Pharmaceutical composition combining aquaporin inhibitor and oxidative phosphorylation inhibitor for treating brain glioma and application of pharmaceutical composition

Abstract

The invention belongs to the field of biological medicine, and discloses a pharmaceutical composition combining an aquaporin inhibitor and an oxidative phosphorylation inhibitor for treating brain glioma, and the pharmaceutical composition comprises an AQP4 inhibitor AER-270 and an oxidative phosphorylation inhibitor Gboxin. 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. According to the composition provided by the invention, through combined administration of the AER270 and the Gboxin, the inhibition effect on the brain glioma is obviously better than that of single AER270 or single Gboxin, the combination of the AER270 and the Gboxin has a synergistic effect on treatment of the brain glioma, and an effective drug combination strategy is provided for treatment of the 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 and an oxidative phosphorylation inhibitor, and its application. Background Technology

[0002] Gliomas are the most aggressive and common primary malignant tumors of the central nervous system. They exhibit strong tumor heterogeneity and rapid malignant progression. Current treatments primarily involve radiotherapy and chemotherapy, which target proliferating tumor cells and produce strong toxic side effects by damaging normal proliferating cells. Temozolomide (TMZ) is a first-line drug for glioma treatment, but its biggest drawback is its significant side effects and the tendency for drug resistance to develop. Studies have shown that due to the activity of MGMT within gliomas, approximately 48-58% of glioma patients are insensitive to TMZ. Therefore, there is an urgent need for researchers and clinicians to develop new treatment strategies and more effective drugs for glioblastoma to improve the quality of life for patients.

[0003] AQP4 is a membrane protein highly expressed in gliomas and is positively correlated with malignancy. The AQP4 inhibitor AER270 can inhibit AQP4 expression. Current research on AQP4 inhibitors mainly focuses on reducing cerebral edema after cerebral infarction or hemorrhage and treating Alzheimer's disease. To prolong the survival time of glioblastoma patients, more and more researchers are exploring combination therapies for anti-tumor drugs (e.g., Chinese patent CN117752656A discloses a combination drug composition for treating glioma containing JJH201601 and temozolomide). Relatively quiescent cancer stem cells in gliomas may evade traditional treatment strategies. Cancer stem cells can possess metabolic characteristics that distinguish them from tumors and somatic cell proliferation. Although proliferating tumor cells rely on aerobic glycolysis, slowly circulating tumor cells may rely more on mitochondrial respiration as their primary energy source.

[0004] Oxidative phosphorylation (OXPHOS) plays a crucial role in cellular energy. The OXPHOS respiratory chain consists of over 90 proteins encoded by both the nuclear and mitochondrial genomes. Gboxin is an OXPHOS inhibitor that rapidly and irreversibly impairs oxygen consumption in glioblastoma cells. Gboxin works by influencing the activity of F0F1 ATP synthase through its positively charged mitochondrial oxidative phosphorylation complex, which depends on the proton gradient of the inner mitochondrial membrane. Gboxin is a small molecule isolated from high-throughput chemical screening that specifically inhibits the growth of primary mouse and human glioblastoma cells. It targets and inhibits glioblastoma growth (Targeting Mitochondrial Metabolism and RNA Polymerase POLRMT to Overcome Multidrug Resistance in Cancer) (Li, Xinnan, et al. "Discovery of a Novel, Potent, Orally Active, and Safe Inhibitor Targeting Human Mitochondrial RNA Polymerase." Journal of Medicinal Chemistry (2023).), and importantly, it is not toxic to normal proliferating cells.

[0005] However, there are currently no research reports on the combined treatment of glioma with AQP4 inhibitors and Gboxin. Summary of the Invention

[0006] The purpose of this invention is to provide a pharmaceutical composition combining an aquaporin inhibitor and an oxidative phosphorylation inhibitor, and its application. The combined administration of AER270 and Gboxin has a significantly better inhibitory effect on glioma than AER270 or Gboxin alone. The combination of the two has a synergistic effect on the treatment of glioma, providing an effective drug combination strategy for the treatment of glioma.

[0007] This invention provides the following technical solution: A first aspect of the present invention provides a pharmaceutical composition for use in combination with an aquaporin inhibitor and an oxidative phosphorylation inhibitor, the pharmaceutical composition comprising the AQP4 inhibitor AER-270 and the oxidative phosphorylation inhibitor (OXPHOS) Gboxin.

[0008] 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. Gboxin accumulates in the mitochondria of glioma cells and rapidly and irreversibly inhibits the activity of Complex V, a key enzyme in oxidative phosphorylation, on the inner mitochondrial membrane, leading to glioma cell death. This invention is the first to discover that the combination of AER270 and Gboxin has a synergistic effect in the treatment of gliomas.

[0009] The Gboxin structure is shown in equation (Ⅰ).

[0010] (I).

[0011] The concentration ratio of the AQP4 inhibitor AER-270 to the oxidative phosphorylation inhibitor Gboxin is 8 μM: (8 μM-16 μM).

[0012] The AQP4 inhibitor AER-270 and the oxidative phosphorylation inhibitor Gboxin are either single-agent formulations stored independently or compound formulations mixed together.

[0013] The pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0014] The pharmaceutically acceptable carrier is a filler, wetting agent, binder, disintegrant, or lubricant.

[0015] A second aspect of this invention also provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0016] The dosage form of the pharmaceutical preparation is oral tablets, granules, injections, or capsules.

[0017] A third aspect of the present invention also provides the use of the above-described pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for treating glioma.

[0018] Compared with the prior art, the present invention has the following superior effects: the combined administration of AER270 and Gboxin has a significantly better inhibitory effect on glioma cells (especially glioblastoma) than the single administration of AER270 or Gboxin. The combination of the two has a synergistic effect on the treatment of glioma, providing an effective drug combination strategy for the treatment of glioma. Attached Figure Description

[0019] Figure 1 The results of AER270, Gboxin and the composition in Example 1 on the inhibition of glioma cell proliferation are shown. Figure 2The results of the efficacy of AER270, Gboxin and the combination against glioma cells in Example 2 are shown. Figure 3 The results of the effects of AER270, Gboxin and the composition on the mitochondrial membrane potential of glioma cells in Example 3 are shown. Figure 4 The results of treating glioma cells with AER270, Gboxin and the composition in Example 4 show the effects of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation of ACC-79. Figure 5 The results of the effects of AER270, Gboxin and the composition on the Ki67 proliferation index of glioma cells in Example 5 are shown. Figure 6 The Western blot results for the expression levels of AQP4 protein in glioma cells by AER270, Gboxin, and the composition in Example 6 are shown. Figure 7 The results show the therapeutic effects of AER270, Gboxin, and the combination in Example 7 on an orthotopic mouse glioma model. Detailed Implementation

[0020] 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.

[0021] Example 1: Inhibition of glioma cell proliferation by AER-270, Gboxin, and their combination 1. Experimental Method: Glioma cells were seeded in 96-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 48 hours. The effects of AER-270 (0 μM, 1 μM, 2 μM, 4 μM, 8 μM) and Gboxin (0 μM, 2 μM, 4 μM, 8 μM, 16 μM) alone on the viability of glioma cells were then detected by CCK8 assay. Glioma cells were then seeded into 96-well plates. The plates were supplemented with AER-270 and Gboxin at concentrations of (8 μM + 2 μM, 8 μM + 4 μM, 8 μM + 8 μM, 8 μM + 16 μM) and a control group containing 0.1 μM DMSO. Cells were cultured at 37°C, 5% CO2, and 90% humidity for 48 hours. The effects of different concentrations of AER-270 and / or Gboxin in combination on glioma cell viability were then assessed using a CCK8 assay.

[0022] 2. Experimental Results: The results of cell viability testing are as follows: Figure 1 As shown in the figure, the results indicated that after 48 hours of treatment with either Gboxin or AER270 on glioma cells, the inhibitory effect on cell proliferation significantly increased with increasing drug concentration. Furthermore, the combined use of AER270 and Gboxin on glioma cells for 48 hours showed a significantly higher inhibitory effect on cell proliferation than the single-drug groups, demonstrating a synergistic inhibitory effect.

[0023] Example 2: The therapeutic effects of AER270, Gboxin, and their combination on glioma cells 1. Experimental Method: 100 μL of 5000 glioblastoma cells per 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), Gboxin (16 μM), and a combination of AER270 (8 μM + 16 μM), respectively. Calcein-AM / PI staining working solution was added, and the killing effect on glioblastoma cells was observed using a fluorescence microscope.

[0024] 2. Experimental Results: Experimental results are as follows Figure 2 As shown, Gboxin and AER270 have a weak killing effect on glioma cells, while the combined use of Gboxin and AER270 significantly enhances the killing effect on glioma cells.

[0025] Example 3: Effects of AER270, Gboxin, and their composition on mitochondrial membrane potential in different glioma cells Experimental methods: Glioma cells were used at a rate of 1×10 5Cells were seeded at a density of 10 cells / well in 6-well plates and cultured. After cell adhesion, the original culture medium was discarded, and the cells were treated with AER270 (8 μM), TMZ (16 μM), and a combination of AER270 and TMZ (8 μM + 16 μM) for 24 hours. JC-1 staining working solution was added, mixed thoroughly, and incubated at 37°C for 20 minutes. After incubation, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer. Mitochondrial membrane potential was detected using a fluorescence microscope.

[0026] 2. Experimental Results: Experimental results are as follows Figure 3 As shown, the results indicate that the use of Gboxin and AER270 alone has little effect on the mitochondrial membrane potential of glioma cells. The combined use of Gboxin and AER270 significantly reduced the mitochondrial membrane potential of glioma cells, achieving a synergistic effect.

[0027] Example 4: Effects of AER270, Gboxin, and their combinations on AMP-activated protein kinase (AMPK) and phosphorylated ACC-79 in different glioma cells. 1. Experimental Method: Glioma cells were seeded at a density of 500 cells / well in 6-well plates. After cell attachment, the original culture medium was discarded, and the cells were treated with AER270 (8 μM), TMZ (16 μM), or a combination of AER270 and TMZ (8 μM + 16 μM). Cells were then collected by centrifugation at 300g for 5 minutes at 4°C, washed with PBS, and proteins were extracted for Western blotting analysis to detect the expression levels of P-AMPK / P-ACC-79 proteins.

[0028] 2. Experimental Results: The results are as follows Figure 4 As shown, the results indicated that glioma cells treated with Gboxin and its combination exhibited increased phosphorylated AMPK and its targeted phosphorylated ACC-7932. This suggests that Gboxin can inhibit oxidative phosphorylation in glioma cells, thereby activating AMPK. Phosphorylation levels were further increased upon combination with AER270.

[0029] Example 5: Effects of AER270, Gboxin, and their combination on the Ki67 proliferation index of glioma cells 1. Experimental Method: Glioma cells were used at a rate of 1×10 5Cells were seeded at a density of 100 cells / well in 6-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 cells were cultured for 24 hours at 37°C, 5% CO2, and 90% humidity using AER270 (8 μM), TMZ (16 μM), and a combination of AER270 and TMZ (8 μM + 16 μM), respectively. Cells were washed twice with PBS, fixed with 4% PFA at room temperature for 1 hour, and then subjected to Ki67 immunofluorescence assays to specifically label proliferating cells.

[0030] All cell numbers were labeled using DAPI. The Ki67 positivity rate was ultimately obtained for different drug groups.

[0031] 2. Experimental Results: Experimental results are as follows Figure 5 As shown, the combination of Gboxin and AER-270 exhibited the highest inhibition rate of glioma cell proliferation compared to using Gboxin and AER-270 alone.

[0032] Example 6: Effects of AER270, Gboxin, and their combination on AQP4 protein expression levels in glioma cells 1. Experimental Method: Glioma cells were used at a rate of 1×10 5 Cells were seeded at a density of 100 cells / well in 6-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 each with AER270 (8 μM), TMZ (16 μM), and a combination of AER270 and TMZ (8 μM + 16 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.

[0033] 2. Experimental Results: like Figure 6 As shown, compared with the use of Gboxin and AER270 alone, the combined use of AER270 and Gboxin can significantly inhibit the expression of AQP4-related molecules.

[0034] Example 7: Inhibition of glioma cell proliferation in animal models by AER270, Gboxin, and their combination. 1. Experimental Method: Five-week-old nude mice weighing 16-18g with athymium were selected, with three mice per group. U87-Luc cells were used for tumor formation. After anesthetizing the mice, they were fixed on a stereotaxic apparatus. The scalp was incised with a scalpel, and a needle was used to drill a hole in the skull, located 2mm to the right of the midline between the anterior and posterior sides. The needle was inserted to a depth of 2.5mm, withdrawn 1mm, and then injected. Each mouse received 3μL of cells, totaling 1×10⁻⁶ cells. 6 After injection, the needle was withdrawn after 1 minute of rest, and the scalp was sutured with fine sutures. Ten days after modeling, drug treatment began, with the model mice receiving AER270 (8 μM), TMZ (16 μM), or a combination of AER270 and TMZ (8 μM + 16 μM) at the same time each day for 7 consecutive days. In vivo imaging of the mice was performed on days 7, 14, and 21 to record tumor signals. The mice's condition was observed daily, and their survival time was recorded.

[0035] 2. Experimental Results: Experimental results are as follows Figure 7 As shown, the combination of Gboxin and AER270 has a good therapeutic effect on mice with orthotopic glioma, and the tumor volume is significantly reduced and the survival time is significantly prolonged after treatment.

[0036] 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 a combination of an aquaporin inhibitor and an oxidative phosphorylation inhibitor, characterized in that, The pharmaceutical composition includes the AQP4 inhibitor AER-270 and the oxidative phosphorylation inhibitor Gboxin.

2. The pharmaceutical composition according to claim 1, characterized in that, The dosage of the AQP4 inhibitor AER-270 and the oxidative phosphorylation inhibitor Gboxin is 8 μM: (8 μM-16 μM).

3. The pharmaceutical composition according to claim 1, characterized in that, The AQP4 inhibitor AER-270 and the oxidative phosphorylation inhibitor Gboxin are either single-agent formulations stored independently or compound formulations mixed together.

4. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition 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 preparation comprises the pharmaceutical composition according to any one of claims 1-5.

7. The pharmaceutical preparation according to claim 6, 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 7 in the preparation of a drug for treating glioma.

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