KR102018085B1 - Pharmaceutical Composition for Treatment and Inhibiting Metastasis of Brain Tumor Comprising Acteoside - Google Patents

Abstract

The present invention relates to a composition for inhibiting brain tumor treatment / improvement and metastasis that induces death of brain tumor cells, inhibits cell migration, and particularly shows synergistic effects when administered in combination with temozolomide, and more specifically, acteoside It relates to a composition for the treatment / improvement of brain tumors and the inhibition of metastasis, which contains a) as an active ingredient.

Description

Pharmaceutical Composition for Treatment and Inhibiting Metastasis of Brain Tumor Comprising Acteoside

The present invention relates to a composition for inhibiting brain tumor treatment / improvement and metastasis, which induces death of brain tumor cells, inhibits cell migration, and particularly shows synergistic effects when administered in combination with temozolomide.

Brain tumors include tumors that occur in the skull, and cancers originating from the brain itself are called primary brain tumors. Secondary brain tumors, such as lung cancer, breast cancer, and gastrointestinal cancers, which are caused by metastases to the brain, are called metastatic brain tumors. Tumors that occur in the lungs, stomach, breasts, etc. are limited to one or two types of organs and have the same or similar properties. However, brain tumors are caused by various tumors such as glioblastoma, meningioma, schwannoma, pituitary tumor, and germ cell tumor.

Glioblastoma, also called glioma, glioblastoma, glioblastoma, glioblastoma, is a highly malignant tumor of grade IV among carcinomas. Glioblastoma most commonly occurs in the subcortical white matter of the cerebral hemispheres in the cortical temporal lobe of the brain and is histologically accompanied by nuclear atypical, mitotic, proliferative and necrotic endothelial cells. In addition, it has a high potential of movement and diffusion, and thus has a high penetration. This results in a very poor prognosis compared to other tumors, with an average survival of only 14.6 months after the first diagnosis. The 5-year survival rate was 8.0% between 1986 and 1990, and to date, 5-year survival rate is less than 10% despite aggressive treatment including tumor resection. The recurrence rate is also very high, with a 6-month progression-free survival rate of 15-21% and an average survival rate of 25 weeks.

Glioblastoma is almost impossible to be completely removed by surgical operation because the boundary between brain cells and tumor cells is not clear, and the treatment of radiation and chemotherapy after the removal of the lesion is a standard treatment. Chemotherapy is the only one used by Ork's oral anticancer drug, Temodal (component name Temozolomide, hereinafter referred to as "TMZ"). Temodal exhibits cancer cell death by spontaneously degrading in cells to produce 5-aminoimidazole-4-carboximide, an active substance. However, when administered to a patient, temodal responds to the drug to some extent, but the resistance to the drug appears quickly, and once the resistance is developed, the treatment is no longer difficult.

Acteoside (acteoside, verbascoside, caffeoyl phenylethanoid glycoside) is present in various medicinal plants as a compound of the following formula (1), and is known to have antimicrobial and anti-inflammatory activity against microorganisms including Staphylococcus aureus. Thus, there have been attempts to utilize Acteoside by extracting it from plants containing Acteoside. Korean Patent No. 10-1356797 discloses a lemon verbena-derived acteoside-containing extract and a functional cosmetic composition comprising the same, and Patent Publication No. 10-2007-0078656 discloses a method for extracting acteoside from an elderberry leaf and an antioxidant containing the same. For anti-inflammatory pharmaceutical compositions, Korean Patent Application Publication No. 10-2014-0020005 discloses a breeding and fucoidan extract having hair loss prevention and hair growth promoting effect using an extract including acteoside as an active ingredient, and Patent No. 10-0514076 For the composition for the prevention and treatment of degenerative neurological diseases, including theoside, Korean Patent Publication No. 10-2007-0082271 discloses a sulfur extract containing acteoside and its use. However, the therapeutic efficacy of acteosides against brain tumors is unknown.

[Formula 1]

Patent Registration 10-1356797 Patent Publication No. 10-2007-0078656 Patent Publication No. 10-2014-0020005 Patent Registration 10-0514076 Patent Publication No. 10-2007-0082271

It is an object of the present invention to provide a composition for the treatment / improvement and metastasis inhibition of new brain tumors.

It is an object of the present invention to provide a composition for the treatment / improvement and metastasis suppression of brain tumors, which can further enhance the therapeutic efficacy of temozolomide, which is used only as a conventional therapeutic agent for brain tumors.

The present invention for achieving the above object relates to a pharmaceutical composition for treating brain tumors and inhibiting metastasis containing acteoside as an active ingredient.

Acteosides, by themselves, have the effect of inducing death of brain tumor cells, inhibiting cell migration and inhibiting metastasis, but especially when administered in combination with temozolomide, It has a much more pronounced cell death effect and metastasis suppression effect than the efficacy of amide. Therefore, the composition of the present invention is preferably administered in combination with temozolomide. At this time, the molar ratio of temozolomide and acteoside may be 1: 0.001 to 1: 0.1.

In the preliminary experiments, 1 to 200 μM of the acteoside was tested by varying the ratio of acteoside to 1-20 mM of the concentration of temozolomide, which induces apoptosis of 40-60% against C6 cells. When used as a concentration and the molar ratio of temozolomide is 1: 0.0001 to 1: 0.1, the synergistic effect of the combination treatment was shown. If the mote ratio of acteoside is too low, the synergy effect was insignificant, and even if the acteoside mole ratio was further increased, the synergy effect was not improved. In the following examples, the efficacy of theozolomide was fixed at 5 mM and the concentration of acteoside at 50 μM.

The composition of the present invention is useful for the treatment and suppression of metastasis of glioblastoma, meningioma, schwannoma, pituitary tumor, germ cell tumor, etc., which is a tumor occurring in the skull, that is, more particularly in glioblastoma with high malignancy and limited treatment. .

In the composition of the present invention, the acteoside may be a purified form of the compound, but may also be an extract form of the acteoside-containing plant. Acteosides are known to exist in a variety of medicinal plants, for example, but are not limited to, such as, but not limited to, bark wood, lemon barbena, laver, turmeric, breeding, cornerstone. Extract of the plant may be prepared by a conventional method for producing a plant extract. That is, it would be easy for a person skilled in the art to extract the stem, branch, fruit, root, flower or leaf of a plant or a mixture thereof using water or an organic solvent and to carry out additional fractionation to increase the content of acteoside. In addition, processing the extract into a form that is convenient for storage, distribution and use, such as concentration or drying without destroying the acteoside component, may also be appropriately selected and used according to a processing method of a conventional extract.

The composition of the present invention may be prepared by a method known in the pharmaceutical art for use as a medicament, and may be used by itself or in admixture with a pharmaceutically acceptable carrier, forming agent, diluent, and the like. have. The composition of the present invention can be formulated as a preparation for oral or parenteral administration and used as a therapeutic and metastasis inhibitor of brain tumors. The dosage of the active ingredient according to the present invention may be appropriately selected according to the absorbance of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the degree of symptoms, and the like may be administered once a day. It may be administered several times. Typical dosages are 0.001 mg / kgday to 10 g / kgday.

The present invention also relates to a nutraceutical composition for improving brain tumor suppression and metastasis containing acteoside as an active ingredient. Of course, in the dietary supplement composition, the acteoside may be contained in the form of extract of the acteoside-containing plant, not a purified compound. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and the foods to which the composition of the present invention can be added include, for example, various foods, beverages, gums, teas, sweets, Alcoholic beverages, chocolate, vitamin complexes, and health functional foods. The effective dose of the health food composition of the present invention can be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of prolonged intake for the purpose of health and hygiene or for the purpose of health control.

As described above, the composition of the present invention exhibits the effect of inhibiting the death and cell mobility of brain tumor cells by itself, and particularly has the effect of further enhancing the therapeutic efficacy of temozolomide, which is conventionally used as the only therapeutic agent, for the treatment of brain tumors. It can be usefully used for improving and inhibiting metastasis.

1 is a graph showing the effect on cell viability of brain tumor cells.
2 is a Western blot electrophoresis image showing the effect on autophagy in brain tumor cells.
3 is an immunofluorescence image and Western blot electrophoresis image showing the effect on autophagy in brain tumor cells.
4 is a microscopic image showing the effect on the cell mobility of brain tumor cells.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, such an embodiment is only an example for easily describing the content and scope of the technical idea of the present invention, whereby the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention based on these examples.

EXAMPLE

Example 1 Cell Line Culture

C6 cell line, a glioma cell, was used in the United States (ATCC, Rockville, MD, USA). In DMEM medium containing 10% fetal bovine serum (FBS), 4 mM glutamine, 100 U / mL penicillin, and 100 mg / mL streptomycin, the cells were cultured at 95% relative humidity, 37 ° C, and 5% CO ₂ .

Example 2 Evaluation of Effect on Brain Tumor Cell Death

1) Cell viability evaluation

C6 cells cultured in Example 1 were inoculated in a 96-well plate at a concentration of 5 × 10 ³ cells / well and cultured for 24 hours at 5% CO ₂ , 37 ° C. to apply a cell monolayer. Then, the wells were treated with TMZ or Acteoside (AC) or TMZ and Acteoside simultaneously and further incubated for 24 hours. The control group was treated with DMEM containing no TMZ or acteosides. After incubation, the MTT solution was added to each well and incubated at 37 ° C. for 4 hours, the culture solution was removed, and then mixed with DMSO (dimethyl sulfoxide) at room temperature for 30 minutes. Cell viability was measured by absorbance of the reaction solution at 595 nm.

1 is a graph depicting the results, showing that acteosides in themselves exhibit a weak killing effect on glioma cells, but further enhance the effect on glioma cells killing when combined with tamorene. .

2) Assessment of the impact on apoptosis

Since caspase plays an essential role in apoptosis, the effect of acteoside and / or TMZ by induction of caspase activity in C6 cells was confirmed by Western blot.

Specifically, C6 cells were precultured in a culture dish at a concentration of 7 × 10 ⁶ cells / ml. Thereafter, TMZ or Acteoside or TMZ and Acteoside were simultaneously treated and incubated for 24 hours. After incubation, the cells were collected and lysed using lysis buffer and sonicator. Cultured cells were lysed using RIPA buffer (50 mM tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% Na.dioxychloate, 0.5% SDS, protease inhibitors, phosphatase inhibitors), 12,000 rpm, 4 ° C. Protein was obtained by centrifugation for 20 minutes at. Proteins were quantified using bicinchoninic acid kit (Sigma-Aldrich). After separating the same amount from 10% SDS-polyacrylamide gel, the protein was transferred to nictrocellulose membrne using transfer kit, and then pro-caspase-3, Expression levels were measured using cleaved-caspase-3, Bax, Bcl-2, and p-P53 antibodies.

Figure 2 is an electrophoretic image showing the Western blot results, it was confirmed that the amount of Bcl-2, a marker of the pre-apoptosis step when the acteoside alone treatment. In combination with TMZ and acteosides, not only decreased Bcl-2 expression, but also significantly increased the amount of apoptosis inducing factors, cleaved-caspase-3, p-P53, and Bax, compared to TMZ alone treatment. The effect was remarkable and it was confirmed that cell death by apoptosis of C6 cells is activated.

3) Assessment of the impact on autophagy

C6 cells were incubated in sterile glass coverslips. TMZ or Acteoside or TMZ and Acteoside were simultaneously treated and further incubated for 24 hours, and 4% paraformaldehyde cells were fixed. For immunofluorescence analysis, cells were blocked with PBS containing 10% goat serum, stained with LAMP1 primary antibody and LC3 primary antibody diluted 1: 500 and then Alexa 488-binding secondary antibody diluted 1: 1000. (Invitrogen, A-11001) and Alexa 594-binding secondary antibody (Invitrogen, A-11007). Finally, slides were washed three times with PBS, stained with DAPI and placed in mounting medium.

The stained image was observed with a fluorescence microscope and the results are shown in A of FIG. 3. As can be seen in A of FIG. 3, acteoside is weaker than TMZ but has an effect of activating autophagy. Combined treatment with acteoside and TMZ not only significantly increased the expression of LAMP1 and LC3, but also strongly induced the colocalization of LAMP1 and LC3. This suggests that fusion of autophagosomes with lysosomes occurs effectively, resulting in increased actual autophagy.

Western blot expression levels of LC3 and Rab7, which are indicators of autophagy (Fig. 3B), also showed a significant increase in autophagy by the combination of acteoside and TMZ. Showed.

Example 3 Evaluation of Effect on Inhibition of Cancer Cell Metastasis

To analyze the effect of inhibiting brain tumor cell metastasis, cell mobility was analyzed by a wound healing assay. After growing C6 cells in a petri dish, the cells were grown in saturation, treated with TMZ or acteoside or TMZ and acteoside at the same time, and the cells were scraped using a sterile 200 μl pipette tip to wound to a constant width. The debris was washed with PBS, further incubated for 24 hours, and then wound recovery ability was evaluated by measuring the cell migration distance of the wound site using Image J software from the photographs before and after the culture.

4 is a graph showing wound recovery capacity calculated from microscope images (A) and microscope images of culture time 0 and 24 hours (B). Acteoside had a certain degree of cell migration inhibitory effect even when treated alone, but synergistic effects of TMZ and acteoside combined showed that cell migration was very effectively inhibited compared to TMZ alone treatment. . From this it can be seen that acteosides are not only effective in inhibiting cancer metastasis on their own, but can further enhance the cancer metastasis suppression effect of TMZ, especially when combined with TMZ.

Claims (7)

delete A pharmaceutical composition for treating brain tumors and inhibiting metastasis, comprising acteoside as an active ingredient and being administered in combination with temozolomide.
The method of claim 2,
The molar ratio of temozolomide and acteoside is 1: 0.0001 to 1: 0.1 pharmaceutical composition for brain tumor treatment and metastasis suppression, characterized in that.
The method of claim 2 or 3,
The brain tumor is glioblastoma, meningioma, schwannoma, pituitary tumor or germ cell tumor, characterized in that the brain tumor treatment and metastasis pharmaceutical composition.
The method of claim 2 or 3,
The acteoside is a pharmaceutical composition for treating brain tumors and inhibiting metastasis, characterized in that it is contained in the form of extracts of the acteoside-containing plants.
The method of claim 5,
The acteoside-containing plants are brain tumor treatment and metastasis inhibiting pharmaceutical composition, characterized in that at least one selected from the group consisting of bark, lemon barbena, nurilla, yogurt, breeding, cornerstone.
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