KR20130015669A - Pharmaceutical composition for inhibiting cancer recurrence or metastasis - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for suppressing cancer recurrence or metastasis is provided to reduce side effects and to enable effective treatment. CONSTITUTION: A pharmaceutical composition for suppressing cancer recurrence or metastasis contains 10-4000 mg of 2-deoxy-D-gluscose as an active ingredient. The pharmaceutical composition contains a biguanide-based therapeutic agent for treating diabetes or a pharmaceutically acceptable salt thereof, and 2-deoxy-D-gluscose as active ingredients. The therapeutic agent is metformin, phenformin, or buformin. The pharmaceutically acceptable salt of metformin is hydrochloride or acetate. The pharmaceutical composition is an oral formulation or a parenteral formulation. The pharmaceutical composition additionally contains an anticancer agent as an active ingredient. The cancer is colon cancer or breast cancer. [Reference numerals] (AA) Mammosphere formation; (BB) Hydrochloride of metformin; (CC) Hydrochloride of metformin + 2-DG

Description

Pharmaceutical composition for inhibition of recurrence or metastasis of cancer {PHARMACEUTICAL COMPOSITION FOR INHIBITING CANCER RECURRENCE OR METASTASIS}

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions for inhibiting cancer recurrence or metastasis, and more particularly, cancer stem cell reduction, and epithelial-to-mesenchymal transition (ETM) It relates to a pharmaceutical composition that can more effectively inhibit the recurrence or metastasis of cancer cells through inhibition.

Biguanide-based diabetes treatment is an oral diabetes treatment that is used for the treatment of insulin-independent diabetes mellitus (type 2 diabetes) because it has an excellent hypoglycemic effect, does not cause hypoglycemia or hyperinsulinemia, and prevents complications. It is one of the drugs. Recently, many studies on metformin, one of the biguanide-type diabetes treatments, have been published, and a study published that metformin inhibits cancer cell proliferation and tumor growth. The incidence and mortality from cancer were reduced. Specifically, metformin is a substance that inhibits the proliferation of cancer cells by inhibiting the action of the electron transport system Complex I of mitochondria and interfering with the production of energy in the cell, and is being studied as an anticancer agent that regulates metabolism of cancer cells.

Meanwhile, 2-deoxy-D-glucose (hereinafter referred to as 2-DG) is a derivative of glucose and is known as a drug that prevents cancer cell growth by limiting the absorption of sugar, which is an energy source of cancer cells. Cancer cells require a lot of energy to support high-speed cell division, not only cancer cells that divide in the presence of oxygen, but also energy that survives to slightly slow-dividing cancer cells in a hypoxic or anoxic environment. . 2-DG is a form of 6-Glucose 6-phosphate and 6-Fructose 6-phosphate in glycolysis, the process of producing the energy (ATP) required for cell growth and maintenance through D-glucose. By blocking the isomerization step of 6-glucose phosphate, which isomerizes with phosphate, it inhibits the proliferation of cells by blocking the energy supply of cancer cells.

Most anticancer drugs developed to date have been drugs targeting rapidly proliferating cells, and thus, cancer stem cells characterized by slow proliferation could survive chemotherapy with these drugs, resulting in poor prognosis and There was a problem of becoming resistant to chemotherapy. Therefore, there is a demand for the development of a drug that can exert an effect on cancer stem cells, which has been limited by conventional anticancer agents, and can effectively suppress cancer recurrence and / or metastasis.

In this regard, studies on the inhibition of cancer metastasis or recurrence of 2-DG drugs, or the increased effects on the inhibition of cancer metastasis and cancer recurrence through combination therapy with biguanide-based diabetes drug drugs and 2-DG drugs None known.

Accordingly, an object of the present invention is to provide 2-DG as a novel active ingredient that can effectively suppress the recurrence or metastasis of cancer.

In addition, the present invention can effectively inhibit the recurrence or metastasis of cancer with a lower content through the synergistic action of the two active ingredients, including 2-DG and biguanide-based diabetes therapeutic drug as the active ingredient, the probability of occurrence of side effects It is an object of the present invention to provide a pharmaceutical composition useful for the prevention or inhibition of cancer metastasis or cancer recurrence, which can be reduced and the therapeutic effect increased.

In addition, an object of the present invention is to provide a pharmaceutical composition that can further improve the metastasis or prevent recurrence of cancer through synergism by additionally including other active ingredients in addition to the two active ingredients.

As a result of the study of the present inventors, it was confirmed that 2-DG can effectively inhibit cancer metastasis or cancer recurrence, and furthermore, the 2-DG may be combined with a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof. When administered in combination, it has been shown that a surprising synergistic effect on inhibition of cancer metastasis or cancer recurrence can be achieved, resulting in a reduction in the dose of each drug according to the synergistic effects of these two drugs, thereby reducing the probability of side effects occurring, The present invention was completed by confirming that the therapeutic effect can be significantly increased.

Specifically, breast cancer stem cells as an indicator for confirming the effect of inhibiting cancer metastasis or cancer recurrence even when 2-DG is used alone as well as when using a biguanide-based diabetes treatment drug or a pharmaceutically acceptable salt thereof. Inhibition of mammosphere formation and suppression of EMT (Epithelial-to-mesenchymal transition) of cancer cells were found to be excellent. Furthermore, when the biguanide-based diabetes treatment drug and 2-DG were treated alone, Compared with the above two drugs, the effects of inhibiting breast cancer stem cell (mammosphere) formation and EMT inhibition of cancer cells were remarkably increased.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising 2-DG as an active ingredient.

The present invention also provides a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof and 2-DG as an active ingredient.

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

Recent studies have shown that cancer stem cells circulating in the blood have both cancer stem cells and EMT markers, and that cancer stem cells are EMT-induced cancer cells. Therefore, if EMT-induced cancer stem cells are removed, cancer metastasis and cancer recurrence can be suppressed.

Experimental results of the present invention confirm that breast cancer stem cell (mammosphere) formation is inhibited by 2-DG, and at the same time surprisingly higher than expected when combined with the treatment of biguanide-based diabetes therapeutic drugs such as metformin and 2-DG It was confirmed that the inhibitory effect was shown. In addition, it was confirmed that expression of a protein involved in the process of metastasis of cancer epithelial cells related to cancer metastasis to mesenchymal cells (EMT), in particular Snail1, was reduced by 2-DG alone treatment. It was confirmed that the reduction was more pronounced (see Experimental Example 1 and Experimental Example 2).

2-DG is a substance that regulates metabolism of cancer cells by inhibiting glycolysis in cancer cells that require a lot of energy (ATP). Cancer cells, whose glycolysis is inhibited by 2-DG and whose energy supply is restricted, operate the energy supply mechanism using TCA circuit and glutamine, and are supplied with energy (ATP) required for division through the electron transport system of mitochondria. . Metformin, one of the biguanide-based diabetic medications, inhibits the mitochondrial complex I's action, and therefore, the combination of 2-DG and metformin is thought to be synergistic.

Cancer stem cells are cancer cells that have self-renewal or differentiation ability that is unique to stem cells. The failure of conventional chemotherapy and the recurrence of tumors have led to the rapid growth of most anticancer drugs developed in the meantime. Cancer stem cells characterized by slow proliferation because they were drugs targeting cells were able to survive such chemotherapy, resulting in poor prognosis and resistance to conventional chemotherapy.

Epithelial-to-mesenchymal transitions (ETMs), on the other hand, are caused by various cytokines and growth factors, but the TGFβ signaling pathway is particularly important. TGFβ is also involved in the development of cancer stem cells in breast cancer, whereby cells EMT-enriched by TGFβ have the ability to initiate carcinoma while at the same time gaining cell mobility, allowing cancer stem cells to travel far and form tumors in new tissues. You can do it. Therefore, inhibiting and preventing the oncogenic and EMT ability of cancer stem cells may be an important treatment method for treating cancer metastasis and lowering the risk of cancer recurrence.

The pharmaceutical composition of the present invention can exert a remarkable effect on cancer stem cells which had the above-mentioned problems, and also can effectively suppress the recurrence and / or metastasis of cancer as the effective inhibition of EMT ability is possible. An improved prognosis can be expected.

In addition, mesenchymal cells are difficult to treat cancer because they are resistant to radiation therapy or chemotherapy, the combination of biguanide-based diabetes drug and 2-DG prevents cancer cells from differentiating into mesenchymal cells In addition, by increasing the EMT not only inhibits but also shows an improved anti-cancer effect, it is expected to be able to prevent cancer metastasis and recurrence more effectively.

Specific diseases to which the pharmaceutical composition of the present invention may be applied include, for example, uterine cancer, breast cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, thyroid cancer Or various cancers such as liver cancer, and preferably any one selected from the group consisting of uterine cancer, breast cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, blood cancer and liver cancer, and more preferably. Colon cancer or breast cancer.

In addition, in addition to the combination of 2-DG or biguanide-based diabetic therapeutic drug and 2-DG, which are the active ingredients of the present invention, it is expected to further increase cancer metastasis and prevent cancer recurrence by further including other active ingredients.

According to one embodiment of the invention, there is provided a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising 2-deoxy-D-glucose of Formula 1 as an active ingredient.

2-Deoxy-D-glucose (2-DG)

The content of 2-DG can be easily determined by those skilled in the art according to the age, sex, disease severity, and weight of the patient. Preferably, the 2-DG is contained in an amount of 5 to 80 wt% based on the total weight of the unit dosage form.

Also preferably, the 2-DG may be administered once to several times a day in a form containing a total of 10 to 4,000 mg, more preferably once a day in a form containing a total of 100 to 2,500 mg. To several times.

When the content of 2-DG is less than the above range, the effect expression of 2-DG may not reach the effective level, and when it exceeds the above range, side effects due to overdose may be expressed and the mass of the composition itself may increase. The difficulty in administering is undesirable because there is a problem that is not useful as a pharmaceutical composition.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof and 2-DG as an active ingredient .

The biguanide-based diabetic therapeutic drug includes at least one selected from the group consisting of metformin of Formula 2, phenformin of Formula 3, Buformin of Formula 4, and the like.

Metformin

Phenformin

Buformin

As used herein, the phrase “a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof and 2-DG as an active ingredient” is explicitly described. As long as there is no, it is understood that a single dosage form, such as oral or injectables, which are taken or administered at one time, as well as a plurality of unit dosage forms which are administered two or more times may be referred to collectively.

In other words, it is to be understood that both single unit dosage forms comprising these two active ingredients together, as well as two unit dosage forms comprising one active ingredient each, may be referred to together. Thus, when these two unit dosage forms are administered simultaneously or at intervals of no more than a certain time such that the two active ingredients contained in each of these unit dosage forms are present together in the body to produce a synergistic effect, the above two unit dosage forms It is to be referred to as belonging to the category of the above "biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof and a pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising 2-DG as an active ingredient". Can be.

The biguanide-based diabetes therapeutic drug may be used by selecting one or more of metformin, phenformin, and buformin. Preferably metformin or phenformin can be used, most preferably metformin drug.

The daily dosage of the biguanide-based diabetes therapeutic drug may be differently applied according to the type of drug, and more specifically, when the drug is metformin, 100 mg to 4,000 mg, preferably 250 mg to 2,000 mg, and pen It may be administered once to several times per day in the form of 10 to 200 mg, preferably 25 mg to 100 mg, and for buformin, 10 to 500 mg, preferably 25 to 300 mg for formin.

Also preferably, the pharmaceutical composition may contain a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof, and the 2-DG from 1: 400 to 400: 1, more preferably from 1: 100 to 20: 1. It may be included in the weight ratio of.

If it is out of the above range may not reach the effective level required for the expression of the effect of each active ingredient, or side effects due to overdose may be expressed and the mass of the composition itself increases, making it difficult to administer the pharmaceutical composition. There is a problem that is not desirable.

On the other hand, the biguanide-based diabetes therapeutic drug may be in the form of itself or a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may be acid addition salts formed with organic or inorganic acids, which may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, Malonic acid, fumaric acid, succinic acid, succinic monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxy acetic acid, benzenesulfonic acid, p- Toluenesulfonic acid and methanesulfonic acid salts and inorganic acids include, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid salts.

The above-mentioned acid addition salts can be, for example, a) directly mixing the compounds of formulas 2, 3, or 4 and acids, b) dissolving one of them in a solvent or a hydrous solvent, or c) Compounds of formulas (2), (3) or (4) and acids may be prepared by application to a general process for preparing salts which are placed in an acid in a solvent or hydration solvent and mixed.

In addition to the above, additionally saltable forms include, but are not limited to, the salts of gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, Aspartate and the like.

In one embodiment, the pharmaceutically acceptable salt of the biguanide family of diabetes therapeutic drugs is formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid , Monosuccinic acid, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, dichloroacetic acid, aminooxyacetic acid, It may be a salt with an acid selected from the group consisting of hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid, preferably in the form of hydrochloride or acetate, more preferably in the form of hydrochloride.

Meanwhile, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients.

“Pharmaceutically acceptable carrier” as defined above means pharmaceutical additives that are useful in formulating pharmaceutical compositions and which are nontoxic and insensitive under the conditions of use, and the specific content ratio of such carriers may be Solubility and chemical properties, as well as the route of administration chosen, can be determined by standard pharmaceutical practices.

More specifically, the pharmaceutical composition of the present invention is a pharmaceutically acceptable carrier using a pharmaceutical additive such as excipients, disintegrants, sweeteners, binders, coatings, expanding agents, lubricants, lubricants, fragrances, etc. It may be formulated in a form suitable for. In addition, the amount of carrier required per dosage unit may be an amount sufficient to provide a size and dosage form that may increase the subject's compliance.

Formulations may be in the form of oral or parenteral preparations, for example capsules, tablets, granules, powders, troches (including liquid-filled) containing tablets, particulates, liquids or powders. Include, but are not limited to, chews, multi- and nano-particulates, gels, solid solutions, liposomes, films (including muco-adhesives), ovals, sprays and solutions It doesn't work.

Liquids include, but are not limited to, for example, suspensions, solutions, syrups and elixirs.

When formulated in the form of general tablets in oral formulations, it may further comprise a disintegrant in addition to the active ingredient. Examples of the disintegrant include starch or modified starch, such as sodium starch glycolate, corn starch, potato starch, or starch gelatinized starch, clays such as bentonite, montmorillonite, and vegum, and low-substituted hydroxypropyl. Celluloses such as cellulose, alginates such as sodium alginate or alginic acid, crosslinked celluloses such as croscarmellose sodium, crosslinked polymers such as crospovidone, and boiling properties such as sodium bicarbonate and citric acid Formulations and the like may be used in combination, but are not limited thereto. The disintegrant may preferably comprise from about 0.5% to about 30% by weight of the dosage form, more preferably from about 1% to about 20% by weight of the dosage form, but is not limited thereto.

The tablet may also further comprise a binder for imparting tack. Binders include, for example, gelatin, sugars, natural and synthetic gums, polyvinylpyrrolidone (povidone), polyvinyl alcohol, copovidone, starch, hydroxypropylcellulose and hypromellose Etc. may be used but is not limited thereto. The binder may preferably include, but is not limited to, from about 0.1% to about 40% by weight of the dosage form, more preferably from about 0.5% to about 25% by weight of the dosage form.

In addition, the tablet may further comprise a diluent. As the diluent, for example, starch, microcrystalline cellulose, lactose, glucose, mannitol, alginate, alkaline earth metal salt, polyethylene glycol and dicalcium phosphate may be used, but not limited thereto. The diluent may preferably include, but is not limited to, from about 0.5% to about 90% by weight of the dosage form, more preferably from about 2% to about 75% by weight of the dosage form.

In addition, the tablet may further include a lubricant. As the lubricant, for example, talc, stearic acid, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, hydrogenated vegetable oil, polyethylene glycol, and the like may be used, but are not limited thereto. The glidant may preferably include, but is not limited to, from about 0.1% to about 20%, more preferably from about 0.2% to about 10% by weight of the dosage form.

In addition, the tablet may optionally include, for example, surfactants such as sodium lauryl sulfate, polysorbate 80 and glidants such as colloidal silicon dioxide, hydrous silicon dioxide, talc. Preferably the surfactant may comprise about 0.1% to about 20% by weight of the dosage form, and the glidant may include but is not limited to about 0.1% to about 20% by weight of the dosage form. .

Ingredients that may also be included include antioxidants, colorants, flavors, preservatives and taste-blocking agents.

The tablet may be formed by directly compressing the components included in the tablet as described above or by pressing with a roller. Alternatively, the components included in the tablets as described above can be wet-, dry-, melt-granulated or melt-congealed, or extruded prior to tableting. The form of the final formulation may comprise one or more layers, may or may not be coated or encapsulated.

In addition, depending on the timing of drug release, it may be formulated as an immediate release and / or a modified release, wherein the modified release formulation may be delayed-, sustained-, pulsating ( pulsed-, controlled-, targeted and programmed release types.

The matrix base for formulating into a modified release tablet is not particularly limited but may be any one or more components selected from the group consisting of enteric polymers, hydrophobic materials, hydrophilic polymers, and the like.

Enteric polymers include, for example, polymethacrylate copolymers such as polyvinylacetate phthalate, poly (methacrylic acid, methyl methacrylate) copolymers and poly (methacrylic acid, ethylacrylate) copolymers, hypro Melophthalate, hypromellose acetate succinate, shellac, cellulose acetate phthalate, cellulose propionate phthalate, and the like may be used, but are not limited thereto.

As the hydrophobic material, pharmaceutically acceptable, for example, polyvinyl acetate, ethyl cellulose and cellulose acetate, fatty acids and fatty acid esters, fatty alcohols and the like may be used, but is not limited thereto.

More specifically, glyceryl palmitostearate, glyceryl stearate, glyceryl bihenate, cetyl palmitate, glyceryl monooleate and stearic acid may be used as fatty acids and fatty acid esters. Cetostearyl alcohol, cetyl alcohol, stearyl alcohol and the like can be used.

Examples of the hydrophilic polymer include, but are not limited to, sugars, cellulose derivatives, gums, proteins, polyvinyl derivatives, polyethylene derivatives, and carboxyvinyl polymers.

More specifically, as the sugar, dextrin, polydextrin, dextran, pectin and pectin derivatives, alginates, polygalacturonic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose, amylopectin and the like And cellulose derivatives, such as hypromellose, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose sodium, hydroxyethyl methyl cellulose, and the like. Guar gum, locust bean gum, tragacanta, carrageenan, acacia gum, gum arabic, gellan gum, xanthan gum, and the like can be used.As for proteins, gelatin, casein, zein and the like can be selected and used. Polyvinyl alcohol, polyvinyl pyrrolidone, etc. can be used as a polyvinyl derivative. Polyethylene glycol, polyethylene oxide, or the like may be used as the polyethylene derivative, and carbomer or the like may be used as the carboxyvinyl polymer.

In addition, the formulation and the pharmaceutically acceptable carrier of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected according to techniques known in the art. Also effective amounts for the treatment or prevention of various cancer diseases include the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and It may be adjusted according to various factors including the diet, the time of administration, the route of administration and the rate of blood clearance of the composition, the duration of treatment, and the drugs used concurrently. For example, in the case of an adult, the pharmaceutical composition of the present invention may be administered once or several times a day, and a single dose may be administered or taken in an effective amount of a total amount of 20 to 8,000 mg. However, it will be apparent to those skilled in the art that the dosage or dosage of each active ingredient should be such that the content of each active ingredient is too high so as not to cause side effects.

According to another preferred embodiment of the present invention, in addition to containing a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof, and 2-DG as an active ingredient, further comprising an anticancer agent as an active ingredient. Pharmaceutical compositions are provided.

It is not only a single unit dosage form comprising a biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof, and 2-DG as an active ingredient, but also an anticancer agent as another active ingredient, as well as one activity each. Also included are three unit dosage forms comprising the component. That is, these three unit dosage forms are administered concurrently or at intervals of no more than one hour so that the three active ingredients included in each of these unit dosage forms are present together in the body, resulting in synergism, e.g. to alleviate or ameliorate symptoms. Therapeutic effects may be further enhanced in various aspects, such as reducing the extent of disease, delaying or alleviating disease progression, improving, alleviating or stabilizing the disease state, partial or complete recovery, prolonging survival, and other beneficial treatment outcomes.

As the anticancer agent, any known anticancer agent may be used. For example, known chemotherapeutic agents such as alkylating agents, metabolic antagonists, natural agents, hormones and antagonists, and biological agents such as immunotherapy agents and gene therapy agents may be used.

More specifically, for example, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, zefitinib, vandetanib, nirotinib, semasanib, conservinib, axitinib , Cediranib, restautinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, biscumalboom, asparaginase, tretinoin, Hydroxycarbamide, dasatinib, estramastine, gemtuzumab ozogamycin, ibritumab tucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil , Holmium nitrate chitosan, gemcitabine, doxyfluidine, pemetrexed, tegapur, capecitabine, gimerasin, oteraceyl, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine , Enositabine, fluta De, decitabine, mercaptopurine, thioguanine, cladribine, carmofer, raltitrexed, docetaxel, paclitaxel, irinotecan, velotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, tenni Fosid, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temozolomide, laying Pan, Ifosfamide, Cyclophosphamide, Melparan, Altretmin, Dcarbazine, Chiotepa, Nimustine, Chlorambucil, Mitolactol, Leucovorin, Tretonin, Xmestan, Aminoglutesimid At least one drug selected from the group consisting of anagrelide, nabelbin, padrazole, tamoxifen, toremifene, testosterone, anastrozole, letrozole, borosol, bicalutamide, romustine and carmustine Available There.

According to the present invention, the inhibition of breast cancer stem cell (mammosphere) formation and inhibition of EMT of 2-DG has been confirmed the effect of inhibiting the proliferation and cancer metastasis of cancer stem cells, which have not been known so far, Combination of one formformin or its pharmaceutically acceptable salts with 2-DG significantly reduced cancer metastasis inhibition and cancer recurrence compared to the case of using only the active ingredients alone, which is much better based on the same content. It was confirmed that it can exhibit the effect of inhibiting cancer metastasis and recurrence.

Therefore, when using the pharmaceutical composition according to the present invention, it is possible to exhibit a substantially superior effect while reducing the content of each active ingredient, thereby enabling more effective treatment while reducing side effects due to drug overuse and side effects due to pharmacological action. Become. In addition, it is expected to have a more synergistic effect when used in combination with the anticancer drug that showed resistance.

Therefore, the pharmaceutical composition for inhibiting recurrence or metastasis of cancer according to the present invention can be very useful for treating and preventing metastasis or recurrence of various cancer diseases.

1 is a graph showing the breast cancer stem cell (mammosphere) formation efficiency (MFE (%)) measured according to Experimental Example 1 of the present invention.

Hereinafter, preferred experimental examples and examples are provided to help understanding of the present invention, but the following experimental examples and examples are merely to illustrate the present invention, but the scope of the present invention is not limited thereto.

[ Experimental Example  1] breast cancer stem cells ( Mammosphere ) Formation inhibitory effect

Inhibition of breast cancer stem cell (mammosphere) formation effect by the combination of 2-DG alone composition and metformin hydrochloride and 2-DG complex composition in human MCF-7 cell line derived from human breast cancer, A brief test method is as follows.

MCF-7 cells derived from human breast cancer (purchased from Korea Cell Line Bank) in DMEM medium containing 10% (v / v) calf serum (purchased from Gibco Life Technologies (USA)), about 80% of the culture area. After culturing (culturing temperature: 37 ℃, pH: 7.0 ~ 7.4) until it was grown, it was used in experiments to confirm the ability to form breast cancer stem cells. Remove the culture medium from the MCF-7 cells that were incubated, collect the cells, add DMEM / F12 medium without serum (purchased from Gibco Life Technologies, USA) to make MCF-7 cells into single cells, and measure the number of cells. The cells were placed in a 6 well plate (6 well plate) such that the cell number was 9000 cells / well. At this time, the 6 well plate used was an Ultralow attachment 6 well plate (Corning, Cat # 3471) in which cells were not attached to the plate.

In addition, cultures for the formation of breast cancer stem cells were treated with 2% B27 (purchased Invitrogen), 20 ng / ml EGF (purchased Sigma), 5 ug / ml insulin (purchased Sigma) and 1 ug / ml hydrogel in serum-free DMEM / F12 medium. Prepared to contain cortisone (hydrocortisone, purchased from Sigma). After culturing the cells in a CO ₂ incubator for 24 hours, metformin hydrochloride and 2-DG at the concentrations of 0.5 mM, 1 mM, and 3 mM were combined and treated alone, respectively, and the culture medium for forming breast cancer stem cells prepared above. Was added to adjust the final culture volume to 3 ml. As a control, those not treated with metformin hydrochloride and 2-DG were used. Incubated for 8 days in a CO ₂ incubator, and observed the size and efficiency of breast cancer stem cell formation. After 8 days, the breast cancer stem cell formation efficiency (MFE (%)) was calculated by measuring the number of breast cancer stem cells having a size of 50 μm or more. Equation for calculating the breast cancer stem cell formation efficiency (MFE (%)) is as follows.

≪ Equation &

Breast cancer for treatment with metformin hydrochloride and 2-DG alone versus combination treatment of breast cancer stem cell formation efficiency (MFE (%)) in well plates not treated with metformin hydrochloride or 2-DG after 8 days of incubation Stem cell formation efficiency (MFE (%)) compared to breast cancer stem cell formation inhibition rate (%) is shown in Table 1 below, the breast cancer stem cell formation efficiency for each is shown in Table 1 and Figure 1 below. In the combination group, metformin hydrochloride and 2-DG were each treated at a ratio of 1: 1 at the concentrations listed in Table 1 below.

Breast cancer stem cell formation inhibition rate (%) 0 0.5 mM 1 mM 3 mM Metformin hydrochloride Breast Cancer Stem Cell Count 378 ± 3 287 ± 14 197 ± 10 94 ± 17 MFE (%) 4.2 ± 0.03 3.2 ± 0.16 2.2 ± 0.11 1.0 ± 0.19 % Inhibition 0.0 24.0 47.7 75.0 2-DG Breast Cancer Stem Cell Count 378 ± 3 291 ± 12 246 ± 41 237 ± 36 MFE (%) 4.2 ± 0.03 3.2 ± 0.14 2.7 ± 0.45 2.6 ± 0.4 % Inhibition 0.0 22.9 34.9 37.3 Metformin Hydrochloride + 2-DG Breast Cancer Stem Cell Count 378 ± 3 246 ± 19 117 ± 10 11 ± 2 MFE (%) 4.2 ± 0.03 2.7 ± 0.21 1.3 ± 0.11 0.1 ± 0.02 % Inhibition 0.0 34.8 69.1 97.0

As can be seen from Table 1 and FIG. 1, the inhibition rate of breast cancer stem cell formation according to the treatment of each drug in MCF7 cells, as well as metformin alone treatment, in particular, the formation of breast cancer stem cells by 2-DG alone treatment It was confirmed for the first time through this experiment that the drug was significantly inhibited as the concentration increased.

Furthermore, as a combination treatment of metformin hydrochloride and 2-DG, it was confirmed that there is a significant synergistic effect on the inhibition of breast cancer stem cell formation compared to when treated alone. More specifically, when the metformin hydrochloride and 2-DG alone were treated at a concentration of 1 mM each, the inhibition rate of breast cancer stem cell formation was about 47.7% for metformin drugs and about 34.9% for 2-DG drugs, while When the two drugs were used in combination, the inhibition rate of breast cancer stem cell formation was about 69.1%, indicating that the effects were increased by about 1.44 and 1.98 times, respectively, at the same concentration compared to the treatment with each drug alone.

As such, the results of this experiment confirmed the cancer stem cell formation and proliferation inhibitory effect of the 2-DG drug alone treatment, and in addition to the combination treatment of metformin and 2-DG drug, a biguanide-based diabetes drug Due to the synergism of each drug, even lower concentrations of the drugs can be used, resulting in better effects. It was confirmed that it can show the effect of inhibiting the formation and growth of elevated cancer stem cells. Therefore, it can be expected to have an excellent effect in preventing the recurrence of cancer and suppressing metastasis of the cancer in the single treatment and combination treatment of each drug.

[ Experimental Example  2] Snail  Through inhibition of expression EMT  Confirmation of inhibition

In the MCF-7 cell line derived from human breast cancer, we measured the effects of metformin hydrochloride and metformin hydrochloride and 2-DG in combination with EMT, especially Snail, on the suppression of Snail expression. same.

MCF-7 cells derived from human breast cancer (obtained from Korea Cell Line Bank) were cultured in DMEM medium (obtained from Gibco Life Technologies, USA) containing 10% (v / v) calf serum, and the cell count was approximately 5 ×. The cells were placed in a 6 well plate such that 10 ⁵ cells were used, and the cells were cultured in an incubator supplied with 5% CO ₂ (cultivation temperature: 37 ° C., pH: 7.0 to 7.4). The culture solution was treated with 2-DG and metformin hydrochloride alone at 0.1 mM, 1 mM, and 10 mM, respectively, and treated with 2-DG and metformin hydrochloride at 0.1 mM, 1 mM, and 10 mM concentrations, respectively. Incubate for 48 hours. In the combination group, metformin hydrochloride and 2-DG were each treated at a ratio of 1: 1 at the concentrations listed in Table 1 below.

After 48 hours, the cultured cells were treated with 10 mM Tris HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na ₄ P ₂ O ₇ , 2 mM Na ₃ VO ₄ , 1 Lysis using% Triton X-100, 10% glycerol, 0.5% deoxycholate, 1 mM PMSF, and 25 ug of cell lysates were electrophoresed on SDS-PAGE gels by protein quantification. Afterwards, the PMSF (purchased from Amersham Life Science) was transferred to a membrane. The membrane is then rat monoclonal anti-human Snail (rat monoclonal anti-human Snail) obtained from rats (purchased from Cell signaling Technology and used to dilute 1: 1000 in 5% skim milk (skim milk, purchased from Difco)) ) Or monoclonal antibody against mouse β-actin (mouse monoclonal anti-β-actin) (purchased from Santa cruz Biotechnology and diluted 1: 5000 in 5% skim milk (from Difco)) After the reaction, the antibody was left without binding. At this time, anti-β-actin was used as an antibody to show that the amount of protein put in the SDS-PAGE gel is the same.

Again the membrane is a secondary antibody, anti-rat IgG (purchased from Cell signaling Technology and diluted 1: 2000 in 5% skim milk (from Difco)) or anti-mouse IgG (anti-mouse IgG) (purchased from Santa cruz Biotechnology, 5% skim milk (skim milk, purchased at Difco diluted 1: 2000)) was added to the reaction. Finally, antibodies bound to the membrane were detected using the ECL Plus system (purchased from Amersham Life Science).

In addition, after analyzing the degree of snail expressed relative to β-actin using the TINA image analysis program, the control group treated with nothing was 100%, and the degree of snail expression according to metformin or 2-DG treatment was expressed as a percentage (%). It is shown in Table 2 below.

Snail Expression Rate (%) Result 0 0.1 mM 1 mM 10 mM Metformin hydrochloride 100% 103% 73% 11% 2-DG 100% 91% 53% 14% Metformin Hydrochloride + 2-DG 100% 74% 16% N.D.

As shown in Table 2, the treatment of metformin hydrochloride or 2-DG alone showed a decrease in the expression of Snail at a concentration of 1 mM. Specifically, metformin hydrochloride had a 27% decrease in the expression of Snail at 1 mM. , 2-DG was found to be reduced by about 47%.

On the other hand, the combination treatment of metformin hydrochloride and 2-DG had already shown a significant reduction effect from the combination treatment of each drug at a concentration of 0.1 mM, specifically, the combination of each drug at concentrations of 0.1 mM and 1 mM. The treatment showed a 26% and 84% reduction, respectively, compared to the treatment with metformin hydrochloride or 2-DG alone.

Particularly, in the case of metrolemin hydrochloride, the effect obtained by using only 1mM in the single treatment can be obtained by using a concentration of only 0.1mM, which is 0.1 times that of the combination with 2-DG. It was confirmed that the drug usage can be significantly reduced.

In addition, when compared to the case of single and combination treatment based on the concentration of 1mM of each drug, the treatment of metformin hydrochloride and 2-DG alone showed 27% and 47% reduction in Snail expression, respectively. When combined treatment was significantly reduced by 84%, it was found that the reduction effect was increased by about 3 times or more and about 1.79 times or more, respectively.

In particular, the effect of reducing the expression of Snail by 2-DG was confirmed for the first time in this experiment. Furthermore, by treating metformin hydrochloride and 2-DG together, even higher concentrations of Snail expression can be obtained even when each drug is used at a much lower concentration. It was confirmed that the synergy between the two drugs when combined treatment.

As a result of this experiment, it was confirmed that the expression of snail protein could be effectively reduced according to the single or concomitant treatment of each drug. Furthermore, since the snail protein is a marker of EMT, It was also found that 2-DG alone not only inhibited EMT, but also induced much higher EMT inhibition upon combination treatment.

Therefore, the combination of 2-DG drug alone and the combination of biguanide-based diabetes therapeutic drug and 2-DG drug is expected to have an effect of inhibiting EMT and further inhibiting metastasis of cancer through inhibition of Snail expression.

[ Example  One] Metformin hydrochloride  And 2- DG  Preparation of Containing Tablets

500 g of metformin hydrochloride, 300 g of 2-DG and 250 g of microcrystalline cellulose were each sieved through a No. 20 sieve, and then mixed in a V-type mixer (V-mixer, Cheil Works, Korea) for 20 minutes. Separately, 25 g of hydroxypropyl cellulose and 15 g of hard silicic anhydride were sieved through a No. 35 sieve, added to the mixture, and mixed for 10 minutes. Finally, 10 g of stearic acid was sieved through a No. 35 sieve, added to the mixture, and mixed for 3 minutes. Subsequently, the final mixture was compressed into tablets containing metformin hydrochloride and 2-DG, and coated with a film based on 30 g of Opadry OY-C-7000A as a coater (SFC-30N, Sejong Machinery, Korea). A layer was formed to prepare a tablet containing 500 mg of metformin hydrochloride and 300 mg of 2-DG in one tablet.

[ Example  2] Metformin acetate  And 2- DG  Preparation of Containing Tablets

571 g of metformin acetate, 300 g of 2-DG, and 179 g of microcrystalline cellulose were each sieved through a No. 20 sieve, and then mixed in a V-type mixer (V-mixer, Cheil Works, Korea) for 20 minutes. Separately, 25 g of hydroxypropyl cellulose and 15 g of hard silicic anhydride were sieved through a No. 35 sieve, added to the mixture, and mixed for 10 minutes. Finally, 10 g of stearic acid was sieved through a No. 35 sieve, added to the mixture, and mixed for 3 minutes. Subsequently, the final mixture was compressed to prepare a tablet containing metformin acetate and 2-DG. As a high coater (SFC-30N, Sejong Machinery, Korea), 30 g of Opadry OY-C-7000A was used as a coating base. A coating layer was formed to prepare tablets containing 571 mg of metformin acetate and 300 mg of 2-DG in one tablet.

[ Example  3] Metforminnicotinate  And 2- DG  Preparation of Containing Tablets

761.5 g of metforminnicotinate, 300 g of 2-DG, and 138.5 g of microcrystalline cellulose were sieved through a No. 20 sieve, and then mixed in a V-type mixer (V-mixer, Cheil Works, Korea) for 20 minutes. Separately, 25 g of hydroxypropyl cellulose and 15 g of hard silicic anhydride were sieved through a No. 35 sieve, added to the mixture, and mixed for 10 minutes. Finally, 10 g of stearic acid was sieved through a No. 35 sieve, added to the mixture, and mixed for 3 minutes. Subsequently, the final mixture was compressed into tablets containing metforminnicotinate and 2-DG. As a high coater (SFC-30N, Sejong Machinery, Korea), Opadry OY-C-7000A 30g was used as a coating film. A coating layer was formed to prepare tablets containing 761.5 mg of metforminnicotinate and 300 mg of 2-DG in one tablet.

[ Example  4] Metformin Malonate  And 2- DG  Preparation of Containing Tablets

704 g of metformin malonate, 300 g of 2-DG, and 146 g of microcrystalline cellulose were sieved through a No. 20 sieve, and then mixed in a V-type mixer (V-mixer, Cheil Works, Korea) for 20 minutes. Separately, 25 g of hydroxypropyl cellulose and 15 g of hard silicic anhydride were sieved through a No. 35 sieve, added to the mixture, and mixed for 10 minutes. Finally, 10 g of stearic acid was sieved through a No. 35 sieve, added to the mixture, and mixed for 3 minutes. Subsequently, the final mixture was compressed into tablets containing metformin malonate and 2-DG. The film was coated with 30 g of Opadry OY-C-7000A as a coating agent as a high coater (SFC-30N, Sejong Machinery, Korea). A coating layer was formed to prepare a tablet containing 704 mg of metformin malonate and 300 mg of 2-DG in one tablet.

[ Example  5] metformin hydrochloride and 2- DG  contain Liquid  Produce

100 g of metformin hydrochloride and 60 g of 2-DG were placed in a 1 L glass container, and 800 mL of purified water was added thereto, followed by stirring for 30 minutes to completely dissolve the purified water. When the preparation was completed, the pH was adjusted to a range of 3 to 4 with 0.1 N hydrochloric acid solution or 0.1 N sodium hydroxide solution to prepare a solution containing 100 mg of metformin hydrochloride and 60 mg of 2-DG per mL of solution.

[ Example  6] Phenformin hydrochloride  And 2- DG  Preparation of Containing Tablets

Tablets containing 100 mg of penformin hydrochloride and 300 mg of 2-DG were prepared in the same manner as in Example 1, except that 100 g of phenformin hydrochloride was used instead of 500 g of metformin hydrochloride.

[ Example  7] Buformin hydrochloride  And 2- DG  Preparation of Containing Tablets

A tablet was prepared in the same manner as in Example 1 except that 50 g of buformin hydrochloride was used instead of 500 g of metformin hydrochloride, and 50 mg of buformin hydrochloride and 300 mg of 2-DG in one tablet.

Claims (12)

A pharmaceutical composition for inhibiting recurrence or metastasis of cancer, comprising 2-deoxy-D-glucose as an active ingredient.
The method of claim 1,
A pharmaceutical composition comprising said 2-deoxy-D-glucose in an amount of 10 to 4000 mg.
A biguanide-based diabetes therapeutic drug or a pharmaceutically acceptable salt thereof, and 2-deoxy-D-glucose as an active ingredient, a pharmaceutical composition for inhibiting recurrence or metastasis of cancer.
The method of claim 3,
The biguanide-based diabetes therapeutic drug is at least one pharmaceutical composition selected from the group consisting of metformin, phenformin, and buformin
The method of claim 3,
The biguanide family diabetes drug or a pharmaceutically acceptable salt thereof, and the 2-deoxy-D-glucose
1: A pharmaceutical composition comprising a weight ratio of 400 to 400: 1.
The method of claim 3,
Pharmaceutically acceptable salts of the biguanide family antidiabetic drugs include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, Glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxy acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, bromic acid, The pharmaceutical composition is at least one acid addition salt selected from the group consisting of sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid.
The method according to claim 6,
Pharmaceutical composition, characterized in that the pharmaceutically acceptable salt of metformin is hydrochloride or acetate.
The method according to claim 1 or 3,
The pharmaceutical composition is a pharmaceutical composition, characterized in that oral or parenteral.
The method according to claim 1 or 3,
Wherein said pharmaceutical composition further comprises an anticancer agent as an active ingredient.
10. The method of claim 9,
The anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, zefitinib, vandetanib, nirotinib, semasanib, conservinib, axitinib, cediranib, restab Urtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, biscumalboom, asparaginase, tretinoin, hydroxycarbamide, Dasatinib, estramastin, gemtuzumab ozogamycin, ibritumab tucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, Gemcitabine, doxyfluridin, pemetrexed, tegapur, capecitabine, gimerasin, oteraceyl, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enositabine, Flutamide, decitabine , Mercaptopurine, thioguanine, cladribine, carmoper, raltitrexed, docetaxel, paclitaxel, irinotecan, velotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin , Idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temozolomide, busulfan, ifospa Mead, cyclophosphamide, melfaran, altretmin, dacarbazine, cheotepa, nimustine, chlorambucil, mitolactol, leukovorin, tretonin, exemestane, aminoglutesimid, anagrely Pharmaceutical composition, which is at least one drug selected from the group consisting of denavel, navelbine, padrazole, tamoxifen, toremifene, testosterone, anastrozole, letrozole, borosol, bicalutamide, romustine and carmustine .
The method according to claim 1 or 3,
The cancer is any one selected from the group consisting of uterine cancer, breast cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, blood cancer and liver cancer
The method of claim 11,
Pharmaceutical composition characterized in that the cancer is colorectal cancer or breast cancer

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