Aquaporin inhibitor and amphotericin B combined pharmaceutical composition for treating brain glioma and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses a pharmaceutical composition combining an aquaporin inhibitor and amphotericin B for treating brain glioma, and the pharmaceutical composition comprises an AQP4 inhibitor AER-270 and amphotericin B. The invention further discloses a pharmaceutical preparation containing the pharmaceutical composition and application of the pharmaceutical preparation to preparation of drugs for treating brain glioma. The tumor cell inhibition effect of the pharmaceutical composition provided by the invention is obviously better than that of single AER270 or amphotericin B, the pharmaceutical composition has an obvious synergistic effect, and an effective drug combination strategy is provided for treatment of brain glioma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition for treating glioma by combining an aquaporin inhibitor with amphotericin B and its application. Background Technology

[0002] Gliomas are the most common primary brain tumors in adults, accounting for 30% of all primary brain tumors. They are characterized by aggressive growth, high incidence, high recurrence rate, and high mortality. Routine clinical treatments include surgical resection, radiotherapy, and chemotherapy. Despite advances in basic and clinical research, data show that even after surgical resection followed by radiotherapy and chemotherapy, the median survival for patients with advanced gliomas is approximately 14.6 months.

[0003] Traditional comprehensive treatment methods utilize temozolomide (TMZ) to induce DNA damage and kill tumor cells, and its combination with radiotherapy can significantly improve the median survival of patients. Temozolomide has become a first-line chemotherapy drug for gliomas. However, due to the high heterogeneity of tumors and the complexity of disease progression, chemotherapy tolerance to TMZ has become an important factor in glioma recurrence. Therefore, finding drugs with higher targeting specificity and the ability to perform precision treatment is currently a hot topic and an urgent problem to be solved in the field of glioma research. For example, Chinese patent publication number CN119318654A discloses the application of fludarabine and temozolomide in combination for the treatment of gliomas.

[0004] Research on aquaporin 4 (AQP4) inhibitors currently focuses on reducing cerebral edema after cerebral infarction or hemorrhage and treating Alzheimer's disease. Studies have found that AQP4 inhibitors can inhibit the membrane protein AQP4, which is highly expressed in gliomas. To enhance therapeutic effects, more and more researchers are exploring combination therapy with anti-tumor drugs. Amphotericin B is an antifungal drug that has been shown to have good therapeutic effects on gliomas (Wei J, Chen P, Gupta P, et al. Immune biology of glioma-associated macrophages and microglia:functional and therapeutic implications[J]. Neuro-Oncology, 2020, 22(2CD):180-194.DOI:10.1093 / neuonc / noz212.). However, to date, there are no reports on the combined use of AQP4 inhibitors and amphotericin B for the treatment of gliomas. Summary of the Invention

[0005] The purpose of this invention is to provide a pharmaceutical composition for the treatment of glioma, which combines an aquaporin inhibitor with amphotericin B. The pharmaceutical composition provided by this invention has a significantly better inhibitory effect on tumor cells than AER270 or amphotericin B alone, and has a significant synergistic effect, providing an effective combination drug strategy for the treatment of glioma.

[0006] This invention provides the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition for treating glioma by combining an aquaporin inhibitor with amphotericin B, the pharmaceutical composition comprising the AQP4 inhibitor AER-270 and amphotericin B.

[0007] AQP4 is a membrane protein highly expressed in gliomas and is positively correlated with malignancy. AER270 is a major inhibitor of water exchange in glioma cells. Amphotericin B, an antifungal drug, can enhance immunity and inhibit cell growth by activating macrophages and microglia. This invention is the first to discover that the combined drug composition of AER270 and amphotericin B has a significant inhibitory effect on the growth of glioblastoma cells. The combination of AER270 and amphotericin B exhibits a synergistic effect, with significantly better efficacy than using either AER270 or amphotericin B alone, thus showing greater promise for clinical application.

[0008] The structural formula of the amphotericin B is shown in Formula (Ⅰ): (I).

[0009] The dosage of the AQP4 inhibitor AER-270 and amphotericin B is 8 μM: (200-400) μM. The AQP4 inhibitor AER-270 and amphotericin B are either single-agent preparations stored independently or compound preparations mixed together. The pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0010] The pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

[0011] In a second aspect, the present invention also provides a pharmaceutical formulation comprising a pharmaceutical composition of aquaporin inhibitor and amphotericin B.

[0012] The dosage form of the pharmaceutical preparation is oral tablets, granules, injections, or capsules.

[0013] Thirdly, the present invention provides the use of the above-mentioned pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for treating glioma.

[0014] This invention is the first to discover that the combined use of the AQP4 inhibitor AER-270 and amphotericin B exerts a synergistic inhibitory effect on gliomas (especially glioblastomas), meaning that the combination of the two has a significant synergistic effect. The inhibitory effect of this combined drug composition on tumor cells is significantly better than that of AER270 or amphotericin B alone. This invention provides an effective drug combination strategy for the treatment of gliomas and has broad application prospects in the field of glioma treatment drug development. Attached Figure Description

[0015] Figure 1 The results of AER-270, amphotericin B, and the combination of drugs inhibiting the proliferation of glioma cells were presented. Figure 2 Results of the efficacy of AER-270, amphotericin B, and combination drugs against glioma cells; Figure 3 The results show the effects of AER-270, amphotericin B, and their combination on the Ki67 proliferation index of glioma cells. Figure 4 Western blot results of AER-270, amphotericin B, and the combination of drugs on the expression level of AQP4 protein in glioma cells; Figure 5 Immunofluorescence results showing the effect of AER-270, amphotericin B, and their combination on AQP4 in glioma cells; Figure 6 The results show the effects of AER-270, amphotericin B, and combined drug combinations on TNF-α in glioma cells. Figure 7 The results show the effects of AER-270, amphotericin B, and their combination on orthotopic glioma tumors in mice. Detailed Implementation

[0016] The present invention will be further described below with reference to embodiments. The AER-270 used in this invention was donated by Nanjing Simcere Pharmaceutical Co., Ltd. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the invention. Various modifications made by those skilled in the art based on the teachings of this invention should be within the scope of protection of the claims of this application.

[0017] Example 1: Results of the inhibitory effect of AER-270, amphotericin B, and their combination on the proliferation of glioma cells. 1. Experimental Method: (1) 100 μL of 5000 glioblastoma cells / well and 100 μL of 5000 microglia / well were seeded into a 96-well plate and cultured for 24 hours at 37°C, 5% CO2 and 90% humidity. (2) Prepare AER270 solutions of different concentrations (1 μM, 2 μM, 4 μM and 8 μM); (3) Prepare amphotericin B solutions of different concentrations (50 μM, 100 μM, 200 μM and 400 μM); (4) Prepare drug combination solutions of different concentrations of AER270 and amphotericin B: 8 μM AER270 + 50 μM amphotericin B, 8 μM AER270 + 100 μM amphotericin B, 8 μM AER270 + 200 μM amphotericin B, 8 μM AER270 + 400 μM amphotericin B; (5) A culture medium containing DMSO (0.1 μM) was used as the control group; (6) Three replicates of each concentration were added to a 96-well plate and incubated for 24 hours at 37°C, 5% CO2 and 90% humidity. (7) Thaw and centrifuge CCK8 at room temperature, and add 10 μL of CCK-8 solution to each well; (8) Incubate at 37℃, 5% CO2 and 90% humidity for 1 hour, and then measure the absorbance at 450nm using an ELISA reader; (9) Process and analyze the results using Excel and Graphpad Prism.

[0018] 2. Experimental Results: The results of cell viability testing are as follows: Figure 1 As shown in the figure. The results showed that after 48 hours of treatment with TMZ and amphotericin B alone, the inhibitory effect on cell proliferation of gliomas significantly increased with increasing drug concentration. Furthermore, the combination of AER270 and amphotericin B, after 48 hours of treatment with gliomas, exhibited a significantly higher inhibitory effect on cell proliferation than the single-drug groups, demonstrating a synergistic inhibitory effect.

[0019] Example 2: The efficacy of AER-270, amphotericin B, and their combination against glioma cells. 1. Experimental Method: 100 μL of 5000 cells / well was seeded into 96-well plates and cultured for 24 hours at 37°C, 5% CO2, and 90% humidity. After cell attachment, the original culture medium was discarded, and the cells were cultured for 24 hours at 37°C, 5% CO2, and 90% humidity using AER270 (8 μM), amphotericin B (400 μM), and a combination of AER270 and amphotericin B (8 μM + 400 μM), respectively. Calcein-AM / PI staining working solution was added, and the killing effect on glioma cells was observed using a fluorescence microscope.

[0020] 2. Experimental Results: Experimental results are as follows Figure 2 As shown, amphotericin B and AER270 have a weak killing effect on glioma cells, while the combined use of amphotericin B and AER270 significantly enhances the killing effect on glioma cells.

[0021] Example 3: Effect of AER-270, amphotericin B, and their combination on the Ki67 proliferation index of glioma cells after treatment. 1. Experimental Method: (1) Glioma cells were injected at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 6-well plates and cultured for 24 hours at 37°C, 5% CO2 and 90% humidity. (2) After the cells adhered to the wall, the original culture medium was discarded, and the cells were cultured for 24 hours at 37°C, 5% CO2 and 90% humidity using AER270 (8 μM), amphotericin B (400 μM), and a combination of AER270 and amphotericin B (8 μM + 400 μM). (3) Wash the cells twice with PBS, fix them with 4% PFA at room temperature for 1 hour, and then perform Ki67 immunofluorescence assay to specifically label the proliferating cells. (4) All cell numbers were labeled using DAPI. The Ki67 positivity rate of different drug groups was finally obtained.

[0022] 2. Experimental Results: Experimental results are as follows Figure 3 As shown, the combined drug combination exhibited the highest inhibition rate against glioma cell proliferation compared to the use of amphotericin B and AER-270 alone.

[0023] Example 4: Effects of AER-270, amphotericin B, and their combination on AQP4 protein expression levels in glioma cells. 1. Experimental Method: Glioma cells were divided into 1×10 5Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37°C, 5% CO2, and 90% humidity for 24 hours. After cell attachment, the original culture medium was discarded, and the cells were cultured for 24 hours with AER270 (8 μM), amphotericin B (400 μM), or a combination of AER270 and amphotericin B (8 μM + 400 μM). Cells were then collected by centrifugation at 300g for 5 minutes at 4°C, washed with PBS, and analyzed by Western blotting to determine AQP4 protein expression levels.

[0024] 2. Experimental Results: like Figure 4 As shown, compared with the use of amphotericin B and AER-270 alone, the combined use of AER-270 and amphotericin B can significantly inhibit the expression of AQP4-related molecules.

[0025] Example 5: Immunofluorescence results of AQP4 in glioma cells after treatment with AER-270, amphotericin B, and their combination. 1. Experimental Method: (1) Glioma cells were injected at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 6-well plates and cultured for 24 hours at 37°C, 5% CO2 and 90% humidity. (2) After the cells adhered to the wall, the original culture medium was discarded, and the cells were cultured for 24 hours at 37°C, 5% CO2 and 90% humidity using AER270 (8 μM), amphotericin B (400 μM), and a combination of AER270 and amphotericin B (8 μM + 400 μM). (3) Wash the cells twice with PBS, fix them with 4% PFA at room temperature for 1 hour, and then perform an immunofluorescence experiment with AQP4. (4) All cell numbers were labeled using DAPI. The AQP4 positivity rate of different drug groups was finally obtained.

[0026] 2. Experimental Results: Experimental results are as follows Figure 5 As shown, the combined drug combination exhibited the highest inhibition rate against AQP4 glioma cells compared to the use of amphotericin B and AER-270 alone.

[0027] Example 6: Effect of AER-270, amphotericin B, and their combination on TNF-α in glioma cells after treatment. 1. Experimental Method: (1) Seed cells at a density of 5000 / well in a 6-well plate; (2) After the cells adhered to the wall, the original culture medium was discarded and the cells were stimulated for 24 hours with AER270 (8 μM), amphotericin B (400 μM), and a combination of AER270 and amphotericin B (8 μM + 400 μM). (3) After aspirating the cell culture medium, wash once with 2 ml of PBS buffer, add 1 ml of trypsin, and continue until the cells are completely separated from the cell wall and suspended. (4) Add 5 ml of complete culture medium to stop enzyme activity, mix well, and transfer the liquid to a 15 ml centrifuge tube for centrifugation. Centrifuge at 800 rpm for 3 minutes, and then collect the supernatant; (5) After removing the kit from the refrigerator, allow it to equilibrate at room temperature (25-28ºC) for 20 minutes. Add 100 μl of sample to each well, seal the wells with a transparent sealing film, and incubate at room temperature for 120 minutes. (6) Wash the board 5 times, and pat it dry on thick absorbent paper on the last time; (7) Add 100 μl of biotinylated antibody to each well. Seal the wells with a clear sealing film and incubate at room temperature for 60 minutes. (8) Wash the board 5 times, and pat it dry on thick absorbent paper on the last time; (9) Add 100 μl of horseradish peroxidase-labeled Streptavidin to each well, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 minutes. (10) Wash the board 5 times, and pat it dry on thick absorbent paper on the last time; (11) Add 100 μl of TMB solution as a colorimetric reagent per well, seal the reaction wells with a white sealing film, and incubate at room temperature in the dark for 20 minutes. (12) Add 50 μl of stop solution per well, mix well and immediately measure the A450 value.

[0028] 2. Experimental Results: Experimental results are as follows Figure 6 As shown, the results indicated that TNF-α levels increased after treatment of glioma cells with the combined drug combination.

[0029] Example 7: Inhibition of glioma cell proliferation in an animal model by a combination of AER-270 and amphotericin B. 1. Experimental Method: (1) Six-week-old male mice with no thymus were anesthetized and given 1×10 6 U87-Luc cells were injected into the right striatum (1.8 mm transverse, 1 mm longitudinal and 2.5 mm deep). The model was successfully established 10 days after in situ implantation. Three mice were in each of the control group, amphotericin B group, AER270 group and amphotericin B and AER270 combination group. (2) The model mice were treated with AER270 (8 μM), amphotericin B (400 μM), or a combination of AER270 and amphotericin B (8 μM + 400 μM) at the same time every day for 7 consecutive days. The control group was injected with saline according to the same procedure. (3) D-fluorescein (150 mg / kg) was injected intraperitoneally on days 10, 20 and 30, and the bioluminescence intensity of the tumor was recorded 10 minutes later. The survival status of the model mice was observed daily.

[0030] 2. Experimental Results: Experimental results are as follows Figure 7 As shown, the drug combination of AER-270 and amphotericin B has a good therapeutic effect on mice with orthotopic glioma. After treatment, the tumor volume is significantly reduced and the survival time is significantly prolonged.

[0031] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating glioma using an aquaporin inhibitor in combination with amphotericin B, characterized in that, The pharmaceutical composition includes the AQP4 inhibitor AER-270 and amphotericin B.

2. The pharmaceutical composition according to claim 1, characterized in that, The dosage of the AQP4 inhibitor AER-270 and amphotericin B is 8 μM: (200-400) μM.

3. The pharmaceutical composition according to claim 1, characterized in that, The AQP4 inhibitor AER-270 and amphotericin B are either single-agent preparations stored independently or compound preparations mixed together.

4. The pharmaceutical composition according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

6. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises a pharmaceutical composition of aquaporin inhibitors in combination with amphotericin B as described in any one of claims 1-4.

7. The pharmaceutical preparation according to claim 1, characterized in that, The dosage form of the pharmaceutical preparation is oral tablets, granules, injections, or capsules.

8. Use of a pharmaceutical composition according to any one of claims 1-5 or a pharmaceutical preparation according to claim 6 or claim 7 in the preparation of a drug for treating glioma.

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