WO2014065545A1

WO2014065545A1 - Pharmaceutical composition containing lobarstin for preventing or treating brain cancer and combined therapy for treating brain cancer using same

Info

Publication number: WO2014065545A1
Application number: PCT/KR2013/009366
Authority: WO
Inventors: 임정한; 김일찬; 김덕규; 한세종; 정희경
Original assignee: 한국해양과학기술원
Priority date: 2012-10-24
Filing date: 2013-10-21
Publication date: 2014-05-01
Also published as: KR20140052396A; KR101892078B1

Abstract

The present invention relates to a novel use of lobarstin which is a metabolite derived from a natural substance. More particularly, the present invention relates to the use of a composition containing lobarstin or a pharmaceutically acceptable salt thereof for preventing or treating brain cancer, and to the use thereof in combined therapy, in conjunction with temozolomide, for treating brain cancer. Since it has been confirmed that cell cytotoxicity is exhibited when a temozolomide resistant cancer cell line is treated solely with lobarstin, which is a compound according to the present invention, the present invention can be prepared as a drug for overcoming anticancer drug resistance. Further, it has been confirmed that cell death is promoted during combined treatment using lobarstin and temozolomide, which is a cancer drug for standard therapy, and thus, it is possible to develop the present invention as a drug for combined therapy in addition to conventional therapy. Therefore, the present invention is useful.

Description

Pharmaceutical composition for the prevention or treatment of brain cancer containing lovastin and combination therapy for the treatment of brain cancer using the same

The present invention relates to a novel use of the natural product-derived metabolite, lovastin, and more particularly, to the prophylaxis or treatment of brain cancer containing lovastin or a pharmaceutically acceptable salt thereof and with temozolomide for the treatment of brain cancer. It relates to the use to combination therapy.

Lichens are known to produce higher plants and other unique secondary metabolites (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002), and most of the secondary metabolites they produce are depside and depsidone. And dibenzfurane, which compounds are believed to be associated with the low growth rate of lichens (Kumar, KCS et al., J. Nat. Prod ., 62: 817, 1999; Huneck, S. , Naturwissenschaften , 86: 559, 1999), various biological activities were confirmed by the screening process of lichen metabolites, including antibiotics, antimycobacterial, antiviral, analgesic and antipyretic effects (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002; Kumar, KCS et al., J. Nat. Prod ., 62: 817, 1999; C. Seo et al., Bioorg Med Chem Lett , 19: 2801-3, 2009). Therefore, interest in the development of medicines using lipoprotein secondary metabolites is increasing.

Meanwhile, glioblastoma multiforme (GBM) is a malignant carcinoma originating from glial cells and their progenitor cells of the central nervous system and most often occurs in the subcortical white matter of the cerebral hemispheres in the cortical temporal lobe of the brain. (N. Engl. J. Med. (2008) 359: 492). GBM is the most aggressive of the glioblastomas with a poor prognosis (S. Tanaka et al., Nat Rev Clin Oncol , 10: 14-26, 2013). GBM corresponds to Grade IV among glial-derived carcinomas, with a high cell count, active cell division, and associated with vascular proliferation and necrosis (AJCC cancer staging handbook: from the AJCC cancer staging manual.7th ed. 2010, New York: Springer. Xix, 718 p).

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

Temozolomide (TMZ), a drug used in the standard treatment of GBM, is an alkylating agent that causes DNA damage to remove cancer cells (Lancet (2000) 355: 1115). This produces a variety of methyl adducts at the O ⁶ -guanine, N ⁷ -guanine and N ³ -guanine positions on DNA (JNSarkaria et al., Clin Cancer Res , 14: 2900-8, 2008). Higher expression of the DNA repair gene 0 ⁶ -methylguanine-DNA-methyltransferase (MGMT) or a transporter (eg P-glycoprotein) that regulates the drug's intracellular bioavailability (J. Neurochem. (2006) 96: 766; Acta. Neuropathol. (1997) 94: 605). Cytotoxicity of TMZ depends on DNA repair systems such as mismatch repair (MMR), MGMT repair and base excision repair (BER) (M. Grzmil et al., Biochim Biophys Acta , 1834: 1371-80, 2013; M. Nakada et al., Front Oncol , 2:98, 2012; J. Koritzer et al., PLoS One 8 , e64498, 2013).

Indeed, GBM exhibits a complex pattern including a wide variety of genetic mutations, including p53 deletion, overexpression of EGFR and VEGF, and gene amplification of MDM2 and MGMT (US Pharmacist. (2010) 35: 3; J; Neurooncol. (2010) 96: 169). In particular, it is well known that overexpression of the MGMT gene is highly correlated with resistance to TMZ (J. Neurochem. (2006) 96: 766). Therefore, there is a need for the development of therapies that can enhance the effectiveness of current standard therapies.

Accordingly, the present inventors have sought to provide a new substance that is effective in eliminating temozolomide-resistant cancer cells and can enhance brain cancer treatment effects when co-administered with temozolomide. As a result, lovastin according to the present invention alone prevents brain cancer. And not only can be treated, it was confirmed that the anti-cancer effect is enhanced when combined with the anticancer temozolomide used in the standard therapy of brain cancer, the present invention was completed.

Summary of the Invention

An object of the present invention is to provide a new material capable of preventing and treating brain cancer.

Another object of the present invention is to provide a new usage that can enhance the effectiveness of the standard treatment of current brain cancer. Specifically, the present invention provides a method of enhancing the toxic effect of an existing anticancer agent through a combination treatment with an anticancer agent, temozolomide (TMZ), which is used in the standard therapy of brain cancer. In addition, the present invention provides a new use of a natural product-derived metabolite to prepare a drug that can overcome the anticancer drug resistance.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of brain cancer containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

The present invention also provides a combination therapy of co-administering the pharmaceutical composition with temozolomide (TMZ).

The present invention also provides the use of a compound represented by the following formula (1) for the manufacture of a medicament for the prevention or treatment of brain cancer:

Formula 1

The present invention also provides a functional food for the prevention or improvement of brain cancer containing the compound represented by the following formula (1) or a food acceptable salt thereof as an active ingredient:

Formula 1

The present invention also provides a combination therapy in which the functional food is co-administered with temozolomide (TMZ).

1 is a graph showing the anticancer effect of the compound of Formula 1, namely, lovastin's T98G cell line, which is a temozolomide-resistant polymorphic glioblastoma cell line, wherein 1A is a graph showing cytotoxicity according to the concentration of temozolomide against T98G cell line. And 1B is a graph showing the cytotoxicity according to the concentration of the compound of formula 1, ie lovastin, against the T98G cell line.

Figure 2 shows the cell viability of normal cells and T98G cells according to lovastin treatment of Example 3-2, that is, a graph showing the cytotoxicity according to the concentration of lovastin.

Figure 3 is a graph comparing the cytotoxicity of T98G cell line with high concentration of TMZ (750 μM) and 40 μM lovastin of the present invention alone and combined treatment (Robarstin + TMZ) for 3 days and 4 days, respectively.

Figure 4 is a graph comparing the cytotoxicity of the T98G cell line with low concentration of TMZ (500 μM) and 40 μM lovastin of the present invention alone and combined treatment (Robarstin + TMZ) for 3 days and 4 days, respectively.

Figure 5 shows the expression of MGMT and GAPDH of T98G cells of Example 5-4.

FIG. 6 shows cell viability when lovastin and TMZ are used in combination with the concentration of TMZ of Example 5-4, and shows the results of treatment for 3 days and 4 days.

7 shows cell viability when lovastin and TMZ are used in combination with the concentration of TMZ of Example 5-4, where V is a vehicle-administered group and L is a lovastin-administered group.

Figure 8 shows the recovery of TMZ induced DNA damage by lovastin of Example 5-5, (A) shows the time conditions of DNA damage and DNA repair, (B) image obtained by fluorescence microscopy (C) shows the numerical results of the experiment. Here, V is a vehicle administration group, L is a lovastin administration group, T is a TMZ administration group, LT is a lovastin and TMZ administration group, (D) means damage and (R) means repair.

Figure 9 shows the decrease in the expression of MGMT, PARP1, etc. when lovastin is administered for 2 days through the immunoblot (Immunoblot) results of Examples 5-6 (left), MGMT, PARP1 through RT-PCR results Decrease in expression of the back (right). V means vehicle administration group, L means lovastin administration group.

Figure 10 shows the decrease in expression of MGMT, PARP1, etc. through the immunoblot (Immunoblot) results of Example 5-6. Here, V is a vehicle administration group, L is a lovastin administration group, T is a TMZ administration group, LT is a lovastin and TMZ administration group, (D) means damage and (R) means repair.

FIG. 11 shows the reduced expression of DNA repair genes in cells using lovastin and TMZ.

Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

“Co-administration” herein may be achieved by the simultaneous, sequential or separate administration of the individual components of the therapy. By combining two or more drugs at the same time or sequentially, or alternately at regular or indeterminate intervals to obtain a combined therapeutic effect, the combination therapy is, for example, the degree of response, reaction rate, disease progression Efficacy as measured over a period of time or survival is defined as being capable of providing a synergistic effect while being therapeutically superior to the efficacy obtained by administering one or the other of the components of the combination therapy in conventional dosages. In the present invention, the compound of formula 1 of the present invention may be administered before, after, or simultaneously with administering an effective amount of temozolomide.

The anticancer efficacy of the treatment method of the present invention includes, but is not limited to, antitumor efficacy, response rate, duration to disease progression, survival rate, and the like. Antitumor efficacy of the treatment methods of the present invention includes, but is not limited to, tumor growth inhibition, tumor growth delay, tumor regression, tumor shrinkage, increased duration to tumor regrowth upon discontinuation of treatment, delay in disease progression, and the like. When treating animals, including humans, in need of treatment of cancer with the methods of treatment of the invention, the methods of treatment are measured, for example, by one or more of the degree of antitumor efficacy, response rate, duration to disease progression, and survival rate. It is expected to produce a potent efficacy. Anticancer efficacy includes prophylactic treatment as well as treatment of existing diseases.

As used herein, an "extract" of lichens is a substance obtained by dissolving an individual, tissue, or cell of lichens in a solvent, and may be concentrated by distillation or evaporation.

The present invention relates to a pharmaceutical composition for preventing or treating brain cancer containing, as an active ingredient, a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof in one aspect:

Formula 1

In one embodiment of the present invention, a cell proliferation assay is performed after treatment of lovastin, a compound of the natural product-derived metabolite 1, at various concentrations in a T98G cell line, a GBM cell line resistant to anti-cancer drug temozolomide (TMZ). The lovastin concentration dependent apoptosis effect was assayed. In particular, the concentration of lovastin showing apoptosis similar to that of high concentration of TMZ was determined. As a result, about 60% survive at 750 μM at 3 days of TMZ peak treatment concentration, but the compound of Formula 1 of the present invention, namely, lovastin is 50 μM or more toxic to T98G cell line, which is a TMM resistant GBM cell line. Remarkable was confirmed. In particular, the cell death effects of TMZ-resistant GBM cell line T98G against TMZ were assayed through cell proliferation assay to establish conditions that induce cell death in a dose-dependent manner.

This is not only because lovastin according to the present invention can be used alone as an effective drug for the prevention and treatment of brain cancer, but also at low concentrations, especially against brain cancer cell lines having temozolomide resistance, which is currently the only standard therapeutic anticancer agent for brain cancer. Bar suggests that it can be prepared as a drug that can overcome the anticancer drug resistance.

Furthermore, although unexpectedly surprising, the inventors of the present invention use the compound of Formula 1 in combination with temozolomide, a standard therapy anticancer agent for brain cancer, than the case of using either the compound of Formula 1 or temozolomide alone. It has been found that it can provide significantly better efficacy. Accordingly, the present invention also provides a combination therapy of co-administering the pharmaceutical composition with temozolomide (TMZ). Wherein lovastin or a pharmaceutically acceptable salt thereof and temozolomide, respectively, may optionally be administered with a pharmaceutically acceptable excipient or carrier.

In one embodiment of the present invention, by overexpressing MGMT in combination with a natural product-derived metabolite lovastin in combination with TMZ to GBM cell line T98G resistant to the anticancer drug TMZ of the standard therapy to determine the cell death effect through cell proliferation assay It was confirmed that apoptosis was enhanced when combined with TMZ rather than monotherapy.

That is, it was confirmed that apoptosis was enhanced when co-treatment with TMZ which is an anticancer agent of standard therapy, and it was confirmed that it could be developed as a combination therapy for existing treatments.

Meanwhile, the compound of Formula 1, ie lovastin, used in the present invention may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts in the present invention can be prepared by conventional methods in the art, for example, salts with inorganic acids such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, carbonic acid, and the like. Or a pharmaceutically acceptable acid together with an organic acid such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestyic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin) Salts may be formed, or they may be reacted with alkali metal ions such as sodium or potassium to form their metal salts, or may be reacted with ammonium ions to form another form of a pharmaceutically acceptable salt.

Pharmaceutical compositions comprising a compound according to the present invention or a pharmaceutically acceptable salt thereof are oral formulations, external preparations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated in the form of suppositories and sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, Uittepsol, macrogol, Tween 60, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.1 to 1000 mg / kg, preferably 1 to 100 mg / kg. Administration may be administered once a day or may be divided orally. The dosage does not limit the scope of the invention in any aspect.

The functional food of the present invention includes various foods, candy, chocolate, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules or beverages.

The compounds of the present invention or their food acceptable salts (such as sodium salts) can be added to food or beverages for the purpose of preventing brain cancer or improving brain cancer conditions. At this time, the amount of the compound in the food or beverage is generally added to the dietary supplement composition of the present invention to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, the health beverage composition is 100 ml It can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g as a reference.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the compound of the present invention as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of natural carbohydrates are monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, conventional sugars such as polysaccharides such as dextrin, cyclodextrin and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio is generally about 1 to 20 g, preferably about 5 to 15 g per 100 ml of the composition of the present invention, in addition to the above, the functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors. Flavoring agents, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks Carbonic acid used, etc. The functional food of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. It may be used either individually or in combination. It is common ratio of the additive is selected from the range of the composition per 100 parts by weight 0 to about 20 parts by weight of the present invention, but not so important.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Lovastin

1-1: Preparation of Lichen Stereocaulon alpinum Extract

The Stereocaulon alpinum (Hedw. GL Sm.) Was introduced in January 2003 at the Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') in King George Island, Antarctica. The lichens can be easily collected from Barton Peninsula.

50 g of dried stereocaulon alpinum was extracted twice with 1 L of methanol for 24 hours to obtain 3.6 g of methanol extract. The obtained extract was loaded into flash column chromatography (5 × 25 cm) filled with silica gel (C ₁₈ ), 10%, 20%, 30%, 40%, 50%, 60%, 70 Concentration gradients of%, 80%, 90% and 100% (v / v) methanol (Methanol) gave each fraction.

1-2: Preparation of Lovaric Acid from Lichen Stereocaulon alpinum Extract

204.6 mg of the fraction obtained by elution with 80% methanol obtained in Example 1-1 was again subjected to sample injection into flash column chromatography (2.5 × 30 cm) filled with silica gel (C ₁₈ ), followed by TLC analysis. 200 ml of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% methanol and methyl chloride, respectively, to obtain the eight major fractions obtained by Solution and 100% (v / v) methanol were injected to give each fraction.

59 mg of the fraction eluted with 9% methanol was injected again into semi-preparative reverse-phase HPLC, and then acetonitrile (CH ₃ CN) aqueous solution containing 0.1% formic acid was added to 75 to Lobaric acid (22.9 mg; t _R = 39 minutes) was isolated by elution for at least 30 minutes with a concentration gradient of 83%.

Formula 2

1-3: Synthesis of Novel Compounds from Lobaric Acid

50 mg of lobaric acid of Example 1-2 was dissolved in 50 mL of acetone, and 50 mL of water was added thereto, followed by stirring. 0.25 mL of 2 N NaOH was added, stirred for 15 minutes at room temperature, and 0.5 mL of 1N HCl solution was added to terminate the reaction. The reaction mixture was concentrated to obtain a methyl chloride dissolved layer through partitioning between methyl chloride and aqueous solution (pH = 2) and concentrated to yield 50 mg of a novel compound of formula 1, which was then referred to as "Lobarstin". It was named.

Formula 1

Example 2: Test of anticancer effect of lovastin's T98G cell line, the temozolomide resistant polymorphic glioblastoma cell line 1

2-1: Preparation of lovastin stock solution

The lovastin powder, a compound of Formula 1 synthesized in Example 1, was completely dissolved by reconstitution at 100 mM concentration in DMSO (dimethylsulfoxide) solution, and then aliquoted and stored at -70 ° C.

2-2: Culture of T98G Cell Line

Human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA). Cell lines were maintained in a 37 ° C. humidified cell culture medium containing 10% FBS (fetal bovine serum; Welgene) in Dulbecco's modified Eagle's medium (DMEM; Welgene) and 5% CO ₂ .

2-3: Treatment of lovastin and confirmation of cytotoxicity

One day prior to drug treatment, the cells of Example 2-2 were cultured by dispensing at a concentration of 1 × 10 ⁶ cells per 100 mm culture dish (about 55 cm 2). When the cells reached 40-50% confluency the next day, the drug (Lovastin solution of Example 2-1) was diluted to an appropriate concentration in the medium and treated for 3 or 4 days. DMSO, a solvent, was used as a negative control, and temozolomide, an anticancer agent of standard therapy, was treated as a positive control.

Then, cytotoxicity was confirmed using EZ-CyTox cell viability assay kit (Daeil Lab Service, Seoul, Korea). The drug-treated cultured cells and 10 μl of the EZ-CyTox kit reagent were added to a 96-well microplate and incubated in a 37 ° C humidified cell incubator for 1 hour, and the absorbance at 450 nm was absorbed (A ₄₅₀ ). Was measured. Survival of the experimental group is calculated by converting A ₄₅₀ of the negative control to 100%.

As a result, as shown in FIG. 1, T98G, a GBM cell line resistant to the anticancer drug, temozolomide (TMZ), survived about 60% even at 750 μM for 3 days at the highest concentration of TMZ (FIG. 1A). , Compound Rovastin of the formula (1) of the present invention was found to be remarkable when the toxicity against T98G cell line GBM cell line having TMZ resistance is more than 50μM (Fig. 1B).

Example 3 Validation of Anticancer Effect of Tovarog Cell Line, a Tmozolomide-Resistant Polymorphic Glioblastoma Cell Line of Rovastin 2

3-1: Preparation of Lovastin and Cell Culture

Lovastin was synthesized as described in Example 1.

Human glioblastoma T98G cells (Dr. SS Kang, D.) supplemented with 10% fetal bovine serum (JR Scientific, USA), 100 units / ml penicillin and 100 μg / ml streptomycin sulfate (Welljin, Korea) Gyeongsang National University, Korea) and primary human fibroblasts (Dr. JH Lee, Chungnam National University, Korea) were cultured.

3-2: Treatment of lovastin and confirmation of cytotoxicity (cell viability)

Primary human fibroblasts and T98G cells were planted 1000 and 2000 per well in 0.1 ml in 96-well flat plates, respectively, and then cultured overnight at 37 ° C. After drug treatment for an appropriate time, water soluble tetrazolium salt (WST) assay was performed using EZ-Cytox (DoGen, Korea).

Lovastin was administered to T98G glioblastoma cells in various amounts for 3 days. The toxic effects of lovastin in T98G were seen at concentrations as low as 10 μM (n = 5, p = 0.002, Student's t- test). However, lovastin did not affect cell viability in human normal fibroblasts at 40 μM (n = 3, p = 0.108, Student's t- test) (FIG. 2). 40 μM of lovastin is toxic to T98G cells (n = 5, p = 5.16E-05, Student's t- test), but the maximum allowable dose in normal fibroblasts is 40 μM in the experiment. Used as.

Example 4 Validation Effect of Combined Dose with TMZ on T98G Cell Line of Lovastin's Temozolomide-Resistant Polymorphic Glioblastoma Cell Line

In order to confirm the effect of the combination of the compound of the formula 1 according to the present invention lovastin with anti-cancer drug, temozolomide, the experiment was performed on the T98G cell line in the same manner as in Example 2.

First, the T98G cell line was divided into four groups and treated with high concentrations of TMZ (750 μM) or the compound lovastin (40 μM) of Formula 1 alone or in combination (Robarstin + TMZ) for 3 days and 4 days. Solvent DMSO was used. Lovastin was used at a concentration of 40 μM or less with a concentration of 50 μM or less confirming the remarkable effect confirmed in Example 2.

As a result, as shown in FIG. 3, when T98G cell line was treated with high concentrations of TMZ (750 μM) and 40 μM lovastin of the present invention for 3 days and 4 days (Robarstin + TMZ), the toxic effect was higher. It can be seen that it is enhanced.

In addition, the T98G cell line was further divided into four groups and treated with low concentrations of TMZ (500 μM) or Compound 1 lovastin (40 μM) alone or in combination (Robarstin + TMZ) for 3 and 4 days. As a solvent, DMSO was used.

As a result, as shown in Figure 4, even when administered in combination with a low concentration of TMZ when combined treatment (Robarstin + TMZ) for 3 days and 4 days it was confirmed that the toxic effect is improved compared to the single treatment. In particular, the cytotoxic effect of the combination treatment for 4 days is similar to the effect of the high concentration of TMZ (750 mM) alone treatment (see Fig. 3), it is expected to be able to alleviate the side effects for high concentration anticancer drugs.

Example 5 Experimental Effect of Combined Administration with TMZ on T98G Cell Line of Lovastin's Temozolomide-Resistant Polymorphic Glioblastoma Cell Line

5-1: Reverse transcription-polymerase chain reaction (RT-PCR)

RT-PCR was performed (S. Jo et al., Biochem Biophys Res Commun , 416: 86-91, 2011). Briefly, PCR using HiPi Plus Thermostable DNA Polymerase (Elpis Biotech, Korea) followed by total RNA extracted with Solution D was used to make cDNA with M-MLV Reverse Transcriptase (Elpis Biotech, Korea). Primers used for PCR were as follows: MGMT _F, GCAATGAGAGGCAATCCTGT; MGMT _R, GTCGCTCAAACATCCATCCT; GAPDH _F, CTCAGACACCATGGGGAAGGTGA; GAPDH _R, ATGATCTTGAGGCTGTTGTCATA; PARP1- F, GCTCCTGAACAATGCAGACA; PARP1 _R, CATTGTGTGTGGTTGCATGA; LIG3 _F, GTGGATTTGGGCATGTATCC; LIG3_R , GCCCATTCCCCCTATACTGT; XRCC1 _F, GAGGATGAGGCCTCTCACAG; XRCC1 _R, TCCTCTGTGTCCCCAGAATC; MPG _F, TGGCACAGGATGAAGCTGTA; MPG _R, GTGTCCTGCTCAGCCACTCT (PARP1, poly (ADP-ribose) polymerase 1; LIG3, ligase 3; XRCC1, X-ray repair cross-complementing protein 1; MPG, N-methylpurine-DNA-glycosylase).

5-2: Western blot analysis

Western blot analysis was performed (H. Chung et al., Int J Cancer , 122: 816-22, 2008). Antibody to MGMT (sc-33674) (Santa Cruz Biotechnology, USA), PARP1 (9532; Cell Signaling Technology, USA), XRCC1 (2735; Cell Signaling Technology, USA), GAPDH (2118; Cell Signaling Technology, USA), LIG3 (GTX103197; GeneTex, USA), MPG (GTX101916; GeneTex, USA) and HRP-conjugated IgGs (111-035-003 and 115-035-003; Jackson ImmunoResearch Laboratories, USA) were used.

5-3: Comet Assay

Genetic comet assays were performed using the CometAssay Kit (Trevigen, USA). Images obtained by fluorescence microscopy (Olympus IX71; Olympus, Japan) were easy to analyze with Comet Assay IV v4.3 (Perceptive Instruments, UK). Statistical analysis was performed on these results (J. Bright et al., Pharm Stat , 10: 485-93, 2011). Briefly, 50 values of tail intensity (TI,% tail DNA) obtained at the time of administration were normalized and expressed as fold-change compared to the vehicle administration group. The results of three independent experiments were analyzed using one-way ANOVA, followed by a post hoc test using Scheffe to examine the differences between the groups.

5-4: Improved toxicity due to the combination of lovastin and temozolomide

Temozolomide is used as a general chemotherapeutic agent for gliomas, but temozolomide is not associated with patients expressing MGMT, a gene involved in restoring alkylation induced at the O ⁶ position of guanine by TMZ. In comparison, the effect was small. To study the effects of lovastin, T98G cells were used because MGMT is known to express these cell lines (FIG. 5). Thus, it was more resistant to TMZ. There was statistically significant toxicity of TMZ under all conditions, but more pronounced at administration of 500 and 750 μM (FIG. 6). Interestingly, the combination of lovastin with high doses of TMZ resulted in enhanced toxicity (FIG. 7). These results indicate that lovastin can enhance the toxicity of TMZ.

5-5: Reduced recovery of TMZ induced DNA damage by lovastin

DNA damage was quantified by gene comet assay. First, the effect of lovastin on DNA damage was investigated. Treatment with only 40 μM of lovastin for 26 hours showed only minimal effect on DNA damage, indicating that tail strength (TI) is comparable to that of carrier administered cells (compare L and V in FIG. 8). ).

Next, T98G cells were administered with 500 μM of TMZ or lovastin together for 2 hours to investigate the effect on DNA damage. Both showed similar ranges of DNA damage under conditions of 2 hours (comparison of T (D) and LT (D) in FIG. 8), which is sufficient time to cause DNA damage and DNA repair following DNA damage. The time is short enough to prevent the systems from overlapping (A in FIG. 8). Therefore, cells were rinsed 2 hours after drug administration and cultured in fresh culture medium to measure recovery of DNA damage (FIG. 8A). When damaged cells were incubated for 24 hours in fresh medium, cells cultured in lovastin-containing medium showed higher TI than cells administered with a carrier (compare T (R) and LT (R) in FIG. 8). These results indicate that lovastin alone does not induce DNA damage, but that DNA damage induced by TMZ persists in the presence of lovastin.

5-6: Reduction of DNA repair expression by lovastin

Genetic comet analysis showed that the lovastin-administered cells showed more DNA damage, indicating that the DNA repair system is affected by lovastin. Administration of lovastin alone for 2 days to T98G cells resulted in a decrease in the expression of MGMT and PARP1 proteins, related to DNA repair, depending on time (left side of FIG. 9). Reduction in expression was also seen in the translation phase (right side of FIG. 9). In addition, the combination of lovastin and TMZ resulted in lower expression of MGMT, PARP1, LIG3 and XRCC1 (FIG. 10). These results indicate that reduced expression of the DNA repair genes appears as a possible mechanism for enhanced sensitivity by the combination of lovastin.

We investigated the effect of lovastin, a lichen metabolite, to improve the sensitivity of TMZ in the chemically resistant GBM cell line T98G, and observed a decrease in intracellular DNA recovery in combination with lovastin and TMZ. As a mechanism, reduced expression of DNA repair genes could be seen (FIG. 11).

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Lobarstin, a compound derived from stereocaulon alpinum according to the present invention, has been shown to show cytotoxicity when treated alone with temozolomide-resistant cancer cell lines, and thus can be prepared as a drug that can overcome anticancer drug resistance. Confirmed that it can. In addition, lovastin is confirmed that the cell death is enhanced when co-treatment with the anti-cancer drug temozolomide of the standard therapy, it is also useful to be developed as a combination therapy for the existing therapy.

Claims

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

Formula 1
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is for co-administration with temozolomide (TMZ).
Functional food for the prevention or improvement of brain cancer containing the compound represented by the following formula (1) or a food acceptable salt thereof as an active ingredient:

Formula 1
The functional food according to claim 3, wherein the functional food is for co-administration with temozolomide (TMZ).