WO2021100897A1 - Pharmaceutical composition for preventing or treating cancer, comprising biguanide-based compound and ferrocene or ferrocene derivative as active ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a biguanide-based compound and ferrocene or a ferrocene derivative as active ingredients. More specifically, it was confirmed that a significantly higher synergistic anticancer activity was exhibited when a biguanide-based compound and ferrocene or a ferrocene derivative were administered in combination or in admixture, as compared to when a biguanide-based compound, ferrocene, or a ferrocene derivative was administered alone. Thus, the pharmaceutical composition comprising the biguanide-based compound and the ferrocene or ferrocene derivative according to the present invention may be effectively used for preventing or treating cancer.

Description

Pharmaceutical composition for preventing or treating cancer containing biguanide series and ferrocene or ferrocene derivatives as active ingredients

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing a biguanide series, and ferrocene or ferrocene derivatives as an active ingredient.

Cancer is a disease caused by abnormal growth of uncontrolled cells that can spread in contact with tissues or other parts of the body, and cancer cells can form solid tumors in which cancer cells are clustered together or exist as dispersed cells as in leukemia. have. Normal cells differentiate until maturity and then replace damaged or dead cells as necessary, but cancer cells constantly differentiate and eventually push nearby cells and spread to other parts, which is called malignant. Malignant tumor cells spread through the bloodstream or lymphatic system to other parts of the body, where they multiply and form new tumors.

Cancer continues to seriously threaten human health worldwide, despite the development of various treatment methods. Currently, major cancer treatments include surgical surgery, radiation therapy, hormone therapy, and chemotherapy, among which chemotherapy is a method of directly treating cancer or alleviating symptoms using one or more anticancer agents.

Traditional chemotherapeutic agents interfere with cancer cell division and metabolism, or inhibit the biosynthesis of nucleic acids or proteins, thereby exhibiting cytotoxicity to cancer cells. However, these chemotherapeutic agents have a problem in that cancer cells have resistance to anticancer agents and have serious side effects such as toxicity to normal tissues. In particular, substances used as existing anticancer agents affect cancer cells, but they are also toxic to normal cells, causing various side effects in many cases. Therefore, there is a need for an anticancer therapeutic agent that does not exert toxicity to normal cells and exhibits excellent toxicity selectively only to cancer cells and has excellent anticancer activity.

Targeted anticancer agents are one of those that have emerged to solve the side effects and problems of conventional chemotherapy drugs. Since target anticancer agents attack specific targets that are expressed only in cancer cells, it is expected that the treatment effect can be improved while reducing side effects compared to conventional chemotherapy drugs. For example, imatinib (Gleevec) that attacks BCR-ABL, a gene specifically expressed in chronic myelogenous leukemia, gefitinib, erlotinib, afatinib, which are used for the treatment of lung cancer with epithelial growth factor receptor (EGFR) mutations. , Crizotinib used for the treatment of ALK gene mutation lung cancer, trastzumab used for HER2-positive breast cancer and gastric cancer, and Rituximab for treating CD20-positive lymphoma are representative anticancer drugs. However, in the case of targeted anticancer drugs, there is a limitation that the therapeutic effect appears only when a specific therapeutic target is expressed. That is, EGFR inhibitors are effective only for lung cancer with EGFR mutations, and are not effective for lung cancers in which the ALK gene is positive. In addition, there is a problem in that resistance to target therapy occurs after a certain period of time. This is because even if the targeted therapy blocks one cancer cell proliferation signal, the cancer cells find another signal pathway and continue cell proliferation.

Immuno-cancer drugs are intended to solve the problems of these chemo-anticancer agents and target anti-cancer agents. Immune cells attack when abnormal cells appear, and cancer cells attack these immune cells and weaken the function of the immune cells, creating an environment in which cancer cells grow well. Immuno-anti-cancer drugs block the path of cancer cells to attack immune cells, or make immune cells themselves stronger so that immune cells can kill cancer cells. Kitruda (ingredient name pembrolizumab) of multinational pharmaceutical company MSD is an immune checkpoint inhibitor that blocks the point where cancer cells attack immune cells, and Green Cross Cell's immune cell therapy Imuncell LC is a liver cancer treatment. Immunotherapy is a newly started method and is still in the development stage.

On the other hand, metabolic anticancer drugs make use of the difference in metabolic processes between cancer cells and normal cells to make normal cells grow and inhibit the proliferation of cancer cells by metabolic components that cancer cells cannot use. Because the metabolic method of cancer cells does not change, metabolic anticancer drugs are less affected even when genetic mutations occur, so there is less problem of drug resistance that occurs in the existing cancer treatment process. Metabolic anticancer drugs targeting leukemia in 2017 were approved for use in the United States, and additional approvals for other cancers such as breast cancer are expected in 2018.

Metformin, Phenformin, Buformin, or Biguanide is a drug of the same Biguanide family, which inhibits the production of sugar in the liver and is used for our use in peripheral blood vessels. It is still widely used as a facilitating type 2 diabetes treatment. Metformin, phenformin, buformin, or biguanide activates AMPK (AMP protein kinase), a key enzyme in metabolic regulation, inhibits the synthesis of proteins, fat lipids, and glycogen and promotes degradation, and mTOR (mammalian target of rapamycin) is activated. ) And inhibits the production of insulin, IGF1, leptin, and adiponectin. By activating AMPK, metabolism of cancer cells is inhibited and cell division is inhibited. In addition, AMPK activation directly inhibits mTOR and inhibits protein synthesis, thereby inhibiting the proliferation of cancer cells. In particular, metformin has been reported to inhibit the growth of cancer cells by inhibiting the expression of angiogenesis promoting factors. Due to these anticancer mechanisms, metformin and phenformin have been attempted to be used alone or in combination with other anticancer drugs in clinical trials of various types of cancer, but their therapeutic effects have been different, and have not yet been approved as anticancer drugs due to various problems. Is not in a state.

Ferrocene derivatives are compounds made by modifying side chains in the basic structure of ferrocene, and various structures have been synthesized to study anticancer effects. Ferrocene, acetylferrocene, diacetylferrocene, bromohexylferrocene, dimethyl aminomethylferrocene, aminoferrocene, ferrocifen, ferro The anticancer effects of ferroquine, etc. were reviewed. Among them, aminoferrocene, ferrocifene, and ferroquine were reported to have anticancer effects. Ferroquine was developed as a malaria treatment and clinical trials were conducted. In 2017, it was first reported that it has anticancer effects on prostate cancer in cell and animal experiments.

As a result of researching to develop excellent substances with anticancer effect, the present inventors have revealed that the combination of biguanide series and ferrocenc derivatives with anticancer effect shows a remarkable synergy in anticancer effect, thereby creating a new anticancer agent. Came to invent.

[Prior technical literature]

[Patent Literature]

US 2014-0113930 A1 (2014.4.24.)

US 2012-0270851 A1 (2012.10.25.)

US 2012-0258945 A1 (2012.10.11.)

[Non-patent literature]

Lopez, J.; Tait, S.W.G. Mitochondrial apoptosis: Killing cancer using the enemy within. Br. J. Cancer 2015, 112, 957-962.

Yip, K.W.; Reed, J.C. BCL-2 family proteins and cancer. Oncogene 2008, 77, 6398-6406.

Elkholi, R.; Renault, T. T.; Serasinghe, M.N.; Chipuk, J.E. Putting the pieces together: How is the mitochondrial pathway of apoptosis regulated in cancer and chemotherapy? Cancer Metab. 2014, 2, 16.

Fiocchetti, M.; Nuzzo, M. T.; Totta, P.; Acconcia, F.; Ascenzi, P.; Marino, M. Neuroglobin, a pro-survival player in estrogen receptor-positive cancer cells. Cell Death Dis. 2014, 5, e1449.

J. Xu, and W. Mao Overview of Research and Development for Anticancer Drugs, Journal of Cancer Therapy, 2016, 7, 762-772.

Makkouk, A. and Weiner, G.J. (2015) Cancer Immunotherapy and Breaking Immune Tolerance: New Approaches to an Old Challenge. Cancer Research, 75, 5-10.

Faubert B, Vincent EE, Poffenberger MC, Jones RG. The AMPactivated protein kinase (AMPK) and cancer: many faces of a metabolic regulator. Cancer Lett 2015;356:165-70.

Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011;12(1):21-35.

Dallaglio K, Bruno A, Cantelmo AR, Esposito AR, Ruggiero L, Orecchioni S, et al. Paradoxic effects of metformin on endothelial cells and angiogenesis. Carcinogenesis 2014;35:1055-66.

Chekhun VF, Mokhir A, Daum S, Todor IN, Lukianova NY, Shvets YV, Burlaka A. Pharmacological effect of aminoferrocene in mice with L1210 leukemia. Exp Oncol. 2015 Jun;37(2):120-5.

Bruyere C, Mathieu V, Vessieres A, Pigeon P, Top S, Jaouen G, Kiss R. Ferrocifen derivatives that induce senescence in cancer cells: selected examples.. J Inorg Biochem. 2014 Dec;141:144-151.

J Immunol Methods 65, 55 (1983).

J Neurochem 69, 581 (1997).

Substances used as existing anticancer agents affect cancer cells, but are toxic to normal cells, such as skin, mucous membranes, and blood cells that divide rapidly, causing various side effects such as hair loss, diarrhea, and leukopenia. There are many. In addition, in many cancer cells, the expression of antiapoptotic proteins such as BCL-2 is increased, or the expression of proapoptotic proteins such as BAX is suppressed. apoptosis) is often lacking. In addition, in cancer cells, the expression of caspases is low or mutations in the caspase gene appear. In some cases, cancer cells may inhibit apoptosis by inhibiting mitochondrial outer membrane permeabilization (MOMP). Since apoptosis does not occur in many cancer cells, there is a problem that the therapeutic effects of many anticancer drugs that induce apoptosis do not appear.

Therefore, there is an urgent need for an anticancer therapeutic agent having excellent anticancer activity while exhibiting excellent toxicity selectively only to cancer cells without toxic to normal cells.

The present invention is a mixed or combined formulation for use in the prevention or treatment of cancer, comprising: a first component comprising a biguanide series or a pharmaceutically acceptable salt thereof; And ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives) or a pharmaceutical composition for the prevention or treatment of cancer containing as an active ingredient a second component comprising a pharmaceutically acceptable salt thereof does not show toxicity to normal cells , It was found that it exhibits remarkably excellent anticancer effects for cancer cells, and the above problems were solved by providing a pharmaceutical composition containing the first and second ingredients as active ingredients.

In one aspect of the present invention, the biguanide series or a pharmaceutically acceptable salt thereof is metformin or a pharmaceutically acceptable salt thereof, phenformin or a pharmaceutically acceptable salt thereof, bufor It may be selected from the group consisting of buformin or a pharmaceutically acceptable salt thereof, and biguanide or a pharmaceutically acceptable salt thereof.

In one aspect of the present invention, the ferrocene derivative is acetylferrocene, bromohexylferrocene, ferroquine, 1,1'-diacetylferrocene (1,1'-diacetylferrocene), aminoferrocene Consisting of (aminoferrocene), dimethyleaminomethyl-ferrocene, benzoylferrocene, ferrocerone, chloroquine, ferrocifen, and ferrocifen family Can be selected from a group.

In one aspect of the present invention, the biguanide series or a pharmaceutically acceptable salt thereof; And ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives) or a pharmaceutically acceptable salt thereof may be blended in a blending ratio of 1 to 1000 parts by weight: 0.00001 to 10 parts by weight.

In one aspect of the present invention, the cancer is lung cancer, breast cancer, prostate cancer, bowel cancer, pancreatic cancer, stomach cancer, liver cancer, blood cancer, bone cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer. , Colon cancer, fallopian tube cancer, uterine cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, bronchial cancer, myeloma, colon cancer, colon cancer, anal cancer, stellate cancer Cytoma, leukemia, lymphoma, testicular cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, oral cancer, rectal cancer, throat cancer, bladder cancer, head and neck cancer, ovarian cancer, basal cell cancer, squamous cell carcinoma, oral squamous cell carcinoma, It may be selected from the group consisting of colorectal cancer, glioblastoma, endometrial cancer and malignant glioma, specifically lung cancer, breast cancer, prostate cancer, bowel cancer, or pancreatic cancer.

In another aspect of the present invention, a formulation comprising the biguanide series or a pharmaceutically acceptable salt thereof; And ferrocene, ferrocene derivatives (ferrocene derivatives) or a pharmaceutically acceptable salt thereof, comprising a formulation comprising a combination kit for cancer prevention or treatment is also provided.

The formulation may be formulated in a formulation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, solutions, suspensions, liquid solutions, emulsions, syrups, suppositories, vaginal tablets, and pills. It is not limited thereto, and may be formulated in an appropriate formulation as needed.

In another aspect of the present invention, a first component comprising a pharmaceutically effective amount of the biguanide series or a pharmaceutically acceptable salt thereof; And there is also provided a method for preventing, improving, or treating cancer comprising administering to a subject a second component including ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof in combination or in combination.

In another aspect of the present invention, a first component comprising a biguanide series or a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for preventing or treating cancer; And a second component including ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect of the present invention, a first component comprising a biguanide series or a pharmaceutically acceptable salt thereof for use as a health food for preventing or improving cancer; And a second component including ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof as an active ingredient.

The biguanide series according to the present invention and ferrocene or ferrocene derivatives each have a weak anticancer effect when treated alone. However, when mixed or combined treatment, a remarkably high anticancer effect appears in various cancer types. The pharmaceutical composition can be usefully used for preventing or treating cancer. In addition, since it does not show toxicity to normal cells at an effective concentration, side effects are greatly reduced, and an excellent anticancer agent can be provided.

Figure 1 shows the degree of growth inhibition (% growth inhibition) when 5mM metformin, 0.0001-10μM ferroquine, metformin + ferroquine were treated with MCF-7, a breast cancer cell, for 24 and 72 hours. It is a degree. As a control, WISH normal epithelial cells (primary epithelial cells) were used.

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine were used in combination for 24 hours (percent)

③: The degree of growth inhibition after 72 hours treatment (percent)

Figure 2 shows the degree of growth inhibition (% growth inhibition) when 5mM metformin, 0.01-1 μM ferroquine, metformin + ferroquine were treated in breast cancer cells BT474, MDA-MB-231 for 24 hours. Is a diagram showing.

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine are co-treated (percent)

Figure 3 shows the degree of growth inhibition (% growth inhibition) when 5mM metformin, 0.01-1 μM ferroquine, metformin + ferroquine were treated on breast cancer cells SKBr-3 and ZR-75 for 24 hours. It is a diagram shown.

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine are co-treated (percent)

Figure 4 is when 5mM metformin (metformin), 0.01-1 μM ferroquine (ferroquine), metformin + ferroquine (ferroquine) was treated in intestinal cancer cells, HCT116 (colon cancer), lung cancer cells (lung cancer) HCC-1195 for 24 hours It is a diagram showing the degree of growth inhibition (% growth inhibition).

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine are co-treated (percent)

5 shows the degree of growth inhibition when 5mM metformin, 0.01-1 μM ferroquine, metformin + ferroquine were treated with pancreas cancer MIA PACa-2 and AsPC-1 for 24 hours ( % growth inhibition).

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine are co-treated (percent)

6 shows the degree of growth inhibition (% growth inhibition) when 5mM metformin, 0.01-1 μM ferroquine, metformin + ferroquine were treated with prostate cancer LNCaP and DU145 for 24 hours. It is a diagram shown.

①: The degree of inhibition of growth of cancer cells when only metformin or ferroquine was treated (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferroquine are co-treated (percent)

Figure 7 shows the degree of growth inhibition when 5mM metformin, 0.01-1 μM ferrocene derivatives, metformin + ferrocene derivatives were treated with MCF-7 in breast cancer for 24 hours. ) Is a diagram showing. Ferrocene derivatives include ferrocene, acetylferrocene, 1,1'-diacetylferrocene, dimethylaminomethyl-ferrocene, and bromohexylferrocene. (bromohexylferrocene), ferroquine.

①: The degree of inhibition of growth of cancer cells when treated with metformin or ferrocene derivatives alone (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferrocene derivatives are co-treated (percent)

FIG. 8 shows the degree of growth inhibition when 5mM metformin, 0.01-1 μM ferrocene derivatives, metformin + ferrocene derivatives were treated on breast cancer MCF-7 for 24-72 hours (% growth inhibition). As a control, WISH normal epithelial cells (primary epithelial cells) were used. Ferrocene derivatives include ferrocene, acetylferrocene, 1,1'-diacetylferrocene, dimethylaminomethyl-ferrocene, and bromohexylferrocene. (bromohexylferrocene), ferroquine.

①: The degree of inhibition of growth of cancer cells when treated with metformin or ferrocene derivatives alone for 24 hours (percent);

②: The degree of inhibition of growth of cancer cells when metformin and ferrocene derivatives were used in combination for 24 hours (percent)

③: About growth inhibition after 48 hours treatment (percent)

④: The degree of growth inhibition after 72 hours treatment (percent)

9 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated in AsPC-1, a pancreatic cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

10 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated with DU145, a prostate cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

11 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated with MBA-MB-231, a breast cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

12 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated in HCT116, an intestinal cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

13 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated with HCC-1195, a lung cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

14 is a diagram showing the results when 5mM metformin, 0.1-10 μM aminoferrocene, metformin + aminoferrocene were treated on WISH cells as a control for 72 hours

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 15 is a diagram showing the results when 5 mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated in AsPC-1, a pancreatic cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 16 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated on DU145, a prostate cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 17 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated with MBA-MB-231, which is a breast cancer cell, for 72 hours. to be

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 18 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated on HCT116, which is an intestinal cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 19 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated with HCC-1195, a lung cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 20 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen A, metformin + Ferrocifen A were treated on WISH cells as a control for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 21 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated in AsPC-1, a pancreatic cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 22 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated with DU145, a prostate cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 23 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated with MBA-MB-231, which is a breast cancer cell, for 72 hours. to be

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 24 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated with HCT116, which is an intestinal cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

25 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated with HCC-1195, which is a lung cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 26 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen B, metformin + Ferrocifen B were treated on WISH cells as a control for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 27 is a diagram showing the results when 5 mM metformin, 0.1-10 μM Ferrocifen C, and metformin + Ferrocifen C were treated in AsPC-1, a pancreatic cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

Figure 28 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen C, metformin + Ferrocifen C were treated with DU145, a prostate cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

29 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen C, metformin + Ferrocifen C were treated with MBA-MB-231, which is a breast cancer cell, for 72 hours. to be

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 30 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen C, metformin + Ferrocifen B were treated on HCT116, which is an intestinal cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 31 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen C, metformin + Ferrocifen C were treated with HCC-1195, a lung cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 32 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen C, metformin + Ferrocifen C were treated on WISH cells as a control for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

Figure 33 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated with pancreatic cancer cells AsPC-1 for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 34 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated on DU145, a prostate cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 35 is a diagram showing the results when 5 mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated with MBA-MB-231, which is a breast cancer cell, for 72 hours. to be

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 36 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated with HCT116, which is an intestinal cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

37 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated with HCC-1195, a lung cancer cell, for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

FIG. 38 is a diagram showing the results when 5mM metformin, 0.1-10 μM Ferrocifen D, metformin + Ferrocifen D were treated on WISH cells as a control for 72 hours.

A: OD(Optical Density)

B:% growth inhibition

C: Survival rate (% Survival)

Figure 39 shows 2μM doxorubicin, 5μM ferroquine, 20μM metformin, 20μM phenformin, 20μM buformin, 20μM metformin + 5μM ferroquine, 20μM phenformin + It is a diagram showing the degree of growth inhibition (% growth inhibition) when 5 μM ferroquine and 20 μM buformin + 5 μM ferroquine were treated in AsPC-1, a pancreatic cancer cell, for 72 hours.

Hereinafter, the present invention will be described in more detail.

The present invention is a mixed or combined formulation for use in the prevention or treatment of cancer, comprising: a first component including biguanide or a pharmaceutically acceptable salt thereof; And ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives) or a second component containing a pharmaceutically acceptable salt thereof as an active ingredient, to a pharmaceutical composition for the prevention or treatment of cancer.

The present invention also provides metformin or a pharmaceutically acceptable salt thereof, phenformin or a pharmaceutically acceptable salt thereof, buformin or a pharmaceutically acceptable salt thereof as the biguanide series or a pharmaceutically acceptable salt thereof. , A first component which is a biguanide or a pharmaceutically acceptable salt thereof; And it relates to a method for preventing or treating cancer comprising administering ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

In the present invention, metformin is a compound of Formula 1:

[Formula 1]

.

.

In the present invention, phenformin is a compound of Formula 2:

[Formula 2]

.

.

In the present invention, buformin is a compound of the following formula 3:

[Formula 3]

.

.

In the present invention, biguanide is a compound of the following formula (4):

[Formula 4]

In the present invention, ferrocene is a compound of formula (5):

[Formula 5]

.

.

The ferrocene derivatives in the present invention are compounds of the following formulas 6 to 15:

[Chemical Formula 6] Acetylferrocene

,

,

[Chemical Formula 7] Bromohexylferrocene

,

,

[Chemical Formula 8] Ferroquine

,

,

[Chemical Formula 9] 1,1-diacetylferrocene

,

,

[Chemical Formula 10] Aminoferrocene

,

,

[Chemical Formula 11] Dimethylaminomethylferrocene

,

,

[Chemical Formula 12] Benzoylferrocene

,

,

[Chemical Formula 13] Ferrocerone

,

,

[Chemical Formula 14] Chloroquine

및

And

[Chemical Formula 15] Ferrocifene

.

.

In addition, the ferrocifene-based compounds in the present invention are ferrocene derivatives, and are compounds of the following formulas 16 to 19:

[Formula 16]

[In the above formula, R ₁ and R ₂ is H, OH, NH ₂ or O(CH ₂ ) _n N(CH ₃ ) ₂ , where n= 2 to 5, or 8.]

[Formula 17]

[In the above formula, R is NHOH or NH ₂ .]

[Formula 18]

[In the above formula, R ₁ and R ₂ is H, OH or NH ₂ .]

[Formula 19]

[In the above formula, R is NHOH or NH ₂ .]

In one aspect of the present invention, the concentration of the biguanide series or a pharmaceutically acceptable salt thereof may be 0.1mM to 100mM, and the concentration of ferrocene or ferrocene derivatives may be 0.001μM to 1mM.

In one aspect of the present invention, the content of the biguanide series or a pharmaceutically acceptable salt thereof, and ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof in the pharmaceutical of the present invention may be appropriately selected depending on the form of the formulation, etc. . For example, in the medicament of the present invention containing the biguanide family, and ferrocene, or a ferrocene derivative in a single formulation, the content of the biguanide family is generally about 0.01 to about 99.99 wt%, preferably about the total formulation. 0.1 to about 50 wt%, and the content of ferrocene or ferrocene derivative is generally about 0.01 to about 99.99 wt%, preferably about 0.1 to about 50 wt%, based on the total formulation.

In one aspect of the present invention, the content ratio of the biguanide series and ferrocene or ferrocene derivatives in the pharmaceuticals of the present invention may be 1 to 1000 parts by weight: 0.00001 to 10 parts by weight.

In one aspect of the present invention, the content of the additive such as a carrier in the pharmaceutical of the present invention varies depending on the form of the formulation, but may be about 1 to about 99.00 wt%, specifically about 10 to about 90 wt% with respect to the entire formulation .

In one aspect of the present invention, when the biguanide series and ferrocene, or ferrocene derivatives are separately formulated, the content of the biguanide series in the biguanide series-containing formulation is generally about 0.01 to about 100 wt%, specifically about 0.1 to about 90 wt%, and the content of ferrocene or ferrocene derivative in the ferrocene or ferrocene derivative-containing formulation is generally about 0.01 to about 100 wt%, specifically about 0.1 to about 0.1 to the total formulation. It is about 90 wt%.

In one aspect of the present invention, the content of additives such as carriers is as mentioned above.

In one aspect of the present invention, metformin or a pharmaceutically acceptable salt thereof, phenformin or a pharmaceutically acceptable salt of phenformin, buformin or a pharmaceutically acceptable salt of buformin, which is a biguanide family, When a pharmaceutically acceptable salt of biguanide or biguanide and/or a pharmaceutically acceptable salt of ferrocene, or a ferrocene derivative is used, the content in the formulation and the content ratio thereof are the biguanide series, and ferrocene. , Or within the same range as indicated above for the ferrocene derivative.

In one aspect of the present invention, the cancer is lung cancer, breast cancer, prostate cancer, bowel cancer, pancreatic cancer, stomach cancer, liver cancer, blood cancer, bone cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer. , Colon cancer, fallopian tube cancer, uterine cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, bronchial cancer, myeloma, colon cancer, colon cancer, anal cancer, stellate cancer Cytoma, leukemia, lymphoma, testicular cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, oral cancer, rectal cancer, throat cancer, bladder cancer, head and neck cancer, ovarian cancer, basal cell cancer, squamous cell carcinoma, oral squamous cell carcinoma, It may be selected from the group consisting of colorectal cancer, glioblastoma, endometrial cancer and malignant glioma, specifically lung cancer, breast cancer, prostate cancer, bowel cancer, or pancreatic cancer.

In another aspect of the present invention, a formulation comprising the biguanide series, or a pharmaceutically acceptable salt thereof; And ferrocene, ferrocene derivatives (ferrocene derivatives) or a pharmaceutically acceptable salt thereof, comprising a formulation comprising a combination kit for cancer prevention or treatment is also provided.

The formulation may be formulated in a formulation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, solutions, suspensions, liquid solutions, emulsions, syrups, suppositories, vaginal tablets, and pills. It is not limited thereto, and may be formulated in an appropriate formulation as needed.

As used herein, the term "prevention" refers to any action that suppresses the onset or delays the onset by administration of the composition. In the present invention, "improvement" or "treatment" refers to any action in which the symptoms of the disease are improved or advantageously changed by administration of the composition.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. Can be administered. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

It can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butine-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving the compound represented by Formula 1 to Formula 19 in an excessive amount of an aqueous acid solution, and dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, acetone. Alternatively, it can be prepared by precipitation using acetonitrile. In addition, the mixture may be dried by evaporating a solvent or an excess of acid, or may be prepared by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

When formulating the composition, it is prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and such solid preparations include at least one excipient in one or more compounds represented by the formulas 1 to 19 of the present invention. For example, it is prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, or syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives are included. I can.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like may be used.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, and drug activity of the patient. , Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as a combined individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and body weight of the patient, and generally 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per 1 kg of body weight is administered daily or every other day. Alternatively, it can be administered in 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

In one aspect of the present invention, a first component comprising a biguanide series or a pharmaceutically acceptable salt thereof; And ferrocene (ferrocene), ferrocene derivatives (ferrocene derivatives) or a second component comprising a pharmaceutically acceptable salt thereof is administered in combination or combination to a subject in need of cancer treatment. Various cancers, including the cancer diseases mentioned above, can be treated by the method according to the present invention.

In the present invention, the subject is a mammal in need of cancer treatment. Typically the subject is a human cancer patient. In one aspect of the invention, the subject is a non-human mammal such as a non-human primate, an animal used in the model system (e.g., mice and rats used for screening, characterizing and evaluating medicaments), and other mammals. , For example, it may be an ape animal such as a rabbit, guinea pig, hamster, dog, cat, chimpanzee, gorilla, and monkey.

In one aspect of the present invention, the pharmaceutical composition may be used alone or in combination with surgery, hormone therapy, drug therapy, and biological response modifiers for the treatment of cancer patients.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for aiding understanding of the present invention and are not intended to limit the scope of the present invention.

<Example 1> Confirmation of anticancer activity of metformin and ferrocene derivatives

<1-1> MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) for growth inhibition by drugs of breast cancer cell line MCF-7 (breast cancer cell) or other cancer cell line bromide) assay. Cancer cell lines were cultured in a 100mm culture dish using DMEM-10% FBS at 5% CO ₂ , 37°C, inoculated with 20% confluence in each well of a 96 well plate, and cultured for 24 hours. Metformin was treated at a concentration of 5 mM and the ferrocene derivative was treated at a concentration of 0.001-10 μM, and incubated for 24-72 hours in a _{CO 2 incubator.} Remove the culture solution from each well, add 100 μl of a new culture solution, add 10 μl of 12 mM MTT stock solution (5 mg MTT/PBS), incubate at 37°C for 2 hours, and then stop the reaction solution, SDS-HCl solution (1 g SDS/10 ml 0.01 M HCl) 100 μl was added and incubated at 37° C. for 4 hours, and OD was measured at 570 nM using a microplate leader. The% growth inhibition was calculated by comparing the OD of the untreated cells. Other cancer cell lines: BT474 cells, MDA-MB-231 cells, SKBr-3 cells and ZR-75 cells (breast cancer), HCT116 cells (colon cancer), HCC-1195 cells (lung cancer), MIA PaCa-2 cells and AsPC -1 cells (pancreas cancer), LNCaP cells and DU145 cells (prostate cancer) were cultured and analyzed in the same way. As a normal cell control, human normal epithelial cells (WISH) were used.

As a result, when metformin was treated alone, the degree of growth inhibition was 10-30% in breast cancer cells and various cancer cell lines at a concentration of 5 mM, and when treated with ferroquine alone at a concentration of 0.01-1 μM, a breast cancer cell line (BT474 cells, MDA-MB-231 cells, SKBr-3 cells, ZR-75 cells) showed 10-40% growth inhibition.

In breast cancer cell line MCF-7, when ferroquine alone was treated at a concentration of 0.01-1 μM, it showed 40-70% growth inhibition. Other ferrocene derivatives (ferrocene, acetylferrocene, 1,1'-diacetylferrocene, dimethylaminomethyl-ferrocene, bromohexylferrocene) When silver alone was treated at a concentration of 0.01-1 μM, the degree of growth inhibition of the breast cancer cell line MCF-7 was 2-15%.

63-82% growth inhibition in breast cancer cell lines (BT474 cells, MDA-MB-231 cells, SKBr-3 cells, ZR-75 cells) when 5 mM metformin and 0.01-1 μM ferroquine were co-treated Showed significantly synergistically higher cancer cell growth inhibitory activity than metformin or ferroquine alone treatment.

In addition, HCT116 cells (intestinal cancer cells), HCC-1195 cells (lung cancer cells), MIA PaCa-2 cells and AsPC-1 cells (pancreatic cancer cells), LNCaP cells and DU145 cells (prostate cancer cells) contain 5mM metformin and 0.01- Even when 1 μM ferroquine was treated in combination, it showed 30-70% growth inhibition, and thus significantly synergistically higher cancer cell growth inhibitory activity than metformin or ferroquine alone treatment.

In addition, 5 mM metformin and 0.01-1 μM of ferrocene derivatives (ferrocene, acetylferrocene, 1,1'-diacetylferrocene, 1,1'-diacetylferrocene), dimethyl Even when combined treatment with dimethylaminomethyl-ferrocene showed a 30-60% growth inhibition, significantly synergistically higher cancer cell growth inhibitory activity than metformin or ferrocene derivative alone treatment.

When the breast cancer cells MCF-7 were treated with 5 mM metformin and 0.0001-10 μM ferroquine in combination, a synergistic effect was shown at low concentrations of 0.0001-0.01 μM, but 80-90% at concentrations above 0.1 μM. It showed a ceiling effect showing growth inhibition.

Relatively, when 5mM metformin and 0.01-1μM ferrocene derivatives were treated in combination with normal human epithelial cells, the growth inhibitory activity was very weak, 0-8%, so metformin and ferrocene derivatives were used in combination. It was shown that treatment markedly inhibited the growth of cancer cells, but had little effect on normal cells.

In conclusion, metformin and ferrocene derivatives showed synergistic results in cancer cell growth inhibitory activity in various types of cancer cells when co-treated, and in certain cancers, they showed a ceiling effect, and normal cell growth. Showed excellent results in cancer cell growth inhibitory activity while having little effect on.

<1-2> In the same manner as in Example <1-1>, cancer cell lines AsPC-1 (pancreas cancer), DU154 (prostate cancer), MBA-MB-231 (breast cancer), HCT116 (colon cancer) and HCC- 1195 (lung cancer) was treated with 5 mM metformin and/or 0.1-10 μM of ferrocene derivatives, amino ferrocene, and cultured, and then analyzed by MTT assay. WISH was used as a control for normal cells.

As a result, in the pancreatic cancer cell line AsPC-1, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, the OD value decreased, and showed a growth inhibition of 3-34% and a survival rate of 65-96%.

Relatively, when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, the OD value decreased in the pancreatic cancer cell line AsPC-1, and showed 61-93% growth inhibition and 6-38% survival rate, resulting in metformin or aminoferrocene alone. It showed higher cancer cell growth inhibitory activity and lower survival rate than treatment.

In addition, in the prostate cancer cell line DU145, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, the OD value decreased, and showed 5-22% growth inhibition and 77-94% survival rate.

Relatively, when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, the OD value decreased in the prostate cancer cell line DU145, and showed 51-93% growth inhibition and 6-48% survival rate, resulting in metformin or aminoferrocene treatment alone. It showed higher cancer cell growth inhibitory activity and lower survival rate.

In addition, in the breast cancer cell line MBA-MB-231, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, the OD value decreased, and showed 10-34% growth inhibition and 65-89% survival rate.

Relatively, when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, the OD value decreased in the breast cancer cell line MBA-MB-231, and showed a growth inhibition of 60-95% and a survival rate of 4-39%. It showed higher cancer cell growth inhibitory activity and lower survival rate than ferrocene treatment alone.

In addition, in the intestinal cancer cell line HCT116, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, the OD value decreased, showed growth inhibition of 8-33% and survival rate of 66-91%.

Relatively, when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, the OD value decreased in the intestinal cancer cell line HCT116, and showed 42-79% growth inhibition and 20-57% survival rate than metformin or aminoferrocene alone treatment. It showed high cancer cell growth inhibitory activity and low survival rate.

In addition, in the lung cancer cell line HCC-1195, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, the OD value decreased, and showed a growth inhibition of 4-27% and a survival rate of 72-95%.

Relatively, when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, the OD value decreased in the lung cancer cell line HCC-1195, and showed 43-81% growth inhibition and 18-56% survival rate, resulting in metformin or aminoferrocene alone. It showed higher cancer cell growth inhibitory activity and lower survival rate than treatment.

On the other hand, in normal cell WISH, when treated with aminoferrocene alone at a concentration of 0.1-10 μM, there was no significant change in OD value, and showed 2-10% growth inhibition and 89-97% survival rate. Even when 5 mM metformin and 0.1-10 μM concentration aminoferrocene were treated in combination, there was no significant change in the OD value, and it showed 2-9% growth inhibition and 90-97% survival rate when treated with metformin or ferrocene derivative aminoferrocene alone. It was shown that there was no significant difference between and.

In conclusion, the combined treatment of metformin and ferrocene derivatives had little effect on the growth of normal cells, and showed excellent results in cancer cell growth inhibitory activity in various types of cancer cells.

<Example 2> Confirmation of anticancer activity of metformin and ferrocifene derivatives

<2-1> In the same manner as in Example <1-1>, cancer cell lines AsPC-1 (pancreas cancer), DU154 (prostate cancer), MBA-MB-231 (breast cancer), HCT116 (colon cancer) and HCC- 1195 (lung cancer) was treated with 5 mM metformin and/or ferrocifen derivatives of 0.1-10 μM, ferrocifene A, B, C, and D, and cultured and analyzed by MTT assay. WISH was used as a control for normal cells.

As a result, in pancreatic cancer cell line AsPC-1, when ferrocifen A (Ferrocifen A; C ₂₇ H ₂₆ FeO ₂ ) alone was treated at a concentration of 0.1-10 μM, the OD value decreased, and growth inhibition of 13-72% and 27- It showed a survival rate of 86%. Other ferrocifene derivatives, ferrocifene B (C ₃₇ H _{48 FeN 2} O ₂ ), ferrocifene C (C ₂₆ H ₂₄ FeO) and _{ferrocifene D (C 31} H ₃₅ FeNO ₂ ) alone 0.1- When treated with 10 μM concentration, the OD value of the pancreatic cancer cell line AsPC-1 decreased, and growth inhibition of 16-75%, 4-18% and 10-20% and 24-83%, 81-95% and 79-89 respectively. % Survival rate.

Relatively, when 5 mM metformin and ferrocifene A at a concentration of 0.1-10 μM were co-treated, the OD value decreased in the pancreatic cancer cell line AsPC-1, and showed a growth inhibition of 40-90% and a survival rate of 10-50%. When 5mM metformin and other ferrocefene derivatives ferrocifene, ferrocifene C, and ferrocifene D were treated in combination, the OD value decreased, and growth inhibition of 43-93%, 55-80% and 53-85%, respectively. And 6-56%, 19-44%, and 14-46% survival rates were shown, showing higher cancer cell growth inhibitory activity and lower survival rates than metformin or ferrocifene derivatives alone.

In addition, in the prostate cancer cell line DU145, when ferrocifene A alone was treated at a concentration of 0.1-10 μM, the OD value decreased, and showed a growth inhibition of 15-70% and a survival rate of 29-84%. The OD value of the prostate cancer cell line DU145 decreased when the other ferrocifene derivatives, Ferrocifen B, Ferrocifen C, and Ferrocifene D, were treated at a concentration of 0.1-10 μM alone, 52-80% and 6- Growth inhibition of 18% and 5-15% and survival rates of 19-47%, 81-93% and 84-94% were shown.

Relatively, when 5 mM metformin and 0.1-10 μM concentration ferrocifene A were co-treated, the OD value decreased in the prostate cancer cell line DU145, and growth inhibition of 43-94% and survival rate of 5-57% were shown. When 5mM metformin and other ferrocifene derivatives, ferrocifene, ferrocifene C, and ferrocifene D, were treated in combination, the OD value decreased, and growth of 49-97%, 54-85% and 60-87%, respectively. Inhibition and survival rates of 2-50%, 14-45% and 12-39% were shown, showing higher cancer cell growth inhibitory activity and lower survival rates than metformin or ferrocifene derivatives alone.

In addition, in the breast cancer cell line MBA-MB-231, when ferrocifene A alone was treated at a concentration of 0.1-10 μM, the OD value decreased, and growth inhibition of 17-60% and survival rate of 39-82% were shown. When other ferrocifene derivatives, ferrocifene B, ferrocifene C, and ferrocifene D, were treated at a concentration of 0.1-10 μM alone, the OD value of the breast cancer cell line MBA-MB-231 decreased, 51-77%, respectively. , Growth inhibition of 12-24% and 0-21% and survival rates of 22-48%, 75-87% and 78-91%.

Relatively, when 5 mM metformin and 0.1-10 μM concentration ferrocifene A were treated in combination, the OD value decreased in the breast cancer cell line MBA-MB-231, and showed a growth inhibition of 40-97% and a survival rate of 2-59%. When 5mM metformin and other ferrocifene derivatives ferrocifene, ferrocifene C, and ferrocifene D were treated in combination, the OD value decreased, and growth of 43-94%, 56-91% and 62-85%, respectively. Inhibition and survival rates of 5-56%, 8-43% and 14-37% were shown, showing higher cancer cell growth inhibitory activity and lower survival rates than metformin or ferrocifene derivatives alone.

In addition, the intestinal cancer cell line HCT116 decreased the OD value when treated with ferrocifene A alone at a concentration of 0.1-10 μM, and showed a growth inhibition of 11-24% and a survival rate of 75-88%. When other ferrocifene derivatives, ferrocifene B, ferrocifene C, and ferrocifene D, were treated at a concentration of 0.1-10 μM alone, the OD value of the intestinal cancer cell line HCT116 decreased, 17-76% and 13-25, respectively. % And 11-19% growth inhibition and survival rates of 23-82%, 74-86% and 80-88%.

Relatively, when 5 mM metformin and 0.1-10 μM concentration ferrocifene A were treated in combination, the OD value was decreased in the intestinal cancer cell line HCT116, and growth inhibition of 56-86% and survival rate of 13-43% were shown. When 5mM metformin and other ferrocifene derivatives ferrocifene, ferrocifene C, and ferrocifene D were treated in combination, the OD value decreased, and growth of 44-93%, 59-82% and 63-93%, respectively. Inhibition and survival rates of 6-55%, 17-40% and 6-36% were shown, showing higher cancer cell growth inhibitory activity and lower survival rate than metformin or ferrocifene derivative alone treatment.

In addition, when the lung cancer cell line HCC-1195 was treated with ferrocifene A alone at a concentration of 0.1-10 μM, the OD value decreased, showed growth inhibition of 12-22% and survival rate of 77-87%. When other ferrocifene derivatives, ferrocifene B, ferrocifene C, and ferrocifene D, were treated at a concentration of 0.1-10 μM alone, the OD value of the lung cancer cell line HCC-1195 decreased, 8-21% and 10, respectively. Growth inhibition of -22% and 4-12% and survival rates of 78-91%, 77-89% and 87-95% were shown.

Relatively, when 5 mM metformin and 0.1-10 μM concentration ferrocifene A were co-treated, the OD value decreased in the lung cancer cell line HCC-1195, and showed a growth inhibition of 61-94% and a survival rate of 5-38%. When 5mM metformin and other ferrocifene derivatives, ferrocifene B, ferrocifene C, and ferrocifene D, were treated in combination, the OD value decreased, respectively, of 58-90%, 58-93%, and 57-82%. Growth inhibition and survival rates of 7-41%, 6-41% and 17-42% were shown, showing higher cancer cell growth inhibitory activity and lower survival rate than metformin or ferrocifene derivative alone treatment.

On the other hand, in normal cell WISH, there was no significant change in OD value when ferrocifene A to D alone was treated at a concentration of 0.1-10 μM, respectively, 3-13%, 4-15%, 2-5%, and 1-15 % Growth inhibition and survival rates of 86-96%, 84-95%, 94-97% and 84-98%.

There was no significant change in OD value even when 5mM metformin and other ferrocifene derivatives ferrocifene B, ferrocifene C, and ferrocifene D were co-treated, 4-11%, 4-14%, 2-5. %, and 5-15% of growth inhibition and 88-95%, 85-95%, 94-97%, and 84-94% of survival rates. Showed no.

In conclusion, the combination treatment of the biguanard family and the ferrocifene derivative had little effect on the growth of normal cells, and showed excellent results in the inhibition of cancer cell growth in various types of cancer cells.

<Example 3> Confirmation of anticancer activity of biguanide and ferrocene derivatives

<3-1> In the same manner as in Example <1-1>, in the cancer cell line AsPC-1 (pancreas cancer), 2 μM doxorubicin or 20 μM metformin, which is a biguanide family, 20 μM phenformin. , 20 μM buformin and/or 5 μM ferroquine were treated and cultured, and then analyzed by MTT assay.

As a result, when 20 μM of metformin, phenformin and buformin, or 5 μM ferroquine, which are the biguanide family, were treated alone, the survival rate was 80-120%.

Relatively, when combined treatment with 20 μM metformin, phenformin and buformin and 5 μM ferroquine, the pancreatic cancer cell line AsPC-1 showed a survival rate of 50-60%. It showed a low survival rate.

In conclusion, the combination treatment of the biguanard family and ferrocene derivatives showed excellent cancer cell reduction effect in cancer cells.

Claims (14)

As a mixed or combined preparation for use in the prevention or treatment of cancer, A first component comprising the biguanide family or a pharmaceutically acceptable salt thereof; And Containing as an active ingredient a second component including ferrocene, ferrocene derivatives or a pharmaceutically acceptable salt thereof, A pharmaceutical composition for the prevention or treatment of cancer. The method of claim 1, The biguanide series is selected from the group consisting of metformin, phenformin, buformin and biguanide, a pharmaceutical composition for the prevention or treatment of cancer. The method of claim 1, Ferrocene derivatives include acetylferrocene, bromohexylferrocene, ferroquine, 1,1'-diacetylferrocene, aminoferrocene, dimethylamino A pharmaceutical composition selected from the group consisting of methyleaminomethyl-ferrocene, benzylferrocene, ferrocerone, chloroquine, ferrocifen and ferrocifen family . The method of claim 3, The ferrocifene family is selected from the group consisting of compounds of Formulas 16 to 19 below: [Formula 16]

[In the above formula, R ₁ and R ₂ is H, OH, NH ₂ or O(CH ₂ ) _n N(CH ₃ ) ₂ , where n= 2 to 5, or 8.] [Formula 17]

[Wherein, R is NHOH or NH ₂ .] [Formula 18]

[In the above formula, R ₁ and R ₂ is H, OH or NH ₂ .] [Formula 19]

[Wherein, R is NHOH or NH ₂ .] The method of claim 1, Biguanide series or a pharmaceutically acceptable salt thereof; And Ferrocene, ferrocene derivatives or pharmaceutically acceptable salts thereof, 1 to 1000 parts by weight: a blending ratio of 0.00001 to 10 parts by weight, Pharmaceutical composition. The method of claim 1, Cancers include lung cancer, breast cancer, prostate cancer, bowel cancer, pancreatic cancer, stomach cancer, liver cancer, blood cancer, bone cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, Uterine cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, bronchial cancer, myeloma, colon cancer, colon cancer, anal cancer, astrocytoma, leukemia, lymphoma, testicular cancer , Sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, oral cancer, rectal cancer, throat cancer, bladder cancer, head and neck cancer, ovarian cancer, basal cell carcinoma, squamous cell carcinoma, oral squamous cell carcinoma, colorectal cancer, glioblastoma, uterus The pharmaceutical composition, which is selected from the group consisting of endometrial cancer and malignant glioma. The method of claim 1, The cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, bowel cancer and pancreatic cancer. The method of claim 1, The formulation is formulated in a formulation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, solutions, suspensions, solutions, emulsions, syrups, suppositories, vaginal tablets, and pills. Ever composition. Formulations containing the biguanide series or a pharmaceutically acceptable salt thereof; And Including a formulation comprising ferrocene, ferrocene derivatives or a pharmaceutically acceptable salt thereof, Combination kit for cancer prevention or treatment. The method of claim 9, The formulation is formulated in a formulation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, solutions, suspensions, solutions, emulsions, syrups, suppositories, vaginal tablets, and pills. Combination kit for prevention or treatment. A first component comprising a pharmaceutically effective amount of the biguanide family or a pharmaceutically acceptable salt thereof; And administering a second component including ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof to a subject in combination or combination. A first component comprising a pharmaceutically effective amount of the biguanide family or a pharmaceutically acceptable salt thereof; And administering a second component including ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof to a subject in combination or combination. A first component comprising a biguanide series or a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for preventing or treating cancer; And ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof, as an active ingredient. A first component comprising a biguanide series or a pharmaceutically acceptable salt thereof for use as a health food for preventing or improving cancer; And ferrocene, a ferrocene derivative, or a pharmaceutically acceptable salt thereof, as an active ingredient.

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