Use of a transmembrane water exchange protein aqp4 inhibitor in the preparation of an anti-glioma drug

Abstract

The application discloses application of a transmembrane water exchange protein AQP4 inhibitor and a prodrug or a pharmaceutically acceptable salt thereof in preparation of an antitumor drug, and measures a glioma water molecule transmembrane outflow rate k io to noninvasively monitor a treatment effect of the AQP4 inhibitor; and the AQP4 inhibitor, the prodrug or the pharmaceutically acceptable salt thereof can effectively and low-toxicity inhibit glioma tumor cell proliferation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of a transmembrane water exchange protein AQP4 inhibitor in preparation of an anti-glioma drug. BACKGROUND

[0002] Glioma has the characteristics of strong invasiveness, fast malignant progression and low cure rate, and its incidence and mortality rate rank first among central nervous system tumors. Due to the biological characteristics of highly infiltrative proliferation of glioma, surgery is almost unable to ensure complete resection of the tumor, and the postoperative recurrence rate is nearly 100%, therefore, chemotherapy plays an important role in the comprehensive treatment of glioma. Temozolomide is a first-line chemotherapeutic drug for the clinical treatment of glioma at present, but it is still difficult to achieve the purpose of inhibiting the proliferation and recurrence of glioma, about 30-40% of glioma patients are initially drug-resistant, more patients will acquire resistance to temozolomide, or be forced to stop temozolomide due to its hematological toxicity, and cell proliferation is the main reason for the failure of glioma treatment. Therefore, seeking a highly efficient and low-toxic drug is an urgent problem to be solved in the current precise treatment of glioma.

[0003] Current research on AQP4 inhibitors is mainly focused on reducing brain edema after cerebral infarction or cerebral hemorrhage (e.g. Chen B, Kong X, Li Z, et al. Downregulation of NF-κB by Shp-1 alleviates cerebral venous sinus thrombosis-induced brain edema via suppression of AQP4 [published online ahead of print, 2022 May 25) Passchier EMJ, Kerst S, Brouwers E, Hamilton EMC, Bisseling Q, Bugiani M, Waisfisz Q, Kitchen P, Unger L, Breur M, Hoogterp L, de Vries SI, Abbink TEM, Kole MHP, Leurs R, Vischer HF, Brignone MS, Ambrosini E, Feillet F, Born AP, Epstein LG, Mansvelder HD, Min R, van der Knaap MS. Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema. Brain. 2023 Aug 1; 146(8): 3444-3454., improving Parkinson's disease (PD, Si X, Dai S, Fang Y, Tang J, Wang Z, Li Y, Song Z, Chen Y, Liu Y, Zhao G, Zhang B, Pu J. Matrix metalloproteinase-9 inhibition prevents aquaporin-4 depolarization-mediated glymphatic dysfunction in Parkinson's disease. J Adv Res. 2023 Mar 20; S2090-1232(23)00086-3. doi: 10.1016 / j.jare.2023.03.004. Epub ahead of print. PMID: 36940850.), anti-Alzheimer's disease (AD) Harrison IF, Ismail O, Machhada A, Colgan N, Ohene Y, Nahavandi P, Ahmed Z, Fisher A, Meftah S, Murray TK, Ottersen OP, Nagelhus EA, O'Neill MJ, Wells JA, Lythgoe MF (2020) Impaired glymphatic function and clearance of tau in an Alzheimer's disease model. Brain 143(8):2576-2593, etc. Currently, there is no application of AQP4 inhibitors to regulate the proliferation of glioma cells. SUMMARY

[0004] The purpose of the present application is to provide the application of a transmembrane water exchange protein AQP4 inhibitor and its prodrug or its pharmaceutically acceptable salt in the preparation of an anti-glioma drug, which can efficiently and low-toxicity inhibit the proliferation of glioma tumor cells.

[0005] The present application provides the following technical solutions:

[0006] The application of a transmembrane water exchange protein AQP4 inhibitor or its pharmaceutically acceptable salt in the preparation of an anti-tumor drug.

[0007] The AQP4 inhibitor is AER-270 (CAS No.: 978-62-1), TGN-020 (CAS No.: 51987-99-6), ORI-TRN-002.

[0008] The structural formula of AER-270 is:

[0009]

[0010] The tumor is glioma, and the glioma is brain glioma or ependymoma (tumor cells highly express AQP4).

[0011] The technical concept of the present application is that, taking brain glioma as an example, AQP4 is a membrane protein highly expressed on glioma and positively correlated with the degree of malignancy, and the treatment of brain glioma is mainly an inhibitor of water exchange of brain glioma cells, i.e., targeting AQP4 to inhibit the transmembrane water exchange to regulate the proliferation of human brain glioma cells, which has a significant and strong inhibitory effect on the proliferation rate of different brain glioma cells, thereby can be used as a new anti-tumor drug for the treatment of malignant brain glioma.

[0012] Preferably, the glioma is brain glioma, and the AQP4 inhibitor is AER-270. AER-270 provided by the application has the effect of inhibiting the proliferation of brain glioma cells and has small toxic side effects, and can be used as a new type of anti-brain glioma drug in clinical practice.

[0013] Further, the water molecule transmembrane efflux rate k io of the glioma is measured by magnetic resonance MRI imaging to noninvasively monitor the therapeutic effect of the AQP4 inhibitor.

[0014] Taking AER-270 as an example, the magnetic resonance water exchange constant k io (water molecule transmembrane efflux rate k io ) is a noninvasive monitoring imaging marker for dynamic quantification of brain glioma cell proliferation of AER-270. Therefore, the water exchange magnetic resonance AQP4 quantitative imaging technology can noninvasively and dynamically monitor the effect of AER-270 on AQP4 inhibition, and accurately screen and predict the treatment benefit population.

[0015] The application also provides a use of a pharmaceutical composition in the preparation of an antitumor drug, wherein the pharmaceutical composition comprises the AQP4 inhibitor or a pharmaceutically acceptable salt thereof described above, and a pharmaceutically acceptable carrier.

[0016] The application also provides a use of a prodrug of a transmembrane water exchange protein AQP4 inhibitor or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

[0017] The prodrug of the AQP4 inhibitor is AER-271 (CAS No.: 634913-39-6). AER-271 is a phosphonate ester precursor of AER-270, which is metabolized into AER-270 to play a role in vivo.

[0018] The structural formula of AER-271 is:

[0019]

[0020] The tumor is glioma, and the glioma is brain glioma or ependymoma (tumor cells highly express AQP4).

[0021] Further, the water molecule transmembrane efflux rate k io of the glioma is measured by magnetic resonance MRI imaging to noninvasively monitor the therapeutic effect of the AQP4 inhibitor.

[0022] Taking AER-271 as an example, the magnetic resonance water exchange constant k io (water molecule transmembrane efflux rate k ioAER-270 is a non-invasive monitoring image marker for dynamic quantitative glioma cell proliferation of AER-270, so it can also non-invasively monitor the treatment sensitivity and effect of AER-271 prodrug on brain glioma, and accurately screen and predict the benefit population.

[0023] The application also provides a use of a pharmaceutical composition in the preparation of an anti-tumor drug, wherein the pharmaceutical composition comprises the prodrug of the AQP4 inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0024] Compared with the prior art, the application has the beneficial effects that the AQP4 inhibitor can inhibit the proliferation of tumor cells, in particular, AER-270 as a specific AQP4 inhibitor, and any other form of derivative drug (i.e., a prodrug such as AER-271) that is metabolized into AER-270 after entering the body can effectively and low-toxicity inhibit the proliferation of glioma tumor cells; thereby effectively solving the problems of clinical recurrence, treatment resistance and first-line chemotherapy drug temozolomide drug resistance of glioma (in particular, brain glioma). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Results of AER-270 on the inhibition of proliferation of different brain glioma cells.

[0026] Figure 2 Results of AER-270 on the IC 50 of the inhibition of proliferation of different brain glioma cells.

[0027] Figure 3 Results of AER-270 and temozolomide on the inhibition of proliferation and functional connection of U87mg cells.

[0028] Figure 4 Results of AER-270, TGN020 and temozolomide on the inhibition effect of proliferation of different brain glioma cells.

[0029] Figure 5 Results of AER-270 on the proliferation index Ki 67 of U87mg.

[0030] Figure 6 Results of AER-270 on the killing rate of brain glioma organoid cells.

[0031] Figure 7 Results of AER-270 on the inhibition effect of proliferation of temozolomide treatment-resistant brain glioma recurrence cells.

[0032] Figure 8 Results of AER-271 on the treatment effect of in situ animal glioma model.

[0033] Figure 9 To utilize k io Results of monitoring the efficacy of AER-270, temozolomide and TGN-020 on brain glioma. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with the examples. It should be noted that the following examples are only used for illustration and are not intended to limit the application. Various changes made by those skilled in the art based on the teachings of the present application shall fall within the scope of protection of the claims of the present application.

[0035] AER-270 and AER-271 used in the present application are donated by Nanjing Xiansheng Pharmaceutical Co., Ltd. It should be noted that AER-270 can be recorded as AER270, and TGN-020 can be recorded as TGN020.

[0036] Example 1: Inhibition of proliferation of different brain glioma cells by AER-270 in vitro

[0037] After inoculating cells (U87MG, C6) in a 48-well plate, the coverage area and cell number of cells in the control group (0.1% DMSO) and the 8 μM AER-270 group were obtained by microscopy every day.

[0038] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, AER-270 has the effect of inhibiting the proliferation of brain glioma cells, and AER-270 has a significant and strong inhibitory effect on the proliferation of different brain glioma cells.

[0039] Example 2: IC 50

[0040] Different concentrations of AER-270 (1, 2, 4, 8 μM) were used to treat cells (U87MG, C6) in vitro, and the cell number was recorded every day. Graphpad Prism and other software were used to calculate the IC 50 value of the drug on the proliferation of different glioma cells.

[0041] The experimental results are shown in Table 2. Figure 2 As shown in Table 2, the IC 50 <4.5 μM of AER-270 on the proliferation of different brain glioma cells.

[0042] Example 3: Comparison of the efficacy of AER-270 and the only clinically proven effective drug temozolomide (TMZ)

[0043] In vitro, U87mg and C6 cells were treated with different drugs, AER-270 (8 μM) and TMZ (100 μM) respectively. Cell number was recorded at logarithmic proliferation phase (6h, 24h, 48h, 72h and 96h) and the difference of cell proliferation inhibition by different drugs was statistically analyzed.

[0044] The experimental results are shown in Table 1, and the efficacy of AER-270 and TMZ, the only clinically proven effective drug, was compared: AER-270 inhibited glioma cell proliferation faster and had a higher inhibition rate. Figure 3

[0045] Example 4: Comparison of the efficacy of AER-270, TGN020 and TMZ

[0046] After U87MG cells were inoculated, the cells were treated with 8 μM AER-270, 300 μM TGN020 and 100 μM TMZ respectively, and cell morphology and total cell area in the field of view were recorded at the logarithmic proliferation phase. The difference in cell proliferation inhibition rate by different drugs was statistically analyzed.

[0047] After C6 cells were inoculated, the cells were treated with 8 μM AER-270, 300 μM TGN020 and 50 μM TMZ respectively, and cell morphology and total cell area in the field of view were recorded at the logarithmic proliferation phase. The difference in cell proliferation inhibition rate by different drugs was statistically analyzed.

[0048] The experimental results are shown in Table 1, and the efficacy of AER-270 and TMZ, the only clinically proven effective drug, was compared: AER-270 inhibited glioma cell proliferation faster and had a higher inhibition rate. Figure 4

[0049] Example 5: Effect of AER-270 on the expression level of brain glioma proliferation marker Ki67 compared with TMZ

[0050] After U87mg cells were treated with different drugs, AER-270 (8 μM) and traditional anticancer drug TMZ (100 μM) in vitro, the cell samples were fixed with 4% PFA at room temperature for 1 hour, and then subjected to Ki67 immunofluorescence experiment. Fluorescence specifically labeled proliferating cells. DAPI was used to label all cell numbers. Finally, the Ki67 positive rate of different drug groups was obtained.

[0051] The experimental results are shown in Table 1, and the efficacy of AER-270 and TMZ, the only clinically proven effective drug, was compared: AER-270 inhibited glioma cell proliferation faster and had a higher inhibition rate. Figure 5

[0052] ​​​Example 6: Occurrence of dead tumor cells in AER-270 treated glioma compared to TMZ

[0053] 1) Collection of patient specimens: Glioma tissue was harvested and stored in tissue preservation solution at 4°C. After transportation to the laboratory, the tissue was isolated under a stereomicroscope in a laminar flow hood. The resected tumor was cut into small pieces of approximately 0.5-1 mm in diameter using microscissors and rinsed with culture medium to remove necrotic tissue and cell debris.

[0054] 2) In vitro culture of glioma organoids: The cut pieces of glioma tissue were seeded in 6-well plates with 4 ml culture medium per well. The culture medium was prepared as follows: 50% DMEM:F12, 50% Neurobasal, 1x Glutamax, 1x NeaaS, 1x Penstrep, 1x N2 supplement. 1x 2-mercaptoethanol and 2.5 mg / ml human insulin per well were placed on a shaker rotating at 120 rpm. The incubator was set to 37°C, 5% CO2 and 90% humidity. The medium was changed by tilting every 48 hours by 75%. Shedding tumor cells and other cell debris were observed within the first week of culture and changed in time when the medium became turbid.

[0055] 3) AER-270 sensitivity test: Successfully cultured and identified glioma organoids were seeded in 96-well plates and inhibited with TMZ (50 mM) and AER-270 (8 mM), respectively. Cell viability was measured in the drug selection plates after 48 hours.

[0056] The results are shown in Figure 6 AER-270 resulted in the most dead tumor cells in glioma compared to TMZ.

[0057] Example 7: Inhibition of TMZ treatment resistant recurrent glioma cells by AER-270

[0058] In vitro treatment of U87 MG cells with TMZ and TMZ+AER-270: TMZ+AER-270 means that TMZ was used for the first 7 days (148h) and AER-270 was used instead of TMZ after the seventh day. Cell numbers were recorded during the logarithmic proliferation phase and the differences in cell proliferation inhibition by the different drugs were statistically analyzed. Cell morphology and total cell area were recorded daily for samples of the three groups (control, TMZ and TMZ+AER-270).

[0059] The results are shown in Figure 7 AER-270 still strongly inhibited TMZ treatment resistant recurrent glioma cells.

[0060] Example 8: Inhibition of brain glioma cell proliferation in animal models by AER-271

[0061] After 6-week-old male athymic mice were anesthetized, 1x10 6 U87-Luc cells were injected into the right striatum (1.8 mm lateral, 1 mm longitudinal and 2.5 mm deep), and the model was successfully constructed after 10 days of in situ implantation, and the model mice were obtained. The model mice were divided into control group and drug administration group, 6 mice in each group. The model mice in the drug administration group were treated with AER-271 (10 mg / kg) at the same time every day for 7 consecutive days. The control group was injected with normal saline according to the same procedure as the drug administration group. D-luciferin (150 mg / kg) was injected intraperitoneally on days 10, 20 and 30, and the tumor bioluminescence intensity was recorded 10 minutes later. The survival status of the model mice was observed daily.

[0062] The experimental results are shown in Figure 8 AER-271 has a good therapeutic effect on in situ brain glioma model mice, and the tumor volume is significantly reduced after treatment, and the survival time is significantly prolonged.

[0063] Example 9: Non-invasive evaluation of AER-270 on AQP4 inhibition efficacy using brain glioma AQP4 magnetic resonance imaging technology.

[0064] After in vitro treatment of U87mg cells with different drugs AER-270 (8 μM), traditional anticancer drug TMZ (100 μM) and traditional AQP4 inhibitor TGN020 (6.1 μM), cell samples were obtained, and cell suspensions were obtained after trypsin digestion. After cell counting, the final concentration of the contrast agent was 5 mM, and after mixing, centrifugation was performed at a speed of 150 rcf for 5 min. After centrifugation, the supernatant was replaced twice with 200 μl of PBS containing 5 mM contrast agent (PBS5). Then the cell pellet sample was transferred to a nuclear magnetic tube specially designed for cell culture magnetic resonance, and centrifuged at 300 rcf for 2 min. After centrifugation, the supernatant was replaced twice with 200 μl of PBS5; finally, it was transferred to MRI scanning, and the extracellular environment was maintained at 95% CO2+5% O2 until the end of magnetic resonance measurement. The water molecule exchange rate constant (k io ) was obtained by model fitting at the end of magnetic resonance measurement.

[0065] The experimental results are shown in Figure 9 The growth inhibition rate and k io inhibition rate trend are the same: AER270 > TGN020 > TMZ. This indicates that k io is a non-invasive monitoring magnetic resonance technology for accurately quantifying the effect of AER-270 on the treatment of brain glioma.

[0066] Further description of the present application: AER-271 has successfully completed the clinical phase I test, and is now in the clinical phase II test for cerebral infarction, and has no obvious toxic side effects on human body.

[0067] In summary, through the above examples, the water channel protein 4 (AQP4) inhibitor AER-270, and any other form of derivative drug that is metabolized into AER270 after entering the body (e.g. AER-271), can effectively and low-toxicity inhibit glioma tumor cell proliferation; and the water exchange magnetic resonance AQP4 quantitative imaging technology can realize non-invasive monitoring of the efficacy of AER-270 on AQP4 inhibition of glioma, and precise screening and prediction of the benefit population.

[0068] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. Use of an inhibitor of the transmembrane water exchange protein AQP4 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against brain glioma, characterized in that, The AQP4 inhibitor is AER-270.

2. Use according to claim 1, characterized in that, Measuring the water molecule efflux rate k across the cell membrane of a glioma by magnetic resonance MRI imaging io to non-invasively monitor the therapeutic effect of AQP4 inhibitors.

3. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of brain glioma, characterized in that, The pharmaceutical composition comprises the AQP4 inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, and a pharmaceutically acceptable carrier.

4. Use of a prodrug of an inhibitor of the transmembrane water exchange protein AQP4 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against brain glioma, characterized in that, The prodrug of the AQP4 inhibitor is phosphate precursor AER-271.

5. Use according to claim 4, characterized in that, Measuring the water molecule efflux rate k across the cell membrane of a glioma by magnetic resonance MRI imaging io to non-invasively monitor the therapeutic effect of AQP4 inhibitors.

6. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of brain glioma, characterized in that, The pharmaceutical composition comprises the prodrug of the AQP4 inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 4-5, and a pharmaceutically acceptable carrier.

Images