Use of SN38 and AZ20 in the preparation of drugs for treating brainstem glioma

Abstract

The present application provides the use of SN38 and AZ20 in the preparation of a medicament for treating brainstem glioma. The experiments of the present application have demonstrated that the combination of the two effectively inhibits the proliferation of TP53 mutant brainstem glioma cell lines at the cellular level, has obvious targeting characteristics, and does not affect the activity of normal brainstem stem cells; at the animal model level, intraperitoneal administration combined with intrathecal injection can effectively inhibit the tumor growth of mice with orthotopic brainstem tumors and prolong the survival period. It provides a new treatment option for the treatment of TP53 mutant brainstem glioma.

Description

Field of the Invention

[0001] This application belongs to the field of tumor treatment. Specifically, this application provides the use of SN38 and AZ20 in the preparation of drugs for treating brainstem glioma. Background Art

[0002] Brainstem glioma is the most common type of brainstem tumor, especially diffuse midline glioma carrying the H3K27M mutation. The overall survival of current patients receiving radiotherapy and surgery is only 10 - 12 months, and the overall prognosis is extremely poor. In addition to the H3K27M mutation, the presence of the TP53 mutation also significantly affects tumor characteristics. The TP53 mutation is the most common mutation form co - occurring with H3K27M in diffuse midline glioma, and patients with the co - occurring TP53 mutation have a worse prognosis. Although there have been multiple studies on drug screening for brainstem glioma, the efficacy has not yet been clearly associated with specific mutation subtypes (such as TP53). Therefore, there is an urgent need to explore effective treatment regimens for the TP53 mutation subtype to extend the survival time of patients and relieve the pain of their families.

[0003] The TP53 pathway affects the proliferation and apoptosis of tumor cells, leading to tumor development and deterioration. The p53 protein encoded by the TP53 gene is an important tumor suppressor factor, participating in cell cycle regulation, DNA repair, and induction of apoptosis; after mutation, the function of p53 is impaired, and it cannot effectively control the growth of abnormal cells, thus accelerating tumor progression. Topoisomerase I assists in the repair of DNA single - strand damage. We have previously demonstrated that topoisomerase I inhibitors can inhibit DNA single - strand repair, thereby causing DNA damage to activate the TP53 signaling pathway and induce apoptosis. However, brainstem glioma with the TP53 mutation will repair DNA damage through the ATR bypass, escaping apoptosis induced by TP53 activation, and thus rendering the treatment ineffective. Therefore, the combined use of appropriate topoisomerase I inhibitors and ATR inhibitors may be a potential treatment method. Summary of the Invention

[0004] To achieve the above - mentioned object, the present invention provides the use of SN38 and AZ20 in the preparation of drugs for treating brainstem glioma.

[0005] On the other hand, this application provides a drug for treating brainstem glioma, which contains SN38 and AZ20 as active ingredients.

[0006] Further, the brainstem glioma is a TP53 - mutant brainstem glioma.

[0007] Further, the brainstem glioma is a brainstem glioma with co - occurring H3K27M mutation and TP53 mutation.

[0008] Further, the drug is an injection.

[0009] Furthermore, the drug is an intravenous injection or an intrathecal injection.

[0010] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0011] Furthermore, the solvent of the drug is 0.9% v / w sodium chloride solution or 5% v / w glucose solution.

[0012] Furthermore, when SN38 is used for intravenous injection, the equivalent dose used is 30 mg / m 2 ; when the AZ20 compound is used for intravenous injection, the equivalent dose used is 25 mg / m 2 ; when the SN38 compound is used for intrathecal injection, the dosage range used is 150 μg to 600 μg.

[0013] Furthermore, the concentration of SN38 in the drug is 40 mg / L to 120 mg / L (the solvent is 0.9% v / w sodium chloride solution), and the concentration of AZ20 is 50 mg / L (the solvent is 0.9% v / w sodium chloride solution).

[0014] In the drug, SN38 and AZ20 should exist independently in different solutions or be a solid mixture; SN38 and AZ20 can be placed in the same package or in different packages as independent solutions or solid forms, and used in combination according to the instructions.

[0015] The structure of the SN38 compound is shown in Formula (1), and the CAS number is: 86639-52-3.

[0016]

[0017] The structure of the AZ20 compound is shown in Formula (2), and the CAS number is: 1233339-22-4.

[0018]

[0019] By using the SN38 compound and the AZ20 compound in combination, the present invention effectively inhibits the proliferation of TP53 mutant brainstem glioma cell lines at the cellular level, has obvious targeting characteristics, and does not affect the activity of normal brainstem stem cells; at the animal model level, intraperitoneal administration combined with intrathecal injection can effectively inhibit the tumor growth of mice with orthotopic brainstem tumors and prolong the survival period. Therefore, the present invention provides a new treatment plan for the treatment of TP53 mutant brainstem glioma. Brief Description of the Drawings

[0020] Figure 1 Results of drug screening for ATR pathway inhibitors conducted in vitro

[0021] Figure 2 Results of relative cell viability in in vitro screening of ATR pathway inhibitor drugs;

[0022] Figure 3 Is the half - inhibitory concentration curve of the AZ20 compound (unit: uM), showing the sensitivity of the brainstem glioma cell line measured in vitro to the AZ20 compound;

[0023] Figure 4 Is the proliferation curve of the brainstem glioma cell line, showing that the SN38 compound effectively inhibits the proliferation of the brainstem glioma cell line (NC: control; Combine: combined administration);

[0024] Figure 5 Is the proliferation curve of the brainstem glioma cell line, showing that combined administration does not increase the efficacy of brainstem gliomas with retained TP53 function (NC: control; Combine: combined administration);

[0025] Figure 6 Is the proliferation curve of the brainstem glioma cell line, showing that combined administration does not increase the efficacy of wild - type TP53 brainstem gliomas (NC: control; Combine: combined administration);

[0026] Figure 7 Is the BLISS model diagram of combined administration of TP53 - mutant brainstem gliomas;

[0027] Figure 8 Is the drug synergy index CI value;

[0028] Figure 9 Is the BLISS model diagram of combined administration of wild - type TP53 brainstem gliomas;

[0029] Figure 10 Is the BLISS model diagram of combined administration of brainstem gliomas with retained TP53 function;

[0030] Figure 11 Shows the growth of tumor fluorescence intensity after administration of the in - vivo animal brainstem orthotopic tumorigenesis model of the present invention;

[0031] Figure 12 Shows the survival period of the in - vivo animal brainstem orthotopic tumorigenesis model of the present invention after administration. Detailed implementation methods

[0032] Example 1

[0033] The experimental process is as follows:

[0034] 1) Prepare a 10 mM stock solution of SN38 compound (MCE, HY-13704) with DMSO and store it at -20 °C; prepare a 50 mM stock solution of AZ20 compound (MCE, HY-15557) with DMSO and store it at -20 °C.

[0035] 2) Culture of primary brainstem glioma cell lines and normal human brainstem stem cells: The primary cell lines derived from patient tumor tissues (obtained by puncture biopsy or surgical resection) are cultured using the primary brainstem glioma cell line medium via the traditional primary cell line culture method. Before passage, the culture flask needs to be coated with 1:1000 diluted Matrigel (Corning, 356234) at 37 °C overnight. Among them, the basal medium of the primary medium is DMEM high-glucose medium (Gibco, 11995073), and it also includes: 20 ng / mL EGF cytokine (Saintu SC102), 20 ng / mL bFGF cytokine (Saintu SC107-10ug), 100 U / mL penicillin and 100 ug / mL streptomycin (100×, Thermo Fisher 10378016), 1×N2 (50×, Saintu S60314017A), 1×B27 (50×, Saintu S60314015A), and 1×ITS (100×, Saintu SC25800). After culturing and passing to the 10th generation, the successful establishment of the cell line is confirmed. The H3K27M and TP53 mutation conditions of the brainstem glioma cell line obtained by whole exome sequencing are as Figure 1 shown.

[0036] The culture method and medium of normal human brainstem stem cells derived from the posterior brain tissue of aborted fetuses are the same as those of the primary brainstem glioma cell line.

[0037] Table 1 H3K27M and TP53 mutation conditions of brainstem glioma cell lines

[0038]

[0039]

[0040] 3) Seed TT190326 cells in logarithmic growth phase at 2000 cells / well in a 384-well plate and culture overnight: ① Use the Echo Sonar automated drug addition system to dispense drugs from the ATR pathway inhibitor drug compound library (MCE) in vitro and add them to the cell culture medium to make the final concentration of each drug 1 μmol / L; ② First, use the Echo Sonar automated drug addition system to dispense the SN38 compound and add it to the cell culture medium to make the final drug concentration 10 nmol / L, then use the Echo Sonar automated drug addition system to dispense drugs from the ATR pathway inhibitor drug compound library (MCE) and add them to the cell culture medium to make the final concentration of each drug 1 μmol / L; After continued culture for 72 h, use the Cell-titer method (Promega, ) Measure the fluorescence intensity with a microplate reader, and calculate the cell viability through the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. The drug screening results are as Figure 1 , 2 shown, showing that the ATR inhibitor AZ20 has a significant killing effect on TP53 mutant brainstem glioma, and the combination with the SN38 compound has the best effect, and at the same time does not kill normal brainstem stem cells.

[0041] Example 2

[0042] 1) Select the primary brainstem glioma cell lines in Table 1, seed the cells in logarithmic growth phase at 3000 cells / well in a 96-well plate and culture overnight. Dilute the 10 mmol / L AZ20 compound stock solution in vitro at a 1:3 gradient, dispense the drug and add it to the cell culture medium to make the final drug concentration in each well 50 μmol / L, 16.67 μmol / L, 5.56 μmol / L, 1.85 μmol / L, 0.62 μmol / L, 0.21 μmol / L, 0.068 μmol / L, 0.023 μmol / L, 0.0076 μmol / L, 0 μmol / L. After continued culture for 72 h, use the Cell-titer method (Promega, ) Measure the fluorescence intensity with a microplate reader, and calculate the cell viability through the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Use the Graphpad Prism 9 software to fit and plot to obtain the average half-maximal inhibitory concentration (IC50) of the AZ20 compound for brainstem glioma as 1 μmol / L, as Figure 3 shown, among which the IC50 for TP53 mutant brainstem glioma is the lowest, and there is no significant effect on normal brainstem stem cells (PPC).

[0043] 2) Select the primary cell line of brainstem glioma in Table 1. Seed the logarithmically growing TT190326 brainstem glioma cells at 5000 cells / well into a 96-well plate and culture overnight. Add the SN38 compound and the AZ20 compound jointly to the cell culture medium so that the final concentration of each drug in each well is 10 nmol / L. Continue the culture for 72 h. At four time points of 0 h, 24 h, 48 h, and 72 h, use the Cell-titer method (Promega, ) to measure the fluorescence intensity under an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell viability through the formula: relative cell viability (%) = fluorescence intensity at a certain culture time / fluorescence intensity at 0 h × 100%. And use the Graphpad Prism 9 software to draw a graph as Figure 4 shown. It shows that the combined drug use has a significant ability to inhibit the proliferation of TP53 mutant brainstem glioma at low concentrations.

[0044] Comparative Example 1

[0045] According to the experimental method in 2) of Example 2, apply the same dose combination of the SN38 compound and the AZ20 compound as that on TT190326 to DIPG17 (combined with H3K27M mutation and TP53 mutation, but the TP53 mutation in this cell does not cause the TP53 pathway to lose responsiveness). Its proliferation curve is as Figure 5 shown. It can be seen that the combined drug administration cannot increase the therapeutic effect on brainstem glioma with retained TP53 function.

[0046] Comparative Example 2

[0047] According to the experimental method in 2) of Example 2, apply the same dose combination of the SN38 compound and the AZ20 compound as that on TT190326 to TT150714 (combined with H3K27M mutation, TP53 wild type). Its proliferation curve is as Figure 6 shown. It can be seen that the combined drug administration cannot increase the therapeutic effect on brainstem glioma with wild-type TP53.

[0048] Example 3

[0049] Select the primary cell line of brainstem glioma in Table 1. Seed the logarithmically growing TT190326 brainstem glioma cells at 3000 cells / well into a 96-well plate and culture overnight. Take the SN38 compound and dilute it in a gradient of 1:10 from 10 μmol / L and add the drug column by column; take the AZ20 compound and dilute it in a gradient of 1:3 from 50 μmol / L and add the drug row by row, so that the wells with a concentration of 0 for both drugs coincide. The concentrations of the SN38 compound from top to bottom in each column are: 10 μmol / L, 0 μmol / L, 0.1 μmol / L, 0.01 μmol / L, 0.001 μmol / L, 0 μmol / L. The concentrations of the AZ20 compound from left to right in each row are: 50 μmol / L, 16.67 μmol / L, 5.56 μmol / L, 1.85 μmol / L, 0.62 μmol / L, 0.21 μmol / L, 0.068 μmol / L, 0.023 μmol / L, 0.0076 μmol / L, 0 μmol / L. Continue to culture for 72 h, use the Cell-titer method (Promega, ) to measure the fluorescence intensity under a microplate reader, and calculate the cell viability through the formula: relative cell viability (%) = fluorescence intensity of a certain drug-administered well / fluorescence intensity of the 0-concentration well × 100%. And use Synergy Finder 3.0 (https: / / synergyfinder.fimm.fi) to calculate the BLISS model as shown in Figure 7 , where the area with a score greater than 10 points is the area of obvious synergistic effect. Figure 8 It shows that the combination of drugs has the ability to synergistically inhibit the proliferation of TP53-mutant brainstem glioma under a wide range of concentration combinations.

[0050] Comparative Example 3

[0051] Seed the logarithmically growing TT150714 brainstem glioma cells at 3000 cells / well into a 96-well plate and culture overnight. Take the SN38 compound and the AZ20 compound and dilute them in a gradient and add the drugs according to the method in Example 3. Continue to culture for 72 h, and the analysis method is the same as that in Example 3. The BLISS model is as shown in Figure 9 , showing that the combination of drugs does not have a synergistic effect on TP53 wild-type brainstem glioma under a wide range of concentration combinations.

[0052] Comparative Example 4

[0053] Seed the logarithmically growing DIPG17 brainstem glioma cells at 3000 cells / well into a 96-well plate and culture overnight. Take the SN38 compound and the AZ20 compound and dilute them in a gradient and add the drugs according to the method in Example 3. Continue to culture for 72 h, and the analysis method is the same as that in Example 3. The BLISS model is as shown in Figure 10As shown, it shows that the combination of drugs does not have a synergistic effect on brainstem gliomas with TP53 mutations but retained function under a wide range of concentration combinations.

[0054] Example 4

[0055] Six- to eight-week-old female Balb / c nude mice were selected for orthotopic implantation of primary brainstem glioma cell lines (TT190326) with TP53 mutations: The primary brainstem glioma cell lines were prepared and slowly and uniformly injected stereotactically at a rate of 100,000 cells / 5 μL into the mouse brainstem position (positioned approximately 1 mm posterior to the intersection of the lambda suture and approximately 1 mm off the midline of the mouse skull) within 2 minutes. One week after inoculation, the status of the mice was observed and the fluorescence value of orthotopic tumor formation in the brainstem was measured using an in vivo fluorescence imager (IVIS Lumina Series III). The tumor-bearing mice were selected and evenly divided into four groups according to the fluorescence value of orthotopic tumor formation, namely the control group, the SN38 single-drug administration group, the AZ20 single-drug administration group, and the combination drug treatment group.

[0056] SN38 compound treatment group: The SN38 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of the SN38 compound was adjusted to 400 nmol / L. Stereotactic injection into the lateral ventricle of the mouse was performed every 4 days to simulate intrathecal injection, and a total of 5 μL of freshly prepared SN38 solution was injected each time (drug concentration in cerebrospinal fluid: 10 nmol / L).

[0057] AZ20 compound treatment group: The AZ20 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of the AZ20 compound was adjusted to 1 mg / mL. 200 μL of freshly prepared AZ20 solution was injected intraperitoneally each time (dosage: 10 mg / kg).

[0058] Combined treatment group: The SN38 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of the SN38 compound was adjusted to 400 nmol / L. Stereotactic injection into the lateral ventricle of the mouse was performed every 4 days to simulate intrathecal injection, and a total of 5 μL of freshly prepared SN38 solution was injected each time (drug concentration in cerebrospinal fluid: 10 nmol / L). The AZ20 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of the AZ20 compound was adjusted to 1 mg / mL. 200 μL of freshly prepared AZ20 solution was injected intraperitoneally each time (dosage: 10 mg / kg).

[0059] Control group: 5 μL of 0.9% (v / w) sodium chloride solution was injected intrathecally each time, and 200 μL of 0.9% (v / w) sodium chloride solution was injected intraperitoneally.

[0060] Four groups were administered simultaneously, with each administration once every 4 days as a cycle, and a total of 4 cycles were administered. On the first day of each cycle, the SN38 compound was administered, and on the next day, the AZ20 compound was administered. The body weight of the mice was measured every three days to track the toxic and side effects of the drugs. During the drug administration period, the body weight of the mice did not show a significant decrease, and no mouse death caused by the drugs occurred; the fluorescence value of in-situ tumorigenesis in the brainstem was measured once a week during the drug administration period, and the fluorescence intensity was recorded; the overall survival period of the four groups of mice was tracked, and the total tracking duration was 8 weeks. After the tracking ended, the mice that did not die were euthanized.

[0061] The changes in fluorescence intensity are as Figure 11 shown, and the survival curve is as Figure 12 shown. It can be seen that the combination of SN38 and AZ20 compounds significantly inhibited the tumor growth in the in-situ tumorigenesis model of TP53 mutant in vivo, prolonged the survival period, and after combination drug treatment, the TP53 mutant brainstem glioma had a significantly better potential prognosis.

Claims

1. Use of SN38 and AZ20 in the preparation of a drug for treating brainstem glioma; the brainstem glioma is a brainstem glioma combined with H3K27M mutation and TP53 mutation, and the brainstem glioma has a P53 pathway defect.

2. The use according to claim 1, wherein the medicine is an injection.

3. The use according to claim 2, wherein the medicine is an intravenous injection or an intrathecal injection.

4. The use according to claim 1, wherein the medicine further comprises a pharmaceutically acceptable excipient.

5. A drug for treating brainstem glioma, the drug comprising SN38 and AZ20 as active ingredients, the brainstem glioma is a brainstem glioma with combined H3K27M mutation and TP53 mutation, and the brainstem glioma has a P53 pathway defect. The use according to claim 5, wherein the medicine is an injection.

7. The use according to claim 6, wherein the medicine is an intravenous injection or an intrathecal injection.

8. The use according to claim 5, wherein the medicine further comprises a pharmaceutically acceptable excipient.

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