KR20140008644A - Pharmaceutical composition for the preventing or treating brain tumor or glioblastoma having resistance of temodal containing benzydamine as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancers such as brain tumors (especially, glioblastoma) or Temodal-resistant glioblastoma, containing benzydamine as an active ingredient. The benzydamine according to the present invention effectively suppresses the proliferation of glioblastoma, which is a type of brain tumor, and exhibits an excellent therapy effect even on glioblastoma cancer cells having resistance to Temodal (temozolomide), which is a conventional agent for treating glioblastoma. Therefore, the benzydamine according to the present invention can be useful as an active ingredient of a pharmaceutical composition for preventing or treating cancers such as brain tumors (especially, glioblastoma) and Temodal-resistant glioblastoma. [Reference numerals] (AA) Inhibition ratio (%)

Description

Pharmaceutical composition for the preventing or treating brain tumor or glioblastoma having resistance of Temodal containing Benzydamine as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating cancer or temodal resistant glioblastoma, such as a brain tumor (particularly glioblastoma), which contains benzidamine as an active ingredient.

Tumors on glial cells that supply nerve cells are called glioma. These gliomas are classified histologically based on nuclear atypical cell death, cell necrosis, vascular endothelial cell proliferation, and mitosis and are classified as the most malignant grade 4 and diagnosed as glioblastoma multiform (GBM). Newlands ES et al. Eur. J. Cancer 32A, 22362241, 1996].

Glioblastoma is the most malignant and most common brain tumor among glioma, accounting for 25% of adult glioma and 15% of pediatric glioma. Currently, there is no medicament other than surgery and radiation therapy, and the prognosis is significantly worse than that of other tumors. The average survival time after diagnosis is only 14 months [Chakravarti A. et al. Clin Cancer Res. 12, 47384746, 2007].

Recently, extensive gene expression studies of glioblastoma have revealed that there are four types of glioblastoma subtypes. The four types of glioblastoma subtypes are the proneural subtype, the mesenchymal subtype, the classical subtype, and the neural subtype, respectively. Life expectancy, mean age of patients, and responsiveness to existing therapies have been found (HS Phillips HS et al., Cancer Cell, 9, 157173, 2006).

Glioblastoma subtypes differ greatly in overexpression or mutation patterns of important genes (Cancer Genome Atlas Research Network, Nature, 455, 10611068, 2008). In the classical subtype, the epidermal growth factor receptor (EGFR) is abnormally overexpressed, and unlike the other three subtypes, there is no mutation in the cancer suppressor gene 'p53'. Patients with classical subtypes are known to have a better course of treatment than other subtypes. Unlike the classical subtypes, there is a high percentage of mutations in the 'p53' gene and a high percentage of mutations in the isocitrate dehydrogenase (IDH1) and platelet-derived growth factor receptor-α (PDGFRA) genes. Mutations in these genes are very important for the proliferation of cancer cells, and mutations in the platelet-derived growth factor receptor-α (PDGFRA) gene appear only in the systemic subtype. Patients with systemic subtypes are younger than other subtypes, but do not increase their survival with current therapies. In mesenchymal subtypes, mutation rates of NF1 and PTEN cancer suppressor genes as well as mutations of the 'p53' gene are high. Nerve subtypes are observed mutations in genes found in other subtypes, but do not characteristically show high or low mutation rates.

Currently, the most common method of treating glioblastoma is surgical removal of cancer, followed by radiation therapy and chemotherapy with temodal (temozolomide).

While no effective drug has yet been developed to treat glioblastoma, temodal is the only oral anticancer drug currently used. When temodal is administered to a patient, it initially responds to the drug to some extent, but the resistance to the drug appears quickly, and once the resistance occurs, the treatment is no longer difficult. Therefore, there is an urgent need for the development of effective anticancer agents that can treat brain tumors, especially for the treatment of tumors resistant to temodal. In addition, the four subtypes of glioblastoma have different genetic backgrounds, and therefore, it is necessary to develop drugs that specifically respond to each subtype.

Temodal is spontaneously degraded within the cell to yield 5- (3-methyltriazin-1-yl) -imidazole-4-carboxamide [5- (3-methyltriazine-1-yl) -imidazole-4-carboxamide , MITC] and MITC again produces 5-aminoimidazole-4-carboximide, an active substance. 5-aminoimidazole-4-carboximide can methylate the purine base of DNA, specifically the N7 position (70%) of guanine, N3 position (9%) of adenine and O6 position (6%) of guanine Methylate. In particular, methylation of the guanine O6 position is very important for cancer cell toxicity of temodal. DNA bases modified with O6-methyl guanine induce inconsistencies between DNA strands during DNA replication and result in cell death [Newlands ES, et al. Cancer Treat. Rev. 23, 3561, 1997].

On the other hand, DNA repair protein MGMT (O6-me-G methyltransferase) serves to remove the methyl residue from the methylated guanine O6 position. There is a 300-fold difference in MGMT protein activity among cancer patients [Silber JR et al., Clin Cancer Res. 5, 807814, 1999]. Epigenetic methylation of the MGMT gene promoter inhibits MGMT gene expression and is found in about 45% of glioma patients [Hegi ME et al., N Engl J Med. 352, 9971003, 2005]. It is known that the therapeutic effect of temodal is good in patients whose gene expression is suppressed due to methylation of the MGMT gene promoter. On the other hand, patients with overexpression of MGMT develop temodal resistance and treatment often fails [Bobola MS et al., Clin Cancer Res. 2, 735741, 1996.

In order to overcome the resistance of the alkylating agent by MGMT activity, non-toxic fake substrates are also used to inhibit MGMT protein activity. Fake substrates include O6-benzylguamine [Dolan ME et al., Proc Natl Acad Sci USA 87, 53685372, 1990] or O6-4- (bromothenyl) guanine [O6- (4-bromothenyl) guanine], which inactivate enzymes by transferring benzyl or bromobenzyl residues to the cysteine amino acids of the enzymes (Clemons M. et al., Brit J Can 93, 11521156, 2005). O6-4- (bromothenyl) guanine has been reported to be of limited use due to myelosuppression when treated in combination with DNA alkylating agents [Gerson SL et al., J Clin Oncol 20, 23882399, 2002] . In addition to MGMT enzymatic activity, the activity of MMR (Mismatch repair) protein complexes that repair DNA strand mismatches is closely related to temodal resistance (Esteller M. and Herman JG Oncogene 23, 18, 2004). The higher the activity of the MMR protein complex, the more likely DNA modification by temodal is recognized by the cells, resulting in easier DNA strand break and subsequent cell death. On the other hand, if MMR activity is deteriorated due to genetic or environmental factors, the DNA modification caused by temodal does not lead to cell death, but rather the increase in temodal resistance [Palombo F. et al., Science 268, 19121914, 1995]. .

Benzidamine (3- (1-benzyl-1H-indazol-3-yloxy) -N, N-dimethylpropan-1-amine) is a nonsteroidal anti-inflammatory drug that binds specifically to inflamed tissue. The mechanism of action is unclear, but it is believed to act as an inhibitor of prostaglandin synthetase, reducing the secretion of substances that induce inflammatory responses. [Anand, JS et al., Clinical Toxicology 45, 198199, 2007] .

In fact, it has been reported that treatment with benzidamine reduces inflammation-induced cytokines such as TNF-alpha and IL-1beta when inflammatory reactions are caused by external stimuli. TNF-alpha is known to activate several types of cytokines and proteases required for the inflammatory response, and TNF-alpha produced in cancer cells is known to play an important role in cancer formation and metastasis [Sironi M. et al. , Cytokine 8, 710-716, 1996].

Therefore, the researchers of the present invention, while studying a compound that can replace the conventional modal treatment glioblastoma, benzidamine (Benzydamine), known as a nonsteroidal anti-inflammatory drug, inhibits the proliferation of glioblastoma, a type of brain tumor In addition, the present invention has been found to effectively work with glioblastoma cancer cells exhibiting resistance to temodal, thereby completing the present invention.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, such as brain tumors (particularly glioblastoma), including benzidamine or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of temodal resistant glioblastoma containing benzydamine or a pharmaceutically acceptable salt thereof as an active ingredient.

It is another object of the present invention to provide a compound or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of cancer, such as brain tumors (especially glioblastoma).

Another object of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of temodal resistant glioblastoma.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, such as brain tumors (particularly glioblastoma), including a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient do.

[Formula 1]

The present invention also provides a pharmaceutical composition for preventing or treating temodal (Tmodal, temozolomide) resistant glioblastoma containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof used in the prevention or treatment of cancers such as brain tumors (especially glioblastoma).

The present invention also provides a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof used in the prevention or treatment of temodal resistant glioblastoma.

Benzidamine according to the present invention not only effectively inhibits the proliferation of glioblastoma, a type of brain tumor, but also has an excellent therapeutic effect on glioblastoma cancer cells that exhibit resistance to conventional glioblastoma therapies (Temodal, temozolomide). Therefore, it may be useful as an active ingredient of a pharmaceutical composition for preventing or treating cancer, such as brain tumors (especially glioblastoma) and temodal resistant glioblastoma.

1 is a graph showing the cell growth inhibition concentration-response curves by benzidamine according to Formula 1 of the present invention (in FIG. 1, '559T' is a systemic subtype glioblastoma and '592T' is a mesenchymal subtype Glioblastoma, '626T' is a classical subtype of glioblastoma, 'NHA' is a normal astrocytic cell, 'U87MG' is a glioblastoma cell line, and 'A549' is a lung cancer cell line).
2 is a diagram showing that the compound of Comparative Example 1 (temodal) inhibits the formation of neurospheres in glioblastoma cells.
3 is a diagram showing that the compound of formula 1 (benzidamine) of the present invention inhibits the formation of neurospheres in glioblastoma cells.

Hereinafter, the present invention will be described in detail.

The present invention relates to a compound that inhibits the proliferation of glioblastoma multiform (GBM), which is a malignant brain tumor. It is intended to be used as a new treatment for glioblastoma, a type of brain tumor, and especially for the treatment of glioblastoma, which is resistant to conventional glioblastoma therapeutic temodal (Temodal, temozolomide).

The present invention provides a pharmaceutical composition for preventing or treating cancer containing the compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition according to the present invention can be used for the treatment of cancers such as brain tumors, and is preferably used for brain tumors, and more preferably used for glioblastoma among the brain tumors.

Here, the subtypes of glioblastoma can be applied to proneural subtype, mesenchymal subtype, classical subtype and neural subtype, and other unclassified It is also applicable to glioblastomas, which are preferred but not limited to mesenchymal subtypes or classical subtypes.

The compound (benzidamine) according to the present invention is to be treated to general glioblastoma cell lines and lung cancer cell lines other than glioblastoma cancer cells derived from patients, to glioblastoma cancer cells derived from patients, to glioblastoma cancer cells derived from patients. In the case of inhibiting the growth of cancer cells, while not toxic to normal astrocytic cells but not cancer cells (see Experimental Example 1).

As described above, benzidamine according to the present invention effectively inhibits the proliferation of glioblastoma, which is a kind of brain tumor, and thus is useful as an active ingredient of a pharmaceutical composition for preventing or treating cancer, such as glioblastoma (glioblastoma). can do.

The present invention also provides a pharmaceutical composition for preventing or treating temodal (temozolomide) resistant glioblastoma containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Currently, the general method of treating glioblastoma is surgical removal of cancer, followed by radiation therapy and chemotherapy with temodal (temozolomide). When patients are treated using temodal, many patients initially respond to the drug to some extent, but the reality is that most patients develop drug resistance and are difficult to treat in case of tumors with temodal resistance.

Therefore, there is an urgent need for the development of effective anticancer drugs that can treat brain tumors (especially glioblastomas), particularly for the treatment of tumors resistant to temodal (temozolomide).

In the case of the compound (benzidamine) according to the present invention, three of four classical subtypes of glioblastoma showed a strong neurosphere inhibitory effect and a strong activity in two cells of unclassified glioblastoma. In particular, the compound (benzidamine) according to the present invention has been shown to have an anticancer effect that inhibits the formation of neurospheres in glioblastoma cancer cells that are resistant to the compound (temodal) of Comparative Example 1. These results indicate that the compound (benzidamine) according to the present invention has a broader anti-cancer effect on glioblastoma cancer cells than the compound (temodal) of Comparative Example 1, and may inhibit the formation of neurospheres of cancer cells having temodal resistance. The facts suggest the possibility of being used as an effective treatment for patients with temodal resistance (see Experimental Example 2).

Accordingly, the pharmaceutical composition containing benzidamine according to the present invention as an active ingredient may be administered to a patient in need of glioblastoma treatment alone or in combination with temodal (temozolomide).

As described above, benzidamine according to the present invention has an excellent therapeutic effect on glioblastoma cancer cells showing resistance to conventional glioblastoma temodal (Temodal, temozolomide), thus preventing or preventing temodal resistant glioblastoma. It may be useful as an active ingredient of a therapeutic pharmaceutical composition.

The composition including the compound represented by Chemical Formula 1 of the present invention preferably includes 0.1 to 50% by weight of the composition, based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

 In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and any parenteral administration method may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Furthermore, the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof used in the prevention or treatment of cancers such as brain tumors (especially glioblastoma).

[Formula 1]

.

.

The compound represented by the formula (1) according to the present invention can be used for the treatment of cancers such as brain tumors, it is preferable to use for brain tumors, more preferably used for glioblastoma among the brain tumors.

Here, the subtypes of glioblastoma can be applied to proneural subtype, mesenchymal subtype, classical subtype and neural subtype, and other unclassified It is also applicable to glioblastomas, which are preferred but not limited to mesenchymal subtypes or classical subtypes.

The present invention also provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof used in the prevention or treatment of Temodal (temozolomide) resistant glioblastoma.

[Formula 1]

The compound represented by Formula 1 according to the present invention may be administered to a patient in need of treatment of glioblastoma alone or in combination with temodal (temozolomide).

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. From non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

Acid addition salt according to the present invention is a conventional method, for example, a precipitate formed by dissolving a compound represented by the formula (1) in an organic solvent, for example methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, and adding an organic or inorganic acid The solvent may be prepared by filtration, drying, or by distillation under reduced pressure of the solvent and excess acid, followed by drying or crystallization under an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, the present invention includes not only the compound of Formula 1 and a pharmaceutically acceptable salt thereof, but also possible solvates, hydrates, and the like that can be prepared therefrom.

Furthermore, the present invention administers a pharmaceutical composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to a patient in need of treatment for cancer or temodal (temozolomide) resistant glioblastoma. It provides a treatment method characterized in that.

Here, the cancer is a brain tumor or the like, preferably applied to a brain tumor, more preferably applied to glioblastoma among the brain tumors.

Also, among the subtypes of glioblastoma, it can be applied to proneural subtype, mesenchymal subtype, classical subtype and neural subtype, and other unclassified. It is also applicable to glioblastomas, which are preferred but not limited to mesenchymal subtypes or classical subtypes.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

<Production Example 1> 3- (1-benzyl--1H- indazol-3-yloxy) -N, N- dimethyl-1-amine [3- (1-benzyl-1H -indazol-3-yloxy) - preparation of N, N-dimethylpropan-1- amine] ( benzimidazolyl damin, Benzydamine)

Benzidamine was purchased from Sigma Aldrich (Reagent No. B5524) and used without further purification.

< Comparative Example  1> Of temodal (temozolomide)  Ready

Temodal was purchased from Sigma Aldrich (Reagent No. A4638) and used without further purification.

< Example  1> Evaluation of cell proliferation inhibitory effect

In order to determine the effect of the compound of formula 1 (benzidamine) according to the present invention to inhibit the proliferation of glioblastoma cancer cells, it was tested as follows.

Specifically, cell proliferation inhibitory effect was measured by WST-1 cell proliferation assay (http://www.roche-applied-science.com/PROD_INF/MANUALS/CELL_MAN/apoptosis_087_088.pdf). The compound of formula 1 was placed in a 384-well cell culture vessel, and three types of glioblastomas (proneural subtype (559T), mesenchymal subtype (592T), classical subtype) subtype, 626T)], normal human astrocyte (NHA), lung cancer cell line (A549) and glioblastoma cell line (U87MG), each of the cells were dispensed at 4.5 x 10 ³ cells per well and filled to 50 μl. At this time, the concentration of DMSO, the solvent of the compound of Formula 1, was not to exceed 0.5%. After incubating the cells for 72 hours, the effect of the compound of Formula 1 on cell proliferation was added to 5 μl of 5 mg / ml WST-1 reagent, and then reacted for 3 to 4 hours, followed by measurement of absorbance at 450 nm. It was.

The results of this experiment are shown in Table 1 and FIG. 1.

Cell line % Inhibition GI ₅₀ (μM) 10 μM 3.2 [mu] M 1.0 μM 0.32 μM 0.1 μM 0.032 μM 559T 33.8 -1.0 -5.7 -7.3 -10.0 -18.6 > 10 592T 88.3 62.8 25.6 15.7 7.0 0.6 2.8 626T 58.0 43.4 41.4 28.9 34.0 33.2 9.0 NHA 3.4 9.7 1.5 1.7 -2.2 -5.0 > 10 U87MG 26.6 9.9 4.2 17.9 -7.0 -3.0 > 10 A549 34.3 25.6 19.5 14.5 13.1 -1.2 > 10

1 is a graph showing the cell growth inhibition concentration-response curves by benzidamine according to Formula 1 of the present invention (in FIG. 1, '559T' is a systemic subtype glioblastoma and '592T' is a mesenchymal subtype Glioblastoma, '626T' is a classical subtype of glioblastoma, 'NHA' is a normal astrocytic cell, 'U87MG' is a glioblastoma cell line, and 'A549' is a lung cancer cell line).

As shown in Table 1 and Figure 1, the compound of formula 1 (benzidamine) 50% growth inhibitory concentration as a result of treatment of whole body (559T), mesenchymal (592T) and classical (626T) subtype glioblastoma cancer cells ( GI ₅₀ ) were> 10, 2.8, and 9.0 μM, respectively, indicating that the cell proliferation inhibitory effect was highest for mesenchymal subtype glioblastoma. On the other hand, no toxicity was confirmed for normal astrocytic cells, not cancer cells, and the compound of formula 1 (benzidamine) was effective in general glioblastoma cell line (U87MG) instead of glioblastoma cancer cells derived from patients. appear. In addition, it was confirmed that there is some effect against lung cancer cell line (A549), not glioblastoma.

Therefore, the compound of formula 1 (benzidamine) according to the present invention has an excellent effect of inhibiting the proliferation of glioblastoma, in particular, because of the effect of significantly inhibiting the growth of mesenchymal subtype cancer cells among various subtypes of glioblastoma, It may be useful as an active ingredient of a pharmaceutical composition for preventing or treating cancer such as brain tumors (particularly, glioblastoma).

< Example  2> Neurosphere ( Neurosphere A) inhibition of formation

In order to determine whether the compound of Formula 1 (benzidamine) of the present invention and the compound of Comparative Example 1 (temodal) exhibit an anticancer effect, the extent of inhibiting neurosphere formation in glioblastoma cells was examined.

Specifically, the neurosphere formation ability was confirmed by a method of measuring the number of newly formed neurospheres by re-distributing the same number of glioblastoma cells in a culture vessel after a certain period of culture. First, while subcultured with glioblastoma patient-derived cells, 20, 50, and 200 cells were mixed in 50 μl of culture medium in 96-well cell culture vessels, and the compounds of formula 1 (Benz) 50 [mu] l of each of the compounds (temodal) and Damin) and Comparative Example 1 were finally treated to 1, 3 and 10 [mu] M. Thereafter, each of the compound of Formula 1 (benzidamine) and the compound of Comparative Example 1 (temodal) were incubated for about 7-23 days in 50 μl of the cell culture medium once a week with the same concentrations as above. When 50 μm or more of neurospheres were formed in a control group not treated with drugs, the number of neurospheres present in each well was measured to determine the number of neurospheres by the compound of Formula 1 (benzidamine) and the compound of Comparative Example 1 (temodal). The reduction was compared.

In this case, glioblastoma patient-derived cells were measured for inhibition of neurosphere formation using a total of 10 glioblastoma cells derived from the patient. The glioblastomas used in the experiment were classified into four types of A, B, C and D according to the gene expression form, and the methylation of MGMT (O6-me-G methyltransferase) gene, which is a DNA repair protein of each cell line, was confirmed. Form A [GBM # 1, # 2, # 3] is the Proneural subtype, Form B [GBM # 4, # 5, # 6, # 7] is the Classical subtype, Form C [GBM # 8] is a mesenchymal subtype and the D type [GBM # 9, # 10] is an unclassified glioblastoma.

The experimental results are shown in Table 2 below and FIGS. 2-3.

Cell line Classification Experiment period
(day) Temodal Azathioprine MGMT Gene Methylation 20 μM 3 [mu] M 10 μM GBM # 1 A 7 S R S M GBM # 2 A 15 R R S UM GBM # 3 A 21 R I I UM GBM # 4 B 9 S S S M / UM GBM # 5 B 21 S S S M <UM GBM # 6 B 23 S S S M> UM GBM # 7 B 23 S R R ** GBM # 8 C 15 R R I UM GBM # 9 D 21 S S S M> UM GBM # 10 D 10 R S S UM

(In Table 2,

A: Proneural subtype,

B: Classical subtype,

C: Mesenchymal subtype,

D: unclassified glioblastoma,

S: strong effect,

I: the effect is moderate,

R: weak effect,

M: gene promoter methylation,

UM: gene promoter demethylation,

**: not rated)

2 is a diagram showing that the compound of Comparative Example 1 (temodal) inhibits the formation of neurospheres in glioblastoma cells.

3 is a diagram showing that the compound of formula 1 (benzidamine) of the present invention inhibits the formation of neurospheres in glioblastoma cells.

As shown in Table 2 and Figures 2-3,

The compound of Comparative Example 1 (temodal) did not inhibit neuronal formation for glioblastomas (marked as "UM" in the MGMT gene methylation list in Table 2) that are believed to have high MGMT activity because the gene promoter was not methylated. In the case of glioblastoma carcinoma cells with relatively high methylation of MGMT genes, temodal showed a tendency to significantly inhibit neuronal formation.

Furthermore, in the case of the compound of formula 1 (benzidamine), at 3 μM concentration, three of four subtypes of classical subtype glioblastoma classified as B-type showed strong neuronal inhibitory effect, and even two cells classified as D-type were strong. Showed activity. Relatively weak effects were found in systemic and mesenchymal subtype glioblastomas (forms A and C). However, when the concentration was increased to 10 μM, all glioblastoma cancer cells except GBM # 7 showed moderate or superior effects.

In particular, the compound of formula 1 (benzidamine) at 10 μM concentrations of neurosphere formation also in GBM # 2, # 3, # 8, # 10 glioblastoma cancer cells that are resistant to the compound of Comparative Example 1 (temodal) It has been shown to have an anticancer effect. These results indicate that the compound of Formula 1 (benzidamine) has a broader anticancer effect on glioblastoma cancer cells than the compound of Comparative Example 1 (temodal), and can also inhibit the formation of neurospheres of cancer cells having temodal resistance. Offers the potential to be used as an effective treatment for patients with temodal resistance.

Therefore, the compound of formula 1 (benzidamine) according to the present invention exhibits a wide range of therapeutic effects on various types of glioblastoma cancer cells, and has a therapeutic effect on glioblastoma cancer cells showing resistance to the compound (temodal) of Comparative Example 1, It may be useful as an active ingredient in a pharmaceutical composition for preventing or treating cancer, such as a glioblastoma (especially glioblastoma), and as an active ingredient for preventing or treating glioblastoma, which is resistant to temodal (temozolomide). Can be useful.

Meanwhile, the compound represented by Formula 1 according to the present invention can be formulated into various forms according to the purpose. The following are some examples of formulation methods containing the compound represented by Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

< Formulation example  1> Preparation of Pharmaceutical Formulations

1-1. Sanje  Produce

500 mg of the compound represented by Formula 1 of the present invention

Lactose 100 mg

10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

500 mg of the compound represented by Formula 1 of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Manufacture of capsules

500 mg of the compound represented by Formula 1 of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

500 mg of the compound represented by Formula 1 of the present invention

Sterile sterilized water for injection

pH adjuster

(2 ml) per ampoule in accordance with the usual injection method.

1-5. Liquid  Produce

100 mg of the compound represented by Formula 1 of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The liquid is prepared by sterilization.

Claims (7)

A pharmaceutical composition for preventing or treating cancer containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.
The method of claim 1,
The cancer is a pharmaceutical composition, characterized in that the brain tumor.
3. The method of claim 2,
The brain tumor is a pharmaceutical composition, characterized in that glioblastoma.
The method of claim 3,
The subtypes of glioblastoma are proneural subtype, mesenchymal subtype, classical subtype, or neural subtype.
A pharmaceutical composition for preventing or treating temodal (temozolomide) resistant glioblastoma containing a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.
The method of claim 5,
The pharmaceutical composition is a pharmaceutical composition, characterized in that administered alone or in combination with Temodal (Temodal, temozolomide).
The method of claim 5,
The subtypes of glioblastoma are proneural subtype, mesenchymal subtype, classical subtype, or neural subtype.

Images