Application of SN38 compound in preparation of medicine for treating brainstem glioma
By using SN38 compounds for intravenous and intrathecal injection, the problem of brainstem gliomas being insensitive to existing chemotherapy drugs was solved, and effective inhibition and survival cycle of TP53 wild-type brainstem gliomas were achieved, providing targeted A new treatment plan for brainstem glioma.
Patent Information
- Application Number
- CN202411407464.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2044-10-10
AI Technical Summary
Brainstem glioma is insensitive to existing chemotherapy drugs, resulting in limited treatment effects. The existing drug screening results are not clearly associated with specific mutation subtypes, and there is a lack of effective treatment methods for brainstem gliomas.
Drugs that use SN38 compound as active ingredient, and are used to treat TP53 wild-type brainstem glioma through intravenous injection and intrathecal injection, significantly inhibiting tumor growth and prolonging survival cycle, and not affecting the activity of normal brainstem stem cells.
SN38 compounds showed obvious dose-dependent and targeted characteristics in in vitro and in vivo experiments, effectively inhibiting the proliferation and tumor growth of TP53 wild-type brainstem glioma, prolonging survival cycle, and providing targeted brainstem gliomas. New treatment options.
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Figure CN119235857A_ABST
Abstract
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 compound in the preparation of drugs 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 show 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, and 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 mutation, brainstem gliomas can also have multiple hotspot mutations, including TP53 mutation, PI3KCA mutation, PPM1D mutation, etc. There have been many studies on drug screening for brainstem gliomas, but the current screening results have failed to clearly associate drug efficacy with specific mutation subtypes, and the problem of drug targeting cannot be explained. Therefore, it is urgent to explore effective treatment methods for clear mutation subtypes of brainstem gliomas to prolong the survival of patients and alleviate the pain of children and their families.
[0004] Primary cell lines are cell lines that are established from patient tumor tissues and can be permanently propagated in vitro. They retain the genetic characteristics of the patient's tumor and can characterize key mutations such as H3K27M. Preclinical experiments on drugs through drug screening and in vitro and in vivo experiments on primary cell lines will create a more realistic environment and conditions for clinical transformation. Summary of the invention:
[0005] The purpose of the present invention is to provide a novel drug application scheme for treating brainstem gliomas, and to determine the pathological subtypes of brainstem gliomas for which the drug is effective, in order to improve the level of chemotherapy treatment of 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 a 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 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 inhibits the proliferation of multiple cell lines of TP53 wild-type brainstem glioma 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, intraperitoneal administration and intrathecal injection can effectively inhibit the tumor growth of mice with brainstem orthotopic tumors and prolong the survival period. Therefore, the present invention provides a new treatment option for the treatment of TP53 wild-type brainstem glioma. Description of the drawings:
[0020] Figure 1 The results of in vitro drug screening for topoisomerase I inhibitors;
[0021] Figure 2 The results of the 150714 cell line viability in the in vitro topoisomerase I inhibitor drug screening;
[0022] Figure 3 The half-maximal inhibitory concentration curve of the SN38 compound shows the sensitivity of the brainstem glioma cell line to the SN38 compound determined in vitro;
[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 brain stem 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 the animal brainstem orthotopic tumor model (intraperitoneal injection of SN38, TT150630);
[0026] Figure 7 The survival of animals after administration of SN38 compounds in the orthotopic brainstem tumor model (intraperitoneal injection of SN38, TT150630);
[0027] Figure 8 The growth of tumor fluorescence intensity after administration of SN38 compound in the animal brainstem orthotopic tumor model (intraperitoneal injection of SN38, TT190326);
[0028] Fig. 9 The survival of animals after administration of SN38 compounds in the orthotopic brainstem tumor model (intraperitoneal injection of SN38, TT190326);
[0029] Fig.10 The growth of tumor fluorescence intensity after administration of SN38 compound in the animal brainstem orthotopic tumor model (simulated intrathecal injection of SN38, TT150630);
[0030] Fig.11 This is the survival status of animals after administration of SN38 compound in the 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. Therefore, simulating different modes of administration is very important for the treatment of brainstem glioma. 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 SN38 compounds on patient-derived brainstem glioma cell lines at different drug concentrations; multiple modes of administration, such as systemic administration and intrathecal injection, are selected in vivo to verify the in vivo effectiveness and safety of SN38 compounds.
[0033] The experimental process is as follows:
[0034] 1) SN38 compound (MCE, HY-13704) was prepared into a 10 mM stock solution with 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 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 Table 1.
[0036] 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.
[0037] Table 1 H3K27M and TP53 mutations in brainstem glioma cell lines
[0038]
[0039]
[0040] 3) The cells in logarithmic growth state were plated at 2000 cells / well in 384-well plates and cultured overnight. The drugs were taken from the topoisomerase I inhibitor drug compound library (MCE) using the Echo sonar automatic dosing system in vitro and added to the cell culture medium to make the final concentration of each drug 1 umol / 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 , 2As shown, it was shown that topoisomerase I inhibitors had a significant killing effect on TP53 wild-type brainstem gliomas, and among them, SN38 compound had the best effect, while not killing normal brainstem stem cells.
[0041] Example 2
[0042] 1) Selection Figure 1 The primary brainstem glioma cell line was cultured overnight in a 96-well plate at 3000 cells / well in logarithmic growth state. The 10mmol / L SN38 compound stock solution was diluted in vitro in a gradient of 1:3. The drug was taken and added to the cell culture medium to make the final drug concentration in each well 10umol / L, 3.33ummol / L, 1.11ummol / L, 0.37ummol / L, 0.12ummol / L, 0.41ummol / L, 0.14ummol / L, 0.046ummol / L, 0.0015ummol / L, 0ummol / L. After continued culture for 72h, 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 SN38 compound for TP53 wild-type brainstem glioma was 10nmol / L using Graphpad Prism 9 software fitting and drawing. Figure 3 shown.
[0043] 2) Select the primary brainstem glioma cell line in the table, plate TT150714 brainstem glioma cells in logarithmic growth state at 5000 cells / well to 96-well plate and culture overnight, take SN38 compound and add it to the cell culture medium, so that the final drug concentration in each well is 15nmol / L of the half-inhibitory concentration of SN38 compound for TT150714 cell line and 2 times (30nmol / L) and 4 times (60nmol / L) of the half-inhibitory concentration. Continue to culture for 72h, and use 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%. Graphpad Prism 9 software was used to draw the graph as shown in Figure 4 As shown. It shows that the SN38 compound has a significant ability to inhibit the proliferation of TP53 wild-type brainstem glioma at the half inhibitory concentration.
[0044] Comparative Example 1
[0045] According to the same experimental method as in 2) of Example 2, the same dose of SN38 compound as that of TT150714 was applied to the human normal brain stem stem cell line PPC, 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 normal human brain stem stem cells within the dosing window.
[0046] Example 3
[0047] Select 6-8 week old female Balc nude mice for brainstem in situ implantation of TP53 wild-type brainstem glioma primary cell line (TT150630) to form tumors: prepare brainstem glioma primary cell line and inject 100,000 cells / 5ul stereotaxically and slowly and evenly into the mouse brainstem within 2 minutes (located about 1mm behind the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). Observe the state of the mice 1 week after inoculation and use an in vivo fluorescence imager (IVIS Lumina Series III) to measure the fluorescence value of brainstem in situ tumors. Select mice with tumors and divide them into two groups according to the fluorescence value of 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, the concentration of SN38 compound was adjusted to 1 mg / mL, and 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] The two groups were given drugs at the same time, once a week, for a total of 4 weeks. The mice were weighed every three days to track the drug's toxicity and side effects. During the drug administration period, there was no significant weight loss in the mice, 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 overall survival of the two groups of mice 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] Select 6-8 week old female Balc nude mice for in situ implantation of TP53 mutant brainstem glioma primary cell line (TT190326) in the brainstem to form tumors: prepare the brainstem glioma primary cell line and inject it 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). Observe the state of the mice 1 week after inoculation and use an in vivo fluorescence imager (IVIS Lumina Series III) to measure the fluorescence value of the brainstem in situ tumor. Select the mice with tumors and divide them into two groups equally 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, the concentration of SN38 compound was adjusted to 1 mg / mL, and 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] The two groups were given drugs at the same time, once a week, for a total of 4 weeks. The mice were weighed every three days to track the drug's toxicity and side effects. During the drug administration period, there was no significant weight loss in the mice, 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 overall survival of the two groups of mice was tracked.
[0057] Fluorescence intensity changes as Figure 8 The survival curve is shown as Fig. 9 As shown, it can be seen that the SN38 compound cannot inhibit tumor growth in the in vivo TP53 mutant orthotopic tumor model and cannot improve survival.
[0058] Example 4
[0059] Select 6-8 week old female Balc nude mice for brainstem in situ implantation of TP53 wild-type brainstem glioma primary cell line (TT150630) to form tumors: prepare brainstem glioma primary cell line and inject 100,000 cells / 5ul stereotaxically and slowly and evenly into the mouse brainstem within 2 minutes (located about 1mm behind the intersection of the lambdoid suture of the mouse skull and about 1mm beside the midline). Observe the state of the mice 1 week after inoculation and use an in vivo fluorescence imager (IVIS Lumina Series III) to measure the fluorescence value of brainstem in situ tumors. Select mice with tumors and divide them into two groups according to the fluorescence value of 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, and 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] The two groups were given drugs at the same time, once every 4 days, for a total of 4 times. After each drug administration, the mice were observed for half an hour to see if they had neurological symptoms such as opisthotonos, slow movements, and unsteady walking to track the neurotoxic side effects of the drug. No such symptoms were observed. The mice were weighed every three days to track the drug toxicity. During the drug administration period, there was no significant weight loss in the mice, 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 overall survival of the two groups of mice was tracked.
[0063] Fluorescence intensity changes as Fig.10 The survival curve is shown as Fig.11 As shown, it can be seen that 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 SN38 compound in the preparation of drugs for treating brainstem glioma.
2. A drug for treating brainstem glioma, characterized in that: The medicine contains the SN38 compound as an active ingredient.
3. The use according to claim 1 or the medicine according to claim 2, wherein the brainstem glioma is a TP53 wild-type brainstem glioma.
4. The use or medicine according to claim 3, wherein the brainstem glioma is a TP53 wild-type brainstem glioma with H3K27M 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, which is an intravenous injection or an intrathecal injection.
7. The use according to claim 1 or the medicine according to claim 2, wherein the content of SN38 compound in the medicine is 90-110 mg / L.
8. The use according to claim 1 or the medicine according to claim 2, further comprising a pharmaceutically acceptable excipient.
9. The use or medicine according to claim 3, wherein the pharmaceutically acceptable excipient is 0.9% v / w sodium chloride or 5% v / w glucose.
10. The use according to claim 1 or the drug according to claim 2, wherein the drug is used for intravenous injection at an equivalent dose of 30 mg / m 2 When used for intrathecal injection, the dosage range is 150ug to 600ug, the solvent is 5ml 0.9% sodium chloride solution, and the concentration range after dissolution is 30mg / L to 120mg / L.