KR20160109926A - Pharmaceutical composition for preventing or treating brain cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a glucose uptake inhibitor and a biguanide-based compound.
The pharmaceutical composition according to the present invention can effectively prevent cancer cell proliferation and prevent and / or treat cancer.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer,

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a glucose uptake inhibitor and a biguanide-based compound.

Cancer is one of the most common causes of death worldwide. Approximately 10 million new cases occur each year, accounting for approximately 12% of all deaths and the third leading cause of death.

Among various types of cancer, brain cancer, especially in children, occurs more frequently than other cancers. Brain cancer refers to brain cancer, secondary brain cancer that has spread from cancer of the brain and other parts of the body, such as brain cancer that occurs in the brain surrounding the brain. Such brain cancer is often distinguished from cancer that occurs in other organs. First, the cancer that develops in the lungs, stomach, and breast is limited to one or two kinds of organs, and their properties are the same or similar. However, there are many different types of cancer in the brain. For example, glioblastoma, malignant glioma, lymph node, bladder, and metastatic tumor.

Among them, glioma, especially glioblastoma (GBM), is the most malignant and aggressive disease with very poor prognosis and a very fatal disease with an average survival time of less than 2 years after diagnosis. Since the boundary between brain cells and tumor cells is not clear, it is almost impossible to completely remove the GBM from the surgeon.

Despite advances in the field of cancer therapy, currently the leading treatment is surgery, radiation and chemotherapy. Chemotherapeutic approaches are used primarily to treat metastatic or particularly aggressive cancers. Most of the currently used cancer chemotherapeutic agents are cytotoxic agents. Cytotoxic agents work by damaging or killing fast-growing cells.

Ideal cytotoxic agents should have specificity for cancer and tumor cells, but not for normal cells. However, such ideal cytotoxic agents have not been found to date, and instead, only agents that specifically target rapidly differentiating cells (both tumor cells and normal cells) are being used. Therefore, substances that are cytotoxic to cancer cells are highly desirable, while only normal cells have a mild effect. In fact, many recent studies have focused on the development of alternative anticancer agents that can specifically inhibit the proliferation of tumor cells.

Therefore, although development of chemical therapeutic agents other than surgical treatment is urgent, effective therapeutic methods have not been developed yet, and research and development are required.

It is an object of the present invention to provide a pharmaceutical composition capable of effectively preventing cancer cell proliferation and preventing and / or treating cancer.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The inventors of the present invention have found that when 2-deoxyglucose (2-DG), a glucose uptake inhibitor, and metformin, a biguanide compound, are administered in combination, It is possible to effectively prevent the proliferation and prevent and / or treat cancer. Thus, the present invention has been completed.

Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an active ingredient of a glucose uptake inhibitor and a biguanide-based compound.

In one embodiment of the present invention, the glucose uptake inhibitor may be 2-deoxyglucose (2-DG).

In one embodiment of the invention, the biguanide-based compound may be metformin.

In one embodiment of the present invention, the glucose uptake inhibitor and the biguanide-based compound may be contained in a weight ratio of 1: 0.5 to 100, preferably 1: 0.5 to 20, or 1: 0.5 to 10, or 1: To 5 weight percent.

In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of cervical cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, blood cancer and liver cancer, The brain cancer may be a glioblastoma (GBM). Herein, the glioblastoma may include tumorsphere.

The pharmaceutical composition for preventing or treating cancer according to the present invention may further be used in combination with other anticancer drugs to thereby effectively treat cancer cells and thereby be used as a pharmaceutical composition for inhibiting cancer recurrence or metastasis have.

Meanwhile, the pharmaceutical composition of the present invention is not limited to these, but may be formulated in the form of an oral preparation, an external preparation, a suppository and a sterile injection solution in the form of powders, granules, capsules, tablets, Can be used. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, , Sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may be administered orally or parenterally depending on various factors including the activity, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated, And the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and is preferably from 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention may be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The pharmaceutical composition according to the present invention can be used for effectively preventing the proliferation of cancer cells to prevent and / or treat cancer.

1 (a) to 1 (c) show results of a Western blot assay according to an embodiment of the present invention.
2 (a) and 2 (b) show results of GBM sphere formation assay according to an embodiment of the present invention.
3 (a) and 3 (b) show results of a three-dimensional invasion assay according to an embodiment of the present invention.
Figure 4 shows the ¹⁸ F- fluoro Lodi oxy-glucose positron emission tomography ^{(18 F-fluorodeoxyglucose positron emission tomography} ) result according to an embodiment of the present invention.
FIG. 5 shows the results of gene expression microarray and class comparison according to an embodiment of the present invention.
FIGS. 6 (a) and 6 (b) show results of observation of gliomagenesis and tumor cell infiltration in an orthotopic xenograft animal model according to an embodiment of the present invention.
Figure 7 shows the survival analysis results of GBM tumor cell-transplanted mice according to one embodiment of the present invention.
FIG. 8 (a) is a photograph of a breast cancer cell and a glioblastoma cell according to an embodiment of the present invention, and FIG. 8 (b) shows a change in size of a tumor according to each treatment condition for an MCF7 cell line.
In the present invention, "control" in each drawing means a control group, "2DG" means 2-deoxyglucose, "M" or "MET" means metformin, "TMZ" means temozolomide (Temozolomide).

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Glioblastoma ( glioblastoma , CPD Separation of single cells from patients

Samples of patients with glioblastoma were collected from Yonsei University Severance Hospital. Separation of tumor sphere cells from the obtained samples was carried out by the following method.

First, the tumor was removed using a mechanical separation method and the cells were isolated within 2 hours. Specifically, the collected samples were separated and pulverized using a scalpel in Dulbecco's modified Eagle medium (nutrient mixture F-12 (DMEM / F-12; Mediatech, Manassas, Va., USA) Diameter nylon mesh cell filters (BD Falcon, Franklin Lakes, NJ, USA). The cell suspension that had passed through the filter was washed twice with DMEM / F-12 and then treated with B27 (1 ×; Invitrogen, San Diego, Calif., USA), 20 ng / ml basic fibroblast growth factor (Sigma, St. Louis, MO, USA), 20 g / ml epidermal growth factor (EGF) and 50 U / ml penicillin / 50 mg / ml streptomycin . In the following experiments, GSC11 and / or TS13-20 were used as GBM tumor cells obtained as described above.

Lentivirus  Vector transduction and expression

For cell counting, the previously isolated GBM tumor cells were cultured in complete medium, and GFP-expressing lentivirus supernatant was added to prepare GFP-GBM tumor cells. Thereafter, polybrene (Sigma) was added to a final concentration of 8 占 퐂 / ml, followed by culturing for 18 hours. After the lentivirus infection described above, cells were added to fresh growth medium and cultured according to the usual method. To remove the uninfected cells, the cells were treated with 1 mg / ml of puromycin (Life Technologies Korea, Seoul, Korea) to finally obtain stable GFP-GBM tumor cells.

Western Blot  Western blot assay

GBM tumor cells were lysed using lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, phosphate inhibitor cocktail solution, protease inhibitor cocktail solution) , Centrifuged at 14,000 rpm for 15 minutes, and the obtained supernatant was used as a cell extract for Western blot analysis.

For western blot analysis of the proteins, samples were separated by SDS-polyacrylamide gel electrophoresis at 120V and transferred to a nitrocellulose membrane. The membranes from which the protein was transferred were blocked with PBS / Tween solution containing 5% skim milk for 1 hour, then reacted with primary antibody (1: 1000 dilution) for 3 hours, then diluted with secondary antibody (1: 3000 dilution ) Was reacted for 1 hour. For visualization, the chemiluminescence method described in Amersham Bioscience was used.

Western blot analysis was performed using AMPK (mammalian target of rapamycin) -related proteins (Raptor, Rictor), nestin, CD133, Oct3 / 4, (EMT) -related markers, such as, for example, plasmids, Sox-2, Notch1 and Notch2, snail, Zeb1, beta -catenin and N-cadherin, The results are shown in Figs. 1 (a) to (c).

As shown in FIG. 1 (a), when the combination of 2-DG and metformin was administered, the expression of mTOR and its related protein (raptor) was suppressed. As shown in FIGS. Similarly, when 2-DG and metformin were co-administered, the down-regulation of the stem cell gene was observed.

It is shown that the combined administration of 2-DG and metformin has a greater effect on apoptosis than the administration of 2-DG and metformin, respectively.

CPD Tumor zone Formability  analysis( CPD  TS formation assay)

(100x, Gibco, Invitrogen Korea, Seoul, South Korea) was inoculated into DMEM / F-12 with 2% 1XB27, 20 ng / ml 0.02% bFGF, 20 ng / ml 0.02% EGF and 50 U / ml penicillin / 50 mg / ml streptomycin GSC11 and TS13-20 cells were cultured in the medium to obtain tumor cells, and the tumor cells were cultured for 3 weeks under different conditions. The shape and size of GBM tumor cells cultured using an inverted phase microscope (IX71 Inverted Microscope, Olympus, Tokyo, Japan) were observed, and the formation ability (%) of each tumor relative to the control group was evaluated. As shown in FIG. In addition, photographs of cells after culturing were taken with DP controller software (Olympus) using a digital camera (DP70 Digital Microscope Camera; Olympus), and the results are shown in FIG. 2 (b).

As shown in FIGS. 2 (a) and 2 (b), the tumor regeneration ability was remarkably decreased when the combination of 2-DG and metformin alone was administered. It is shown that the combined administration of 2-DG and metformin has a greater effect on apoptosis than the administration of 2-DG and metformin, respectively.

Three-dimensional invasion assay model

GFP-stable GSC11 cells grown in spheroids were cultured in collagen I matrix using polydimethylsiloxane (PDMS) -based micro-wells (diameter and depth of micro-wells: 6 mm, 500 μm). The micro-wells were treated with 1% poly (ethyleneimine) (Sigma-Aldrich, St. Louis, Mo., USA) solution for 10 minutes and then washed with 0.1% glutaraldehyde (Sigma- For 30 min, and washed with PBS overnight. The collagen I matrix at 4 mg / ml was prepared according to the general method using high concentration of rat tail collagen I (BD Bioscience, CA, USA). Of 10x phosphate buffered saline (PBS), 1N NaOH, sterile dH ₂ O and collagen Ⅰ were mixed such that the desired concentration. The mixed solution was mixed and maintained at 4 캜 until use. Subsequently, 10 μl of collagen I solution (4 mg / ml) was pipetted into the micro-well, and a single G-GSC11 spoloid was transferred from the culture plate to the collagen I matrix and 10 μl of collagen I solution (4 mg / ml ) Was dropped on the G-GSC11 sphereoid. The platform was incubated for 30 minutes under 37 ℃ and 5% CO ₂ atmosphere. Analysis of cell viability was performed by staining G-GSC11 spheroids with 8 占 에 ethidium homodimer-1 (Invitrogen Korea, Seoul, South Korea) for 30 minutes at 37 占 폚 before implantation into the collagen matrix. After complete gelation, 2% 1XB27, 20 ng / ml 0.02% bFGF, 20 ng / ml 0.02% EGF, 1% antibiotic-antifungal solution (100x, Gibco, Invitrogen Korea, Seoul, South Korea ) Was added to the culture supernatant. To confirm each drug effect, the drug was mixed into the medium considering the final concentration of each drug. GS-11 spheroids were observed with a reversed phase-contrast microscope (Nikon Ti-E, Tokyo, Japan) at 72 hours after the drug mixture, and the results are shown in FIG. In addition, the invasiveness of the GBM tumor was measured using the following formula, and the results are shown in FIG. 3 (b). Data were analyzed using software image J (NIH, Bethesda, Maryland, USA).

Invasiveness (%) = (Obscured area at specific time / Initial spheroid area) X 100

As shown in Figs. 3 (a) and 3 (b), it was confirmed that the combination inhibition effect of 2-DG and metformin was remarkably excellent when they were administered in combination.

¹⁸ F- Fluorodioxyglucose  Positron Emission Tomography ( ¹⁸ F-fluorodeoxyglucose (FDG) positron emission tomography )

The GBM cell line was added to a 12-well plate at a concentration of 3 × 10 ⁵ cells / well, followed by culturing for 24 hours. 2-DG, metformin, a combination thereof and temozolomide were added, followed by culturing for another 3 days. Subsequently, the medium was replaced with glucose-free DMEM medium (Gibco) supplemented with 0.5 uCi of ¹⁸ F-FDG, followed by incubation for 15 minutes, washing three times with phosphate-buffered saline, and adding 0.1 ml Lysis buffer was added. The dissolved cells were collected and the degree of radioactivity was analyzed using a gamma-counter (Wallac 148 Wizard 3; Perkin Elmer). The results are shown in FIG. The degree of radioactivity measured was normalized by protein content.

As shown in FIG. 4, when FDG absorption rate was remarkably decreased when 2-DG and metformin alone were administered in combination with them. It is shown that the combined administration of 2-DG and metformin has a greater effect on apoptosis than the administration of 2-DG and metformin, respectively.

Gene expression Microarray  And Gene expression microarray  and class comparison)

The total RNA (total RNA) was extracted according to the general method using Qiagen miRNA kit. Expression profiles were performed using an Illumina human HT-12 v24 expression bead chip (Illumian, Inc., San Diego, Calif.). The raw data was extracted using software supplied by Illumina Studio v2011.1 (Gene Expression Module v1.9.0). The data was transformed by logarithm and normalized by the quantile method. Adhesion junctions, adhesion molecules (CAMs), ECM-receptor interactions, focal adhesion, actin cytoskeleton regulation; SNARE interaction in vesicle transport; The genes associated with TGF-beta signaling (defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway gene set) were filtered and analyzed. All analyzes were performed using the BRB-ArrayTools and BRB-Array Tools development team developed by Dr. Richard Simon. Using a random-variance t-test, we identified genes that were differentially expressed between two taxa (control and 2-DG plus metformin), and genes were statistically significant (p <0.001) . In order to compare the differences in the expression profiles between the two groups, a global test was performed on expression profiles with differences between groups by arranging the array levels matching each group with permutation. For each permutation, the p value was recalculated and the number of gene significance at p value of 0.001. The percentage of permutations given at least as important as genes in actual data was evaluated as the level of importance of global testing. The results are shown in Fig.

FIG. 5 shows a gene expression heat map of a gene set. When 2-DG and metformin were co-administered, the genes associated with the epithelial mesenchymal transition were down-regulated in comparison with the control group.

Animal xenograft model Orthotopic xenograft  animal model)

4-8 week-old male athymic nude mice (Central Lab Animal Inc., Seoul, Korea) were used in the following experiments. Male rats were raised in micro-isolator cages under sterile conditions and observed until one week prior to the experiment to maintain desirable health conditions. Light, temperature and humidity were adjusted centrally. Male rats were injected intraperitoneally with Zoletil® (30 mg / kg; Virbac Korea, Seoul, South Korea) and xylazine (10 mg / kg; Bayer Korea, After anesthesia, a GBM tumor was implanted in the right frontal lobe of nude mice using a guide-screw system. 5 × 10 ⁵ GBM tumor cells were injected into male rats at a depth of 4.5 mm using a Hamilton syringe (Dongwoo Science Co., Seoul, South Korea). GBM tumor cells were simultaneously injected into 3 male rats at a rate of 0.5 μl / min using a multiple micro-injection syringe pump (Harvard Apparatus, Holliston, Mass., USA). Thereafter, 500 mg / kg of 2-DG, 500 mg / kg of metformin, a combination thereof and 66.7 mg / kg of temozolomide were injected daily into the abdominal cavity of the male rats described above. Male rats were weighed daily and euthanized if they lost more than 15% of their original weight. When the mouse died, the brain was carefully removed, gliomagenesis was observed using H / E staining, and tumor cell infiltration was observed using Zebl staining. The results are shown in FIGS. 6 (a) and 6 (b).

As shown in FIG. 6 (a), when compared with the case of treatment with 2-DG and metformin alone, the size of the tumor was significantly reduced when the combination was administered.

As shown in FIG. 6 (b), when the combination of 2-DG and metformin was administered alone, the invasiveness of the glioblastoma was markedly decreased, and the treatment with temozolomide It was found that the effect of suppressing infiltration was superior to that of the case

Statistical analysis

Survival analysis of the obtained GBM tumor cell-transplanted mice was performed using a Kaplan-Meier estimator. Data were expressed as means ± SD. All statistical analyzes and lapping were performed using SPSS version 18.0KO software (SPSS Korea, Seoul, Korea). The results are shown in FIG. 7 as a graph (P < 0.001).

As shown in FIG. 7, the survival time of rats was increased when the combination of 2-DG and metformin was administered alone, as compared with the case where 2-DG and metformin alone were administered.

Breast Cancer and Glioblastoma glioblastoma Comparative analysis of effect

(100x, Gibco, Invitrogen Korea, Seoul, South Korea) was inoculated into DMEM / F-12 with 2% 1XB27, 20 ng / ml 0.02% bFGF, 20 ng / ml 0.02% EGF and 50 U / ml penicillin / 50 mg / ml streptomycin The breast cancer cell line MCF7, MDA-MB 231 and glioblastoma GSC11 cells were cultured in the medium. The above cells were cultured for 1 week under different conditions and the photographs of the tumor obtained after culturing were observed with an inverted phase microscope (IX71 Inverted Microscope; Olympus, Tokyo, Japan) , And the results are shown in Fig. 8 (b). However, the photographs of cells after culturing were taken with DP controller software (Olympus) using a digital camera (DP70 Digital Microscope Camera; Olympus).

As shown in FIG. 8 (a), MDA-MB 231 did not form tumor parenchyma. In contrast to the case of treatment with 2-DG and metformin alone, Was small.

On the other hand, in the case of MCF7, the tumor size reduction effect due to the combination of 2-DG and metformin was found to be insignificant when compared with the glioblastoma GSC11.

Furthermore, as shown in FIG. 8 (b), in the case of MCF7, when the 2-DG and metformin were co-administered, the size of the tumor increased compared with the case of 2-DG alone.

Accordingly, it can be seen that the pharmaceutical composition comprising 2-DG and metformin according to the present invention is effective for prevention or treatment of cancer, particularly, brain cancer, preferably glioblastoma.

Claims (8)

A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a glucose uptake inhibitor and a biguanide-based compound. The method according to claim 1,
Wherein the glucose uptake inhibitor is 2-deoxyglucose (2-DG). The method according to claim 1,
RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; wherein the biguanide-based compound is metformin. The method according to claim 1,
Wherein the glucose uptake inhibitor and the biguanide based compound are included in a weight ratio of 1: 0.5-100. The method according to claim 1,
Wherein the cancer is at least one selected from the group consisting of cervical cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, blood cancer and liver cancer. The method according to claim 1,
Wherein said cancer is brain cancer. The method according to claim 6,
Wherein said brain cancer is a glioblastoma. 8. The method of claim 7,
Wherein the glioblastoma comprises tumor spheres.

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