Application of SN38 and AZ20 in preparation of medicine for treating brainstem glioma

Abstract

The invention provides an application of SN38 and AZ20 in preparation of a medicine for treating brainstem glioma. Experiments prove that the combination of the two can effectively inhibit the proliferation of a TP53 mutant brainstem glioma cell line at the cellular level, has obvious targeting characteristics, and does not influence the activity of normal brainstem stem cells; on the animal model level, through combination of intraperitoneal administration and intrathecal injection, tumor growth of brainstem in-situ tumorigenesis mice can be effectively inhibited, and the life cycle can be prolonged. A new treatment choice is provided for the treatment of the TP53 mutant brainstem glioma.

Description

Field of the Invention

[0001] The present application belongs to the field of tumor treatment. Specifically, the present 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 H3K27M mutation. The current overall survival of patients receiving radiotherapy and surgery is only 10-12 months, and the overall prognosis is extremely poor. In addition to H3K27M mutation, the presence of TP53 mutation also significantly affects tumor characteristics. TP53 mutation is the most commonly combined mutation form of H3K27M in diffuse midline glioma, and patients with combined TP53 mutation have a worse prognosis. Although there are many studies on drug screening for brainstem glioma, it has not yet been possible to clearly associate efficacy with specific mutation subtypes (such as TP53). Therefore, there is an urgent need to explore effective treatment options for TP53 mutation subtypes to prolong patient survival and alleviate the suffering 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 that participates in cell cycle regulation, DNA repair, and induction of apoptosis. After mutation, p53 function is impaired and cannot effectively control the growth of abnormal cells, thereby accelerating tumor progression. Topoisomerase I assists in the repair of DNA single-strand damage. In the past, we have confirmed that topoisomerase I inhibitors can inhibit DNA single-strand repair, thereby causing DNA damage to activate the TP53 signaling pathway and cause cell apoptosis. Brainstem gliomas with TP53 mutations repair DNA damage through the ATR bypass, escaping TP53 activation-guided apoptosis, and thus rendering the treatment ineffective. Therefore, the combination of appropriate topoisomerase I inhibitors and ATR inhibitors may be a potential treatment method. Summary of the invention

[0004] In order to achieve the above object, the present invention provides the use of SN38 and AZ20 in preparing a drug for treating brainstem glioma.

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

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

[0007] Furthermore, the brainstem glioma is a brainstem glioma combined with H3K27M mutation and TP53 mutation.

[0008] Furthermore, the medicine is an injection.

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

[0010] Furthermore, the medicine 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 the 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 is 150ug to 600ug.

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

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

[0016]

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

[0019]

[0020] The present invention effectively inhibits the proliferation of TP53 mutant brainstem glioma cell lines at the cellular level by combining SN38 compound and AZ20 compound, 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 brainstem orthotopic tumor-forming mice 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

[0021] Figure 1 Results of in vitro screening of ATR pathway inhibitors

[0022] Figure 2 The relative cell viability results of the ATR pathway inhibitor drug screening in vitro;

[0023] Figure 3 is the half-maximal inhibitory concentration curve of AZ20 compound (unit: uM), showing the sensitivity of brainstem glioma cell line to AZ20 compound determined by in vitro experiments;

[0024] Figure 4 The proliferation curve of brain stem glioma cell line shows that SN38 compound effectively inhibits the proliferation of brain stem glioma cell line (NC: control; Combine: combined administration);

[0025] Figure 5 This is the proliferation curve of brainstem glioma cell line, showing that combined administration does not increase the efficacy of brainstem glioma with preserved TP53 function (NC: control; Combine: combined administration);

[0026] Figure 6 This is the proliferation curve of brainstem glioma cell line, showing that combined administration does not increase the efficacy of TP53 wild-type brainstem glioma (NC: control; Combine: combined administration);

[0027] Figure 7 This is a diagram of the BLISS model after combined drug administration for TP53 mutant brainstem glioma;

[0028] Figure 8 is the drug synergy index CI value;

[0029] Fig. 9 This is a diagram of the BLISS model after combined drug administration in TP53 wild-type brainstem glioma;

[0030] Fig.10 Diagram of the BLISS model after combined drug administration for brainstem glioma with preserved TP53 function;

[0031] Fig.11 The growth of tumor fluorescence intensity in the in vivo animal brainstem in situ tumor model of the present invention after drug administration;

[0032] Fig.12 This is the survival period of the in vivo animal brainstem orthotopic tumor model after administration of the present invention. DETAILED DESCRIPTION

[0033] Example 1

[0034] The experimental process is as follows:

[0035] 1) The SN38 compound (MCE, HY-13704) was prepared into a 10 mM stock solution with DMSO and stored at -20°C; the AZ20 compound (MCE, HY-15557) was prepared into a 50 mM stock solution with DMSO and stored at -20°C.

[0036] 2) Culture of primary brainstem glioma cell lines and normal human brainstem stem cells: Primary cell lines derived from patient tumor tissues (obtained by biopsy or surgical resection) were cultured using brainstem glioma primary cell line culture medium using traditional primary cell line culture methods. Before subculturing, the culture flasks were coated with 1:1000 diluted Matrigel (Corning, 356234) at 37°C overnight; the basal culture medium of the primary culture medium was DMEM high glucose medium (Gibco, 11995073), which also included: 20 ng / mL EGF cytokine (Syntop SC102), 20 ng / mL bFGF cytokine (Synthet SC107-10ug), 100U / mL penicillin and 100ug / mL streptomycin (100×, Thermo Fisher 10378016), 1×N2 (50×, Synthet S60314017A), 1×B27 (50×, Synthet S60314015A) and 1×ITS (100×, Synthet SC25800). After the 10th generation of culture, the establishment of the line was confirmed to be successful. The H3K27M and TP53 mutations of the brainstem glioma cell line obtained by whole exome sequencing are shown in the figure. Figure 1 shown.

[0037] The culture method and culture medium of normal human brainstem stem cells derived from the posterior brain tissue of aborted fetuses are the same as those of primary brainstem glioma cell lines.

[0038] Table 1 H3K27M and TP53 mutations in brainstem glioma cell lines

[0039]

[0040]

[0041] 3) TT190326 cells in logarithmic growth state were plated at 2000 cells / well in 384-well plates and cultured overnight: ① In vitro, the Echo sonar automatic dosing system was used to take drugs from the ATR pathway inhibitor drug compound library (MCE) and added to the cell culture medium, so that the final concentration of each drug was 1umol / L; ② The SN38 compound was first taken using the Echo sonar automatic dosing system and added to the cell culture medium to make the final concentration of the drug 10nmol / L, and then the Echo sonar automatic dosing system was used to take drugs from the ATR pathway inhibitor drug compound library (MCE) and added to the cell culture medium to make the final concentration of each drug 1umol / L; After continuing to culture for 72 hours, the Cell-titer method (Promega, ) The fluorescence intensity was measured by an ELISA instrument, and the cell viability was calculated by the formula: relative cell viability (%) = fluorescence intensity of the drug-added group / fluorescence intensity of the control group × 100%. Figure 1 , 2 As shown, the ATR inhibitor AZ20 has a significant killing effect on TP53 mutant brainstem gliomas, and the effect is best when used in combination with the SN38 compound, while it does not kill normal brainstem stem cells.

[0042] Example 2

[0043] 1) Primary brainstem glioma cell lines in Table 1 were selected, and 3000 cells in logarithmic growth state were plated into 96-well plates and cultured overnight. 10mmol / L AZ20 compound stock solution was diluted in vitro in a gradient of 1:3, and the drug was taken and added to the cell culture medium to make the final drug concentration in each well 50umol / L, 16.67ummol / L, 5.56ummol / L, 1.85ummol / L, 0.62ummol / L, 0.21ummol / L, 0.068ummol / L, 0.023ummol / L, 0.0076ummol / L, 0ummol / L, and then cultured for 72h and the cell titer method (Promega, ) The fluorescence intensity was measured under an ELISA instrument, 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%. The average half-maximum inhibitory concentration (IC50) of AZ20 compound for brainstem glioma was obtained by fitting and drawing using Graphpad Prism 9 software, which was 1umol / L. Figure 3 As shown, the IC50 was the lowest for TP53 mutant brainstem glioma, while it had no significant effect on normal brainstem stem cells (PPC).

[0044] 2) The primary brainstem glioma cell lines in Table 1 were selected, and TT190326 brainstem glioma cells in logarithmic growth state were plated at 5000 cells / well in a 96-well plate and cultured overnight. The SN38 compound and the AZ20 compound were co-added to the cell culture medium so that the final concentration of the two drugs in each well was 10 nmol / L. The culture was continued for 72 hours, and the Cell-titer method (Promega, ) The fluorescence intensity was measured by an ELISA instrument, and the cell viability was calculated by the formula: relative cell viability (%) = fluorescence intensity at a certain culture time / fluorescence intensity at 0 h × 100%. GraphpadPrism 9 software was used to draw the graph. Figure 4 As shown. It shows that the combination of drugs has the ability to significantly inhibit the proliferation of TP53 mutant brainstem glioma at low concentrations.

[0045] Comparative Example 1

[0046] According to the experimental method of 2) in Example 2, DIPG17 (combined with H3K27M mutation and TP53 mutation, but the TP53 mutation of the cells does not cause TP53 pathway failure) was treated with the same dose of SN38 compound and AZ20 compound as that of TT190326, and the proliferation curve was as follows: Figure 5 As shown, combined administration does not increase the efficacy of brainstem gliomas with preserved TP53 function.

[0047] Comparative Example 2

[0048] According to the experimental method of 2) in Example 2, TT150714 (combined with H3K27M mutation, TP53 wild type) was treated with the same dose of SN38 compound and AZ20 compound combination as TT190326, and its proliferation curve was as follows Figure 6 As shown, combined administration does not increase the efficacy of TP53 wild-type brainstem glioma.

[0049] Example 3

[0050] The primary brainstem glioma cell lines in Table 1 were selected, and TT190326 brainstem glioma cells in logarithmic growth state were plated into 96-well plates at 3000 cells / well and cultured overnight. The SN38 compound was diluted from 10umol / L at a rate of 1:10 and added by column; the AZ20 compound was diluted from 50umol / L at a rate of 1:3 and added by row, so that the wells with 0 drug concentrations overlapped. The concentrations of SN38 compounds from top to bottom in each column are: 10umol / L, 0umol / L, 0.1umol / L, 0.01umol / L, 0.001umol / L, 0umol / L, and the concentrations of AZ20 compounds from left to right in each row are: 50umol / L, 16.67ummol / L, 5.56ummol / L, 1.85ummol / L, 0.62ummol / L, 0.21ummol / L, 0.068ummol / L, 0.023ummol / L, 0.0076ummol / L, 0ummol / L. The culture was continued for 72h and the cells were cultured using the Cell-titer method (Promega, ) The fluorescence intensity was measured by an ELISA instrument, and the cell viability was calculated by the formula: relative cell viability (%) = fluorescence intensity of a certain dosing well / fluorescence intensity of 0 concentration well × 100%. Synergy Finder 3.0 (https: / / synergyfinder.fimm.fi) was used to calculate the BLISS model. Figure 7 As shown, the areas with scores greater than 10 are areas of obvious synergy. Figure 8 The combination therapy was shown to have the ability to synergistically inhibit the proliferation of TP53 mutant brainstem gliomas under a wide range of concentration combinations.

[0051] Comparative Example 3

[0052] TT150714 brainstem glioma cells in logarithmic growth state were plated in 96-well plates at 3000 cells / well and cultured overnight. SN38 compound and AZ20 compound were diluted and added according to the method of Example 3, and cultured for 72 hours. The analysis method was the same as that of Example 3. The BLISS model was as follows: Fig. 9 As shown, the combination therapy showed no synergistic effect on TP53 wild-type brainstem gliomas under a wide range of concentration combinations.

[0053] Comparative Example 4

[0054] DIPG17 brainstem glioma cells in logarithmic growth state were plated in 96-well plates at 3000 cells / well and cultured overnight. SN38 compound and AZ20 compound were diluted and added according to the method of Example 3, and cultured for 72 hours. The analysis method was the same as that of Example 3. The BLISS model was as follows: Fig.10As shown, the combination therapy was not synergistic in a wide range of concentration combinations against brainstem gliomas with TP53 mutations but preserved function.

[0055] Example 4

[0056] Female Balc nude mice aged 6-8 weeks were selected for in situ implantation of TP53 mutant brainstem glioma primary cell line (TT190326) in the brainstem to form tumors: the primary brainstem glioma cell line was prepared and injected into the mouse brainstem at a slow and uniform rate of 100,000 cells / 5ul stereotaxically within 2 minutes (located about 1mm behind the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). The state of the mice was observed 1 week after inoculation, and the fluorescence value of the in situ tumor in the brainstem was measured using an in vivo fluorescence imager (IVIS Lumina Series III). Mice with tumors were selected and divided into four groups according to the fluorescence value of the in situ tumors, namely the control group, SN38 monotherapy group, AZ20 monotherapy group and combined therapy group.

[0057] SN38 compound treatment group: SN38 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of SN38 compound was adjusted to 400 nmol / L. Stereotactic injection was completed into the lateral ventricle of mice 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).

[0058] AZ20 compound treatment group: AZ20 compound was dissolved in 0.9% (v / w) sodium chloride solution, the concentration of AZ20 compound was adjusted to 1 mg / mL, and 200 μL of freshly prepared AZ20 solution was intraperitoneally injected each time (dosage: 10 mg / kg).

[0059] Combined treatment group: SN38 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of SN38 compound was adjusted to 400nmol / L. Stereotactic injection was completed once every 4 days in the lateral ventricle of mice to simulate intrathecal injection, and 5μL of freshly prepared SN38 solution was injected each time (drug concentration in cerebrospinal fluid: 10nmol / L). AZ20 compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of AZ20 compound was adjusted to 1mg / mL. 200μL of freshly prepared AZ20 solution was injected intraperitoneally each time (dosage: 10mg / kg).

[0060] Control group: 5 μl of 0.9% (v / w) sodium chloride solution was injected intrathecally and 200 μl of 0.9% (v / w) sodium chloride solution was injected intraperitoneally each time.

[0061] The four groups were given drugs at the same time, with one cycle being every 4 days, for a total of 4 cycles. The SN38 compound was given on the first day of each cycle, and the AZ20 compound was given on the next day. The mice were weighed every three days to track the toxic and side effects of the drugs. There was no significant weight loss during the drug administration period, and no drug-induced mouse deaths occurred. During the drug administration period, the fluorescence value of the brainstem in situ tumor was measured once a week, and the fluorescence intensity was recorded. The total survival of the four groups of mice was tracked for a total of 8 weeks, and the mice that did not die after the end of the tracking were euthanized.

[0062] Fluorescence intensity changes as Fig.11 The survival curve is shown as Fig.12 As shown, the combination of SN38 and AZ20 compounds significantly inhibited the tumor growth of the in vivo TP53 mutant orthotopic tumor model and prolonged the survival period. After combined drug treatment, TP53 mutant brainstem glioma achieved a significantly better potential prognosis.

Claims

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

2. A drug for treating brainstem glioma, characterized in that: The medicine contains SN38 and AZ20 as active ingredients.

3. The use according to claim 1 or the medicine according to claim 2, wherein the brainstem glioma is a TP53 mutant brainstem glioma.

4. The use or medicine according to claim 3, wherein the brainstem glioma is a brainstem glioma combined with H3K27M mutation and TP53 mutation.

5. The use according to claim 1 or the medicine according to claim 2, which is an injection.

6. The use or medicine according to claim 5, wherein the medicine is an intravenous injection or an intrathecal injection.

7. The use according to claim 1 or the medicine according to claim 2, further comprising a pharmaceutically acceptable excipient.

8. The use or medicine according to claim 7, wherein the solvent of the medicine is 0.9% v / w sodium chloride solution or 5% v / w glucose solution.

9. The use according to claim 1 or the medicine according to claim 2, wherein the equivalent dose of SN38 used for intravenous injection 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 is 150ug to 600ug.

10. The use or medicine according to claim 8, wherein the concentration of SN38 in the medicine is 40 mg / L to 120 mg / L, and the concentration of AZ20 is 50 mg / L.

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