Application of aurora kinase B inhibitor in preparation of medicine for treating brainstem glioma

Abstract

The invention provides an application of an aurora kinase B inhibitor for preparing a medicine for treating brain stem glioma. In-vitro experiments of various cell lines and actual animal model verification prove that the aurora kinase B inhibitor, especially Barsertib-HQPA, has a good treatment effect on the brain stem glioma, especially the brain stem glioma combining H3F3A mutation and RELN mutation.

Description

Field of the invention:

[0001] The present application belongs to the field of tumor treatment. Specifically, the present application provides the use of an Aurora kinase B inhibitor for preparing a drug for treating brainstem glioma. Background technology:

[0002] Brainstem glioma is the most common type of brainstem tumor, especially diffuse midline glioma carrying H3K27M mutation. The current overall survival of patients receiving radiotherapy and surgery is only 10-12 months, and the overall prognosis is extremely poor. Although H3K27M mutant brainstem glioma has been clearly defined as an independent molecular subtype (WHO grade IV), the dilemma of lack of therapeutic targets has not been alleviated - except for epigenetic regulation related to H3K27M, no other effective targeting strategies have been found. Through whole-exome sequencing, our research team discovered for the first time the presence of in-frame deletion mutations in the RELN gene in tumor cell lines derived from brainstem glioma patients. Although the mutation has not been reported in the literature, it is co-expressed with H3K27M, suggesting that the two may synergistically drive the malignant progression of the tumor. Therefore, there is an urgent need to explore new targeted treatment options.

[0003] Reelin, encoded by the RELN gene, is a core regulator of central nervous system development and function. During neural development, Reelin regulates neuronal migration and positioning, and maintains normal cellular morphology and function in the adult brain. Its mechanism of action relies on the Reelin-Dab1-PI3K signaling cascade: Reelin binds to the ApoER2 / VLDLR receptor, inducing phosphorylation of the adaptor protein Dab1, which in turn activates the PI3K / AKT pathway. This, in turn, inhibits glycogen synthase (GSK3β) and reduces Tau phosphorylation, thereby stabilizing the microtubule network and promoting dendritic development. Recent studies have revealed a potential link between Reelin's epigenetic regulation and global hypomethylation caused by H3K27M mutations. Loss of H3K27me3 may relieve RELN gene silencing, leading to Reelin overexpression, which in turn aberrantly activates the PI3K / AKT pathway and promotes tumor cell invasion.

[0004] In H3K27M-mutant brainstem gliomas, the expression levels of Aurora A / B kinases are significantly higher than in normal tissues. Aurora-B regulates chromosome alignment and cytokinesis by binding to microtubules, and its abnormally high expression is closely associated with a poor prognosis in malignant tumors. Current research on Aurora kinase inhibitors is significantly biased: inhibitors targeting Aurora-A (such as Alisertib) are well developed, while inhibitors targeting Aurora-B (such as Barasertib) are relatively scarce. Although studies have shown that Aurora-B inhibitors can induce G2 / M arrest in high-grade pediatric astrocytomas, the potential targeting mechanism of Aurora-B through non-canonical pathways (such as RELN) has not been reported due to limited sample availability and the lack of systematic drug screening processes. In recent years, although the academic community has begun to use combination strategies (such as AURKA + PLK1 inhibitors) to enhance therapeutic efficacy, the mechanism of action and applicable population remain unclear due to the limited cell models used and the lack of mutation typing association analysis. In addition, the reported median effective dose of Aurora kinase inhibitors is generally high (all exceeding 1μM), which may cause serious toxic side effects and limit clinical translation. Therefore, the present invention is dedicated to the development of Aurora kinase B inhibitors with the significance of targeting brainstem glioma mutations for therapeutic use, in order to achieve clearer and more significant therapeutic effects for clinical translation. Summary of the invention:

[0005] The present invention proposes the use of the Aurora kinase B inhibitor Barasertib-HQPA in the treatment of brainstem gliomas carrying RELN mutations. Through in vitro drug screening experiments, the present invention evaluated the effects of various Aurora kinase inhibitors on brainstem glioma cell lines. The results showed that this class of inhibitors had a significant killing effect on brainstem glioma cells with RELN-H3K27M co-mutations, and Aurora kinase B inhibitors were more effective than Aurora kinase A inhibitors. Barasertib-HQPA exhibited the best anti-tumor activity. At the cellular level, Barasertib-HQPA effectively inhibited the proliferation of RELN-H3K27M co-mutated brainstem glioma cells, showing a clear dose-dependent and targeted nature, while having no significant effect on the activity of normal brainstem stem cells. In an animal orthotopic brainstem tumor model, intraperitoneal administration effectively inhibited tumor growth and significantly prolonged the survival of tumor-bearing mice. Therefore, the present invention provides a novel treatment strategy for RELN-H3K27M co-mutated brainstem gliomas through the use of Barasertib-HQPA.

[0006] In one aspect, the present application provides the use of an Aurora kinase B inhibitor for preparing a medicament for treating brainstem glioma.

[0007] Furthermore, the Aurora B inhibitor is Barasertib-HQPA.

[0008] Furthermore, the brainstem glioma is a brainstem glioma with RELN mutation.

[0009] Furthermore, the brainstem glioma is a brainstem glioma combined with H3F3A mutation and RELN mutation.

[0010] Furthermore, the RELN mutation is a frameshift deletion mutation.

[0011] Furthermore, the H3F3A mutation is an H3K27M mutation.

[0012] Furthermore, the medicine is an injection.

[0013] Furthermore, the injection is a water injection or a powder injection

[0014] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0015] In addition to injections, other dosage forms such as oral preparations may also be used. Pharmaceutically acceptable excipients include, but are not limited to, solvents, cosolvents, pH adjusters, isotonic agents, antioxidants, preservatives; fillers, binders, sustained-release agents, lubricants, flavoring agents, taste-masking agents, capsule shells, and coating materials. Those skilled in the art can select appropriate excipients or design new formulations based on their pharmaceutical knowledge and experimental methods.

[0016] Furthermore, the solvent in the injection is 0.9% v / w sodium chloride solution or 5% v / w glucose solution.

[0017] The drug of the present application can be administered by intravenous injection, preferably by intravenous drip, with a single administration time of not less than 2 hours. When used for intravenous injection, the equivalent dose range used is 100 mg to 200 mg.

[0018] The Aurora B inhibitor Barasertib-HQPA described in this application has the structural formula shown in Formula I below and the CAS number 722544-51-6. It is also known as AZD1152-HPQA, balacetib-HQPA, etc. These names are used interchangeably and have the same meaning:

[0019]

[0020] Beneficial effects:

[0021] Experiments in this application demonstrate that the Aurora B inhibitor Barasertib-HQPA effectively inhibits the proliferation of RELN-mutant brainstem glioma cell lines at the cellular level, exhibiting clear targeting characteristics without affecting the activity of normal brainstem stem cells. In an animal model, systemic intraperitoneal administration of the drug effectively inhibits tumor growth and prolongs survival in mice with orthotopic brainstem tumors. This invention provides a new treatment option for RELN-mutant brainstem gliomas. Description of the drawings:

[0022] Figure 1 The results of the in vitro screening of RELN pathway inhibitors performed by the present invention (normalized by cell line) are shown. Note: PAN in Targets: B has multiple targets in addition to Aurora kinase; DUAL-AB in Selectivity: Simultaneous inhibition of Aurora kinases A and B; DUAL-BC: Simultaneous inhibition of Aurora kinases B and C;

[0023] PAN: Simultaneously inhibits Aurora kinases A, B, and C.

[0024] Figure 2 The results of the in vitro screening of RELN pathway inhibitors conducted in this invention (normalized by drug). Note: PAN in Targets indicates multiple targets in addition to Aurora kinase; DUAL-AB in Selectivity indicates simultaneous inhibition of Aurora kinases A and B; DUAL-BC indicates simultaneous inhibition of Aurora kinases B and C; PAN indicates simultaneous inhibition of Aurora kinases A, B, and C.

[0025] Figure 3 : is the IC50 curve (Barasertib-HQPA) of the Aurora kinase inhibitor against different brainstem glioma cell lines determined by in vitro experiments of the present invention.

[0026] Figure 4 : is the IC50 curve of the Aurora kinase inhibitors (Alisertib, LY3295668) on different brainstem glioma cell lines determined by in vitro experiments of the present invention.

[0027] Figure 5 1 is the IC50 curve of the Aurora kinase inhibitor on different brainstem glioma cell lines determined by in vitro experiments of the present invention (SP-96).

[0028] Figure 6 These are the results of an EDU proliferation experiment on a brainstem glioma cell line under administration of an Aurora kinase B inhibitor determined in vitro by the present invention.

[0029] Figure 7 This is the growth of tumor fluorescence intensity after drug administration in the in vivo animal brainstem in situ tumor model of the present invention.

[0030] Figure 8 This is the survival period of the in vivo animal brainstem in situ tumor model after administration of the present invention.

[0031] Figure 9 This is the IC50 curve of the RELN knockout cell line constructed in vitro according to the present invention after administration of the Aurora kinase B inhibitor. Specific implementation method:

[0032] Example 1 Basic experimental methods and reagents

[0033] Aurora kinase inhibitors were prepared as 10 mM stock solutions in DMSO and stored at -20°C.

[0034] Culture of primary brainstem glioma cell lines and normal human brainstem stem cells: Primary cell lines derived from patient tumor tissue obtained by biopsy or surgical resection were cultured using brainstem glioma primary cell line culture medium using traditional primary cell line culture methods. Before passaging, the culture flasks were coated with 1:1000 diluted Matrigel (Corning, 356234) at 37°C overnight. The primary culture medium contained the basal medium of DMEM high glucose medium (Gibco, 11995073), as well as 20 ng / mL EGF cytokine (Synthes SC102), 20 ng / mL bFGF cytokine (Ceintu SC107-10 μg), 100 U / mL penicillin and 100 μg / mL streptomycin (100×, Thermo Fisher Scientific 10378016), 1×N2 (50×, Ceintu S60314017A), 1×B27 (50×, Ceintu S60314015A) and 1×ITS (100×, Ceintu SC25800). The successful establishment of the cell line was confirmed after culture and passage to the 10th passage. The H3K27M and RELN mutations of the brainstem glioma cell line obtained by whole exome sequencing are shown in the figure below. Figure 1 The culture method and culture medium for human normal brainstem stem cells derived from aborted fetal hindbrain tissue are the same as those for primary brainstem glioma cell lines.

[0035] Table 1 H3K27M and RELN mutations in brainstem glioma cell lines

[0036]

[0037] Logarithmically growing cells were plated at 2000 cells / well in 384-well plates and cultured overnight. Drugs were taken from the Aurora Kinase Inhibitor Drug Compound Library (MCE) (including ARUKA / AURKB inhibitors) using the Echo Sonar automated dosing system and added to the cell culture medium to a final concentration of 1 μmol / L for each drug. After 72 h of culture, the cells were titrated using the Cell-titer method (Promega, ) The fluorescence intensity was measured under a microplate reader, and the cell viability was calculated by the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Figure 1 (normalized by cell line), Figure 2 As shown (normalized by drug), AURK inhibitors have a significant killing effect on RELN mutant brainstem glioma cell lines, while not killing normal brainstem stem cells. Among them, the selective Aurora kinase B inhibitor Barasertib-HQPA is the most preferred, showing the most significant targeted killing differentiation effect at a single concentration.

[0038] Example 2 Effects of Aurora Kinase Inhibitors on Different Brainstem Glioma Cell Lines

[0039] Logarithmically growing cells were plated at 3000 cells / well in 96-well plates and cultured overnight. 50 mmol / L Barasertib-HQPA compound (MCE, HY-10126) stock solution was diluted in vitro in a 1:3 gradient. The drug was taken and added to the cell culture medium to make the final drug concentration in each well 50 μmol / L, 16.67 μmmol / L, 5.56 μmmol / L, 0.62 μmmol / L, 0.21 μmmol / L, 0.069 μmmol / L, 0.023 μmmol / L, 0.0076 μmmol / L, 0.0025 μmmol / L, and 0 μmmol / L. After further culture for 72 h, the cells were titrated using the Cell-titer method (Promega, ) Fluorescence intensity was measured on a microplate reader, and cell viability was calculated using the formula: Relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Graphpad Prism 9 software was used to fit the plot and show that the average half-maximal inhibitory concentration (IC50) of the Barasertib-HQPA compound for RELN mutant brainstem gliomas was 5 nmol / L, while the average half-maximal inhibitory concentration (IC50) for RELN wild-type brainstem gliomas was approximately 10 μmol / L. The efficacy between different mutation types differed by more than 2000 times, confirming the drug's unique targeted therapeutic characteristics, such as Figure 3 shown.

[0040] 3000 cells / well in logarithmic growth state were plated into 96-well plates and cultured overnight. Alisertib, an inhibitor of Aurora kinase A, and LY3295668 were diluted in a 1:3 gradient in vitro. The drugs were taken and added to the cell culture medium to make the final drug concentrations in each well 50 μmol / L, 16.67 μmmol / L, 5.56 μmmol / L, 0.62 μmmol / L, 0.21 μmmol / L, 0.069 μmmol / L, 0.023 μmmol / L, 0.0076 μmmol / L, 0.0025 μmmol / L, and 0 μmmol / L. After further culture for 72 h, the cells were titrated using the Cell-titer method (Promega, ) The fluorescence intensity was measured on a microplate reader, and the cell viability was calculated using the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Graphpad Prism 9 software was used to fit and plot the IC50 curves of Alisertib and LY3295668 compounds for various brainstem glioma cell lines. It can be seen that compared with the Aurora kinase B inhibitor Barasertib-HQPA, the Aurora kinase A inhibitor has a poorer effect on the discrimination and killing of RELN mutations, and can also kill other types of brainstem glioma cells at lower concentrations (such as 150728), confirming the advantages of the Aurora kinase B inhibitor Barasertib-HQPA in targeted therapy. Figure 4 shown.

[0041] Logarithmically growing cells were plated at 3000 cells / well in 96-well plates and cultured overnight. 50 mmol / L Aurora kinase B inhibitor SP-96 compound stock solution was diluted in vitro in a 1:3 gradient. The drug was taken and added to the cell culture medium to make the final drug concentration in each well 50 μmol / L, 16.67 μmmol / L, 5.56 μmmol / L, 0.62 μmmol / L, 0.21 μmmol / L, 0.069 μmmol / L, 0.023 μmmol / L, 0.0076 μmmol / L, 0.0025 μmmol / L, and 0 μmmol / L. After further culture for 72 h, the cells were titrated using the Cell-titer method (Promega, ) Fluorescence intensity was measured on a microplate reader, and cell viability was calculated using the formula: Relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Graphpad Prism 9 software was used to fit and plot the IC50 curves of the SP-96 compound against various brainstem glioma cell lines. Compared to the Aurora kinase B inhibitor Barasertib-HQPA, SP-96, also an Aurora kinase B inhibitor, maintained its ability to discriminate against RELN mutations, but its IC50 was slightly higher than that of Barasertib-HQPA, confirming that Barasertib-HQPA is a more preferred Aurora kinase B inhibitor. Figure 5 shown.

[0042] Example 3 EDU proliferation experiment

[0043] An appropriate number of brainstem glioma cells were cultured in 6-well plates. After overnight culture, the AURKB inhibitor Barasertib-HQPA (DMSO negative control, with a dosing gradient of IC50 and 2x IC50) was added. A 2X EdU working solution was prepared, preheated to 37°C, and added to the 6-well plate in equal volumes to achieve a final EdU concentration of 1X. The cells were incubated until the cell cycle reached 1 / 10, approximately 3-4 hours. After EdU labeling, the culture medium was removed and 1 ml of fixative solution was added for 15 minutes. The fixative solution was removed and the cells were washed three times with 1 ml of wash solution per well for 3-5 minutes each. The wash solution was removed and the cells were incubated with 1 ml of permeabilization solution (PBS containing 0.3% Triton X-100) per well at room temperature for 10-15 minutes. The permeabilization solution was removed and the cells were washed one to two times with 1 ml of wash solution per well for 3-5 minutes each. The Click Additive Solution and Click Reaction Solution were prepared. The sample must be used within 15 minutes of preparation. Add 0.5 ml of Click reaction solution to each well and gently shake the culture plate to ensure that the reaction mixture evenly covers the sample. Incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes. Stain the cell nuclei (Biyuntian, C0071S). Finally, develop the image using a fluorescence microscope. The results are as follows: Figure 6 It can be seen that the AURKB inhibitor Barasertib-HQPA significantly inhibited the proliferation of RELN mutant brainstem glioma cells (190326) and killed tumor cells, but did not affect other types of brainstem glioma cells and normal control cells PPC.

[0044] Example 4 Animal brainstem in situ tumor model experiment

[0045] The experiment used female BALB / c nude mice aged 6-8 weeks to establish a brainstem orthotopic tumor model using the RELN mutant brainstem glioma primary cell line (190326). The specific method is as follows: a brainstem glioma primary cell suspension (100,000 cells / 5μL) was prepared and stereotactically injected into the mouse brainstem within 2 minutes, with the injection rate maintained uniformly and slowly. The injection site was located approximately 1mm posterior to the intersection of the lambdoid suture and approximately 1mm lateral to the midline of the mouse skull. One week after inoculation, the general condition of the mice was observed, and the fluorescence signal intensity of the brainstem orthotopic tumor was detected using an in vivo fluorescence imager (IVIS Lumina Series III). Subsequently, the tumor-bearing mice were randomly divided into two groups based on the size of the fluorescence signal: a control group and a Barasertib-HQPA treatment group to ensure balanced tumor burden in the two groups.

[0046] Aurora kinase B inhibitor Barasertib-HQPA treatment group: Barasertib-HQPA compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of Barasertib-HQPA compound was adjusted to 2.5 mg / mL. 200 μL of freshly prepared Barasertib-HQPA solution was injected intraperitoneally each time (dosage: 25 mg / kg).

[0047] Control group: 200 μL of 0.9% (v / w) sodium chloride solution was injected intraperitoneally each time.

[0048] Both groups received the drug simultaneously, once weekly, for a total of four weeks. Mice were weighed every three days to monitor drug toxicity and side effects. No significant weight loss was observed during the drug-induced period, and no drug-induced mortality occurred. Fluorescence measurements of brainstem in situ tumors were performed weekly during the drug-induced period, and fluorescence intensity was recorded. Overall survival of both groups was tracked.

[0049] Fluorescence intensity changes as Figure 7 The survival curve is shown as Figure 8 As shown, the Barasertib-HQPA compound significantly inhibited tumor growth in the in vivo RELN mutant orthotopic tumor model and prolonged survival.

[0050] Example 5 Effects on RELN Knockout Cell Lines Constructed in Vitro

[0051] Construction of a RELN-KO gene knockout brainstem glioma cell line (150728H3) transfected with lentivirus K27M / Reln - / - ) and the control supratentorial H3K27 wild-type glioblastoma cell line (U87_MG H3 WT / Reln - / -); Whole-exome sequencing confirmed that the RELN gene underwent a "stop-gained" mutation after knockout, resulting in loss of function.

[0052] Logarithmically growing cells were plated at 3000 cells / well in 96-well plates and cultured overnight. 50 mmol / L Barasertib-HQPA compound (MCE, HY-10126) stock solution was diluted in vitro in a 1:3 gradient. The drug was taken and added to the cell culture medium to make the final drug concentration in each well 50 μmol / L, 16.67 μmmol / L, 5.56 μmmol / L, 0.62 μmmol / L, 0.21 μmmol / L, 0.069 μmmol / L, 0.023 μmmol / L, 0.0076 μmmol / L, 0.0025 μmmol / L, and 0 μmmol / L. After further culture for 72 h, the cells were titrated using the Cell-titer method (Promega, ) Fluorescence intensity was measured on a microplate reader, and cell viability was calculated using the formula: Relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Graphpad Prism 9 software was used to fit the plot and show that the IC50 of the Barasertib-HQPA compound was significantly downregulated for RELN-KO-knockout brainstem gliomas, which was significantly different from the unknockout cell line. However, there was no sensitization phenomenon after RELN-KO in the control supratentorial H3K27 wild-type glioblastoma cell line, confirming the unique targeted therapy of the Barasertib-HQPA compound for RELN-mutant brainstem gliomas. Figure 9 shown.

Claims

1. Use of an Aurora kinase B inhibitor for the preparation of a medicament for treating brainstem glioma.

2. The method according to claim 1, wherein the Aurora B inhibitor is Barasertib-HQPA.

3. The use according to claim 2, wherein the brainstem glioma is a brainstem glioma with RELN mutation.

4. The use according to claim 3, wherein the brainstem glioma is a brainstem glioma combined with H3F3A mutation and RELN mutation.

5. The use according to claim 4, wherein the RELN mutation is a frameshift deletion mutation.

6. The use according to claim 5, wherein the H3F3A mutation is an H3K27M mutation.

7. The use according to any one of claims 1 to 6, wherein the medicine is an injection.

8. The use according to claim 7, wherein the injection is a water injection or a powder injection.

9. The use according to claim 8, wherein the medicine further comprises a pharmaceutically acceptable excipient.

10. The use according to claim 9, wherein the solvent in the injection is 0.9% v / w sodium chloride solution or 5% v / w glucose solution.

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