A pharmaceutical composition for treating neuroblastoma

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a pharmaceutical composition for treating neuroblastoma, which comprises ethacrynic acid, or a precursor of ethacrynic acid, or a pharmaceutically acceptable salt of ethacrynic acid, or a pharmaceutically acceptable carrier of ethacrynic acid; further comprises an antitumor drug, or a precursor of the antitumor drug, or a pharmaceutically acceptable salt of the antitumor drug, or a pharmaceutically acceptable carrier of the antitumor drug; the antitumor drug is quazatin, doxorubicin or cisplatin. The pharmaceutical composition provided by the application can not only significantly induce SH-SY5Y and SK-N-B2 neuroblastoma death in vitro to enhance the antitumor activity of quazatin, but also can significantly inhibit the growth of neuroblastoma subcutaneous transplanted tumors of nude mice in vivo, and induce the death of transplanted tumor cells, and the enhanced autophagy activity plays an important role in the death of neuroblastoma induced by quazatin combined with ethacrynic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating neuroblastoma. BACKGROUND

[0002] Neuroblastoma (NB) is different from most other tumors in that it is most common in children, is an embryonic tumor originating from the neural crest, is composed of undifferentiated sympathetic nerve cells, and has an incidence of about 1 / 100,000 in children under 15 years old. It is the most common malignant tumor in children under 1 year old in clinical practice. Due to the update and improvement of the diagnosis method and treatment means for neuroblastoma in recent years, the overall survival rate of neuroblastoma patients has been greatly improved, but the survival rate and quality of life of high-risk children have not been significantly improved.

[0003] Clinically, neuroblastoma is divided into low-risk group, medium-risk group and high-risk group. The high-risk group refers to clinical stage 4, age > 1 year old; or clinical stage 3, > 1 year old, Shimada poor prognosis type, MYCN gene amplification or serum ferritin elevation; or clinical stages 3, 4, 4S, any age + MYCN amplification; or clinical stages 1, 2, age > 1 year old + MYCN amplification. Due to the high degree of malignancy, early bone marrow and / or bone metastasis, and the presence of minimal residual disease (MRD), the effect of surgical treatment is very limited. At present, the treatment effect and patient tolerance of the traditional chemotherapy regimen for neuroblastoma have reached the maximum limit. The CiE (cisplatin + etoposide) and CDV (cyclophosphamide + daunorubicin + vincristine) alternating chemotherapy regimen is the first-line chemotherapy regimen for high-risk neuroblastoma, but the complete remission (CR) and very good partial remission (VGPR) are still less than 40%.

[0004] At present, exploring a more effective drug regimen based on molecular targeted drugs is the top priority for the treatment of high-risk neuroblastoma, in order to improve the treatment effect and cure rate, and improve the quality of life of clinical children. SUMMARY

[0005] The purpose of the present application is to provide a pharmaceutical composition for treating neuroblastoma, in order to solve the above technical problems.

[0006] One aspect of the present application relates to a pharmaceutical composition for treating neuroblastoma, comprising etanidazole, or a precursor of etanidazole, or a pharmaceutically acceptable salt of etanidazole, or a pharmaceutically acceptable carrier of etanidazole; and further comprising an antitumor drug, or a precursor of the antitumor drug, or a pharmaceutically acceptable salt of the antitumor drug, or a pharmaceutically acceptable carrier of the antitumor drug; the antitumor drug is quazatin, doxorubicin or cisplatin.

[0007] In the present application, Ethacrynic acid (EA), also known as Diuretic acid, is an organic compound, CAS NO: 58-54-8, with chemical formula C 13 H 12 Cl2O4, is a diuretic, mainly used for congestive heart failure, acute pulmonary edema, renal edema, ascites due to cirrhosis, ascites due to hepatocellular carcinoma, ascites due to schistosomiasis, cerebral edema and other edematous diseases, and can relieve edema symptoms caused by heart failure and kidney failure. However, there is no record of using Ethacrynic acid for neuroblastoma treatment.

[0008] Quizartinib (AC220) has a molecular formula of C 29 H 32 N6O4S, a molecular weight of 560.7, and a CAS No. 950769-58-1. It inhibits wild-type FLT3 and mutant FLT3 ITD autophosphorylation and is a highly selective and effective second-generation type II FLT3 (type II FLT3) tyrosine kinase inhibitor with oral activity. Quizartinib can significantly inhibit the proliferation of some tumor cells, but the proliferation inhibition effect of Quizartinib mainly depends on its significant cycle arrest and cannot effectively induce tumor cell death. Therefore, the effect of Quizartinib in preclinical animal experiments is not ideal.

[0009] The pharmaceutical composition provided by the present application can be made into any conventional preparation form by a conventional method. The dosage form can be various, as long as the active ingredient can effectively reach the mammalian body. For example, it can be selected from: injection, infusion, tablet, capsule, and pill. Ethacrynic acid or quizartinib can exist in a suitable solid or liquid carrier or diluent.

[0010] Ethacrynic acid and quizartinib can be administered orally and intravenously, intramuscularly or subcutaneously, or ethacrynic acid and quizartinib are mixed and then administered orally and intravenously, intramuscularly or subcutaneously. The pharmaceutical forms suitable for injection include: sterile aqueous solutions or dispersions and sterile powders (for the preparation of sterile injection solutions or dispersions). In all cases, these forms must be sterile and must be fluid to facilitate the discharge of the syringe. They must be stable under the conditions of manufacture and storage and must be protected from the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, alcohol (such as glycerol, propylene glycol and liquid polyethylene glycol), their appropriate combinations and vegetable oils.

[0011] If necessary, ethacrynic acid and quizartinib can also be administered in combination with other active ingredients or drugs.

[0012] Through long-term and extensive research, it is accidentally found that the combination of etanidazole and quizartinib has excellent effect on treating neuroblastoma.

[0013] In specific embodiments of the present application, some compositions of etanidazole and quizartinib are given, for example, in one embodiment, a dosing regimen of etanidazole and quizartinib in a weight ratio of 5:3 is proposed in animal experiments on mice, specifically, etanidazole 5mg / kg, quizartinib 3mg / kg.

[0014] Another aspect of the present application relates to the use of a pharmaceutical composition of etanidazole and quizartinib in the treatment of neuroblastoma.

[0015] Another aspect of the present application relates to the use of a pharmaceutical composition of etanidazole and doxorubicin in the treatment of neuroblastoma.

[0016] Another aspect of the present application relates to the use of a pharmaceutical composition of etanidazole and cisplatin in the treatment of neuroblastoma.

[0017] The present application is combined with various natural compounds to screen out substances that can cooperate with each other to enhance the anti-tumor effect. Through cell experiments, it is proved that AC220 combined with doxorubicin and AC220 combined with cisplatin can significantly inhibit the proliferation of SH-SY5Y cells. After a large amount of screening work, it is found that etanidazole and quizartinib combined treatment of neuroblastoma can not only significantly induce SH-SY5Y, SK-N-B2 neuroblastoma death in vitro, but also enhance the anti-tumor activity of quizartinib, and can also significantly inhibit the growth of neuroblastoma in vivo. In addition, it can induce the death of transplanted tumor cells, and the inhibition of mitochondrial autophagy activity plays an important role in the death of neuroblastoma induced by quizartinib combined with etanidazole. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 4 is a graph showing the effect of AC220+EA combination on the inhibition of SH-SY5Y cell proliferation; wherein A: SH-SY5Y cells treated with DMSO for 24h; B: SH-SY5Y cells treated with 75μM EA for 24h; C: SH-SY5Y cells treated with 10μM AC220 for 24h; D: SH-SY5Y cells treated with 10μM AC220 for 2h, then combined with 75μM EA for 24h.

[0019] Figure 2 Figure 6 is a flow cytometry detection statistical chart of PI uptake.

[0020] Figure 3 Figure 8 is a flow cytometry detection statistical chart of mitochondrial ROS.

[0021] Figure 4 Results of pyroptosis of SH-SY5Y cells induced by the AC220+EA combination group.

[0022] Figure 5 Fig. 27 is a small-animal live imaging system imaging image of mice on day 27 after tumor transplantation.

[0023] Figure 6 Fig. 30 is an image of tumor tissue of mice ex vivo on day 30 after tumor transplantation. Among them, A: control group: Saline group tumor, the mice in this group were injected with a control solvent every day after tumor transplantation; B: experimental group 1: AC220 group, the mice in this group were injected with 3 mg / kg of AC220 every day after tumor transplantation; C: experimental group 2: EA group, the mice in this group were injected with 5 mg / kg of EA every day after tumor transplantation; D: experimental group 3: EA+AC220 group, the mice in this group were injected with 3 mg / kg of AC220 and 5 mg / kg of EA every day after tumor transplantation.

[0024] Figure 7 Fig. 31 is a tumor weight statistical chart.

[0025] Figure 8 Fig. 32 is a graph of changes in body weight of mice after tumor transplantation.

[0026] Figure 9 Fig. 33 is a Ki67 staining result chart of tumor tissue sections.

[0027] Figure 10 Fig. 34 is a Cleaved-caspase3 staining result chart of tumor tissue sections.

[0028] Figure 11 Fig. 35 is an effect chart of the inhibitory effect of the AC220+doxorubicin combination group on SH-SY5Y cell proliferation. Among them, A: SH-SY5Y cells treated with DMSO for 24 h; B: SH-SY5Y cells treated with 2.5 μM doxorubicin for 24 h; C: SH-SY5Y cells treated with 10 μM AC220 for 24 h; D: SH-SY5Y cells treated with 10 μM AC220 for 2 h, then combined with 2.5 μM doxorubicin for 24 h.

[0029] Figure 12 Fig. 36 is an effect chart of the inhibitory effect of the AC220+Cisplatin combination group on SH-SY5Y cell proliferation. Among them, A: SH-SY5Y cells treated with DMSO for 24 h; B: SH-SY5Y cells treated with 10 μM Cisplatin for 24 h; C: SH-SY5Y cells treated with 10 μM AC220 for 24 h; D: SH-SY5Y cells treated with 10 μM AC220 for 2 h, then combined with 10 μM Cisplatin for 24 h. Detailed Implementation

[0030] The following detailed description illustrates the specific implementation method:

[0031] I. Cell Experiments

[0032] 1. Cell Culture

[0033] The human neuroblastoma cell line SH-SY5Y was purchased from the American Type Culture Collection (ATCC). SH-SY5Y cells were cultured in DMEM high-glucose medium (purchased from Wuhan Pronosei Biotechnology Co., Ltd.), with the addition of 10% fetal bovine serum (purchased from OriCell) and 1% penicillin-streptomycin (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.). Cells were cultured in 6-well plates, 1.5 mL of medium per well, in a 37°C, 5% CO2 saturated humidity incubator. AC220 and EA were dissolved in DMSO before use, aliquoted appropriately, and stored at -20°C protected from light.

[0034] 2. Drug treatment

[0035] Prepare a 10 mM stock solution by dissolving AC220 in DMSO, and aliquot it for use. Prepare a 75 mM stock solution by dissolving EA in DMSO, and aliquot it for use. Store at -80°C protected from light.

[0036] Prepare to administer the drug when the cell density reaches 60-70%.

[0037] Experimental groups: DMSO group, EA group, AC220 group, and AC220+EA combined group.

[0038] After the AC220+EA combined group of cells was pretreated with 10 μM MAC220 for 2 h, each group was treated with DMSO, 10 μM AC220, 75 μM EA, and 10 μM MAC220 combined with 75 μM EA, respectively. After 24 h, the inhibitory effect of each group of drugs on the proliferation of human neuroblastoma cell line SH-SY5Y was observed.

[0039] Figure 1 The diagram shows the inhibitory effect of the AC220+EA combination on the proliferation of SH-SY5Y cells; where A: SH-SY5Y cells treated with DMSO for 24 h; B: SH-SY5Y cells treated with 75 μM EA for 24 h; C: SH-SY5Y cells treated with 10 μM MAC220 for 24 h; D: SH-SY5Y cells pretreated with 10 μM MAC220 for 2 h, followed by treatment with 75 μM EA for 24 h. Figure 1 As shown, compared with other groups, the AC220+EA combination group can significantly inhibit the proliferation of SH-SY5Y cells.

[0040] 3. PI uptake detection

[0041] (1) Digestion collection of cells after (two) treatment. Culture supernatant in 5 mL EP tube. Take 0.5 mL of 4°C pre-cooled PBS buffer to wash the cells twice, and then transfer the PBS to the corresponding EP tube. Then add 0.5 mL of trypsin without EDTA to each well, and then place it in a 37°C incubator for 5 min. Gently blow the cells down and transfer them to the corresponding EP tube to terminate digestion, and then wash the well plate with 0.5 mL of PBS and transfer it to the corresponding EP tube. Centrifuge at 1000 rpm for 5 min at 4°C. Discard the supernatant, and then quickly add 1 mL of pre-cooled PBS to the EP tube to wash the cell pellet, and mix gently several times. Centrifuge at 1000 rpm for 5 min at 4°C. Repeat this step once. Obtain single suspended cells.

[0042] (2) Take 1000 μL of PBS and add 3 μL of PI dye (kit purchased from Biyun Tian Bio, ST511). Resuspend the washed cells with 100 μL of diluted PI dye, filter with a 200 mesh filter, and then transfer to a flow tube. Store on ice in the dark, and then perform machine detection on the 561 nm channel of the flow cytometer (BD Biosciences, FACSCelesta). Record the PI positive proportion, which is the proportion of dead cells.

[0043] Figure 2 PI uptake flow cytometry detection chart. As shown in Figure 2 compared with other groups, it can be observed that the PI uptake of the AC220+EA combined group is significantly increased, indicating that the AC220+EA combined group has stronger killing effect on SH-SY5Y than other groups.

[0044] 4. MitoROS detection

[0045] Take the cells treated with (two) drugs, and obtain single suspended cells by the method in (three) (1). Take 1000 μL of PBS, and add 0.5 μL of MitoSOX (M36008, Invitrogen) stock solution to make the final concentration of the dye solution 1 μM. Resuspend the washed cells with 100 μL of diluted MitoSOX dye, and then filter with a 200 mesh filter and transfer to a flow tube. Store on ice in the dark, and then perform machine detection on the 561 nm channel of the flow cytometer (BD Biosciences, FACSCelesta). Record the MitoSOX positive proportion.

[0046] Figure 3 MitoROS flow cytometry detection chart. As shown inFigure 3 As shown, a significant increase in mitoROS production can be observed in the combination group, indicating an increase in mitochondrial reactive oxygen species.

[0047] 5. Expression detection of pyroptosis execution protein GSDME

[0048] Western Blot was performed on cells treated with (two) drugs, and the specific operation was as follows:

[0049] (1) Sample preparation: the treated cells were washed with cold PBS twice, and the culture medium was removed. Then, according to the cell amount, an appropriate amount of protein lysis solution was added, collected into a 1.5 mL centrifuge tube, and placed in a 100℃ metal bath for heating for 10 min. The precipitate was removed by centrifugation. 5x loading buffer was added, and the sample was heated in a 100℃ metal bath for 5 min.

[0050] (2) SDS-PAGE electrophoresis: prepare 10% separating gel and 5% concentrated gel, use 1.5 mm glass plate and comb. After the gel is completely solidified, load the sample. The upper gel electrophoresis parameters are constant voltage 80V, 30min, and the lower gel electrophoresis parameters are constant voltage 120V, 80min, until the bromophenol blue strip approaches the low end of the gel.

[0051] (3) Membrane transfer: prepare a PVDF membrane of appropriate size, activate it with anhydrous methanol, and then place the gel and PVDF membrane in a pre-cooled membrane transfer solution. From negative to positive, the order is sponge-filter paper-gel-PVDF membrane-filter paper-sponge, and make sure there is no air bubble between each layer. Place the membrane transfer system in an ice bath, use constant current 400mA, and transfer for 90min.

[0052] (4) Blocking: after membrane transfer is completed, take out the PVDF membrane with the front side up, and place it in 5% skimmed milk prepared with TBST, and incubate in a shaker for 1h.

[0053] (5) Antibody incubation: after blocking is completed, place the PVDF membrane on the shaker, and rinse with TBST for 3 times, 10min each time. After washing, add GSDME antibody, and incubate overnight in a 4℃ shaker. The next day, recover the antibody, and rinse with TBST for 3 times, 10min each time. Then add the corresponding secondary antibody, and incubate on the shaker at room temperature for 1h. Then recover the secondary antibody, and rinse with TBST for 3 times, 10min each time.

[0054] (6) Machine development: take the developing liquid reagent A: reagent B = 1:1, and mix well. Drop it on the PVDF membrane, and use a coating rod to evenly spread the developing liquid. Reflect fully, and then place it in a chemiluminescence instrument for detection and recording.

[0055] Figure 4 Results of SH-SY5Y cell pyroptosis induced by AC220+EA combination group. As shown in Figure 4 Figure 6, GSDME cleavage was not obvious in other groups, while GSDME cleavage was significantly increased in the combination group, indicating that AC220+EA combination group treatment promoted GSDME cleavage, thereby promoting the pyroptosis of cells.

[0056] 6. Other anticancer drugs combined with AC220 treatment of SH-SY5Y cells

[0057] AC220 was dissolved with DMSO to prepare a stock solution with a final concentration of 10 mM, and was aliquoted for use. Cisplatin was dissolved with sterile water to prepare a stock solution of 1 mg / mL, and was aliquoted for use. Doxorubicin was dissolved with DMSO to prepare a stock solution of 5 mM, and was stored at 4°C in the dark. After 2 h of pre-treatment of SH-SY5Y cells with 10 μM AC220, DMSO, doxorubicin 2.5 μM (or cisplatin 10 μM), AC220 10 μM, and 10 μM AC220+doxorubicin combination group 2.5 μM (or cisplatin 10 μM) were used for treatment, respectively, and the proliferation inhibition effect of each group of drugs on SH-SY5Y cells was observed after 24 h.

[0058] Figure 11 Figure 6 is a diagram of the proliferation inhibition effect of AC220+doxorubicin combination group on SH-SY5Y cells. Among them, A: SH-SY5Y cells treated with DMSO for 24 h; B: SH-SY5Y cells treated with 2.5 μM doxorubicin for 24 h; C: SH-SY5Y cells treated with 10 μM AC220 for 24 h; D: SH-SY5Y cells treated with 10 μM AC220 for 2 h, and then treated with 2.5 μM doxorubicin for 24 h. Figure 12 Figure 7 is a diagram of the proliferation inhibition effect of AC220+Cisplatin combination group on SH-SY5Y cells. Among them Figure 12 A is SH-SY5Y cells treated with DMSO for 24 h, Figure 12 B is SH-SY5Y cells treated with 10 μM Cisplatin for 24 h, Figure 12 C is SH-SY5Y cells treated with 10 μM AC220 for 24 h, Figure 12 D is SH-SY5Y cells treated with 10 μM AC220 for 2 h, and then treated with 10 μM Cisplatin for 24 h.

[0059] As shown in Figure 11 , Figure 12As shown, it can be observed that the AC220+doxorubicin combination group 2.5 μΜ (or Cisplatin 10 μΜ) significantly inhibited the proliferation of SH-SY5Y cells.

[0060] II. Animal Experiments

[0061] 1. Construction of Luciferase-labeled neuroblastoma cell SH-SY5Y cell strain

[0062] (1) Take 6 x 10 4 logarithmic growth phase SH-SY5Y cells, resuspend the cells with serum-free, double-antibody-free medium, inoculate in a 96-well plate, add 10 μL of EFla-mCherry-Luc Lentivirus (GMABIO, GM-0220IV208) with a concentration of 1 x 10 8 TU / mL. After 16 h of transfection, change the medium every two days, and subculture when the cells grow to 90%. Until transferred into a 10 cm dish, collect the cells for flow sorting, and screen out cells with mCherry label, which are the successfully infected cells, denoted as SH-SY5Y-Luc.

[0063] (2) 5-7 x 10 6 SH-SY5Y-Luc cells were cultured in a 10 cm dish using DMEM medium containing 10% fetal bovine serum and 0.1 mg / mL penicillin-streptomycin.

[0064] 2. In situ injection of SH-SY5Y-Luc cells into mouse adrenal glands

[0065] (1) Subdivide the Matrigel and place it in the ice box at 4°C for 24 h in advance. Subdivide the PBS and precool it separately. Place the high-pressure 1.5 mL centrifuge tube, 1 mL syringe, 200 μL syringe, and insulin syringe in -20°C for precooling. Change the medium for the cells one day in advance.

[0066] (2) On the day of tumor inoculation, prepare the ice box cells in advance and irradiate them with ultraviolet light for 30 min. Place the Matrigel on ice throughout the process. Trypsinize the cells with a density of 80-90%, wash them twice with 4°C PBS, resuspend the cells with 1 mL of PBS, and count them. Dilute the cells with cold PBS and Matrigel at a ratio of 1:1, and resuspend the cells with a density of 1 x 10 7 mL in 2 mL of PBS-Matrigel suspension. Subdivide the resuspended cells into 5 tubes and place them on ice.

[0067] (3) After weighing, NCG mice (purchased from Cyagen Biosciences Co., Ltd.) were anesthetized. Once the mice were stable under anesthesia, surgery was performed. The fur on the right side of the mouse's back was shaved and disinfected with alcohol. An incision was made at the lower edge of the right rib to avoid excessive bleeding and to expose the adrenal gland. The incision was opened with forceps to expose the injection area. 0.02 mL of the inverted and mixed cell suspension was drawn up using an insulin needle and injected into each mouse. After injection, the syringe was held for a few seconds to ensure successful injection and prevent leakage. After injection, the muscle and skin layers were sutured with 4-0 sutures, and appropriate antibiotics were administered. The mice were then returned to a clean cage. This was day 0 of tumor implantation.

[0068] (4) Three days after tumor implantation, in vivo imaging of mice was performed. The procedure was as follows: 0.1 mL of 20 mg / mL D-luciferin potassium salt, a substrate of luciferase, was injected into the mouse intraperitoneally. Seven minutes later, the anesthetized mouse was placed in a PE LμMina III (purchased from PerkinElmer) imaging system for imaging.

[0069] They were randomly divided into 4 groups:

[0070] Control group, experimental group 1, experimental group 2 and experimental group 3.

[0071] Control group: The tumors in the Saline group were injected with blank solvent daily after tumor transplantation in mice in this group.

[0072] Experimental Group 1: The AC220 group, in which mice were injected with 3 mg / kg AC220 per day after tumor transplantation.

[0073] Experimental group 2: namely the EA group, mice in this group were injected with 5 mg / kg EA per day after tumor transplantation.

[0074] Experimental group 3: namely the EA+AC220 group (combined group), mice in this group were injected daily with 3 mg / kg AC220 and 5 mg / kg EA after tumor transplantation.

[0075] After grouping, the mice were given the first dose, which was recorded as day 1. The mice were then given the first dose daily, and weighed each day.

[0076] Figure 8 This is a graph showing the changes in body weight in mice after tumor transplantation. Figure 8 As shown, there were no significant differences between the groups, and imaging was performed on days 3, 6, 9, 12, 15, 18, 21, 24, and 27 after drug administration to record changes in the tumor.

[0077] Figure 5 This is an in vivo imaging image of a small animal on day 27 after tumor transplantation. Figure 5As shown in the figure, the tumor size in the EA+AC220 combination group was significantly smaller than that in the other three groups.

[0078] (5) On the 30th day after administration, the mice were sacrificed by cervical dislocation, and the kidney tissue and tumor tissue were taken. The tumor tissue was weighed, and the results are shown in the figure. Figure 6 As shown in the figure, Figure 6 A is the control group: the saline group tumor, the mice in this group were injected with blank solvent every day after tumor transplantation, Figure 6 B is experimental group 1: AC220 group, the mice in this group were injected with 3 mg / kg AC220 every day after tumor transplantation, Figure 6 C is experimental group 2: EA group, the mice in this group were injected with 5 mg / kg EA every day after tumor transplantation, Figure 6 D is experimental group 3: EA+AC220 group, the mice in this group were injected with 3 mg / kg AC220 and 5 mg / kg EA every day after tumor transplantation. The tumor tissue was separated from the kidney tissue and weighed, and the results are shown in the figure. Figure 7 As shown in the figure, the tumor tissue volume and weight in the EA+AC220 group were significantly inhibited after administration.

[0079] (6) The separated tumor tissue was fixed, dehydrated, wax-embedded, and then cut into 4-6 microns thick slices using a microtome. Then, the steps of dewaxing, rehydrating, antigen repairing, removing endogenous peroxidase, blocking, antibody incubation, secondary antibody, tertiary antibody incubation, and color development were performed in turn. Immunohistochemical detection of Ki67 and cleaved-caspase3 expression in the tissue was performed.

[0080] Figure 9 The figure is the Ki67 staining result of tumor tissue sections. Figure 10 The figure is the Cleaved-caspase3 staining result of tumor tissue sections. As shown in the figure, Figure 9 and Figure 10 The results show that the expression of Ki67 in the tissue decreases and the expression of Cleaved-caspase3 increases in the AC220+EA combination group.

[0081] In summary, the cell experiment proves that AC220 combined with doxorubicin and AC220 combined with cisplatin can significantly inhibit the proliferation of SH-SY5Y cells. Through cell experiments and animal experiments, it is proved that the combination of etanercept and quinazoline can not only significantly induce the death of SH-SY5Y and SK-N-B2 neuroblastoma cells in vitro, enhance the anti-tumor activity of quinazoline, but also significantly inhibit the growth of neuroblastoma subcutaneous transplanted tumors in nude mice in vivo, and induce the death of transplanted tumor cells. The enhanced autophagy activity plays an important role in the death of neuroblastoma induced by the combination of etanercept and quinazoline.

[0082] The preferred embodiments of the application have been described above in detail. It should be understood that modifications and variations to the preferred embodiments could be made by those skilled in the art in light of the teachings above. It is therefore contemplated that the application can encompass other variations and modifications that fall within the scope of the claims.

Claims

1. A pharmaceutical composition for treating neuroblastoma, characterized in that, It includes ethacrynic acid and an antitumor drug; the antitumor drug is quezartinib.

2. The pharmaceutical composition for treating neuroblastoma according to claim 1, characterized in that, Ethanoic acid 5 mg / kg, quezartinib 3 mg / kg.

3. The use of pharmaceutical compositions including ethacrynic acid and quezartinib in the preparation of drugs for the treatment of neuroblastoma.

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