WO2013019058A2 - Pharmaceutical composition for inhibiting cancer recurrence or metastasis - Google Patents

Pharmaceutical composition for inhibiting cancer recurrence or metastasis Download PDF

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Publication number
WO2013019058A2
WO2013019058A2 PCT/KR2012/006096 KR2012006096W WO2013019058A2 WO 2013019058 A2 WO2013019058 A2 WO 2013019058A2 KR 2012006096 W KR2012006096 W KR 2012006096W WO 2013019058 A2 WO2013019058 A2 WO 2013019058A2
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WIPO (PCT)
Prior art keywords
acid
cancer
pharmaceutical composition
metastasis
recurrence
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PCT/KR2012/006096
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French (fr)
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WO2013019058A3 (en
Inventor
Sung-Wuk Kim
Chang-Hee Min
Ja-Seong Koo
Sang-Ouk Sun
Yong-Eun Kim
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Hanall Biopharma Co., Ltd.
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Publication of WO2013019058A2 publication Critical patent/WO2013019058A2/en
Publication of WO2013019058A3 publication Critical patent/WO2013019058A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a pharmaceutical composition for inhibiting cancer recurrence or metastasis and a method for inhibiting cancer recurrence or metastasis using the same. More specifically, the present invention relates to a pharmaceutical composition and a method for effectively inhibiting the recurrence or metastasis of a cancer cell through the decrease of cancer stem cell and the inhibition of epithelial-to-mesenchymal transition (EMT, below).
  • EMT epithelial-to-mesenchymal transition
  • a biguanide based antidiabetic agent is used as an oral preparation for the treatment of diabete millitus, and shows an excellent hypoglycemic effect, causes no glycopenia or hyperinsulinemia and can prevent complications. Thus, it is one of the agents that are typically used for the treatment of non-insulin-dependent diabete mellitus(Type 2 diabetes). Recently, many researches on metformin— one of biguanide based antidiabetic agents— have been made. As a result thereof, an article reporting that metformin inhibits the cancer cell proliferation and tumor growth was published, and it was verified through an epidemiological investigation that cancer incidence and mortality due to the cancer decrease in the patients who take metformin.
  • metformin inhibits the action of Complex I in the electron transport system of mitochondria to disturb the energy production in the cell, whereby it acts as a substance inhibiting the cancer cell proliferation. This is why it is studied as an anticancer agent that controls the metabolism of a cancer cell.
  • 2-deoxy-D-glucose (2-DG, below) is a glucose derivative and known as a drug inhibiting the cancer cell growth by the restriction of sugar uptake which is the energy source of cancer cells.
  • Cancer cells are in need of lots of energy for supporting the rapid cell division. As well as the cancer cells which are divided under the aerobic condition, even the cancer cells which are somewhat slowly divided under the low concentration of oxygen or anaerobic condition require the energy for their survival.
  • 2-DG blocks the isomerization step of glucose 6-phosphate— i.e., isomerization of glucose 6-phosphate to fructose 6-phosphate— in the glycolysis which is a procedure of producing energy (ATP) required for the growth and maintenance of the cell from D-glucose, whereby it interrupts the energy supply to the cancer cell and ultimately inhibits the cell proliferation.
  • ATP energy
  • 2-DG as a novel active ingredient that can effectively inhibit cancer recurrence or metastasis.
  • compositions useful for the prevention or inhibition of cancer recurrence or cancer metastasis comprising 2-DG and a biguanide based antidiabetic agent as active ingredients.
  • This composition can effectively inhibit cancer recurrence or metastasis using a smaller amount thereof to reduce the probability of side effect development and to increase the therapeutic effect through the synergistic action of said two active ingredients.
  • it is another object of the present invention to provide a pharmaceutical composition comprising a further active ingredient in addition to said two active ingredients, which can more improve the synergistic effect for inhibiting cancer recurrence or metastasis.
  • the present invention provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising 2-DG as an active ingredient.
  • the present invention also provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients.
  • the present invention also provides a method for inhibiting cancer recurrence or metastasis, which comprises administering an effective amount of said pharmaceutical composition.
  • cancer stem cell circulating in the blood has both a cancer stem cell and an EMT marker, and the cancer stem cell has a correlation with EMT from a fact that most cancer cells from which EMT is derived are cancer stem cells. Thus, if the cancer stem cell from which EMT is derived is removed, cancer recurrence or metastasis may be inhibited.
  • 2-DG is a substance controlling the metabolism of a cancer cell by inhibiting the glycolysis in a cancer cell which requires a lot of energy (ATP).
  • the cancer cell which has a limited amount of energy due to the glycolysis suppression by 2-DG sets the energy supply mechanism based on TCA cycle, glutamine, etc. so that it gets a supply of the required amount of energy (ATP) for its division from the electron transport system of mitochondria.
  • ATP energy
  • Cancer stem cells refer to the cancer cells which have the unique ability of stem cells of self-regeneration or differentiation.
  • the reason why the existing chemotherapies fail to treat cancers and result in tumor recurrences is that most of the anticancer agents developed hitherto target only the rapidly proliferated cells and thus the cancer stem cells characterized by their slow proliferation can survive such chemotherapies. Accordingly, the prognosis is not good and the patients become to show resistance to the existing chemotherapies.
  • TGF ⁇ also participates in the development of cancer stem cells in the breast cancer.
  • the cells subjected to EMT by TGF ⁇ become to acquire the ability of cancer initiation and cell mobility at the same time, whereby the cancer stem cells can move a long distance and form tumors in a new tissue.
  • the suppression and prevention of cancer initiation and EMT ability of cancer stem cells may be an important therapy in reducing the risk of cancer metastasis and cancer recurrence.
  • the pharmaceutical composition of the present invention can exhibit a superior effect on the cancer stem cells having the above stated problems. Also, cancer recurrence and/or metastasis may be effectively inhibited according to the effective suppression of EMT ability, whereby a remarkably improved prognosis can be expected.
  • the specific diseases to which the pharmaceutical composition of the present invention can be applied may include various cancers, such as for example, uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, renal cancer, hematologic malignancy, pancreatic cancer, prostate cancer, thyroid cancer, liver cancer, etc.
  • the disease may be any one selected from the group consisting of uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, hematologic malignancy and liver cancer, and more preferably it may be colon cancer or breast cancer.
  • composition of the present invention may further include other active ingredients in addition to 2-DG or the combination of 2-DG and a biguanide based antidiabetic agent to show more synergistic effect for the inhibition of cancer metastasis and recurrence.
  • One embodiment of the present invention provides a pharmaceutical composition for inhibiting cancer recurrence or metastasis, comprising 2-deoxy-D- glucose of the following formula 1 as an active ingredient:
  • the content of 2-DG may be easily determined by a person skilled in the art depending on the age, gender, severity of disease, body weight, etc. of the patient. Preferably, it is contained in the amount of 5 to 80% by weight based on the total weight of the unit dosage.
  • 2-DG may be administered once or several times a day in a form of formulation containing total 10 to 4,000 mg, and more preferably it may be administered once or several times a day in a form of formulation containing total 100 to 2,500 mg.
  • Another embodiment of the present invention provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients.
  • Said biguanide based antidiabetic agent includes one or more selected from the group consisting of metformin of the following formula 2, phenformin of the following formula 3 and buformin of the following formula 4:
  • a pharmaceutical composition for the inhibition of cancer recurrence or metastasis comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients
  • a pharmaceutical composition for the inhibition of cancer recurrence or metastasis comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients
  • the two unit dosage forms each of which comprises one active ingredient, as well as a single dosage form comprising these two active ingredients together.
  • the two unit dosage forms may be comprehensively understood to be within the scope of "a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients.”
  • metformin As the biguanide based antidiabetic agent, one or more selected from metformin, phenformin and buformin may be used. Metformin or phenformin may be preferably used, and metformin may be most preferably used.
  • the daily dose of the biguanide based antidiabetic agent may differ depending on the kind of drug. Specifically, the preparation containing 100 mg ⁇ 4,000 mg, preferably 250 mg ⁇ 2,000 mg, of metformin, the preparation containing 10 mg ⁇ 200 mg, preferably 25 mg ⁇ 100 mg, of phenformin, or the preparation containing 10 mg ⁇ 500 mg, preferably 25 mg ⁇ 300 mg, of buformin may be administered once or over several times a day.
  • the pharmaceutical composition may comprise a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG in the weight ratio of 1 : 400 to 400 : 1 , more preferably in the weight ratio of 1 : 100 to 20 : 1.
  • the biguanide based antidiabetic agent may be used in itself or in the form of its pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt may be an acid addition salt formed by using an organic acid or inorganic acid wherein the organic acid includes, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifiuoroacetic 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 and methanesulfonic acid, and the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid.
  • the above mentioned acid addition salt can be prepared by the conventional salt forming method of, such as for example, a) directly mixing the compound of formula 2, 3 or 4 with an acid, b) dissolving one of these compounds in a solvent or a water-containing solvent and then mixing, or c) placing the compound of formula 2, 3 or 4 and the acid in a solvent or in an acid among the hydrated solvent and then mixing.
  • the possible salt may further include GABA salt, gabapentin salt, pregabalin salt, nicotinate, adipate, hemimalonate, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornitine salt, aspartate, etc.
  • the pharmaceutically acceptable salt of said biguanide based antidiabetic agent may include the salt with an acid selected from the group consisting of formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifiuoroacetic 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, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, dichloroacetic acid, aminooxy acetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid.
  • the salt may preferably be hydrochloride
  • composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the above mentioned active ingredients.
  • the "pharmaceutically acceptable carrier” described above means a pharmaceutical additive that is useful for formulating the pharmaceutical composition and non-toxic and unsusceptible under the condition of use.
  • the specific ratio of content of such a carrier may be determined by the standard pharmaceutical practices, as well as the solubility and chemical characteristics of the active ingredients and the administration route selected.
  • the pharmaceutical composition of the present invention may be formulated to a form suitable for the desired administration method using such additives as excipient, disintegrator, sweetner, binder, coating agent, expansion agent, lubricant, flavor, etc. as a pharmaceutically acceptable carrier.
  • the amount of carrier needed per a unit dosage may be the amount enough for providing the size and administration form by which the compliance of the subject can be increased.
  • the preparations may be oral or parenteral forms, such as for example, tablet, particulate, capsule containing liquid or powder, pill, granule, powder, troches (including filled with liquid), chew, multi- and nano-particle, gel, solid solution, liposome, film (including mucosa-adhesive), ovule, spray and liquid, but are not limited thereto.
  • the liquid for example, includes suspension, solution, syrup and elixir, but is not limited thereto.
  • the tablet may further comprise a disintegrator besides the active ingredients.
  • a disintegrator include starches or modified starches such as sodium starch glycolate, corn starch, potato starch, pregelatinized starch, etc., clays such as bentonite, montmorillonite, veegum, etc., celluloses such as low substituted hydroxypropyl cellulose, etc., alginates such as sodium alginate, alginic acid, etc., cross-linked celluloses such as sodium croscarmellose, etc., cross-linked polymers such as crospovidone, etc., effervescent agents such as sodium bicarbonate, citric acid, etc. or their mixtures.
  • the disintegrator occupies preferably about 0.5 to about 30% by weight, more preferably about 1 to about 20% by weight, of the dosage form, but not limited thereto.
  • a tablet may further comprise a binder for the purpose of having adhesive power.
  • a binder for the purpose of having adhesive power.
  • Unlimited examples of the binder include, for example, gelatin, sugar, natural or synthetic gum, polyvinylpyrrolidone (povidone), polyvinyl alcohol, co-povidone, starch, hydroxypropyl cellulose, hypromellose, etc.
  • the binder occupies preferably about 0.1 to about 40% by weight, more preferably about 0.5 to about 25% by weight, of the dosage form, but not limited thereto.
  • a tablet may further comprise a diluent.
  • diluent include, for example, starch, microcrystalline cellulose, lactose, glucose, mannitol, alginate, alkaline earth metal, polyethylene glycol, dicalcium phosphate, etc.
  • the diluent occupies preferably about 0.5 to about 90% by weight, more preferably about 2 to about 75% by weight, of the dosage form, but not limited thereto.
  • a tablet may further comprise a lubricant.
  • a lubricant include, for example, talc, stearic acid, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, hydrogenated vegetable oil, polyethylene glycol, etc.
  • the lubricant occupies preferably about 0.1 to about 20% by weight, more preferably about 0.2 to about 10% by weight, of the dosage form, but not limited thereto.
  • a tablet may selectively comprise a surfactant such as sodium lauryl sulfate and polysorbate 80, and a glidant such as colloidal silicon dioxide, hydrated silicon dioxide and talc.
  • the surfactant preferably occupies about 0.1 to about 20% by weight of the dosage form, and the glidant preferably occupies about 0.1 to about 20% by weight of the dosage form, but not limited thereto.
  • ingredients also able to be included are antioxidant, coloring agent, flavor, preservative, taste masker, etc.
  • a tablet may be formed by a direct compression of the ingredients contained in a tablet as mentioned above or by a roller compression. Otherwise, the above stated ingredients contained in a tablet may be wet-, dry- or melt-granulated, melt-congealed, or extruded, prior to the tablet formulation.
  • the form of final preparation may include one or more layers, be coated or not coated, or be capsulated.
  • modified release form may be formulated as an immediate release and/or modified release form, and this modified release form includes delayed-, sustained-, pulsed-, controlled-, targeted- or programmed release form.
  • the matrix base used for preparing the modified release tablet is not particularly limited, but may be one or more selected from the group consisting of enteric polymers, hydrophobic materials, hydrophilic polymers, etc.
  • enteric polymers include polyvinyl acetate phthalate, polymethacrylate copolymers such as poly(methacrylic acid, methyl methacrylate)copolymer and poly(methacrylic acid, ethyl acrylate)copolymer, hypromellose phthalate, hypromellose acetate succinate, shellac, cellulose acetate phthalate, cellulose propionate phthalate, etc.
  • the hydrophobic materials should be pharmaceutically acceptable, and unlimited examples thereof include polyvinyl acetate, ethyl cellulose and cellulose acetate, fatty acid and fatty acid esters, fatty alcohols, etc.
  • glyceryl palmitostearate glyceryl stearate, glyceryl behenate, cetyl palmitate, glyceryl monooleate, stearic acid, etc.
  • cetostearyl alcohol, cetyl alcohol, stearyl alcohol, etc. may be used as the fatty alcohols.
  • hydrophilic polymers examples include sugars, cellulose derivatives, gums, proteins, polyvinyl derivatives, polyethylene derivatives, carboxyvinyl polymers, etc.
  • the sugars may include dextrin, polydextrin, dextran, pectin and pectin derivatives, alginate, polygalactronic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose, amylopectin, etc.
  • the cellulose derivatives may include hypromellose, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl methyl cellulose, etc.
  • the gums may include guar gum, locust bean gum, tragacanth, carrageenan, acacia gum, arabic gum, gellan gum, xanthan gum, etc.
  • the proteins may selectively include gelatin, casein, zein, etc.
  • the polyvinyl derivatives may include polyvinyl alcohol, polyvinyl pyrrolidone, etc.
  • the polyethylene derivatives may include polyethylene glycol, polyethylene oxide, etc.
  • the carboxyvinyl polymers may include carbomer, etc.
  • Formulations and pharmaceutically acceptable carriers of the pharmaceutical composition according to the present invention are not specially limited to the above, and may be suitably selected based on the techniques known in the art.
  • the effective amount for the treatment or prevention of various cancers may be adjusted depending on various factors such as disease type, severity of disease, active ingredient contained in the composition, kind and content of other ingredient, formulation type, age, weight, general hygienic status, gender and diet of patient, administration time, administration route, plasma clearance of the composition, treatment period, co-used drug, etc.
  • the pharmaceutical composition of the present invention may be administered once to several times a day at an effective dose of total of 20 to 8,000 mg at one time.
  • the dose or amount of administration of each active ingredient should be adequate not to cause any side effect.
  • Another embodiment of the present invention provides a pharmaceutical composition further comprising an anticancer agent as an active ingredient, in addition to a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2- DG.
  • It includes three unit dosage forms which comprise the active ingredients, respectively, as well as a single unit dosage form comprising an anticancer agent as another active ingredient in addition to a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG.
  • these three unit dosage forms are administered simultaneously or at different times having some intervals so that the three active ingredients contained in each of these unit dosage forms are present in the body together with each other and produce a synergistic action.
  • the effect of treatment may be more improved in various aspects such as alleviation or amelioration of symptom, decrease of scope of disease, delay or alleviation of disease progression, improvement, alleviation or stabilization of disease condition, partial or complete recovery, survival extension, other advantageous result of treatment, etc.
  • any known anticancer agent may be used.
  • known chemotherapeutic agents such as alkylating agent, metabolic antagonist, natural preparation, hormone, antagonist, etc. and biological products such as immunotherapeutic agent, genetherapeutic agent, etc. may be mentioned.
  • one or more drugs selected from the group consisting of the following may be used: nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semaxanib, bosutinib, axxtinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuxumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylamino
  • Another embodiment of the present invention provides a method for inhibiting cancer recurrence or metastasis, which comprises administering an effective amount of said pharmaceutical composition.
  • the pharmaceutical composition may comprise 2-DG alone as an active ingredient, and preferably further comprise a biguanide based antidiabetic agent or its pharmaceutically acceptable salt. It may additionally comprise various known anticancer agents as an active ingredient besides the above, and preferable type and content thereof have been described above in connection with the pharmaceutical composition.
  • the above method comprises the administration step of said 2-deoxy- D-glucose in the amount of 10 to 4000 mg.
  • the above method preferably comprises the administration step of said biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-deoxy-D-glucose in the weight ratio of 1 : 400 to 400 : 1.
  • the method further comprises the step of confirming the subject in need of the inhibition of cancer recurrence or metastasis.
  • Said subject may preferably be mammals including a human being, more preferably be a human being.
  • the present invention identified the activity of 2-DG for the inhibition of mammosphere formation and EMT, indicating its inhibitory effect against proliferation of cancer stem cell and cancer metastasis, which has not been known hitherto.
  • the present invention also identified that the combined use of metformin— one of biguanide based antidiabetic agents— or its pharmaceutically acceptable salt and 2-DG can inhibit cancer metastasis and steeply decrease cancer recurrence in comparison to the single use of said active ingredients, resulting in a superior effect in the inhibition of cancer metastasis and recurrence when the same dosage is applied.
  • the pharmaceutical composition of the present invention can be used to show substantially more excellent effect with less amount of each active ingredient, reducing the side effects due to the use of excess drug and due to their pharmacological action, and providing more efficacious treatment. Moreover, when it is used along with an anticancer agent to which resistance is shown, it is expected that more synergistic effect can be exhibited. Therefore, the pharmaceutical composition for inhibiting cancer recurrence or metastasis according to the present invention can be used very advantageously for the treatment and prevention of metastasis or recurrence of various cancer diseases.
  • Figure 1 is a graph representing mammosphere forming efficiency (MFE(%)) measured by Experimental examples 1 of the present invention.
  • MCF-7 cell line derived from the human breast cancer the inhibitory effect against mammosphere formation of the exclusive composition comprising 2-DG alone and the combined composition comprising metformin hydrochloride— one of biguanide based antidiabetic agents— and 2-DG was measured, and the brief experimental procedure is as follows.
  • MCF-7 cells derived from the human breast cancer were maintained (culture temperature: 37 ° C, pH: 7.0 ⁇ 7.4) in a DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco Life Technologies (USA)) to the confluence of 80% of the culture dish area, and subsequently they were used for the experiment of identifying the ability for the mammosphere formation.
  • the culture media of the MCF-7 cell culture was removed to harvest cells to which was added serum free DMEM/F12 culture media (Gibco Life Technologies (USA)) to make MCF-7 cells a single cell.
  • the cells were counted and introduced to a 6 well plate at a density of 9000 cells/well.
  • the 6 well plate used herein was Ultralow attachment 6 well plate (Corning, Cat # 3471) wherein the cells do not adhere to the plate.
  • the culture media for the mammosphere formation was prepared to contain 2% B27 (Invitrogen), 20 ng/mL EGF (Sigma), 5 / g/mL insulin (Sigma) and 1 pg/mL hydrocortisone (Sigma) in DMEM/F12 media.
  • Cells were grown in a C0 2 incubator for 24 h and treated with 0.5 raM, 1 mM or 3 raM metformin hydrochloride and 2-DG, both as single or combined manner. Then, the culture media for the mammosphere formation as prepared above was added to make the total culture volume of 3 mL. As the control, cells treated with neither metformin hydrochloride not 2-DG were used.
  • MFE(%) The equation for calculating the mammosphere forming efficiency (MFE(%)) is as follows.
  • MFE(%) (Number of Mammosphere formed / Number of Cells Seeded) x 100
  • the mammosphere forming efficiencies (MFE(%)) in the well plates of single or combined treatment of metformin hydrochloride and 2- DG were compared with the mammosphere forming efficiency (MFE(%)) in the well plate not treated with metformin hydrochloride or 2-DG to represent the inhibition (%) of mammosphere formation in the following Table 1 and to represent the mammosphere forming efficiency for each group in the following Table 1 and Figure 1.
  • metformin hydrochloride and 2-DG were used in the ratio of 1 :1 at the concentrations described in Table 1, respectively.
  • metformin hydrochloride and 2-DG exhibits a superior synergistic effect in the inhibition of mammosphere formation to the single use of each drug. More specifically, when metformin hydrochloride and 2-DG were used independently at a concentration of 1 mM, the inhibition (%) of mammosphere formation was about 47.7% in the case of metformin alone, about 34.9% in the case of 2-DG, but about 69.1% in the case of combined use thereof at the same concentration. Thus, it was demonstrated that the combined use showed an effect increased by about 1.44 fold and about 1.98 fold compared with the single use of each drug at the same concentration.
  • the present experimental results show the inhibitory effect against formation and proliferation of cancer stem cells by the single treatment of 2-DG, as well as the superior effect by the combined use of metformin— a biguanide based antidiabetic agent— and 2-DG to the single use of each at a lower concentration due to their synergistic action. Consequently, the present invention demonstrated that the amount of each drug used can be reduced with the decrease of probability of side effect occurrence and simultaneously with the remarkably improved inhibitory effect against the formation and proliferation of cancer stem cells. Thus, a superior effect may be expected in the inhibition of cancer recurrence and metastasis by the single and combined use of each drug as mentioned above.
  • MCF-7 cells derived from the human breast cancer were maintainted in a DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco Life Technologies (USA)), introduced into a 6 well plate at a density of about 5xl0 5 cells/well, and grown in a 5% C0 2 incubator (culture temperature: 37 ° C, pH: 7.0 ⁇ 7.4).
  • the cells were treated with 2-DG or metformin hydrochloride alone at concentrations of 0.1 mM, 1 mM and 10 mM, respectively, and also treated with the combination of metformin hydrochloride and 2-DG at concentrations of 0.1 mM, 1 mM and 10 mM, respectively, and then incubated for 48 h.
  • metformin hydrochloride and 2-DG were used in the ratio of 1 : 1 at concentrations as represented in the following Table 2.
  • the cultivated cells were lysed by 10 mM Tris HC1 (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na 4 P 2 0 7 , 2 mM Na 3 V0 4 , 1% Triton X-100, 10% glycerol, 0.5% deoxycholate and 1 mM PMSF, and 25 ⁇ & of cell lysate by the protein assay was subjected to electrophoresis on SDS-PAGE gel and transferred to a PMSF (Amersham Life Science) membrane.
  • said membrane was reacted with a rat monoclonal anti-human Snail (purchased from Cell signaling Technology and diluted in 5% skim milk (Difco) at a ratio of 1 :1000) or a mouse monoclonal anti-P-actin (purchased from Santa cruz Biotechnology and diluted in 5% skim milk (Difco) at a ratio of 1:5000), and the remaining antibodies that were not bound were removed.
  • the anti-P-actin was used as an antibody to show that the amounts of proteins loaded on the SDS-PAGE gel were same.
  • the membrane was reacted with an anti-rat IgG as a secondary antibody (purchased from Cell signaling Technology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000) or an anti-mouse IgG (purchased from Santa cruz Biotechnology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000).
  • an anti-rat IgG purchased from Cell signaling Technology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000
  • an anti-mouse IgG purchased from Santa cruz Biotechnology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000.
  • ECL Plus system Anamersham Life Science
  • the amount of drug used may be remarkably reduced since the same effect as is obtained by 1 mM of metformin hydrochloride alone can be achieved by its combined use with 2-DG at a concentration of 0.1 mM which corresponds to only one tenth of 1 mM.
  • the single use and combined use are compared with each other on the basis of 1 mM of each drug, the single use of metformin hydrochloride and 2-DG reduces the Snail expression rate by about 27% and 47%, respectively, whereas the combined use thereof remarkably reduces the Snail expression rate by about 84%, which indicates an increase of effect by a factor of about 3 or more and about 1.79 or more compared with their single use.
  • the single or combined use of the drugs effectively decreases the expression of Snail protein.
  • the Snail protein is a marker for EMT.
  • a biguanide based drug or 2-DG alone inhibits EMT, but their combined use can induce much more synergistic EMT inhibition.
  • 2-DG and the combined use of a biguanide based antidiabetic drug and 2-DG can show the inhibition of EMT through the suppression of Snail expression, and furthermore an effect of inhibiting cancer metastasis.
  • metformin hydrochloride 500 g of metformin hydrochloride, 300 g of 2-DG and 250 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery, Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35- sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin hydrochloride and 2-DG.
  • a film coating layer was formed using 30 g of opadry OY-C-7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 500 mg of metformin hydrochloride and 300 mg of 2-DG in one tablet.
  • a tablet comprising metformin acetate and 2-
  • a film coating layer was formed using 30 g of opadry OY-C- 7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 571 mg of metformin acetate and 300 mg of 2-DG in one tablet.
  • a tablet comprising metformin nicotinate and
  • metformin nicotinate 300 g of 2-DG and 138.5 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery., Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35- sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin nicotinate and 2-DG.
  • a film coating layer was formed using 30 g of opadry OY-C-7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 761.5 mg of metformin nicotinate and 300 mg of 2-DG in one tablet.
  • a tablet comprising metformin malonate and
  • a film coating layer was formed using 30 g of opadry OY-C- 7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 704 mg of metformin malonate and 300 mg of 2-DG in one tablet.
  • SFC-30N Sejong Pharmatech Co., Ltd., Korea
  • Example 2 The same procedure as Example 1 was carried out except that 100 g of phenformin hydrochloride was used instead of 500 g of metformin hydrochloride to give a tablet comprising 100 mg of phenformin hydrochloride and 300 mg of 2-DG in one tablet.
  • Example 2 The same procedure as Example 1 was carried out except that 50 g of buformin hydrochloride was used instead of 500 g of metformin hydrochloride to give a tablet comprising 50 mg of buformin hydrochloride and 300 mg of 2-DG in one tablet.

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Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer recurrence or metastasis and a method for inhibiting cancer recurrence or metastasis using the same. The present invention identified the activity of 2-deoxy-D-glucose (2-DG, below) for the inhibition of mammosphere formation and EMT, indicating its inhibitory effect against proliferation of cancer stem cell and cancer metastasis, which has not been known hitherto. The present invention also identified that the combined use of a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG can inhibit cancer metastasis and steeply decrease cancer recurrence in comparison to the single use of said active ingredients, resulting in a superior effect in the inhibition of cancer metastasis and recurrence when the same dosage is applied. Therefore, the pharmaceutical composition of the present invention can be used to show substantially more excellent effect with less amount of each active ingredient, reducing the side effects due to the use of excess drug and due to their pharmacological action, and providing more efficacious treatment. Moreover, when it is used along with an anticancer agent to which resistance is shown, it is expected that more synergistic effect can be exhibited. Therefore, the pharmaceutical composition and method for inhibiting cancer recurrence or metastasis according to the present invention can be used very advantageously for the treatment and prevention of metastasis or recurrence of various cancer diseases.

Description

[SPECIFICATION]
[TITLE OF THE INVENTION]
PHARMACEUTICAL COMPOSITION FOR INHIBITING CANCER RECURRENCE OR METASTASIS
[TECHNICAL FIELD]
The present invention relates to a pharmaceutical composition for inhibiting cancer recurrence or metastasis and a method for inhibiting cancer recurrence or metastasis using the same. More specifically, the present invention relates to a pharmaceutical composition and a method for effectively inhibiting the recurrence or metastasis of a cancer cell through the decrease of cancer stem cell and the inhibition of epithelial-to-mesenchymal transition (EMT, below).
[BACKGROUND ART]
A biguanide based antidiabetic agent is used as an oral preparation for the treatment of diabete millitus, and shows an excellent hypoglycemic effect, causes no glycopenia or hyperinsulinemia and can prevent complications. Thus, it is one of the agents that are typically used for the treatment of non-insulin-dependent diabete mellitus(Type 2 diabetes). Recently, many researches on metformin— one of biguanide based antidiabetic agents— have been made. As a result thereof, an article reporting that metformin inhibits the cancer cell proliferation and tumor growth was published, and it was verified through an epidemiological investigation that cancer incidence and mortality due to the cancer decrease in the patients who take metformin. Specifically, metformin inhibits the action of Complex I in the electron transport system of mitochondria to disturb the energy production in the cell, whereby it acts as a substance inhibiting the cancer cell proliferation. This is why it is studied as an anticancer agent that controls the metabolism of a cancer cell.
On the other hand, 2-deoxy-D-glucose (2-DG, below) is a glucose derivative and known as a drug inhibiting the cancer cell growth by the restriction of sugar uptake which is the energy source of cancer cells. Cancer cells are in need of lots of energy for supporting the rapid cell division. As well as the cancer cells which are divided under the aerobic condition, even the cancer cells which are somewhat slowly divided under the low concentration of oxygen or anaerobic condition require the energy for their survival. 2-DG blocks the isomerization step of glucose 6-phosphate— i.e., isomerization of glucose 6-phosphate to fructose 6-phosphate— in the glycolysis which is a procedure of producing energy (ATP) required for the growth and maintenance of the cell from D-glucose, whereby it interrupts the energy supply to the cancer cell and ultimately inhibits the cell proliferation.
Most anticancer agents developed up to now were drugs targeting the rapidly proliferating cells. Thus, cancer stem cells having the feature of slow proliferation may survive the chemotherapy using such drugs, which results in the problem of causing unsatisfactory prognosis and resistance to the earlier chemotherapy. Thus, there is a keen need to develop a drug that can exhibit a therapeutic effect for cancer stem cells to which the earlier anticancer agents have a limit, and furthermore that can effectively inhibit the cancer recurrence and/or metastasis.
In this regard, researches for the inhibition of cancer recurrence or metastasis using 2-DG or for the synergistic inhibition of cancer recurrence or metastasis through the combined therapy of a biguanide based antidiabetic agent and 2-DG have not been reported at all until now. [CONTENTS OF THE INVENTION]
[PROBLEMS TO BE SOLVED]
Thus, it is an object of the present invention to provide 2-DG as a novel active ingredient that can effectively inhibit cancer recurrence or metastasis.
Also, it is another object of the present invention to provide a pharmaceutical composition useful for the prevention or inhibition of cancer recurrence or cancer metastasis, comprising 2-DG and a biguanide based antidiabetic agent as active ingredients. This composition can effectively inhibit cancer recurrence or metastasis using a smaller amount thereof to reduce the probability of side effect development and to increase the therapeutic effect through the synergistic action of said two active ingredients.
Also, it is another object of the present invention to provide a pharmaceutical composition comprising a further active ingredient in addition to said two active ingredients, which can more improve the synergistic effect for inhibiting cancer recurrence or metastasis.
Also, it is another object of the present invention to provide a method for inhibiting cancer recurrence or metastasis, which comprises administering said pharmaceutical composition.
[TECHNICAL MEANS]
As a result of studies by the present inventors, it has been verified that 2-DG can effectively inhibit cancer metastasis or cancer recurrence. Furthermore, the present inventors have surprisingly confirmed that a synergistic effect can be shown for the inhibition of cancer metastasis or cancer recurrence when 2-DG is administered along with a biguanide based antidiabetic agent or its pharmaceutically acceptable salt. It has also been confirmed that the dose of each drug can be decreased, the probability of side effect development can be reduced and the therapeutic effect can be remarkably increased due to the synergistic effect of the two drugs, whereby the present invention has been completed.
Specifically, when 2-DG is used alone as well as when a biguanide based antidiabetic agent or its pharmaceutically acceptable salt is used alone, it was confirmed that an excellent effect in the inhibition of mammosphere formation and epithelial-to-mesenchymal transition of a cancer cell as an indicator of the inhibitory effect against cancer metastasis or cancer recurrence is shown. Furthermore, in comparison to the cases when 2-DG or a biguanide based antidiabetic agent is used alone, their combined use shows a surprisingly synergistic effect in the inhibition of mammosphere formation and epithelial-to-mesenchymal transition of a cancer cell.
Thus, the present invention provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising 2-DG as an active ingredient.
The present invention also provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients.
The present invention also provides a method for inhibiting cancer recurrence or metastasis, which comprises administering an effective amount of said pharmaceutical composition.
Hereinafter, the present invention is explained more in detail.
The recent studies confirmed that a cancer cell circulating in the blood has both a cancer stem cell and an EMT marker, and the cancer stem cell has a correlation with EMT from a fact that most cancer cells from which EMT is derived are cancer stem cells. Thus, if the cancer stem cell from which EMT is derived is removed, cancer recurrence or metastasis may be inhibited.
From the experimental results by the present inventors, it has been confirmed that the mammosphere formation is inhibited by 2-DG, and at the same time surprisingly confirmed that a synergistic inhibitory effect more than expected is shown when 2-DG is used in combination with a biguanide based antidiabetic agent such as metformin. Furthermore, it has been confirmed that the single use of 2-DG can decrease the expression of a protein— in particular, Snail 1— participating in the process (EMT) of changing a cancer epithelial cell to a mesenchymal cell, which is correlated with cancer metastasis, and it has been confirmed that the combined use of metformin and 2-DG can more definitely decrease this expression (see Experimental examples 1 and 2).
2-DG is a substance controlling the metabolism of a cancer cell by inhibiting the glycolysis in a cancer cell which requires a lot of energy (ATP). The cancer cell which has a limited amount of energy due to the glycolysis suppression by 2-DG sets the energy supply mechanism based on TCA cycle, glutamine, etc. so that it gets a supply of the required amount of energy (ATP) for its division from the electron transport system of mitochondria. It is considered that the combined use of 2-DG and metformin can show the synergistic action since metformin, one of biguanide based antidiabetic agents, suppresses the action of Complex I in the mitochondrial electron transport system.
Cancer stem cells refer to the cancer cells which have the unique ability of stem cells of self-regeneration or differentiation. The reason why the existing chemotherapies fail to treat cancers and result in tumor recurrences is that most of the anticancer agents developed hitherto target only the rapidly proliferated cells and thus the cancer stem cells characterized by their slow proliferation can survive such chemotherapies. Accordingly, the prognosis is not good and the patients become to show resistance to the existing chemotherapies.
While, the epithelial-to-mesenchymal transition (EMT, below) occurs by various cytokines and growth factors, but particularly TGF β signaling pathway plays the most important role. TGF β also participates in the development of cancer stem cells in the breast cancer. The cells subjected to EMT by TGF β become to acquire the ability of cancer initiation and cell mobility at the same time, whereby the cancer stem cells can move a long distance and form tumors in a new tissue. Thus, the suppression and prevention of cancer initiation and EMT ability of cancer stem cells may be an important therapy in reducing the risk of cancer metastasis and cancer recurrence.
The pharmaceutical composition of the present invention can exhibit a superior effect on the cancer stem cells having the above stated problems. Also, cancer recurrence and/or metastasis may be effectively inhibited according to the effective suppression of EMT ability, whereby a remarkably improved prognosis can be expected.
Mesenchymal cells show resistance against radiation therapy or chemotherapy, which makes the treatment of cancer difficult. However, the combination therapy of a biguanide based antidiabetic agent and 2-DG disturbs the differentiation of cancer cells to mesenchymal cells and increases their susceptibility to drugs, resulting in the EMT suppression and more improved anticancer effect. Thus, it is expected that cancer metastasis and recurrence may be more effectively prevented. The specific diseases to which the pharmaceutical composition of the present invention can be applied may include various cancers, such as for example, uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, renal cancer, hematologic malignancy, pancreatic cancer, prostate cancer, thyroid cancer, liver cancer, etc. Preferably, the disease may be any one selected from the group consisting of uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, hematologic malignancy and liver cancer, and more preferably it may be colon cancer or breast cancer.
Also, the composition of the present invention may further include other active ingredients in addition to 2-DG or the combination of 2-DG and a biguanide based antidiabetic agent to show more synergistic effect for the inhibition of cancer metastasis and recurrence.
One embodiment of the present invention provides a pharmaceutical composition for inhibiting cancer recurrence or metastasis, comprising 2-deoxy-D- glucose of the following formula 1 as an active ingredient:
Formula 1
Figure imgf000009_0001
2-Deoxy-D-glucose (2-DG)
The content of 2-DG may be easily determined by a person skilled in the art depending on the age, gender, severity of disease, body weight, etc. of the patient. Preferably, it is contained in the amount of 5 to 80% by weight based on the total weight of the unit dosage.
Also, preferably, 2-DG may be administered once or several times a day in a form of formulation containing total 10 to 4,000 mg, and more preferably it may be administered once or several times a day in a form of formulation containing total 100 to 2,500 mg.
When the content of 2-DG is less than the above range, the effect of 2-DG may not reach the therapeutic level. When the content of 2-DG exceeds the above range, it may produce some unfavorable side effects due to the overdose and the administration may not be easily done due to the weight increase of the composition per se, which results in the problem that the pharmaceutical composition is not useful and accordingly not desirable.
Another embodiment of the present invention provides a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients.
Said biguanide based antidiabetic agent includes one or more selected from the group consisting of metformin of the following formula 2, phenformin of the following formula 3 and buformin of the following formula 4:
Formula 2: Metformin
Figure imgf000010_0001
Formula 3: Phenformin
Figure imgf000011_0001
Formula 4: Buformin
Figure imgf000011_0002
The phrase "a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients" as used herein is interpreted to comprehensively refer to a single dosage form such as an oral or injectable preparation that is taken or administered at one time or plural unit dosage forms that are separately administered for twice or more, unless otherwise specified.
That is, said phrase is interpreted to refer to the two unit dosage forms each of which comprises one active ingredient, as well as a single dosage form comprising these two active ingredients together. Thus, when the two unit dosage forms are administered simultaneously or at different times having some intervals so that the two active ingredients contained in these unit dosage forms are present in the body together with each other and produce a synergistic action, the two unit dosage forms may be comprehensively understood to be within the scope of "a pharmaceutical composition for the inhibition of cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG as active ingredients."
As the biguanide based antidiabetic agent, one or more selected from metformin, phenformin and buformin may be used. Metformin or phenformin may be preferably used, and metformin may be most preferably used.
The daily dose of the biguanide based antidiabetic agent may differ depending on the kind of drug. Specifically, the preparation containing 100 mg ~ 4,000 mg, preferably 250 mg ~ 2,000 mg, of metformin, the preparation containing 10 mg ~ 200 mg, preferably 25 mg ~ 100 mg, of phenformin, or the preparation containing 10 mg ~ 500 mg, preferably 25 mg ~ 300 mg, of buformin may be administered once or over several times a day.
Also, preferably, the pharmaceutical composition may comprise a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG in the weight ratio of 1 : 400 to 400 : 1 , more preferably in the weight ratio of 1 : 100 to 20 : 1.
When the weight ratio is out of the above mentioned range, it may not reach the therapeutic level which is required for the achievement of treatment desired by the respective active ingredient, or any side effect may be caused due to the excess administration. Otherwise, the medication may not be easy due to the weight increase of the composition per se, which makes the composition unuseful as a pharmaceutical composition and thus not desirable. On the other hand, the biguanide based antidiabetic agent may be used in itself or in the form of its pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be an acid addition salt formed by using an organic acid or inorganic acid wherein the organic acid includes, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifiuoroacetic 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 and methanesulfonic acid, and the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid.
The above mentioned acid addition salt can be prepared by the conventional salt forming method of, such as for example, a) directly mixing the compound of formula 2, 3 or 4 with an acid, b) dissolving one of these compounds in a solvent or a water-containing solvent and then mixing, or c) placing the compound of formula 2, 3 or 4 and the acid in a solvent or in an acid among the hydrated solvent and then mixing.
Besides the above, the possible salt may further include GABA salt, gabapentin salt, pregabalin salt, nicotinate, adipate, hemimalonate, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornitine salt, aspartate, etc.
In one embodiment, the pharmaceutically acceptable salt of said biguanide based antidiabetic agent may include the salt with an acid selected from the group consisting of formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifiuoroacetic 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, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, dichloroacetic acid, aminooxy acetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid. The salt may preferably be hydrochloride or acetate, and more preferably hydrochloride.
On the other hand, the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the above mentioned active ingredients.
The "pharmaceutically acceptable carrier" described above means a pharmaceutical additive that is useful for formulating the pharmaceutical composition and non-toxic and unsusceptible under the condition of use. The specific ratio of content of such a carrier may be determined by the standard pharmaceutical practices, as well as the solubility and chemical characteristics of the active ingredients and the administration route selected.
More specifically, the pharmaceutical composition of the present invention may be formulated to a form suitable for the desired administration method using such additives as excipient, disintegrator, sweetner, binder, coating agent, expansion agent, lubricant, flavor, etc. as a pharmaceutically acceptable carrier. The amount of carrier needed per a unit dosage may be the amount enough for providing the size and administration form by which the compliance of the subject can be increased.
The preparations may be oral or parenteral forms, such as for example, tablet, particulate, capsule containing liquid or powder, pill, granule, powder, troches (including filled with liquid), chew, multi- and nano-particle, gel, solid solution, liposome, film (including mucosa-adhesive), ovule, spray and liquid, but are not limited thereto.
The liquid, for example, includes suspension, solution, syrup and elixir, but is not limited thereto.
When the drug is formulated into a tablet, a typical form of oral preparation, the tablet may further comprise a disintegrator besides the active ingredients. Unlimited examples of the disintegrator include starches or modified starches such as sodium starch glycolate, corn starch, potato starch, pregelatinized starch, etc., clays such as bentonite, montmorillonite, veegum, etc., celluloses such as low substituted hydroxypropyl cellulose, etc., alginates such as sodium alginate, alginic acid, etc., cross-linked celluloses such as sodium croscarmellose, etc., cross-linked polymers such as crospovidone, etc., effervescent agents such as sodium bicarbonate, citric acid, etc. or their mixtures. The disintegrator occupies preferably about 0.5 to about 30% by weight, more preferably about 1 to about 20% by weight, of the dosage form, but not limited thereto.
Also, a tablet may further comprise a binder for the purpose of having adhesive power. Unlimited examples of the binder include, for example, gelatin, sugar, natural or synthetic gum, polyvinylpyrrolidone (povidone), polyvinyl alcohol, co-povidone, starch, hydroxypropyl cellulose, hypromellose, etc. The binder occupies preferably about 0.1 to about 40% by weight, more preferably about 0.5 to about 25% by weight, of the dosage form, but not limited thereto.
Also, a tablet may further comprise a diluent. Unlimited examples of the diluent include, for example, starch, microcrystalline cellulose, lactose, glucose, mannitol, alginate, alkaline earth metal, polyethylene glycol, dicalcium phosphate, etc. The diluent occupies preferably about 0.5 to about 90% by weight, more preferably about 2 to about 75% by weight, of the dosage form, but not limited thereto.
Also, a tablet may further comprise a lubricant. Unlimited examples of the lubricant include, for example, talc, stearic acid, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, hydrogenated vegetable oil, polyethylene glycol, etc. The lubricant occupies preferably about 0.1 to about 20% by weight, more preferably about 0.2 to about 10% by weight, of the dosage form, but not limited thereto.
Besides the above, a tablet may selectively comprise a surfactant such as sodium lauryl sulfate and polysorbate 80, and a glidant such as colloidal silicon dioxide, hydrated silicon dioxide and talc. The surfactant preferably occupies about 0.1 to about 20% by weight of the dosage form, and the glidant preferably occupies about 0.1 to about 20% by weight of the dosage form, but not limited thereto.
The ingredients also able to be included are antioxidant, coloring agent, flavor, preservative, taste masker, etc.
A tablet may be formed by a direct compression of the ingredients contained in a tablet as mentioned above or by a roller compression. Otherwise, the above stated ingredients contained in a tablet may be wet-, dry- or melt-granulated, melt-congealed, or extruded, prior to the tablet formulation. The form of final preparation may include one or more layers, be coated or not coated, or be capsulated.
Depending on the time of drug release, it may be formulated as an immediate release and/or modified release form, and this modified release form includes delayed-, sustained-, pulsed-, controlled-, targeted- or programmed release form. The matrix base used for preparing the modified release tablet is not particularly limited, but may be one or more selected from the group consisting of enteric polymers, hydrophobic materials, hydrophilic polymers, etc.
Unlimited examples of the enteric polymers include polyvinyl acetate phthalate, polymethacrylate copolymers such as poly(methacrylic acid, methyl methacrylate)copolymer and poly(methacrylic acid, ethyl acrylate)copolymer, hypromellose phthalate, hypromellose acetate succinate, shellac, cellulose acetate phthalate, cellulose propionate phthalate, etc.
The hydrophobic materials should be pharmaceutically acceptable, and unlimited examples thereof include polyvinyl acetate, ethyl cellulose and cellulose acetate, fatty acid and fatty acid esters, fatty alcohols, etc.
More specifically, glyceryl palmitostearate, glyceryl stearate, glyceryl behenate, cetyl palmitate, glyceryl monooleate, stearic acid, etc. may be used as the fatty acids and fatty acid esters, and cetostearyl alcohol, cetyl alcohol, stearyl alcohol, etc. may be used as the fatty alcohols.
Unlimited examples of the hydrophilic polymers include sugars, cellulose derivatives, gums, proteins, polyvinyl derivatives, polyethylene derivatives, carboxyvinyl polymers, etc.
More specifically, the sugars may include dextrin, polydextrin, dextran, pectin and pectin derivatives, alginate, polygalactronic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose, amylopectin, etc., and the cellulose derivatives may include hypromellose, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl methyl cellulose, etc. The gums may include guar gum, locust bean gum, tragacanth, carrageenan, acacia gum, arabic gum, gellan gum, xanthan gum, etc., the proteins may selectively include gelatin, casein, zein, etc., and the polyvinyl derivatives may include polyvinyl alcohol, polyvinyl pyrrolidone, etc. Also, the polyethylene derivatives may include polyethylene glycol, polyethylene oxide, etc., and the carboxyvinyl polymers may include carbomer, etc.
Formulations and pharmaceutically acceptable carriers of the pharmaceutical composition according to the present invention are not specially limited to the above, and may be suitably selected based on the techniques known in the art. Also, the effective amount for the treatment or prevention of various cancers may be adjusted depending on various factors such as disease type, severity of disease, active ingredient contained in the composition, kind and content of other ingredient, formulation type, age, weight, general hygienic status, gender and diet of patient, administration time, administration route, plasma clearance of the composition, treatment period, co-used drug, etc. For example, as for an adult, the pharmaceutical composition of the present invention may be administered once to several times a day at an effective dose of total of 20 to 8,000 mg at one time. However, it is obvious for a skilled artisan that the dose or amount of administration of each active ingredient should be adequate not to cause any side effect.
Another embodiment of the present invention provides a pharmaceutical composition further comprising an anticancer agent as an active ingredient, in addition to a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2- DG.
It includes three unit dosage forms which comprise the active ingredients, respectively, as well as a single unit dosage form comprising an anticancer agent as another active ingredient in addition to a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-DG. In other words, these three unit dosage forms are administered simultaneously or at different times having some intervals so that the three active ingredients contained in each of these unit dosage forms are present in the body together with each other and produce a synergistic action. According to this synergistic action, the effect of treatment may be more improved in various aspects such as alleviation or amelioration of symptom, decrease of scope of disease, delay or alleviation of disease progression, improvement, alleviation or stabilization of disease condition, partial or complete recovery, survival extension, other advantageous result of treatment, etc.
As the anticancer agent, any known anticancer agent may be used. For example, known chemotherapeutic agents such as alkylating agent, metabolic antagonist, natural preparation, hormone, antagonist, etc. and biological products such as immunotherapeutic agent, genetherapeutic agent, etc. may be mentioned.
More specifically, for example, one or more drugs selected from the group consisting of the following may be used: nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semaxanib, bosutinib, axxtinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuxumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastin, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonine, exemestane, aminoglutethimide, anagrelide, navelbine, fadrazol, tamoxifen, toremifen, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine and carmustine.
Another embodiment of the present invention provides a method for inhibiting cancer recurrence or metastasis, which comprises administering an effective amount of said pharmaceutical composition.
The pharmaceutical composition may comprise 2-DG alone as an active ingredient, and preferably further comprise a biguanide based antidiabetic agent or its pharmaceutically acceptable salt. It may additionally comprise various known anticancer agents as an active ingredient besides the above, and preferable type and content thereof have been described above in connection with the pharmaceutical composition.
In the case that the pharmaceutical composition has 2-deoxy-D-glucose as an active ingredient, the above method comprises the administration step of said 2-deoxy- D-glucose in the amount of 10 to 4000 mg.
Also, in the case that the pharmaceutical composition has a biguanide based antidiabetic agent or its pharmaceutically acceptable salt together with 2-DG, the above method preferably comprises the administration step of said biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-deoxy-D-glucose in the weight ratio of 1 : 400 to 400 : 1.
Preferably, prior to the administration step of said pharmaceutical composition, the method further comprises the step of confirming the subject in need of the inhibition of cancer recurrence or metastasis.
Said subject may preferably be mammals including a human being, more preferably be a human being.
[EFFECT OF THE INVENTION]
The present invention identified the activity of 2-DG for the inhibition of mammosphere formation and EMT, indicating its inhibitory effect against proliferation of cancer stem cell and cancer metastasis, which has not been known hitherto. The present invention also identified that the combined use of metformin— one of biguanide based antidiabetic agents— or its pharmaceutically acceptable salt and 2-DG can inhibit cancer metastasis and steeply decrease cancer recurrence in comparison to the single use of said active ingredients, resulting in a superior effect in the inhibition of cancer metastasis and recurrence when the same dosage is applied.
Therefore, the pharmaceutical composition of the present invention can be used to show substantially more excellent effect with less amount of each active ingredient, reducing the side effects due to the use of excess drug and due to their pharmacological action, and providing more efficacious treatment. Moreover, when it is used along with an anticancer agent to which resistance is shown, it is expected that more synergistic effect can be exhibited. Therefore, the pharmaceutical composition for inhibiting cancer recurrence or metastasis according to the present invention can be used very advantageously for the treatment and prevention of metastasis or recurrence of various cancer diseases.
[BRIEF DESCRIPTION OF DRAWINGS]
Figure 1 is a graph representing mammosphere forming efficiency (MFE(%)) measured by Experimental examples 1 of the present invention.
[DETAILED DESCRIPTION OF THE INVENTION]
Hereinafter, the following preferable Experimental examples and examples are provided only to assist the understanding of the present invention. However, it should be understood that they are intended to illustrate the present invention but not in any manner to limit the scope of the present invention.
[Experimental example 1] Identification of inhibition of mammosphere formation
In MCF-7 cell line derived from the human breast cancer, the inhibitory effect against mammosphere formation of the exclusive composition comprising 2-DG alone and the combined composition comprising metformin hydrochloride— one of biguanide based antidiabetic agents— and 2-DG was measured, and the brief experimental procedure is as follows.
MCF-7 cells derived from the human breast cancer (Korean Cell Line Bank) were maintained (culture temperature: 37 °C, pH: 7.0 ~ 7.4) in a DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco Life Technologies (USA)) to the confluence of 80% of the culture dish area, and subsequently they were used for the experiment of identifying the ability for the mammosphere formation. The culture media of the MCF-7 cell culture was removed to harvest cells to which was added serum free DMEM/F12 culture media (Gibco Life Technologies (USA)) to make MCF-7 cells a single cell. The cells were counted and introduced to a 6 well plate at a density of 9000 cells/well. The 6 well plate used herein was Ultralow attachment 6 well plate (Corning, Cat # 3471) wherein the cells do not adhere to the plate.
The culture media for the mammosphere formation was prepared to contain 2% B27 (Invitrogen), 20 ng/mL EGF (Sigma), 5 / g/mL insulin (Sigma) and 1 pg/mL hydrocortisone (Sigma) in DMEM/F12 media. Cells were grown in a C02 incubator for 24 h and treated with 0.5 raM, 1 mM or 3 raM metformin hydrochloride and 2-DG, both as single or combined manner. Then, the culture media for the mammosphere formation as prepared above was added to make the total culture volume of 3 mL. As the control, cells treated with neither metformin hydrochloride not 2-DG were used. Culture was performed for 8 days in a C02 incubator, during which the mammosphere forming efficiency and size were observed. After 8 days, the mammospheres having the size of 50 or more were counted to calculate the mammosphere forming efficiency (MFE(%)). The equation for calculating the mammosphere forming efficiency (MFE(%)) is as follows.
<Equation>
Mammosphere Forming Efficiency (MFE(%)) = (Number of Mammosphere formed / Number of Cells Seeded) x 100
After cultivation of 8 days, the mammosphere forming efficiencies (MFE(%)) in the well plates of single or combined treatment of metformin hydrochloride and 2- DG were compared with the mammosphere forming efficiency (MFE(%)) in the well plate not treated with metformin hydrochloride or 2-DG to represent the inhibition (%) of mammosphere formation in the following Table 1 and to represent the mammosphere forming efficiency for each group in the following Table 1 and Figure 1. In the case of combined treatment group, metformin hydrochloride and 2-DG were used in the ratio of 1 :1 at the concentrations described in Table 1, respectively.
Table 1
Results of inhibition (%) of mammosphere formation
Figure imgf000024_0001
As Table 1 and Figure 1 show, the experiment for studying the inhibition (%) of mammosphere formation by the treatment of each drug in MCF7 cells confirmed for the first time that the mammosphere formation is significantly suppressed as the drug concentration increases necessarily by the treatment of metformin alone and even by the treatment of 2-DG alone.
It was furthermore confirmed that the combined use of metformin hydrochloride and 2-DG exhibits a superior synergistic effect in the inhibition of mammosphere formation to the single use of each drug. More specifically, when metformin hydrochloride and 2-DG were used independently at a concentration of 1 mM, the inhibition (%) of mammosphere formation was about 47.7% in the case of metformin alone, about 34.9% in the case of 2-DG, but about 69.1% in the case of combined use thereof at the same concentration. Thus, it was demonstrated that the combined use showed an effect increased by about 1.44 fold and about 1.98 fold compared with the single use of each drug at the same concentration.
As reviewed above, the present experimental results show the inhibitory effect against formation and proliferation of cancer stem cells by the single treatment of 2-DG, as well as the superior effect by the combined use of metformin— a biguanide based antidiabetic agent— and 2-DG to the single use of each at a lower concentration due to their synergistic action. Consequently, the present invention demonstrated that the amount of each drug used can be reduced with the decrease of probability of side effect occurrence and simultaneously with the remarkably improved inhibitory effect against the formation and proliferation of cancer stem cells. Thus, a superior effect may be expected in the inhibition of cancer recurrence and metastasis by the single and combined use of each drug as mentioned above.
[Experimental example 2] Identification of EMT inhibition through the suppression of Snail expression In MCF-7 cell line derived from the human breast cancer, the EMT inhibition effect, particularly the suppression of Snail expression effect, by the combined composition comprising metformin hydrochloride— one of biguanide based antidiabetic agents— and 2-DG was measured, and the brief experimental procedure is as follows.
MCF-7 cells derived from the human breast cancer (Korean Cell Line Bank) were maintainted in a DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco Life Technologies (USA)), introduced into a 6 well plate at a density of about 5xl05 cells/well, and grown in a 5% C02 incubator (culture temperature: 37 °C, pH: 7.0 ~ 7.4).
The cells were treated with 2-DG or metformin hydrochloride alone at concentrations of 0.1 mM, 1 mM and 10 mM, respectively, and also treated with the combination of metformin hydrochloride and 2-DG at concentrations of 0.1 mM, 1 mM and 10 mM, respectively, and then incubated for 48 h. In the case of combined treatment group, metformin hydrochloride and 2-DG were used in the ratio of 1 : 1 at concentrations as represented in the following Table 2.
After 48 h, the cultivated cells were lysed by 10 mM Tris HC1 (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P207, 2 mM Na3V04, 1% Triton X-100, 10% glycerol, 0.5% deoxycholate and 1 mM PMSF, and 25 μ& of cell lysate by the protein assay was subjected to electrophoresis on SDS-PAGE gel and transferred to a PMSF (Amersham Life Science) membrane. Then, said membrane was reacted with a rat monoclonal anti-human Snail (purchased from Cell signaling Technology and diluted in 5% skim milk (Difco) at a ratio of 1 :1000) or a mouse monoclonal anti-P-actin (purchased from Santa cruz Biotechnology and diluted in 5% skim milk (Difco) at a ratio of 1:5000), and the remaining antibodies that were not bound were removed. Here, the anti-P-actin was used as an antibody to show that the amounts of proteins loaded on the SDS-PAGE gel were same.
Again, the membrane was reacted with an anti-rat IgG as a secondary antibody (purchased from Cell signaling Technology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000) or an anti-mouse IgG (purchased from Santa cruz Biotechnology and diluted in 5% skim milk (Difco) at a ratio of 1 :2000). Finally, the antibodies bound to the membrane were detected using ECL Plus system (Amersham Life Science).
Furthermore, after the degree of Snail expression compared with β-actin was analyzed using ΤΓΝΑ-image analysis program, the degree of Snail expression according to metformin or 2-DG treatment was shown in the following Table 2 as a percent (%) with respect to 100% of the control group which was not treated at all.
Table 2
Results of Snail expression rate (%)
Figure imgf000027_0001
As the above Table 2 shows, it was demonstrated that the single use of metformin hydrochloride or 2-DG induces the decrease of Snail expression rate at the concentration of 1 mM. Specifically, the Snail expression rate falls by about 27% at 1 mM of metformin hydrochloride and falls by about 47% at 1 mM of 2-DG.
On the other hand, in the case of combined use of metformin hydrochloride and
2-DG, a significant decrease of expression is shown even at the concentration of 0.1 mM of each drug. Specifically, the combined use of both drugs at concentrations of 0.1 mM and 1 mM exhibits a remarkable decrease of expression by about 26% and 84%, respectively, in comparison to the single use thereof.
Particularly, the amount of drug used may be remarkably reduced since the same effect as is obtained by 1 mM of metformin hydrochloride alone can be achieved by its combined use with 2-DG at a concentration of 0.1 mM which corresponds to only one tenth of 1 mM.
When the single use and combined use are compared with each other on the basis of 1 mM of each drug, the single use of metformin hydrochloride and 2-DG reduces the Snail expression rate by about 27% and 47%, respectively, whereas the combined use thereof remarkably reduces the Snail expression rate by about 84%, which indicates an increase of effect by a factor of about 3 or more and about 1.79 or more compared with their single use.
Particularly, the effect in the decrease of Snail expression by 2-DG was confirmed by this experiment for the first time. Moreover, it was confirmed that more synergistic effect in the decrease of Snail expression can be obtained by the combined use of metformin hydrochloride and 2-DG even though each drug is used at a much lower concentration, which indicates the mutual synergistic action of the two drugs when they are combined.
As was confirmed by the present experiment, the single or combined use of the drugs effectively decreases the expression of Snail protein. The Snail protein is a marker for EMT. Thus, it was also confirmed that a biguanide based drug or 2-DG alone inhibits EMT, but their combined use can induce much more synergistic EMT inhibition. In sum, it is expected that the single use of 2-DG and the combined use of a biguanide based antidiabetic drug and 2-DG can show the inhibition of EMT through the suppression of Snail expression, and furthermore an effect of inhibiting cancer metastasis.
[Example 1] Preparation of a tablet comprising metformin hydrochloride and 2-DG
500 g of metformin hydrochloride, 300 g of 2-DG and 250 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery, Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35- sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin hydrochloride and 2-DG. A film coating layer was formed using 30 g of opadry OY-C-7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 500 mg of metformin hydrochloride and 300 mg of 2-DG in one tablet. [Example 2] Preparation of a tablet comprising metformin acetate and 2-
DG
571 g of metformin acetate, 300 g of 2-DG and 179 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35 sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin acetate and 2-DG. A film coating layer was formed using 30 g of opadry OY-C- 7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 571 mg of metformin acetate and 300 mg of 2-DG in one tablet. [Example 3] Preparation of a tablet comprising metformin nicotinate and
2-DG
761.5 g of metformin nicotinate, 300 g of 2-DG and 138.5 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery., Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35- sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin nicotinate and 2-DG. A film coating layer was formed using 30 g of opadry OY-C-7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 761.5 mg of metformin nicotinate and 300 mg of 2-DG in one tablet. [Example 4] Preparation of a tablet comprising metformin malonate and
2-DG
704 g of metformin malonate, 300 g of 2-DG and 146 g of microcrystalline cellulose were passed through a No. 20 sieve, respectively, and then mixed for 20 min in a V-mixer (Cheil Machinery, Korea). Separately, 25 g of hydroxypropyl cellulose and 15 g of light anhydrous silicic acid were passed through a No. 35 sieve, which were then added to the above mixture and mixed for 10 min. Finally, 10 g of stearic acid was passed through a No. 35 sieve, added to the above mixture, and mixed for 3 min. Subsequently, the final mixture was compressed to give a tablet comprising metformin malonate and 2-DG. A film coating layer was formed using 30 g of opadry OY-C- 7000A as coating material in a hi-coater (SFC-30N, Sejong Pharmatech Co., Ltd., Korea) to give a tablet containing 704 mg of metformin malonate and 300 mg of 2-DG in one tablet. [Example 5] Preparation of an oral solution comprising metformin hydrochloride and 2-DG
100 g of metformin hydrochloride and 60 g of 2-DG were introduced to a 1L volume glass vessel, 800 mL of purified water was added, and the mixture was stirred for 30 min to be thoroughly dissolved. Purified water was added to precisely make 1 L. After preparation, O.lN-hydrochloric acid solution or O.lN-sodium hydroxide solution was added to adjust pH 3-4, giving an oral solution comprising 100 mg of metformin hydrochloride and 60 mg of 2-DG per 1 mL of the solution. [Example 6] Preparation of a tablet comprising phenformin hydrochloride and 2-DG
The same procedure as Example 1 was carried out except that 100 g of phenformin hydrochloride was used instead of 500 g of metformin hydrochloride to give a tablet comprising 100 mg of phenformin hydrochloride and 300 mg of 2-DG in one tablet.
[Example 7] Preparation of a tablet comprising buformin hydrochloride and 2-DG
The same procedure as Example 1 was carried out except that 50 g of buformin hydrochloride was used instead of 500 g of metformin hydrochloride to give a tablet comprising 50 mg of buformin hydrochloride and 300 mg of 2-DG in one tablet.

Claims

[CLAIMS]
1. Pharmaceutical composition for inhibiting cancer recurrence or metastasis, comprising 2-deoxy-D-glucose as an active ingredient.
2. The pharmaceutical composition according to Claim 1, wherein 2-deoxy-D- glucose is contained in the amount of 10 to 4000 mg.
3. Pharmaceutical composition for inhibiting cancer recurrence or metastasis, comprising a biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-deoxy-D-glucose as active ingredients.
4. The pharmaceutical composition according to Claim 3, wherein the biguanide based antidiabetic agent is one or more selected from the group consisting of metformin, phenformin and buformin.
5. The pharmaceutical composition according to Claim 3, wherein the biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-deoxy-D- glucose are contained in the weight ratio of 1 : 400 to 400 : 1.
6. The pharmaceutical composition according to Claim 3, wherein the pharmaceutically acceptable salt of biguanide based antidiabetic agent is one or more acid addition salt of which acid is selected from the group consisting of 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, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid.
The pharmaceutical composition according to Claim 6, wherein the pharmaceutically acceptable salt of metformin is hydrochloride or acetate.
The pharmaceutical composition according to Claim 1 or 3, which is an oral or parenteral preparation.
The pharmaceutical composition according to Claim 1 or 3, which further comprises an anticancer agent as an active ingredient.
The pharmaceutical composition according to Claim 9, wherein the anticancer agent is one or more selected from the group consisting of nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semaxanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuxumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastin, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonine, exemestane, aminoglutethimide, anagrelide, navelbine, fadrazol, tamoxifen, toremifen, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine and carmustine.
The pharmaceutical composition according to Claim 1 or 3, wherein the cancer is one or more selected from the group consisting of uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, hematologic malignancy and liver cancer.
The pharmaceutical composition according to Claim 11 , wherein the cancer is colon cancer or breast cancer.
13. Method for inhibiting cancer recurrence or metastasis, which comprises administering an effective amount of a pharmaceutical composition comprising 2-deoxy-D-glucose as an active ingredient.
The method according to Claim 13, wherein 2-deoxy-D-glucose is administered in the amount of 10 to 4000 mg.
15. The method according to Claim 13, wherein the pharmaceutical composition further comprises a biguanide based antidiabetic agent or its pharmaceutically acceptable salt as an active ingredient.
The method according to Claim 15, wherein the biguanide based antidiabetic agent or its pharmaceutically acceptable salt and 2-deoxy-D-glucose are administered in the weight ratio of 1 : 400 to 400 : 1.
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