaded with 7 μM of FX-11 was compared with the case of intraperitoneal treatment of FX-11 and IACS-010759 alone. It was confirmed that it showed a higher anti-cancer effect and had a cancer cell death rate of 80%. Therefore, it was confirmed that loading the drug into exosomes showed higher anticancer efficiency even at a lower drug concentration than when not loading the drug into exosomes (Figure 24B).

6) Confirmation of apoptosis effect of FX-11 and IACS-010759 loaded in exosomes on pancreatic cancer cell line (MIA PaCa-2)

Exosomes loaded with FX-11 (10 μM, 120 μM) and exosomes loaded with IACS-010759 (100 nM) were simultaneously administered intraperitoneally to a pancreatic cancer cell line (MIA PaCa-2) purchased from the National Cancer Center Hepatobiliary and Pancreatic Center. In the case of treatment, exosomes loaded with FX-11 (10 μM, 120 μM) or exosomes loaded with IACS-010759 (100 nM) are treated intraperitoneally alone and gemcitabine (0.5 μM) It was confirmed that the anticancer effect increased compared to the case of treatment (Figure 25).

7) Comparison of the tumor growth inhibition effects of FX-11 and IACS-010759 and sorafenib

FX-11, IACS-010759, and sorafenib were tested against the Huh-SR cell line acquired from the Department of Life Sciences at Seoul National University, which is resistant to sorafenib (sorafenib, Santa Cruz, CAS 284461-73-0), a standard treatment for liver cancer patients. The purpose was to confirm the tumor suppressive effect. 20 Balb-c nude mice were divided into 5 control groups, 5 sorafenib administered groups, 5 FX-11 + IACS-010759 administered groups, and 5 exosome-loaded FX-11 + exosome-loaded IACS-010759 administered groups. The experiment was conducted separately, and ^the sorafenib-resistant Huh-SR cell line ( administered. Each drug was administered intraperitoneally once a day, and the concentration of each drug was 30 mg/kg for sorafenib, 2 mg/kg for FX-11, and 2 mg/kg for IACS-010759 for 3 weeks. Afterwards, the tumor volume of each animal at the time of first administration of each drug and the relative ratio of tumor volume at various administration times of each drug were analyzed. As a result, it was confirmed that the most powerful tumor suppressing effect was observed in the group administered FX-11 loaded on exosomes + IACS-010759 loaded on exosomes, and FX-11 loaded on exosomes + IACS-010759 loaded on exosomes It was confirmed that the administration group showed a stronger tumor growth inhibition effect than the FX-11 + IACS-010759 administration group (FIG. 26).

8) Comparison of the inhibitory effects of FX-11 and IACS-010759 on ATP and lactate production with sorafenib

As a result of comparing the degree of inhibition of ATP production in tumor tissue of the control group and the FX-11 loaded on exosomes + IACS-010759 administered group loaded on exosomes using the experimental method of Example 2.(2)7), the exosomes It was confirmed that ATP production in tumor tissue was significantly suppressed in the group administered with FX-11 + IACS-010759 loaded on exosomes compared to the control group (FIG. 27). In other words, it was confirmed that ATP, the source of energy production in cancer cells, was inhibited, and that the blockage of energy production by simultaneous administration of the glycolysis inhibitor and mitochondrial respiration process inhibitor also occurred within tumor tissues of animals after drug administration.

In addition, as a result of comparing the degree of inhibition of lactate acid production in the tumor tissue of the control group and the FX-11 + exosome-loaded IACS-010759-administered group, FX-11 + exosomes loaded It was confirmed that lactate acid production in tumor tissue was significantly suppressed in the loaded IACS-010759 administration group compared to the control group (FIG. 28).

9) Comparison of toxicity confirmation between FX-11 and IACS-010759 and sorafenib

By the experimental method of Example 2. (2) 7), the drug was administered to the control group, the sorafenib-administered group, the FX-11 + IACS-010759-administered group, and the FX-11 + IACS-010759-administered group loaded on exosomes. As a result of checking the change in body weight over time after drug administration to confirm the possibility of toxicity, after starting drug administration in both the FX-11 + IACS-010759 administration group and the exosome-loaded FX-11 + exosome-loaded IACS-010759 administration group, No significant weight loss was observed, and rather an increase in body weight was observed, indicating a low possibility of toxicity due to the drug (FIG. 29).

Claims (16)

A pharmaceutical composition for preventing or treating cancer containing a glycolysis inhibitor and a mitochondrial function inhibitor.
The method of claim 1, wherein the glycolysis inhibitor is dichloroacetic acid (DCA), FX-11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propyl-naphthalene-1-carboxylic acid), A composition that is at least one selected from the group consisting of 2-DG (2-Deoxy-d-glucose), iodoacetate, and 3-bromopyruvate.
The method of claim 1, wherein the mitochondrial function inhibitor is metformin, IACS-010759 (4-(methylsulfonyl)-1-[3-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]- 1,2,4-oxadiazol-5-yl]-1H-1,2,4-triazol-1-yl]methyl]phenyl]-piperidine), rotenone, Piericidin A, A composition comprising at least one selected from the group consisting of Streptozotocin and Antimycin A.
The composition of claim 1, wherein the glycolysis inhibitor is administered at 1 to 200 mg/kg.
The composition of claim 1, wherein the mitochondrial function inhibitor is administered at 1 to 300 mg/kg.
The composition according to claim 1, wherein the glycolysis inhibitor and the mitochondrial function inhibitor are administered at a mass ratio of 60:1 to 1:5.
The composition according to claim 1, wherein the composition inhibits ATP production in cancer cells.
The composition of claim 1, wherein the intracellular or extracellular environment of the cancer is a glucose-deficient environment.
The method according to claim 1, wherein the composition inhibits PI3K/Akt/mTORC1 signaling in cancer cells.
The composition according to claim 1, wherein the composition enhances the expression of REDD1 (regulated in development and DNA damage responses 1) protein in cancer cells.
The method of claim 1, wherein the composition is selected from the group consisting of cyclin D1, Ki-67, p-Akt, mTOR, proliferating cell nuclear antigen, and mitochondrial respiratory complex I in cancer cells. A composition that inhibits the expression of one or more proteins.
The composition according to claim 1, wherein the glycolysis inhibitor and the mitochondrial function inhibitor are loaded on extracellular endoplasmic reticulum.
The composition according to claim 12, wherein the glycolysis inhibitor and the mitochondrial function inhibitor are each loaded into separate extracellular vesicles or simultaneously loaded into one extracellular vesicle.
The composition of claim 12, wherein the extracellular vesicles are one or more selected from the group consisting of exosomes, microvesicles, ectosomes, and apoptotic bodies.
The method of claim 1, wherein the cancer is glioblastoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, and ureteral cancer. , a composition that is one or more selected from the group consisting of bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.
Health functional food for preventing or improving cancer containing glycolysis inhibitors and mitochondrial function inhibitors.