A kind of medicine composition and application of anti-malignant glioma

Abstract

The present invention relates to an anti-malignant glioma pharmaceutical composition and application, and relates to the field of biomedicine technology. The application of indole alkaloids in the preparation of anti-malignant glioma drugs, wherein the indole alkaloids are N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide or / and a pharmaceutically acceptable salt. The present invention finds that N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can effectively inhibit the growth, migration and invasion of human brain astroglioma U87-MG cells through CCK-8, Transwell cell migration and invasion experiments, thereby inhibiting the malignant progression of gliomas, providing a new drug for glioma treatment, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-malignant glioma drug composition and application thereof. Background Art

[0002] Glioma is one of the most common primary tumors of the central nervous system, accounting for 81% of malignant tumors of the central nervous system, and mainly originates from glial tissue. According to the new classification of central nervous system tumors by the World Health Organization in 2021, gliomas are divided into grades 1-4. Among them, glioblastoma (GBM) belongs to the most malignant grade 4, which is highly invasive and has a high mortality rate. The five-year survival rate of patients is the lowest among all cancers, and the prognosis is poor and prone to recurrence. At present, the clinical treatment of glioma is mainly surgical resection, combined with radiotherapy, chemotherapy and targeted therapy. However, surgical treatment is difficult to completely remove the lesions, and radiotherapy and chemotherapy are prone to drug resistance. Therefore, the survival of most patients is still very short, especially GBM patients, whose survival period is only about fifteen months, and there is a lack of effective intervention measures.

[0003] Toad venom is a traditional Chinese medicine. It is made from the dried white venom secreted from the skin glands and parotid glands of the Chinese giant toad or the black-framed toad of the Toad family. The Chinese Pharmacopoeia records that toad venom is pungent and warm in nature, has the functions of detoxification, analgesia, awakening the mind, and is widely used as an anti-inflammatory drug, analgesic, cardiotonic, antibacterial drug and local anesthetic. Toad venom has clear anti-cancer activity and has a good tumor inhibition effect on lung cancer, prostate cancer, liver cancer, cervical cancer and other diseases. Toad venom extract-related marketed drugs such as Huachansu Tablets, Anke Capsules, and Jinpu Capsules have been widely used in the treatment of various cancer patients including liver cancer, lung cancer, colon cancer and pancreatic cancer. The chemical components in toad venom are complex and diverse. According to different separation and identification methods, its main chemical components have been determined to be bufadienolides, indole alkaloids, sterols and other compounds.

[0004] At present, the research on the anti-tumor effect of the active ingredients of toad venom is mainly focused on toad venom compounds. However, whether in traditional applications or the preparation of clinical preparations, such as cinobuccal injection, cinobuccal oral solution, cinobuccal tablets, etc., water extraction is the main process, so it contains a large amount of hydrophilic components. Indole alkylamine alkaloids are one of the important hydrophilic active ingredients of toad venom, with the characteristics of special structure, strong drug-like properties, high selectivity and small toxic side effects. The activity in anti-tumor has attracted widespread attention from drug developers. Many indole anti-tumor drugs have been used in the clinical treatment of various cancers, including drug-resistant and even multi-drug-resistant tumors. However, there are few studies on the anti-tumor effects of indole alkaloids in toad venom. At the same time, although toad venom and its preparations have a wide range of anti-tumor effects, the tumor inhibition mechanism is not very clear to a certain extent, resulting in unstable effects, and its toxicity also limits its promotion and application to a certain extent. Therefore, it is very important to develop highly efficient and low-toxic toad venom anticancer drugs for the prevention and treatment of gliomas. In view of this, the present invention provides an anti-malignant glioma drug composition and application. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a drug composition for preventing and treating malignant gliomas and its application. The purpose is to inhibit the growth, migration and invasion of glioma cells by N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide, and to provide a new drug and method for the prevention and treatment of gliomas.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, an indole alkaloid is used in the preparation of a drug for treating malignant glioma, wherein the indole alkaloid is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide or / and a pharmaceutically acceptable salt thereof.

[0008] Among them, N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide, referred to as Bufoserotonin C, has a molecular formula of C 18 H 19 N3O3, molecular weight is 325.36, CAS number is 1002722-87-3, the structural formula is as follows:

[0009]

[0010] The beneficial effect of the present invention is that: the present invention uses N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide to intervene in human brain astroglioma U87-MG cells, and through CCK-8 cell viability experiments, Transwell cell migration experiments and invasion experiments, it is found that N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide can effectively inhibit the growth, migration and invasion of human brain astroglioma U87-MG cells, thereby inhibiting the malignant progression of gliomas, providing a new drug for glioma treatment, and having good application prospects.

[0011] Based on the above technical solution, the present invention can also be improved as follows.

[0012] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the growth of human brain astrocytic glioblastoma.

[0013] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the migration of human brain astrocytic glioblastoma.

[0014] Furthermore, the N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the invasion of human brain astrocytes.

[0015] In a second aspect, a pharmaceutical composition for treating malignant glioma comprises N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

[0016] Further, the half inhibitory concentration IC of N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide in human brain astrocytoblastoma U87-MG cells in vitro is 50 It is 45.11μM.

[0017] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and auxiliary components.

[0018] Furthermore, the pharmaceutical composition also includes at least one of a filler (diluent), a lubricant (glidant or anti-adhesive agent), a dispersant, a wetting agent, a binder, a regulator, a solubilizer, an antioxidant, an antibacterial agent, an emulsifier, and a disintegrant.

[0019] Furthermore, the dosage form of the pharmaceutical composition is a clinically acceptable pharmaceutical preparation.

[0020] Furthermore, the dosage form of the pharmaceutical composition includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

[0021] The pharmaceutical composition of the present invention can be prepared as a pharmaceutical composition according to methods known in the art, and can be made into any dosage form suitable for human or animal use by combining the drug of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0022] The actual dosage of the active ingredient in the pharmaceutical composition of the present invention (N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide of the present invention) should be determined according to a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, gender, and weight. Therefore, the above dosage should not limit the scope of protection of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the inhibition of U87-MG cell viability by Bufoserotonin C in an embodiment of the present invention;

[0024] Figure 2 The figure is a diagram of Bufoserotonin C inhibiting U87-MG cell migration in the embodiment of the present invention; wherein A is a cell picture corresponding to the cell migration experiment; B is the statistical result of the number of cell migration, and the statistical result is expressed as the mean ± standard deviation of 3 independent experiments, **P<0.01, ***P<0.001;

[0025] Figure 3 : This is a diagram of Bufoserotonin C inhibiting U87-MG cell invasion in an embodiment of the present invention, wherein A is a cell picture corresponding to the cell invasion experiment; B is the statistical result of the number of cell invasions, and the statistical results are expressed as the mean ± standard deviation of 3 independent experiments, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0027] Example

[0028] 1. Test materials and instruments

[0029] 1.1 Test materials

[0030] Human astrocytic glioblastoma U87-MG cells (purchased from Wuhan Punosai Biotechnology Co., Ltd.); MEM culture medium (purchased from Gibco); fetal bovine serum FBS (purchased from Sigma); N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide (Bufoser otonin C, purchased from Chengdu Desit Biotechnology Co., Ltd.); DMSO (purchased from Shanghai Sulai Biotechnology Co., Ltd.; CCK-8 detection kit (purchased from White Shark Biotechnology Co., Ltd.); 1% crystal violet dye (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.); transwell chamber (8μm) PC membrane (purchased from corning); Matrigel matrix (purchased from corning).

[0031] 1.2 Test equipment

[0032] Experimental instruments: CO2 cell culture incubator (model Thermo3111; manufacturer Thermo Fisher Scientific); fully automatic cell counter (model CellDrop BF; manufacturer De novix); continuous wavelength multifunctional microplate detector (model Spark; manufacturer TECAN); inverted fluorescence microscope (model CKX53; manufacturer OLYMPUS).

[0033] 2 Test methods

[0034] 2.1 Cell culture and treatment

[0035] Bufoserotonin C was dissolved in 77 μL DMSO to obtain a 200 mM Bufoserotonin C solution, and Bufoserotonin C was diluted with MEM medium containing 10% fetal bovine serum to final concentrations of 5 μM, 25 μM, 50 μM, 100 μM, 150 μM and 200 μM, respectively.

[0036] U87-MG cells were cultured in MEM medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator. When the cell density reached 80-90%, 1 mL of trypsin was used to digest the cells. After the cells became round under a microscope, 4 mL of MEM medium containing 10% FBS was added to terminate the digestion and the cells were counted.

[0037] 2.2 Detection methods of cell viability assay, Transwell cell migration and invasion assay

[0038] (1) Cell viability assay

[0039] In this example, the effect of Bufoserotonin C on the viability of U87-MG cells was detected by CCK-8 assay.

[0040] Press 1×10 4 U87-MG cells were inoculated into 96-well plates at 100 μg / well and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. The cells were randomly divided into 7 groups. After the old culture medium was removed, MEM culture medium (containing 10% fetal bovine serum) containing 0 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 150 μM and 200 μM Buf oserotonin C solution was added to each group. Cell viability was detected after 48 h of cell culture. Another group containing only blank culture medium was set up as a blank control.

[0041] Use CCK-8 detection kit to evaluate cell viability: add 10 μL CCK-8 reagent to 100 μL culture medium and incubate for 1 hour at 37°C and 5% CO2. Read the absorbance value at 450nm using continuous wavelength multifunctional microplate reader. The results are as follows Figure 1 shown.

[0042] (2) Cell migration assay

[0043] In this example, the effect of Bufoserotonin C on the migration ability of U87-MG cells was detected by Transwell cell migration assay.

[0044] The density of U87-MG cells was adjusted to 1.5 × 10 cells / mL using MEM medium containing 1% fetal bovine serum. 5 / mL, add 800μL of MEM medium containing 10% fetal bovine serum to the lower chamber of the 24-well plate, take 100μL of cell suspension and add it to the Transwell chamber. Remove the bubbles, then put the chambers into the culture plate, place them in a 37℃, 5% CO2 cell culture incubator for 24h, and randomly divide them into 4 groups. After removing the culture medium in the upper chamber, add 100μL of MEM medium (containing 1% fetal bovine serum) containing 0μM, 25μM, 50μM, and 100μM Bufoserotonin C solution to each group, and test them after 48h of cell culture.

[0045] Remove the Transwell chamber, discard the culture medium in the well, and wash twice with 1×PBS. Add 4% paraformaldehyde to cover the cell surface and fix at room temperature for 10 minutes. Discard the fixative, wash twice with PBS, discard PBS, add 1% crystal violet staining for 10 minutes, wash 2-3 times with PBS, and gently wipe off the upper layer of non-migrated cells with a cotton swab. Use an inverted fluorescence microscope to take pictures and count the cells that migrated to the lower layer of the microporous membrane. Select 5 fields of view for each sample to take pictures and calculate the number of migrated cells.

[0046] (3) Cell invasion assay

[0047] In this example, the effect of Bufoserotonin C on the invasion ability of U87-MG cells was detected by Transwell cell invasion assay.

[0048] Use pre-cooled MEM serum-free medium to prepare matrigel matrix to a working concentration of 300ug / ml, and then add 100μL to the Transwell chamber. After spreading the gel, place it in a 37℃ incubator for 1 hour. If there is liquid culture medium on the upper layer after taking it out, carefully discard the liquid culture medium on the upper layer before use. Use MEM medium containing 1% fetal bovine serum to adjust the density of U87-MG cells to 2×10 5 / mL, add 800μL of MEM medium containing 10% fetal bovine serum to the lower chamber of the 24-well plate, take 100μL of cell suspension and add it to the Transwell chamber. Remove the bubbles, then put the chambers into the culture plate, place them in a 37℃, 5% CO2 cell culture incubator for 24h, and randomly divide them into 4 groups. After removing the culture medium in the upper chamber, add 100μL of MEM medium (containing 1% fetal bovine serum) containing 0μM, 25μM, 50μM, and 100μM Bufoserotonin C solution to each group, and test them after 48h of cell culture.

[0049] Remove the Transwell chamber, discard the culture medium in the well, and wash twice with 1×PBS. Add 4% paraformaldehyde to cover the cell surface and fix at room temperature for 10 minutes. Discard the fixative, wash twice with PBS, discard PBS, add 1% crystal violet staining for 10 minutes, wash 2-3 times with PBS, and gently wipe off the upper layer of non-migrated cells with a cotton swab. Use an inverted fluorescence microscope to take pictures and count the cells that migrated to the lower layer of the microporous membrane. Select 5 fields of view for each sample to take pictures and calculate the number of migrated cells.

[0050] 2.3 Statistical analysis

[0051] The experiment was repeated three times, and the results are expressed as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad, California, USA). One-way ANOVA was used to evaluate the differences between groups, followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant, where * P < 0.05; ** P < 0.01; *** P < 0.001.

[0052] 3. Experimental results

[0053] The results are as follows Figure 1-3 As shown:

[0054] (1) By Figure 1 It was found that Bufoserotonin C inhibited the viability of U87-MG cells. The effects of 0μM, 5μM, 10μM, 25μM, 50μM, 100μM, 150μM and 200μM Bufoserotonin C intervention on the viability of U87-MG cells were detected. The CCK-8 results showed that with the increase of Bufoserotonin C treatment concentration, cell viability decreased, and when the treatment concentration was 45.11μM, cell viability decreased to 50%.

[0055] (2) From Figure 2 As shown in A and B, Bufoserotonin C can effectively inhibit the migration of U87-MG cells. The effects of 25μM, 50μM and 100μM Bufoserotonin C intervention on the migration ability of U87-MG cells were detected. The results of the cell migration assay showed that with the increase of Bufoser otonin C treatment concentration, the number of migrated cells gradually decreased.

[0056] (3) From Figure 3 As shown in A and B, Bufoserotonin C can effectively inhibit the invasion of U87-MG cells. The effects of 25μM, 50μM and 100μM Bufoserotonin C intervention on the invasion ability of U87-MG cells were detected. The results of the cell invasion assay showed that with the increase of Bufoser otonin C treatment concentration, the number of invaded cells gradually decreased.

[0057] In summary, through CCK-8 cell viability assay, Transwell cell migration assay and invasion assay, it was found that Bufoserotonin C can effectively inhibit the growth, migration and invasion of human brain glioblastoma U87-MG cells, thereby inhibiting the malignant progression of glioma, providing a new drug for the treatment of glioma with good application prospects.

[0058] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. The use of indole alkaloids in the preparation of drugs for treating malignant gliomas, characterized in that: The indole alkaloid is N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide or / and a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the growth of human brain astroglioma cells.

3. The use according to claim 1, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the migration of human brain astroglioma cells.

4. The use according to claim 1, characterized in that: The N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide inhibits the invasion of human brain astrocytoblastoma cells.

5. A pharmaceutical composition for treating malignant glioma, characterized in that: The pharmaceutical composition includes N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide.

6. The pharmaceutical composition for treating malignant glioma according to claim 5, characterized in that: The half inhibitory concentration IC of N-[5-acetyl-1-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-1H-pyrrol-3-yl]acetamide in human brain astrocytoblastoma U87-MG cells in vitro 50 It is 45.11μM.

7. The pharmaceutical composition for treating malignant glioma according to claim 6, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and auxiliary components.

8. The pharmaceutical composition for treating malignant glioma according to claim 6, characterized in that: The pharmaceutical composition further comprises at least one of a filler, a lubricant, a dispersant, a wetting agent, a binder, a regulator, a solubilizer, an antioxidant, an antibacterial agent, an emulsifier, and a disintegrant.

9. The pharmaceutical composition for treating malignant glioma according to claim 8, characterized in that: The dosage form of the pharmaceutical composition is a clinically acceptable pharmaceutical preparation.

10. The pharmaceutical composition for treating malignant glioma according to claim 9, characterized in that: The dosage form of the pharmaceutical composition includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

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