KR20200134519A - Composition for treatment of brain tumor or temozolomide-resistant glioma comprising mg624 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain cancer containing MG624, wherein the composition of the present invention has effects of inhibiting the formation and metastasis ability of brain cancer cells by inhibiting STAT3 activity, and has effects of inhibiting the onset and recurrence of brain cancer by weakening resistance with respect to temozolomide drugs, thereby being able to be usefully used as the pharmaceutical composition for preventing or treating brain cancer, or a food composition for preventing or alleviating brain cancer.

Description

A pharmaceutical composition for preventing or treating brain cancer containing MG624 as an active ingredient {COMPOSITION FOR TREATMENT OF BRAIN TUMOR OR TEMOZOLOMIDE-RESISTANT GLIOMA COMPRISING MG624}

The present invention relates to a pharmaceutical composition for preventing or treating brain cancer comprising MG624 as an active ingredient.

Glioblastoma or Glioblastoma multiforme (GBM) is a malignant carcinoma originating from glial cells and their progenitor cells of the central nervous system. The white matter of the cerebral hemisphere in the cortical temporal lobe of the brain Most commonly occurs in (N. Engl. J. Med. (2008) 359:492). As the most common malignant brain cancer, accounting for about 12-15% of brain tumors, glioblastoma is the most malignant with a very fast growth rate and a dangerous potential in the grade Ⅰ to Ⅳ according to the cancer malignancies of the World Health Organization (WHO). It is a high tumor and is classified as grade IV. The number of cells is large, which is vigorous cell division, accompanied by vascular proliferation and necrosis (AJCC cancer staging handbook: from the AJCC cancer staging manual. 7th ed. 2010, New York: Springer. xix, 718 p).

GBM is a very fatal carcinoma with an average survival rate of only 14.6 months after surgical removal of the lesion, followed by radiotherapy and chemotherapy (N. Engl. J. Med. (2005) 3520:987). ). GBM has a very high recurrence rate, with a 6-month progression-free survival (PFS) of 15-21% and an average survival rate of 25 weeks, with a very poor prognosis (J. Clin. Oncol. (1999) 17:2572).

STATs (Signal transducers and activators of transcription) are transcription factors and are observed in many cancer cells and cancer tissues in which STATs are continuously activated. STAT3 protein present in the cytoplasm is phosphorylated and activated. Activated STAT3 moves to the cell nucleus and regulates the transcription of genes related to cell proliferation, transformation, and apoptosis.

Temozolomide (TMZ), a drug used in GBM's standard treatment regimen, is the only oral anticancer drug, and is a kind of alkylating agent that causes DNA damage to remove cancer cells (Lancet (2000) 355:1115). However, most of the patients develop resistance to temozolomide, and there are no further treatment options after resistance has developed. Therefore, it is urgent to develop a therapeutic agent for temozolomide-resistant glioblastoma.

Accordingly, as a result of the present inventors' efforts to solve the above problems, the substance that inhibits STAT3 activity MG624 exhibits an effect of inhibiting the proliferation of glioblastoma resistant to temozolomide, and thus has a preventive and therapeutic effect on brain cancer. By confirming that the present invention was completed.

Sandeep S., et al. "Nicotinic acetylcholine receptor signaling in tumor growth and metastasis." Journal of Oncology (2011). Melissa K. B, "The use of nicotinic acetylcholine receptor antagonists to target breast tumor-initiating cells." M. Sc. Thesis (2014). Carlos A., et al. "Activation of the macrophage α7 nicotinic acetylcholine receptor and control of inflammation." J Neuroimmune Pharmacol. 10(3) (2015): 468-476.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating brain cancer comprising MG624 as an active ingredient.

Specifically, one object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain cancer resistant to temozolomide containing MG624 as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving brain cancer comprising MG624.

Specifically, one object of the present invention is to provide a food composition for preventing or improving brain cancer resistant to temozolomide containing MG624 as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating brain cancer, comprising administering MG624 to an individual.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating brain cancer comprising MG624 as an active ingredient.

The MG624 is N,N,N-triethyl-2-(4- trans- stilbenoxy)ethylammonium iodide {N,N,N-Triethyl-2-(4- trans- stilbenoxy)ethylammonium iodide} As a compound It is represented by Formula 1.

[Formula 1]

That is, the present invention provides a pharmaceutical composition for preventing or treating brain cancer comprising the compound represented by Formula 1 as an active ingredient.

The MG624 of the present invention can be chemically synthesized or separated from natural substances, and can also be purchased and used commercially at home and abroad.

The present inventors were the first to find out that the MG624 of the present invention is excellent in inhibiting the proliferation of glioblastoma that is resistant to temozolomide, and thus, the use of MG624 for preventing or treating brain cancer was first identified.

Specifically, as a result of treatment with MG624 on temozolomide-resistant glioblastoma cells in the examples of the present invention, it was confirmed that STAT3 activity was remarkably reduced (FIG. 3). As a result, it was confirmed that the sphere formation of glioblastoma cells, which is another characteristic of cancer stem cells, was also significantly reduced (FIG. 2), thereby confirming the effect of preventing and treating brain cancer.

The term "brain cancer" used in the present invention is an aggressive characteristic in which cells divide and grow in the brain by ignoring normal growth limits, invasive characteristics that penetrate into surrounding tissues, and spread to other parts of the body. It is a generic term for diseases caused by cells having metastatic properties. The brain cancer is also used in the same meaning as a malignant tumor.

In general, in the case of brain tumor treatment, drugs cannot penetrate the blood-brain barrier, so surgery or radiation therapy has been relied on. The Blood Brain Barrier is a barrier that separates cerebrospinal fluid and blood as a kind of protective device between brain capillaries. Due to its high selective permeability, it is responsible for isolating the body's main regulatory center from pathogens that can be carried into the blood such as bacteria and potentially dangerous substances in the blood. If a typical blood vessel is made of vascular endothelial cells, the blood-brain barrier is surrounded by layers of various cells, such as vascular endothelial cells, pericyte, and astrocytes, forming a tight junction. This interferes with the movement of solutes between cells and blocks the passage of polymers and hydrophilic substances. This allows only highly selectively selected substances to pass through the blood-brain barrier through special channels or carrier proteins.

Because of the blood-brain barrier with very low molecular permeability, brain cancer is a difficult drug treatment compared to other common cancers. Therefore, blood-brain barrier permeability is a very important factor in drug development for brain diseases.

In the present invention, the prevention or treatment of brain cancer may consist of inhibiting tumorigenesis or metastasis of the brain cancer by inhibiting STAT3 (signal transducer and activator of transcription 3) activity.

The disease to be prevented, improved, or treated according to the present invention is not specifically limited to a specific disease name as long as it medically falls within the category of brain cancer, but the brain cancer may be preferably a brain cancer resistant to temozolomide, and the brain cancer is It may be a blastoma or an undifferentiated astrocytoma. Further, the brain cancer according to the present invention may be a glioblastoma or an undifferentiated astrocytoma in which Ras-related associated with diabetes (RRAD) is overexpressed.

The term "prevention" used in the present invention refers to any action that inhibits or delays the onset of brain cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms due to brain cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "inhibition of activity" means causing a decrease in the function of a target gene or a target protein.

The composition comprising the MG624 of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the above ingredients.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to MG624.

The kind of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. I can. These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention can be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, and fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , A binder or a lubricant may be additionally added and used.

In the pharmaceutical composition of the present invention, MG624 may be included in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, more preferably 0.1% by weight based on the total weight of the pharmaceutical composition. % To 70% by weight, more preferably 0.1% to 50% by weight, but is not limited thereto, and may be variously changed according to the state of the subject to be administered, the type of specific condition, and the degree of progression. If necessary, it may also be included in the total amount of the pharmaceutical composition.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

The pharmaceutical composition of the present invention is suitable for oral administration or parenteral administration and may be formulated and used in various dosage forms.

Non-limiting examples of formulations for oral administration using the pharmaceutical composition of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft Capsules, syrup, elixirs, and the like.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrants such as corn starch or sweet potato starch; Lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be used, and sweeteners, fragrances, syrups, and the like may also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be additionally used.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, and the like.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. can be used. As the non-aqueous solvents and suspensions, propylene glycol and polyethylene Glycols, vegetable oils such as olive oil, injectable esters such as ethyloleate, and the like may be used.

When the pharmaceutical composition of the present invention is formulated as an injection solution, the pharmaceutical composition of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, and this is used for unit administration of an ampoule or vial. It can be formulated.

When the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like may be blended together with an additive so that the aqueous concentrate or wet powder is dispersed.

When formulating the pharmaceutical composition of the present invention into ointments, creams, powders for application, oils, skin external preparations, etc., animal oils, vegetable oils, waxes, paraffins, starches, tracanth, cellulose derivatives, polyethylene glycols, silicones, bentonite , Silica, talc, zinc oxide, and the like can be used as a carrier.

The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the mode of administration, the time of administration, and/or the route of administration, and the response to be achieved by the administration of the pharmaceutical composition Including the type and degree, the type of the subject to be administered, the age, weight, general health condition, the symptom or degree of the disease, sex, diet, excretion, drugs used concurrently with the subject or at the same time, and other components of the composition. It may vary depending on various factors and similar factors well known in the medical field, and a person of ordinary skill in the art can easily determine and prescribe an effective dosage for a desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once to several times a day.

The administration of the pharmaceutical composition of the present invention may be administered once a day, or may be administered several times. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. Considering all of the above factors, it may be administered in an amount capable of obtaining the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may each be independent, and as long as the pharmaceutical composition can reach the desired site, any route of administration and mode of administration may be followed without particular limitation. The pharmaceutical composition may be administered orally or parenterally.

As a parenteral administration method of the pharmaceutical composition of the present invention, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, etc. can be used, and a method of applying, spraying, or inhaling the composition to a diseased site It can also be used, but is not limited thereto.

The pharmaceutical composition of the present invention may be used in combination with various methods such as hormone therapy and drug therapy in order to prevent, improve or treat brain cancer.

The present invention provides a food composition for preventing or improving brain cancer comprising MG624 as an active ingredient.

The term "improvement" of the present invention means all actions in which brain cancer is improved or beneficially altered by administration of the composition of the present invention.

The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" refers to the structure of the human body. And it means ingestion for the purpose of obtaining a useful effect for health use such as controlling nutrients for function or physiological action.

The food composition of the present invention may contain additional ingredients that are commonly used to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.), disinfectants (bleached and highly bleached, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT) ), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweeteners (dulsin, cyclamate, saccharin, sodium, etc.), Food additives such as flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen stannate, etc.), reinforcing agents, emulsifiers, thickeners (thickening agents), coating agents, gum base agents, foam inhibitors, solvents, improvers, etc. Can be added. The additive may be selected according to the type of food and used in an appropriate amount.

When the food composition of the present invention is used as a food additive, it may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

In the food composition of the present invention, the content of MG624 is not particularly limited, and may be variously changed depending on the state of the subject to be administered, the type of specific condition, and the degree of progression. If necessary, it can also be included in the total amount of the food.

As another aspect of the present invention for achieving the above object, it provides a method for preventing or treating brain cancer, comprising administering to an individual a pharmaceutical composition containing MG624 as an active ingredient.

The MG624, brain cancer, administration, prevention, and treatment are as described above.

In the present invention, the term "individual" may mean all animals including humans who have developed or are likely to develop brain cancer. The animals may be mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. in need of treatment for not only humans but similar symptoms. It may also mean animals other than humans, but is not limited thereto.

The prevention or treatment method of the present invention may specifically include the step of administering the composition in a pharmaceutically effective amount to an individual who has or is at risk of developing brain cancer.

MG624 of the present invention is a pharmaceutical composition for preventing or treating brain cancer by inhibiting STAT3 activity, reducing tumor formation and metastasis ability of brain cancer cells, and resistance to temozolomide drugs to inhibit the onset and recurrence of brain cancer, or It can be usefully used as a food composition for preventing or improving brain cancer.

1 is a graph confirming that temozolomide-resistant glioblastoma cells (LN229-RRAD) have strong resistance to temozolomide (TMZ) compared to maternal LN229-VEC cells.
FIG. 2 is a graph showing the result of remarkably reducing sphere formation in temozolomide-resistant glioblastoma cells (LN229-RRAD) when MG624 was treated with 0.5 μmol/L.
3 is a graph confirming that STAT3 activity was decreased by treatment with MG624 at 5 μmol/L in a state in which STAT3 activity was increased by treatment with growth factor EGF in temozolomide-resistant glioblastoma cells (LN229-RRAD).
Figure 4 is a graph confirming that the IC ₅₀ of MG624 in LN229-RRAD cells is significantly low, 0.24 μmol / L.
5 is a graph confirming that three types of muscarinic acetylcholine receptor inhibitors other than MG624 were tested under the same conditions as MG624, and these drugs did not affect sphere formation at all even if the concentration was increased.
6 is a graph confirming the results that mecamylamine and methyllycaconitine passing through the cerebrovascular barrier are nicotine-based acetylcholine receptors such as MG624, but only MG 624 affects the growth inhibition of temozolomide-resistant glioblastoma cells.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Human glioblastoma cell line LN229 was cultured in DMEM medium containing 5% fetal bovine serum and penicillin/streptomycin (Gibco BRL) at 37°C and 5% CO ₂ .

Example 1. Confirmation of Temozolomide Resistance of RRAD Overexpressing Cell Line

LN229-VEC and LN229-RRAD cells were added to each of 500 in a 6 well plate, and temozolomide was treated for 48 hours by concentration (10-300 νM), and then the culture solution was replaced . LN229-VEC cells are general glioblastoma cells, and LN229-RRAD cells are cells that are resistant to temozolomide (RRAD overexpressing cells).

Clones formation was prevented for 7-14 days after the gastric culture was replaced. Observation was performed with respect to clones of 50 cells or more, counting the number of clones, staining with crystal violet and extracting with 1% SDS, and measuring absorbance at 570 nm.

The results are shown in FIG. 1.

As can be seen in FIG. 1, temozolomide-resistant glioblastoma cells (LN229-RRAD) overexpressing RRAD had strong resistance to temozolomide compared to the control LN229-VEC cells. Specifically, as a result of measuring the half maximal inhibitory concentration (IC ₅₀ ) of temozolomide, it was found that the LN229-RRAD cells were 300 μmol/L or more, whereas the control LN229-VEC cells were 10 μmol/L.

Example 2. Sphere formation assay

To perform the sphere forming assay, cells were suspended in DMEM/F12 (Invotrogen) medium containing EGF (20 ng/mL; Invitrogen), bFGF (20 ng/mL; Invitrogen), and N2 supplement (1X; Invitrogen). Cultured. After 5 to 7 days, the formed spheres were recovered and proteins were extracted for further experiments or separated into single cells with Accutase (Invitrogen) and analyzed. A sphere forming assay was performed for 5-7 days after treatment with MG624 on the glioblastoma cell line.

The results are shown in FIG. 2.

As can be seen in FIG. 2, when MG624 was treated, sphere formation was significantly reduced in temozolomide-resistant glioblastoma cells (LN229-RRAD).

Example 3. STAT3 activity assay

In order to examine the inhibitory effect of the compound of the present invention on STAT3 activity, the following experiment was performed using a dual luciferase assay. In brain cancer cell line (LN229), a plasmid expressing firefly luciferase is increased in proportion to the activity of STAT3 and a plasmid expressing Renilla luciferase is simultaneously transfected. ). Brain cancer cell lines transfected with plasmids were cultured for 16 hours, and then MG624 was treated with 5 μmol/L. After two hours, the cells expressing firefly luciferase and renilla luciferase were washed with lysis buffer, followed by sequential addition of specific separate substrates (Beetle luciferin and coelenterazine). I did. The luminescence intensity by decomposition of the substrate was measured using a luminometer.

The results are shown in FIG. 3.

As can be seen in FIG. 3, the activity of STAT3 induced by EGF (50 ng/ml) in temozolomide-resistant glioblastoma cells (LN229-RRAD) was decreased. Through the above results, it was confirmed that MG624 is very effective in selectively activating STAT3 and inhibiting autoproliferation of glioblastoma against temozolomide-resistant glioblastoma cells.

Example 4. IC of MG624 ₅₀ Measurement and comparison with other drugs

In a 96 well plate in a state in which LN229-RRAD cells were suspended in DMEM/F12 (Invotrogen) medium containing EGF (20ng/mL; Invitrogen), bFGF (20ng/mL; Invitrogen), and N2 supplement (1X; Invitrogen). 700 dogs were cultured. MG624 and other drugs were diluted by a stepwise dilution method, treated with 0.03-10 νM, and further observed for 5-7 days. The number of spheres was counted under a microscope, and a graph was drawn with Prism 5 of GraphPad, and the IC ₅₀ was determined.

The results are shown in FIG. 4.

As can be seen in FIG. 4, as a result of measuring the IC ₅₀ of MG624, it was found that cancer cell proliferation was effectively inhibited even at a low MG624 concentration of 0.24 μmol/L for LN229-RRAD cells.

In addition, the results of experimenting with three types of muscarinic acetylcholine receptor inhibitors as other drugs under the same conditions as MG624 are shown in FIG. 5. The results of FIG. 5 show that even if the concentration of drugs is increased in LN229-RRAD cells, there is no effect on sphere formation. Specifically, the results when AFDX116 (Otenzepad), 4-DAMP, and TriHP (Trihexyphenidyl) are used. It was confirmed that only MG624 can exhibit a therapeutic effect.

In addition, as can be seen in Figure 6, mecamylamine and Methyllycaconitine (MLA), two kinds of acetylcholine receptor inhibitory drugs that pass through the cerebrovascular barrier, are nicotine-based acetylcholine receptors such as MG624, but temozolomide resistance bridge It was confirmed that only MG624 is effective in inhibiting the growth of blastoma cells.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

Claims (8)

A pharmaceutical composition for preventing or treating brain cancer, comprising a compound represented by the following Formula 1 as an active ingredient:
[Formula 1]

The pharmaceutical composition for preventing or treating brain cancer according to claim 1, wherein the brain cancer is temozolomide-resistant brain cancer. The pharmaceutical composition for preventing or treating brain cancer according to claim 1, wherein the brain cancer is glioblastoma or undifferentiated astrocytoma. The pharmaceutical composition for preventing or treating brain cancer according to claim 1, wherein the prevention or treatment of brain cancer is to inhibit tumor formation or metastasis of brain cancer. The pharmaceutical composition of claim 1, wherein the composition further contains a pharmacologically acceptable carrier or adjuvant. A food composition for preventing or improving brain cancer, comprising a compound represented by the following Formula 1 as an active ingredient:
[Formula 1]

The food composition of claim 6, wherein the brain cancer is temozolomide-resistant brain cancer. The food composition according to claim 6, wherein the brain cancer is glioblastoma or undifferentiated astrocytoma.

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