Use of SN38 compound in the preparation of drugs for treating brainstem glioma

Abstract

This application provides the use of the SN38 compound in the preparation of a drug for treating brainstem gliomas and the corresponding drug. Experimental results show that the SN38 compound can effectively inhibit the proliferation of multiple cell lines of TP53 wild-type brainstem gliomas at the cellular level, with obvious dose-dependency and targeting characteristics, and does not affect the activity of normal brainstem stem cells; at the animal model level, both intraperitoneal administration and intrathecal injection can effectively inhibit tumor growth and prolong the survival cycle of mice with brainstem orthotopic tumors; providing a new treatment option for the treatment of TP53 wild-type brainstem gliomas.

Description

Field of the invention:

[0001] The present application belongs to the field of tumor treatment. Specifically, the present application provides the use of the SN38 compound in the preparation of a drug for treating brainstem glioma. Background technology:

[0002] Brainstem gliomas account for the vast majority of brainstem tumors, among which diffuse midline gliomas with H3K27M mutations are the most common. Current studies have shown that the median survival time of patients with brainstem gliomas who receive radiotherapy and / or surgery is 11 months, and the prognosis of brainstem gliomas is extremely poor. To date, the standard treatment for brainstem gliomas is radiotherapy, and other treatment options, including surgery and chemotherapy, have not achieved survival benefits. The insensitivity of brainstem gliomas to currently commonly used chemotherapy drugs such as temozolomide has resulted in extremely limited effectiveness of clinical first-line chemotherapy regimens, which has not fundamentally improved the prognosis of patients with brainstem gliomas, and the survival period has not been substantially improved compared to the past.

[0003] In addition to H3K27M mutations, brainstem gliomas can harbor multiple hotspot mutations, including TP53, PI3KCA, and PPM1D. While numerous studies have examined drug screening for brainstem gliomas, current screening results have failed to clearly correlate drug efficacy with specific mutation subtypes, leaving the question of drug targeting unclear. Therefore, there is an urgent need to explore effective treatments for specific mutation subtypes of brainstem gliomas, prolong patient survival, and alleviate the suffering of patients and their families.

[0004] Primary cell lines are established from patient tumor tissue and can be permanently passaged in vitro. They retain the genetic characteristics of the patient's tumor and can characterize key mutations such as H3K27M. Preclinical drug testing in primary cell lines through drug screening and in vitro and in vivo experiments will create a more realistic environment and conditions for clinical translation. Summary of the invention:

[0005] The purpose of the present invention is to provide a novel drug application scheme for treating brainstem gliomas and determine the pathological subtypes of brainstem gliomas for which the drug is effective, aiming to improve the level of chemotherapy treatment for brainstem gliomas.

[0006] On the one hand, the present application provides the use of SN38 compound in the preparation of a drug for treating brainstem glioma.

[0007] On the other hand, the present application provides a drug for treating brainstem glioma, which contains the SN38 compound as an active ingredient.

[0008] Furthermore, the brainstem glioma is a TP53 wild-type brainstem glioma.

[0009] Furthermore, the brainstem glioma is a TP53 wild-type brainstem glioma with H3K27M mutation.

[0010] Furthermore, the medicine is an injection.

[0011] Furthermore, the medicine is an intravenous injection and an intrathecal injection.

[0012] Furthermore, when the SN38 is used for intravenous injection, the equivalent dose used is 30 mg / m 2 .

[0013] Furthermore, the content of SN38 compound in the drug is 90-110 mg / L.

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

[0015] Furthermore, the pharmaceutically acceptable excipient is 0.9% v / w sodium chloride or 5% v / w glucose.

[0016] Furthermore, when the drug is used for intrathecal injection, the dosage range is 150ug to 600ug, the solvent is 5ml of 0.9% sodium chloride solution, and the concentration range after dissolution is 30mg / L to 120mg / L.

[0017] The structure of the SN38 compound is shown in formula (1), and its CAS number is 86639-52-3.

[0018]

[0019] The SN38 compound effectively inhibited the proliferation of multiple TP53 wild-type brainstem glioma cell lines at the cellular level, demonstrating a clear dose-dependent and targeted nature, without affecting the activity of normal brainstem stem cells. In animal models, both intraperitoneal and intrathecal administration effectively inhibited tumor growth and prolonged survival in mice bearing orthotopic brainstem tumors. Therefore, the present invention provides a new treatment option for TP53 wild-type brainstem gliomas. Description of the drawings:

[0020] Figure 1 The results of in vitro topoisomerase I inhibitor drug screening;

[0021] Figure 2 The results of 150714 cell line viability in in vitro topoisomerase I inhibitor drug screening;

[0022] Figure 3 The half-maximal inhibitory concentration curve of SN38 compound shows the sensitivity of brainstem glioma cell line to SN38 compound determined by in vitro experiments;

[0023] Figure 4 This is the proliferation curve of brainstem glioma cell line under the administration of SN38 compound determined in vitro;

[0024] Figure 5 This is the proliferation curve of normal brainstem stem cells under the administration of SN38 compound determined in vitro;

[0025] Figure 6 The growth of tumor fluorescence intensity after administration of SN38 compound in an animal brainstem orthotopic tumor model (intraperitoneal injection of SN38, TT150630);

[0026] Figure 7 Survival of animals after administration of SN38 compounds in an orthotopic brainstem tumor model (intraperitoneal injection of SN38, TT150630);

[0027] Figure 8 The growth of tumor fluorescence intensity after administration of SN38 compound in an animal brainstem orthotopic tumor model (intraperitoneal injection of SN38, TT190326);

[0028] Figure 9 Survival of animals after administration of SN38 compounds in an orthotopic brainstem tumor model (intraperitoneal injection of SN38, TT190326);

[0029] Figure 10 The increase in tumor fluorescence intensity after administration of SN38 compound in an animal brainstem orthotopic tumor model (simulated intrathecal injection of SN38, TT150630);

[0030] Figure 11 The survival time of animals after administration of SN38 compound in an orthotopic brainstem tumor model (simulated intrathecal injection of SN38, TT150630). Specific implementation method:

[0031] Example 1

[0032] Due to the presence of the blood-brain barrier, it is often difficult for chemotherapy drugs to penetrate into the central nervous system and reach the target area when administered peripherally to central nervous system tumors. Therefore, simulating different modes of administration is very important for the treatment of brainstem gliomas. The present invention conducts cytological experiments (cell-titer experiments) such as topoisomerase I inhibitor drug screening on multiple primary brainstem glioma cell lines in vitro to measure the killing and proliferation inhibition effects of the SN38 compound on patient-derived brainstem glioma cell lines at different drug concentrations; and multiple modes of administration, including systemic administration and intrathecal injection, are used to verify the in vivo effectiveness and safety of the SN38 compound.

[0033] The experimental process is as follows:

[0034] 1) SN38 compound (MCE, HY-13704) was prepared into a 10 mM stock solution in DMSO and stored at -20°C.

[0035] 2) 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 traditional primary cell line culture methods using brainstem glioma primary cell line culture medium. Before passaging, the culture flasks were coated with 1:1000 diluted Matrigel (Corning, 356234) at 37°C overnight. The primary culture medium was DMEM high glucose medium (Gibco, 11995073), which also contained: 20 ng / mL EGF cytokine (Ciinto SC102), 20 ng / mL bFGF cytokine (Ciendo SC107-10 μg), 100 U / mL penicillin and 100 μg / mL streptomycin (100×, Thermo Fisher Scientific 10378016), 1×N2 (50×, Ciendo S60314017A), 1×B27 (50×, Ciendo S60314015A), and 1×ITS (100×, Ciendo SC25800). Successful cell line establishment was confirmed after passage 10. The H3K27M and TP53 mutation profiles of the brainstem glioma cell lines obtained by whole-exome sequencing are shown in Table 1.

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

[0037] Table 1 H3K27M and TP53 mutations in brainstem glioma cell lines

[0038]

[0039]

[0040] 3) Cells in logarithmic growth were plated at 2000 cells / well in 384-well plates and cultured overnight. Drugs were taken from the topoisomerase I inhibitor drug library (MCE) using the Echo sonic 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 assay (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 、 2As shown, topoisomerase I inhibitors have a significant killing effect on TP53 wild-type brainstem gliomas, and among them, SN38 compound has the best effect, while not killing normal brainstem stem cells.

[0041] Example 2

[0042] 1) Selection Figure 1 The primary brainstem glioma cell line in the experiment was cultured. 3000 cells in logarithmic growth state were plated into 96-well plates and cultured overnight. 10 mmol / L SN38 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 10 μmol / L, 3.33 μmmol / L, 1.11 μmmol / L, 0.37 μmmol / L, 0.12 μmmol / L, 0.41 μmmol / L, 0.14 μmmol / L, 0.046 μmmol / L, 0.0015 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 the plot and the average half-maximal inhibitory concentration (IC50) of SN38 compound for TP53 wild-type brainstem glioma was 10 nmol / L. Figure 3 shown.

[0043] 2) Select the primary brainstem glioma cell lines listed in the table, plate TT150714 brainstem glioma cells in logarithmic growth state at 5000 cells / well into 96-well plates and culture overnight. Take the SN38 compound and add it to the cell culture medium so that the final drug concentration in each well is 15 nmol / L, 2 times (30 nmol / L), and 4 times (60 nmol / L) the 50% inhibitory concentration of the SN38 compound for the TT150714 cell line. Continue to culture for 72 hours, and use the Cell-titer method (Promega, ) The fluorescence intensity was measured by microplate reader, 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%. Graphpad Prism 9 software was used to plot the results. Figure 4 As shown. It shows that the SN38 compound has the ability to significantly inhibit the proliferation of TP53 wild-type brainstem glioma at the half-maximal inhibitory concentration.

[0044] Comparative Example 1

[0045] According to the same experimental method as in 2) of Example 2, the human normal brain stem stem cell line PPC was treated with the same dose of SN38 compound as that of TT150714, and the proliferation curve was as follows: Figure 5 As shown, it can be seen that the SN38 compound does not affect the normal proliferation of human normal brainstem stem cells within the dosing window.

[0046] Example 3

[0047] 6-8 week old female Balc nude mice were selected for in situ implantation of TP53 wild-type brainstem glioma primary cell line (TT150630) in the brainstem to form tumors: the brainstem glioma primary cell line was prepared and 100,000 cells / 5ul were stereotactically injected slowly and evenly into the mouse brainstem within 2 minutes (located about 1mm after the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). The mice were observed one week after inoculation and the fluorescence value of the brainstem in situ tumor was measured using an in vivo fluorescence imager (IVIS Lumina Series III). Mice with tumors were selected and divided equally into two groups according to the fluorescence value of the in situ tumors, namely the control group and the SN38 compound treatment group.

[0048] 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 1 mg / mL. 200 μL of freshly prepared SN38 solution was injected intraperitoneally each time (dosage: 10 mg / kg).

[0049] Control group: 200 μL of 0.9% (v / w) sodium chloride solution was injected intraperitoneally each time.

[0050] 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.

[0051] Fluorescence intensity changes as Figure 6 The survival curve is shown as Figure 7 As shown, it can be seen that the SN38 compound significantly inhibited the tumor growth of the in vivo TP53 wild-type orthotopic tumor model and prolonged the survival period. After treatment with the SN38 compound, TP53 wild-type brainstem glioma can obtain a better potential prognosis.

[0052] Comparative Example 2

[0053] 6-8 week old female Balc nude mice 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 stereotactically within 2 minutes (located about 1mm after the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). The mice were observed one week after inoculation and the fluorescence value of the brainstem in situ tumor was measured using an in vivo fluorescence imager (IVIS Lumina Series III). Mice with tumors were selected and divided equally into two groups according to the fluorescence value of the in situ tumors, namely the control group and the SN38 compound treatment group.

[0054] 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 1 mg / mL. 200 μL of freshly prepared SN38 solution was injected intraperitoneally each time (dosage: 10 mg / kg).

[0055] Control group: 200 μL of 0.9% (v / w) sodium chloride solution was injected intraperitoneally each time.

[0056] 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.

[0057] Fluorescence intensity changes as Figure 8 The survival curve is shown as Figure 9 As shown, the SN38 compound cannot inhibit tumor growth in the in vivo TP53 mutant orthotopic tumor model and cannot improve survival.

[0058] Example 4

[0059] 6-8 week old female Balc nude mice were selected for in situ implantation of TP53 wild-type brainstem glioma primary cell line (TT150630) in the brainstem to form tumors: the brainstem glioma primary cell line was prepared and 100,000 cells / 5ul were stereotactically injected slowly and evenly into the mouse brainstem within 2 minutes (located about 1mm after the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). The mice were observed one week after inoculation and the fluorescence value of the brainstem in situ tumor was measured using an in vivo fluorescence imager (IVIS Lumina Series III). Mice with tumors were selected and divided equally into two groups according to the fluorescence value of the in situ tumors, namely the control group and the SN38 compound treatment group.

[0060] 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. A total of 5 μL of freshly prepared SN38 solution was injected each time (drug concentration in cerebrospinal fluid: 10 nmol / L).

[0061] Control group: Intrathecal injection was completed at the same time as the SN38 compound treatment group, with 5 μL of 0.9% (v / w) sodium chloride solution injected intrathecally each time.

[0062] Both groups received the drug simultaneously, once every four days for a total of four doses. Mice were observed for half an hour after each dose for neurological symptoms such as opisthotonos, bradykinesia, and unsteady gait to track drug neurotoxicity, but no such symptoms were observed. Mice were weighed every three days to track drug toxicity, but no significant weight loss was observed during the dosing period, and no drug-related deaths occurred. Fluorescence measurements of brainstem tumors in situ were performed weekly during the dosing period, and the fluorescence intensity was recorded. Overall survival of both groups was tracked.

[0063] Fluorescence intensity changes as Figure 10 The survival curve is shown as Figure 11 As shown, intrathecal injection of SN38 compound significantly inhibited tumor growth in the in vivo TP53 wild-type orthotopic tumor model and prolonged survival. Compared with intraperitoneal administration, intrathecal injection can achieve better prognosis with a smaller dosage.

Claims

1. Use of the SN38 compound in the preparation of a medicament for treating brainstem glioma, wherein the brainstem glioma is a TP53 wild-type brainstem glioma with an H3K27M mutation.

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

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

4. The method according to claim 1, wherein the content of SN38 compound in the medicine is 90-110 mg / L.

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

6. The use according to claim 5, wherein the pharmaceutically acceptable excipient is 0.9% v / w sodium chloride or 5% v / w glucose.

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