KR20120134605A - COMPOSITION COMPRISING 1,2,3,4,6-PENTA-O-GALLOYL-β-D-GLUCOSE AND IMATINIB AS EFFECTIVE COMPONENTS FOR TREATMENT OF CHRONIC MYELOGENOUS LEUKEMIA - Google Patents

Abstract

PURPOSE: A composition containing 1,2,3,4,6-penta-O-galloyl-beta-D-glucose and imatinib is provided to reduce imatinib resistance and to treat chronic myelogenous leukemia. CONSTITUTION: A composition for treating chronic myelogenous leukemia contains 1,2,3,4,6-penta-O-galloyl-beta-D-glucose of chemical formula 1 and imatinib of chemical formula 2 as an active ingredient, which is mixed in a molar ratio of 1:80-1:320. The chronic myelogenous leukemia is caused by imatinib resistant chronic myelogenous leukemia cells. The composition increases apoptotic sub-G1 DNA content and poly(ADP-ribose)polymerase(PARP) division in the imatinib resistant chronic myelogenous leukemia cells.

Description

TECHNICAL FIELD The present invention relates to a composition for treating chronic myelogenous leukemia comprising 1,2,3,4,6-pentanediol, galloyl, beta, di-glucose and imatinib as active ingredients. -PENTA-O-GALLOYL-β-D-GLUCOSE AND IMATINIB AS EFFECTIVE COMPONENTS FOR TREATMENT OF CHRONIC MYELOGENOUS LEUKEMIA}

The present invention relates to a composition for treating chronic myelogenous leukemia comprising 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib as an active ingredient.

Imatinib, which is marketed by Novartis under the trade name Gleevec ^TM , is used as a cancer chemotherapeutic for the treatment of chronic myelogenous leukemia, but it is expensive and has minor side effects such as nausea, vomiting, fluid retention, diarrhea, muscle spasms, Depression, convulsions, heart failure, thrombosis, embolism, hemorrhage, anemia, and thrombocytopenia.

In addition, about 90% of patients with chronic myelogenous leukemia treated with imatinib show resistance, most of the hematologic tumor cells are removed by imatinib, but a small number of mutated cells survive despite treatment with imatinib Eventually, it will cause recurrence of bone cancer. In clinical practice, the cause of tolerance observed in patients with resistance to imatinib is additional chromosomal abnormalities, point mutations in the BCR / ABL gene, and gene amplification in the order of BCR / ABL gene amplification 52.8%, 48.5% and 6.2%, respectively (Leukemia 2002, 16: 2190-96).

In other words, Imatinib is an important chemotherapeutic agent for treating chronic myelogenous leukemia. However, it is expensive and has a high side effect when administered at a high concentration, and resistance to imatinib is low due to resistance to use in chronic myelogenous leukemia.

Therefore, patients who are already predicted to have resistance, or who are initially positive for imatinib susceptibility but are expected to develop resistance, are required to use a high concentration of imatinib and have a high side effect, so that the problem of imatinib resistance is required .

Korean Patent Publication No. 2005-0095287 discloses that 1,2,3,4,6-penta-O-galloyl-beta-di-glucose inhibits cyclooxygenase-2 (COX-2), induces apoptosis And an effect of suppressing tumor growth.

Accordingly, the inventors of the present invention focused on the fact that imatinib resistance can be overcome by administering other inhibitory drugs related to the action mechanism of BCR / ABL protein concurrently. As a result, , And concomitant administration of 4,6-penta-O-galloyl-beta-di-glucose and imatinib increased the sensitivity of imatinib-resistant chronic myelogenous leukemia cell line (K562 / ImaR-1) to imatinib (K562 / ImaR-1), which is an immune-tolerant chronic myelogenous leukemia cell line.

It is an object of the present invention to provide a pharmaceutical composition containing 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib effective to reduce side effects of resistance to imatinib and increase anti- And a composition for treating chronic myelogenous leukemia.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising 1,2,3,4,6-penta-O-gal-β-di-glucose the present invention provides a composition for treating chronic myelogenous leukemia comprising, as an active ingredient, Imatinib of the following formula (2): < EMI ID =

[Chemical Formula 1]

(2)

The 1,2,3,4,6-penta-O-galloyl-beta-di-glucose can be isolated from Galla Rhois. Rhus japonica is a parasitic insect hump formed on the leaves of rhus javanica (Rhus javanica) of Rhus javanica.

The imatinib is obtained by reacting 4 - [(4-methylpiperazin-1-yl) methyl] -N- [4-methyl- 4-methylpiperazin-1-yl) methyl] -N- [4-methyl-3 - [(4- pyridin-3-ylpyrimidin-2-yl) amino] phenyl] benzamide}.

When the combination index of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose is 1 or less, imatinib and 1,2,3,4,6- Penta-O-galloyl-beta-di-glucose, respectively.

According to one embodiment of the present invention, the chronic myelogenous leukemia may be chronic myelogenous leukemia caused by imatinib-resistant chronic myelogenous leukemia cells.

Imatinib-resistant chronic myelogenous leukemia cells can be produced by continuously treating imatinib with chronic myelogenous leukemia cell lines to obtain surviving cells. For example, it can be produced by continuously obtaining the surviving cells in K562 cells (Lozzio, C. B. and Lozzio, B. B. Blood 45: 321-334 (1975)), an emetic-sensitive chronic myelogenous leukemia cell line.

According to one embodiment of the present invention, the above-mentioned emetic-resistant chronic myelogenous leukemia cell may be K562 / ImaR-1 cell strain.

In the cytotoxicity test for imatinib, imatinib-resistant chronic myelogenous leukemia cell K562 / ImaR-1 showed resistance to imatinib without affecting cell viability as compared with imatinib-sensitive K562 (See Fig. 1).

Imatinib aims to inhibit the overexpression of a fusion protein called bcr-abl, which is a cause of chronic myelogenous leukemia. Thus, the imatinib-resistant cells can be confirmed by an increased expression of the bcr-abl protein (see FIG. 2).

The survival group of the chemotherapeutic agent can be confirmed by confirming the expression of the drug resistance marker. Decreased expression of drug resistance markers means increased drug sensitivity. Examples of drug resistance markers resistant to chemotherapeutic agents include MDR1 gene (Multi-Drug Resistance 1) and P-glycoprotein (P-gp). The MDR1 gene induces multiple drug resistance and produces P-glycoprotein, which acts as an efflux pump for anticancer drug preparations. The high concentration of MDR1 gene is closely related to multiple drug resistance, and P- And Pacos and Loannidis reported that P-glycoprotein positivity and histologic necrosis response to drugs were independent of disease progression but were associated with disease progression (Pakos EE, Ioannidis JP. A meta-analysis of the association of pglycoprotein with response to chemotherapy and clinical outcome in patients with osteosarcoma. Cancer 2003; 98: 581-9).

According to an embodiment of the present invention, the composition for treating chronic myelogenous leukemia may reduce the expression of a drug resistance marker on the ion-resistant chronic myelogenous leukemia cells. The ability of the composition for treating chronic myelogenous leukemia to decrease the expression of the drug resistance marker means that the drug sensitivity can be increased.

According to one embodiment of the present invention, the drug resistance marker may be P-glycoprotein. P-glycoprotein is a multifunctional resistance gene.

According to one embodiment of the present invention, the molar ratio of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imanitib contained in the composition for treating chronic myelogenous leukemia is 1:80 To 1: 320. For example, in the composition for treating chronic myelogenous leukemia, the concentration of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib is 250 nM of Imatinib, , 3,4,6-penta-O-galloyl-β-D-glucose 20 μM to Imatinib 125 nM, 1,2,3,4,6-penta-O-galloyl-β-D- Lt; / RTI > In vitro, selective cytotoxicity and increased apoptosis can be identified in the above range of concentrations in the imatinib-resistant chronic myelogenous leukemia cells (K562 / ImaR-1 cell line).

According to one embodiment of the present invention, the composition for treating chronic myelogenous leukemia increases apoptotic sub-G1 DNA content in imatinib-resistant chronic myelogenous leukemia cells, poly (ADP-ribose) Polymerase [poly (ADP-ribose) polymerase; PARP] cleavage and decrease the expression of B-cell lymphoma 2 (Bcl-2), thereby increasing cytotoxicity and apoptosis to imatinib-resistant chronic myelogenous leukemia cells.

Caspase-3 is an important enzyme in the apoptosis of cancer cells, and is a poly (ADP-ribose) polymerase; PARP] is a caspase-3 substrate. When apoptosis of cancer cells is induced, the activity of caspase-3 is increased, and fragmentation of the poly (ADP-ribose) polymerase as its substrate appears. Thus, increasing the activity of caspase-3 and inducing fragmentation of poly (ADP-ribose) polymerase may increase the effects of cancer treatment agents (cytotoxicity and apoptosis).

Bcl-2 is an anti-apototic protein that prevents or delays apoptosis and prolongs cell survival. Thus, decreasing the expression of Bcl-2 may increase apoptosis to the imatinib-resistant chronic myelogenous leukemia cells.

According to one embodiment of the present invention, the composition for treating chronic myelogenous leukemia reduces the expression of p-glycoprotein in the imatinib-resistant chronic myelogenous leukemia cells, and the expression of imatinib-resistant chronic myelogenous leukemia cells It is possible to increase the susceptibility of the virus.

The composition for treating chronic myelogenous leukemia of the present invention may contain additives such as pharmaceutically acceptable diluents, binders, disintegrants, lubricants, pH adjusters, antioxidants, solubilizing aids and the like within the range not impairing the effects of the present invention.

The diluent may be sugar, starch, microcrystalline cellulose, lactose (lactose hydrate), glucose, di-mannitol, alginate, alkaline earth metal salt, clay, polyethylene glycol, anhydrous calcium hydrogen phosphate, or a mixture thereof; The binder may be selected from the group consisting of starch, microcrystalline cellulose, highly disperse silica, mannitol, di-mannitol, sucrose, lactose hydrate, polyethylene glycol, polyvinylpyrrolidone (povidone), polyvinylpyrrolidone copolymer , Hydroxypropylcellulose, natural gum, synthetic gum, copovidone, gelatin, or a mixture thereof.

The disintegrant may be a starch or modified starch such as sodium starch glycolate, corn starch, potato starch or pregelatinized starch; Clays such as bentonite, montmorillonite, or veegum; Cellulose such as microcrystalline cellulose, hydroxypropylcellulose or carboxymethylcellulose; Alginates such as sodium alginate and alginic acid; Crosslinked celluloses such as croscarmellose sodium; Guar gum and xanthan gum; Crosslinked polymers such as crosslinked polyvinylpyrrolidone (crospovidone); Sodium bicarbonate, citric acid and the like, or a mixture thereof.

The lubricant may be selected from the group consisting of talc, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, sodium stearyl fumarate, glyceryl behenate, glyceryl monolate, glyceryl monostearate, Stearate, colloidal silicon dioxide, or a mixture thereof may be used.

The pH adjusting agent may be an acidifying agent such as acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium ethoxide, malic acid, succinic acid, tartaric acid, fumaric acid, citric acid (citric acid) and precipitated calcium carbonate, ammonia water, meglumine, Basic agents such as sodium, magnesium oxide, magnesium carbonate, sodium citrate, and tribasic calcium phosphate can be used.

As the antioxidant, dibutylhydroxytoluene, butylated hydroxyanisole, tocopheryl acetate, tocopherol, propyl gallate, sodium hydrogen sulfite, sodium pyrophosphate and the like can be used. In the prior-release compartment of the present invention, Polyoxyethylene sorbitan fatty acid esters such as sodium lauryl sulfate and polysorbate, docusate sodium, poloxamer and the like can be used.

In addition, an enteric polymer, a water-insoluble polymer, a hydrophobic compound, and a hydrophilic polymer may be included to form a delayed-release preparation.

The enteric polymer is insoluble or stable under acidic conditions below pH 5 and is dissolved or decomposed under specific pH conditions of pH 5 or higher. For example, (Hydroxypropylmethylcellulose phthalate), hydroxymethylethylcellulose phthalate, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate maleate, cellulose benzoate phthalate, cellulose propionate phthalate, methylcellulose phthalate, carboxymethylethylcellulose and An enteric cellulose derivative such as ethylhydroxyethylcellulose phthalate and methylhydroxyethylcellulose; Styrene-acrylic acid copolymers such as styrene-acrylic acid copolymers, acrylic acid methyl-acrylic acid copolymers, acrylic acid methyl methacrylic acid copolymers (e.g., acrylic acid), butyl acrylate-styrene-acrylic acid copolymers, and methyl methacrylate- Said enteric acrylic acid-based copolymer; Poly (methacrylic acid methyl acrylate) copolymers (e.g., Eudragit L100-E), poly (methacrylic acid methyl methacrylate) copolymers such as Eudragit L, Eudragit S, An enteric polymethacrylate copolymer such as < RTI ID = 0.0 > Maleic anhydride copolymers, styrene-maleic anhydride copolymers, styrene-maleic anhydride copolymers, styrene-maleic anhydride copolymers, vinyl styrene-maleic anhydride copolymers, styrene-maleic anhydride copolymers, styrene-maleic anhydride copolymers, Maleic anhydride copolymers such as styrene-maleic anhydride copolymer, maleic anhydride copolymer, maleic anhydride copolymer, maleic anhydride copolymer, maleic anhydride copolymer, maleic anhydride copolymer, maleic anhydride copolymer, And enteric polyvinyl derivatives such as polyvinyl alcohol phthalate, polyvinyl acetal phthalate, polyvinyl butyrate phthalate and polyvinylacetacetal phthalate.

The water-insoluble polymer refers to a polymer that does not dissolve in a pharmaceutically acceptable water to control the release of the drug. For example, the water-insoluble polymer can be selected from the group consisting of polyvinyl acetate (e.g., Colicot SR30D), water insoluble polymethacrylate copolymer (e.g., poly (ethyl acrylate-methyl methacrylate) , Poly (ethyl acrylate-methyl methacrylate-trimethylaminoethyl methacrylate) copolymer (such as Eudragit RSPO), ethyl cellulose, cellulose ester, cellulose ether, cellulose acylate, cellulose diacylate, Cellulose triacylate, cellulose acetate, cellulose diacetate and cellulose triacetate.

The hydrophobic compound refers to a pharmaceutically acceptable water-insoluble substance that controls the release of the drug. For example, fatty acid and fatty acid esters such as glyceryl palmitostearate, glyceryl stearate, glyceryl behenate, cetyl palmitate, glyceryl monooleate, and stearic acid; Fatty acid alcohols such as cetostearyl alcohol, cetyl alcohol and stearyl alcohol; Waxes such as carnauba wax, beeswax, and microcrystalline wax; Talc, precipitated calcium carbonate, calcium monohydrogenphosphate, zinc oxide, titanium oxide, kaolin, bentonite, montmorillonite, and insoluble materials.

The hydrophilic polymer refers to a polymeric substance that dissolves in a pharmaceutically acceptable water to control the release of a drug. For example, there may be mentioned sugars such as dextrin, polydextrin, dextran, pectin and pectin derivatives, alginates, polygalacturonic acid, xylan, arabinozaylan, arabinogalactan, starch, hydroxypropyl starch, amylose and amylopectin ; Cellulose derivatives such as hypromellose, hydroxypropylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose and carboxymethylcellulose sodium; Gums such as guar gum, locust bean gum, tragacanth, carrageenan, acacia gum, gum arabic, gellan gum, and xanthan gum; Proteins such as gelatin, casein, and zein; Polyvinyl derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl acetal diethylaminoacetate; Poly (ethyl acrylate-methyl methacrylate-methyl methacrylate) copolymer (e.g., Eudragit E100, Ebonic, Germany), poly (butyl methacrylate- (2-dimethylaminoethyl) Hydrophilic polymethacrylate copolymers such as triethylaminoethyl-methacrylate chloride) copolymer (e.g., Eudragit RL, RS, Evonik, Germany); Polyethylene derivatives such as polyethylene glycol, and polyethylene oxide; And carbomers.

In addition, the pharmaceutical composition of the present invention can be formulated by selecting pharmaceutically acceptable additives as various additives selected from coloring agents and perfumes.

The range of the additive in the present invention is not limited to the use of the additive, and the additive may be formulated to contain the usual range of capacity by selection.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, oral formulations, external preparations, suppositories or sterilized injection solutions according to a conventional method have.

The present invention also provides a composition for treating chronic myelogenous leukemia comprising 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib as an active ingredient of the present invention, To provide a method for treating chronic myelogenous leukemia. The term "administering" in the present invention means introducing a composition for treating chronic myelogenous leukemia of the present invention into a patient by any appropriate method, and the administration route of the composition for treating chronic myelogenous leukemia of the present invention can reach the target tissue May be administered via any of the usual routes. But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, intranasal administration, intrapulmonary administration, intrarectal administration, intraperitoneal administration, intraperitoneal administration and intrathecal administration.

The composition for treating chronic myelogenous leukemia according to the present invention may be administered at least once a day or at regular intervals.

The dosage of the composition for treating chronic myelogenous leukemia of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

It is to be understood that the dosage of the composition for treating chronic myelogenous leukemia of the present invention should be determined according to the activity, sex, administration time, route of administration, duration and frequency of treatment, etc. of the composition for treating chronic myelogenous leukemia of the present invention, Is not intended to limit the scope of the invention in any way.

The composition for treating chronic myelogenous leukemia comprising 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib as an active ingredient according to the present invention is useful for the treatment of chronic myelogenous leukemia Can be used in the treatment of chronic myelogenous leukemia by reducing the adverse effects of resistance to Jane imatinib and increasing the anti-cancer effect, and it can be particularly useful for imatinib resistant chronic myelogenous leukemia.

FIG. 1 is a graph showing cytotoxicity against the imatinib treatment concentration for the imatinib sensitive cell line K562 and the imatinib resistant cell line K562 / ImaR-1.
FIG. 2 shows Western blotting results of confirming an increase in the expression of bcr-abl after treating imatinib with the imatinib-sensitive cell line K562.
Fig. 3 shows cytotoxicity results for the treatment with imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose, respectively, in the imatinib-resistant cell line K562 / ImaR-1 It is a histogram.
4 shows the combination index of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose for the imatinib-resistant cell line K562 / ImaR-1.
FIG. 5 is a graph showing the effect of 250 nM imatinib and 20 μM of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose on the imatinib-resistant cell line K562 / ImaR- Lt; RTI ID = 0.0 > DNA-1 < / RTI > DNA using a flow cytometer.
6 is a graph showing the effect of 250 nM imatinib and 20 μM of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose on the imatinib-resistant cell line K562 / ImaR- And the ratio of apoptotic cells measured using a flow cytometer, that is, the sub-G1 DNA content after the combined treatment.
FIG. 7 shows the results obtained by treating imatinib-resistant cell line K562 / ImaR-1 with imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di- The results of western blotting confirmed the change in the expression level of the protein, the degree of cleavage of poly (ADP-ribose) polymerase (PARP) and the expression level of bcl-2.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Production of imatinib-resistant chronic myelogenous leukemia cell line

Starting from 0.05 μM of Novartis's Gleevec ^™ with Imatinib in K562 cells (Lozzio, CB and Lozzio, BB Blood 45: 321-334 (1975)), a chronic myelogenous leukemia cell line, And the concentration was increased to 1 μM. Imatinib was used by dissolving in dimethylsulfoxide (DMSO) solvent. After this, 1 μM of imatinib was continuously treated, and the cells surviving for 4 months from the start were named K562 / ImaR-1 cell line.

The resistance of the K562 / ImaR-1 cell line to imatinib was assessed using a cytotoxicity test (see Fig. 1) and bcr-abl antibody (c-Abl (24-11), sc-23, Santa Cruz Biotechnology, Santa Cruz, Calif. USA) using Western blotting (see FIG. 2).

The cytotoxicity test was performed by measuring the cell viability by XTT assay. Imatinib at 0, 0.125, 0.25, 0.5, 1, or 2 μM was treated with imatinib-sensitive chronic myelogenous leukemia cell line K562 and imatinib-resistant chronic myelogenous leukemia cell line K562 / ImaR-1 for 24 or 48 hours. Sodium 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium- (Molecular Devices, Sunnyvale, CA, USA) at 450 nm for 2 hours at 37 ° C, and then the absorbance at 450 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, Calif. CA, USA), and the survival rates were compared and analyzed according to the following formula.

Cell viability (%) = [absorbance (drug treatment group) -absorbance (medium)] - [OD (no treatment) -OD (medium)] 100 The OD represents the optical density.

Imatinib sensitive cell line K562 was treated with imatinib at a concentration of 0.05 μM every 2 weeks starting from 0.05 μM until 1 μM. Thereafter, the increase in the expression of bcr-abl was confirmed by western blotting while 1 μM of imatinib was continuously treated.

The western blotting was performed as follows. All cells were harvested and treated with a protease inhibitor and a solution containing 50 mM TrisHCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% deoxycholate acid, . Protein was quantitated in the supernatant and 20 μg of the protein was electrophoresed on a 10% SDS-polyacrylamide gel. The protein migrated on the polyacrylamide gel was electrophoretically transferred to nitrocellulose membrane and then blocked with 5% bovine serum albumin at room temperature for one hour. (24-11), sc-23, Santa Cruz Biotechnology, Santa Cruz, CA) and beta-actin (Sigma, St. Louis, Mo., USA) as a monoclonal antibody to Bcr-abl The reaction was carried out at 4 ° C for 12 hours and washed three times with a Tris buffered saline (TBS) -T solution containing Tween 20. The cells were reacted with secondary antibodies (horseradish peroxidase (HRP) -conjugated anti-mouse or rabbit secondary antibodies, Invitrogen, Carlsbad, Calif., USA) and developed with ECL system (Amersham Pharmacia, Piscataway, NJ, USA) . Beta-actin was used as a reference to show that the same level of the house keeping gene in all cells was analyzed for the same amount of protein. Increased expression of bcr-abl was confirmed by Western blotting. As a result of comparing the amount of protein expression of Bcr-abl with the existing cell line K562 and the imatinib-resistant cell line, the amount of Bcr-abl expression was significantly increased in the imatinib-resistant cell line compared to the existing cell line, It can be seen that tolerance to Tiniv has occurred.

Example  2. Preparation of 1,2,3,4,6-penta-o- Galois - Isolation of beta-D-Glucose

The shrub dwarf (355 g) was placed in an extraction vessel and refluxed into methanol (730 ml) for three times. The resulting solution was concentrated to give the methanol extract of obsidian (252 g). Water was added to the water layer and the butanol solution at a volume ratio of 1: 1 (vol / vol), and the mixture was partitioned. The butanol solution and the aqueous layer were concentrated to obtain 35 g of butanol extract and 45 g of water extract, respectively. (10 g) was adsorbed onto silica gel, and then CHCl ₃ : methanol (MeOH): H ₂ O = 65: 35:10 and ethyl acetate (EtOAc): methanol (MeOH): H ₂ O = 100: 15.6 : 13.5. The reaction mixture was subjected to reverse phase column chromatography using methanol (MeOH) as a solvent to isolate the compound. The reversed phase column chromatography used Sephadex-HR20 was analyzed by NMR, mass spectrometry and the like to give 1,2,3,4,6-penta-O-galloyl-beta-di-glucose ( Supported, molecular weight 940.67 g / mol).

Example  3. Cytotoxicity and concurrent administration test

The K562 / ImaR-1 cell line was treated with 250 nM imatinib and 10, 20 or 40 μM of 1,2,3,4,6-penta-O-galloyl-beta-di- Respectively. Imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose were used by dissolving in DMSO solvent.

Cell viability was measured by XTT analysis. Sodium 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium- (Molecular Devices, Sunnyvale, CA, USA) at 450 nm for 2 hours at 37 ° C, and then the absorbance at 450 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, Calif. CA, USA), and compared the survival rates.

Imatinib (250 nM) did not show toxicity to K562 / ImaR-1 cells, and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose (10, ) Treatment showed slight toxicity in a concentration dependent manner. The combined treatment of imatinib (250 nM) and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose (10, 20 or 40 μM) significantly increased cytotoxicity 3).

The combination index (CI) of the anticancer synergistic effect due to the combination administration of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose on K562 / ImaR- Respectively. Combination indices were measured by using CalcuSyn software (Biosoft, Ferhuson, MO, USA) as a comparison of the cell survival inhibition effects between the single treatment group and the combined treatment group.

Chou and Talalay's method (Chou TC, Talalay P) were analyzed according to the method of Adv Enzyme Regul 1984, 22: 27-55. FIG. 4 shows the anti-cancer synergistic effect of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose on K562 / ImaR-1 cells by calculating the combination index. In Figure 4, the effect of the x-axis is the OD value at the time of simultaneous treatment of the two drugs in the cytotoxicity test (XTT assay) (circle; 1,2,3,4,6-penta-O- Beta-di-Glucose 10 / Imatinib 0.125, Nemo; 1,2,3,4,6-Penta-O-Galoyl-beta-D- Glucose 20 / Imatinib 0.25, , 4,6-penta-O-galloyl-beta-di-glucose 40 / imatinib 0.5).

More specifically, D is the concentration of the drug, Dm is the concentration of the drug exhibiting the intermediate effect, and log [(1-f) / f] for log [D / Dm] X slice (log IC ₅₀ ) and slope m for each drug and drug combination were calculated by the least squares method on the curve (Median Effect Curve). This value was used to calculate the concentration of each drug and the concentration of the drug combination, which shows stepwise cytotoxicity according to the formula below. f means fractional survival, and the survival rate of the drug treatment plate divided by the survival rate of the control plate), and IC ₅₀ means 50% inhibition concentration.

The drug combination concentration of D (dose of drug) = Dm [(1-f / f] ^{1 / m} ) is expressed as (D) ₁ and (D) ₂ for Drug 1 and Drug 2, The index combination index (CI) indicating cytotoxicity was calculated using the following equation.

_{CI = (D comb) 1 /} (D alone) 1 + (D comb) 2 / (D alone) 2 + α [(D comb) 1 × (D comb) 2] / [(D alone) 1 × (D _{alone 2} ]

(D _{alone) 1} and (D _{alone) 2} refers to the concentration of drug showing the effect (survival rate (fa)) given when only treatment of drug 1 and 2, and (D _{alone) 1} and (D _{alone) 2} is the drug This is the concentration for obtaining the given effect (fa) when combined. And if the actions of the drug are mutually exclusive, α = 0 and mutually exclusive α = 1.

CI = 1, a synergistic effect between two drugs, CI = 1, an additive effect between two drugs, and CI> 1, an antagonistic effect between the two drugs .

4, when imatinib is treated with 1,2,3,4,6-penta-O-galloyl-beta-di-glucose in combination, the CI value is 1 or less, , And 3,4,6-penta-O-galloyl-beta-di-glucose, respectively.

Example  4. Apoptosis  serve- G1 ( apoptotic sub - G1 ) DNA  Content measurement

The K562 / ImaR-1 cell line was treated with 250 nM imatinib and 20 μM 1,2,3,4,6-penta-O-galloyl-beta-di-glucose for 24 hours, respectively. Imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose were used by dissolving in DMSO solvent. After staining with a propidium iodide (PI) solution (Sigma Chemical, St. Louis, Mo., USA), the cell cycle was analyzed using a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) , And apoptotic sub-G1 DNA content indicating apoptotic cells (see Figures 5 and 6).

The combined treatment of imatinib with 1,2,3,4,6-penta-O-galloyl-beta-di-glucoside was found to inhibit apoptotic sub-G1 DNA content in K562 / ImaR-1 cells .

Example  5. Poly ( ADP - Ribos ) Polymerase (PARP) cleavage and bcl -2 expression measurement

The concentration of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose was fixed at 20 μM and either 250 or 500 nM imatinib alone or 1,2,3,4,6 -Penta-O-galloyl-beta-di-glucoside for 24 hours. Imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose were used by dissolving in DMSO solvent. Changes in the amount of expression of p-glycoprotein, the degree of cleavage of poly (ADP-ribose) polymerase (PARP) and the expression level of bcl-2 were confirmed by Western blotting (see FIG. 7).

The imatinib concentration was fixed at 125 nM, and 10, 20 or 40 μM of 1,2,3,4,6-penta-O-galloyl-beta-di- , 6-penta-O-galloyl-beta-di-glucose for 24 hours. Imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose were used by dissolving in DMSO solvent. Changes in the amount of expression of p-glycoprotein, the degree of cleavage of poly (ADP-ribose) polymerase (PARP) and the expression level of bcl-2 were confirmed by Western blotting (see FIG. 7).

The concomitant administration of imatinib with 1,2,3,4,6-penta-O-galloyl-beta-di-gluco increases the poly (ADP-ribose) polymerase (PARP) cleavage and increases the anti-apoptotic protein bcl-2, and at the same time decreased expression of p-glycoprotein, which is a drug resistance marker of cancer cells, in a dose-dependent manner. In particular, a synergistic effect was observed in the combination treatments at the respective concentration treatments, and a reduction effect of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imatinib in a concentration- appear. Therefore, by using concomitant administration of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucoside, it is possible to reduce the resistance exhibited by a previously used dose of imatinib, And the death induction (anti-cancer) effect is further elevated.

According to the embodiments of the present invention, the combination therapy of imatinib and 1,2,3,4,6-penta-O-galloyl-beta-di-glucose is effective for the treatment of imatinib-resistant chronic myelogenous leukemia cell line (K562 / ImaR -1) may increase the susceptibility to imatinib, thereby increasing the anticancer effect against the imatinib-resistant chronic myelogenous leukemia cell line (K562 / ImaR-1).

Claims (8)

(1,2,3,4,6-penta-O-galloyl-β-D-glucose) and imatinib Imatinib) as an active ingredient for the treatment of chronic myelogenous leukemia. The composition for treating chronic myelogenous leukemia according to claim 1, wherein the chronic myelogenous leukemia is chronic myelogenous leukemia caused by imatinib-resistant chronic myelogenous leukemia cells. The composition for treating chronic myelogenous leukemia according to claim 2, wherein the imatinib-resistant chronic myelogenous leukemia cell is a K562 / ImaR-1 cell line. The composition for treating chronic myelogenous leukemia according to claim 1, wherein the composition for treating chronic myelogenous leukemia reduces the expression of a drug resistance marker in an imatinib-resistant chronic myelogenous leukemia cell. The composition for treating chronic myelogenous leukemia according to claim 4, wherein the drug resistance marker is P-glycoprotein (P-gp). The method of claim 1, wherein the molar ratio of 1,2,3,4,6-penta-O-galloyl-beta-di-glucose and imanitib in the composition for treating chronic myelogenous leukemia is 1: 320. ≪ / RTI > The composition for treating chronic myelogenous leukemia according to claim 1, wherein the composition for treating chronic myelogenous leukemia increases the apoptotic sub-G1 DNA content in the imatinib-resistant chronic myelogenous leukemia cells and poly (ADP-ribose) polymerase (ADP-ribose) polymerase; PARP] cleavage and decreased expression of B-cell lymphoma 2 (Bcl-2), thereby increasing cytotoxicity and apoptosis of imatinib-resistant chronic myelogenous leukemia cells A composition for treating chronic myelogenous leukemia. The composition for treating chronic myelogenous leukemia according to claim 1, wherein the composition for treating chronic myelogenous leukemia reduces the expression of p-glycoprotein in an imatinib-resistant chronic myelogenous leukemia cell, and the sensitivity of imatinib to imatinib-resistant chronic myelogenous leukemia cells Wherein the composition is administered to a patient in need thereof.

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