Use of a pharmaceutical composition in the preparation of a medicament for treating H3K27M mutant glioblastoma

Abstract

The application discloses application of a pharmaceutical composition in preparation of a drug for treating H3K27M mutant glioblastoma. The pharmaceutical composition comprises GSK-J4 or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof. Through in vivo orthotopic transplantation tumor mouse models and patient-derived H3K27M mutant glioblastoma spheroid models, it is confirmed that the combined drug use scheme has a significant synergistic anti-tumor effect. The application provides a combined drug use strategy with a clear mechanism and a significant effect for glioma, in particular, H3K27M mutant glioblastoma, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a pharmaceutical composition in the preparation of a drug for treating H3K27M mutant glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is the most common and most malignant primary tumor of the central nervous system, accounting for more than 50% of all gliomas. Glioblastomas with histone H3K27M mutations (including diffuse midline gliomas, diffuse endophytic pontine gliomas (DIPG), and some high-grade gliomas in children) have an extremely poor prognosis, with a median survival of less than one year, making them one of the most aggressive glioma subtypes clinically. H3K27M mutations lead to a significant decrease in global H3K27me3 levels, which in turn triggers extensive epigenetic reprogramming, driving malignant tumor progression. Studies have shown that more than 70%-80% of H3K27M-mutant gliomas also have TP53 mutations; the two synergistically promote tumor development and progression, leading to high resistance to conventional treatments.

[0003] Temozolomide (TMZ) is currently a first-line chemotherapy drug for the clinical treatment of glioblastoma. TMZ induces mismatch repair (MMR) by methylating guanine (O6-meG) on DNA, ultimately leading to DNA double-strand breaks and cell death. However, the clinical efficacy of TMZ is limited by the following factors: (1) MGMT repair mechanism: O6-methylguanine-DNA methyltransferase (MGMT) can repair DNA damage caused by TMZ, leading to drug resistance; (2) MMR deficiency: The loss of function of the mismatch repair system makes tumor cells resistant to TMZ damage; (3) H3K27M mutation-related drug resistance: Epigenetic reprogramming leads to H3K27M mutant gliomas being inherently insensitive to TMZ.

[0004] Therefore, developing novel combination therapy strategies that can enhance the efficacy of TMZ and overcome drug resistance is of great clinical significance.

[0005] KDM6B (also known as JMJD3) is a histone H3K27me3-specific demethylase belonging to the Jumonji family. KDM6B is abnormally overexpressed in various tumors, activating oncogene expression by removing the H3K27me3 inhibitory marker. GSK-J4 is a selective small-molecule inhibitor of KDM6B / KDM6A that competitively binds to the α-ketoglutarate binding region of the KDM6B catalytic site, inhibiting its demethylase activity and thus increasing H3K27me3 levels. Summary of the Invention

[0006] This invention provides the application of a pharmaceutical composition in the preparation of a drug for treating H3K27M mutant glioblastoma. The pharmaceutical composition comprises GSK-J4 and temozolomide (TMZ), and in vitro and in vivo experimental results show that this pharmaceutical combination has a significant inhibitory effect on temozolomide-resistant H3K27M mutant glioblastoma. This invention aims to solve the following technical problems: (1) providing a novel combination therapy regimen that can overcome TMZ resistance in H3K27M mutant glioblastoma; (2) providing a TMZ sensitization strategy supported by a clear epigenetic mechanism; and (3) providing a combination therapy regimen with promising clinical translation prospects based on patient-derived organoid validation.

[0007] This invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a pharmaceutical composition for treating glioma, the pharmaceutical composition comprising GSK-J4 or a pharmaceutically acceptable salt thereof, and temozolomide or a pharmaceutically acceptable salt thereof.

[0009] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipients.

[0010] Preferably, the mass ratio of GSK-J4 to temozolomide in the pharmaceutical composition is 1:(4-24).

[0011] Preferably, the glioma is a glioblastoma.

[0012] Further preferably, the glioblastoma is an H3K27M mutant glioblastoma.

[0013] The pharmaceutical composition of the present invention consists of the following two active ingredients: (1) GSK-J4, which is a small molecule inhibitor of KDM6B (also known as JMJD3), with the chemical name: ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azapyro-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propionate, with the molecular formula C 24 H 27N5O2, with a molecular weight of 417.50; GSK-J4 inhibits the catalytic activity of KDM6B, blocking its demethylation of histone H3K27me3. (2) Temozolomide (TMZ), temozolomide is a first-line chemotherapy drug used in clinical practice to treat glioblastoma. Its chemical name is 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-1,2,3,5-tetraazine-8-carboxamide, with the molecular formula C6H6N6O2 and a molecular weight of 194.15. Temozolomide hydrolyzes under physiological conditions to generate the active metabolite MTIC, which leads to DNA methylation damage and exerts cytotoxic effects.

[0014] This invention demonstrates, through orthotopic glioblastoma xenograft mouse models and patient-derived glioblastoma organoid models, that the combination of GSK-J4 and temozolomide can significantly synergistically inhibit tumor growth and significantly prolong the survival of tumor-bearing mice.

[0015] The mechanism of action elucidated by this invention is as follows: YAF2, as a subunit of the non-classical PRC1 complex, is specifically and highly expressed in glioblastoma. YAF2 promotes H3K27ac deposition by binding to HIF-1α and the transcriptional coactivator EP300, and then to the promoter region of the KDM6B gene, thereby activating KDM6B transcriptionally. KDM6B, as an H3K27me3 demethylase, activates downstream oncogenes by removing H3K27me3 modification, promoting the malignant progression of glioblastoma. GSK-J4 inhibits KDM6B enzyme activity, blocking its demethylation of H3K27me3, leading to an increase in H3K27me3 levels, thereby inhibiting the transcriptional activation of downstream oncogenes. Temozolomide induces tumor cell apoptosis through DNA alkylation damage. Both work synergistically through different and complementary mechanisms: GSK-J4 inhibits tumor malignant progression at the epigenetic level, while temozolomide directly kills tumor cells through DNA damage.

[0016] A second object of the present invention is to provide a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0017] Preferably, the dosage form of the formulation includes an injectable dosage form, an intracavitary dosage form, or a transdermal dosage form.

[0018] A third objective of this invention is the use of the above-described pharmaceutical composition or pharmaceutical preparation in the preparation of a medicament for treating glioma.

[0019] Preferably, the glioma is a glioblastoma.

[0020] Further preferably, the glioblastoma is an H3K27M mutant glioblastoma.

[0021] Preferably, GSK-J4 and temozolomide in the pharmaceutical composition or formulation are administered simultaneously, separately, or sequentially.

[0022] A fourth objective of this invention is to provide a method for inhibiting glioma cell growth and promoting glioma cell apoptosis in vitro for non-therapeutic purposes, the method comprising treating glioma cells with the above-mentioned pharmaceutical composition or pharmaceutical preparation.

[0023] Preferably, the glioma cells include H3K27M mutant glioblastoma cells and / or U87 glioblastoma cells.

[0024] The dosing regimen for the pharmaceutical composition (based on animal and in vitro studies) is as follows:

[0025] (a) Effective concentrations for in vitro cell and organoid experiments

[0026] In U87 glioblastoma cells, 300 nM GSK-J4 showed a significant inhibitory effect on cell proliferation; in patient-derived H3K27M mutant glioblastoma organoids (G33-O), treatment with 50 μM temozolomide combined with 1 μM GSK-J4 for 14 days resulted in significant organoid morphological disintegration and fragmentation.

[0027] (II) Effective dosing regimens for in vivo animal models

[0028] The following effective protocol was verified in the NOD-Scid immunodeficient mouse orthotopic xenograft model: (1) Subjects: NOD-Scid female mice (4-6 weeks old) inoculated with U87 human glioblastoma cells in the right cerebral cortex. (2) Drug preparation: GSK-J4 was first dissolved with a small amount of DMSO and then diluted with sterile physiological saline, while temozolomide was prepared directly with physiological saline. (3) Dosage: GSK-J4 alone or in combination: 10 mg / kg body weight; temozolomide alone or in combination: 40 mg / kg body weight. (4) Route of administration: Intraperitoneal injection. (5) Frequency and cycle of administration: Administration began on the 3rd day after tumor cell inoculation and was administered once every 3 days until the ethical endpoint of the mice. (6) Control group: An equal volume of physiological saline was administered at the same time. Under the above animal dosing regimens, the median survival of the GSK-J4 + temozolomide combination group reached 66 days, which was significantly longer than that of the temozolomide monotherapy group (57 days) and the GSK-J4 monotherapy group (63 days).

[0029] Beneficial effects:

[0030] 1. The pharmaceutical composition of the present invention exhibits significant synergistic antitumor effects (in vivo animal experiments). This invention is the first to demonstrate the synergistic effect of GSK-J4 combined with temozolomide in a mouse model of glioblastoma orthotopic xenografts. Specific data are as follows: Temozolomide monotherapy group (40 mg / kg): median survival 57 days; GSK-J4 monotherapy group (10 mg / kg): median survival 63 days; GSK-J4 + TMZ combination group (10 mg / kg + 40 mg / kg): median survival significantly prolonged to 66 days. The median survival of the combination therapy group was approximately 9 days longer than that of the temozolomide monotherapy group and approximately 3 days longer than that of the GSK-J4 monotherapy group. The difference was statistically significant (P<0.01) according to the Log-rank test, indicating that the combination of the two drugs has a synergistic effect.

[0031] 2. The pharmaceutical composition of the present invention has a significant inhibitory effect on H3K27M mutant glioblastoma (patient-derived organoid model). In patient-derived H3K27M mutant glioblastoma organoids (G33-O), the present invention demonstrated that: treatment with GSK-J4 alone (1 μM) for 14 days partially inhibited organoid growth; combined treatment with GSK-J4 (1 μM) and temozolomide (50 μM) for 14 days resulted in significant morphological disintegration and fragmentation of the organoids, with severely inhibited growth, showing superior efficacy compared to either single agent. However, in H3K27M wild-type organoids (G36-O), GSK-J4 did not show significant efficacy, suggesting that this combination regimen may have a more significant effect on the H3K27M mutant subtype.

[0032] 3. In vivo pathological evidence showed that on day 25 after tumor inoculation, HE staining of the brain tissue of mice in each group revealed that: large invasive tumor lesions were visible in the brains of mice in the control group, with tumor cells extensively infiltrating the surrounding normal brain tissue; the tumor volume was significantly reduced in the GSK-J4 monotherapy group and the TMZ monotherapy group; and the tumor volume was the smallest, the degree of invasion was the least, and the tumor boundary was relatively clear in the combination therapy group.

[0033] 4. The pharmaceutical composition of the present invention has good safety. In the embodiments of the present invention, no treatment-related deaths or serious systemic toxicities were observed in mice during a 30-day co-administration of GSK-J4 (10 mg / kg) and temozolomide (40 mg / kg). Attached Figure Description

[0034] Figure 1 This refers to the inhibitory effect of GSK-J4 on the proliferation of U87 glioblastoma cells in Example 1.

[0035] Figure 2 These are representative images of H&E staining of brain tissue from four groups of mice on day 25 after tumor inoculation in Example 2 (scale bar = 300 μm).

[0036] Figure 3 The Kaplan-Meier survival curves of the four groups of mice in Example 2 (control group, TMZ monotherapy group, GSK-J4 monotherapy group, and combination group) are shown; the statistical comparison of the median survival time of each group is shown (combination group 66 days vs. TMZ monotherapy group 57 days vs. GSK-J4 monotherapy group 63 days).

[0037] Figure 4 These are representative H&E stained images of liver and kidney sections from four groups of mice in Example 2 (control group, TMZ monotherapy group, GSK-J4 monotherapy group, and combination group) (scale bar = 50 μm).

[0038] Figure 5 This is a representative bright-field microscopy image of the H3K27M mutant organoid G33-O after 14 days of treatment with GSK-J4 (1 μM) and / or TMZ (50 μM). The organoids in the combined treatment group showed significant morphological disintegration and fragmentation (scale bar = 800 μm). Detailed Implementation

[0039] To ensure that the objectives, technical solutions, and advantages of this invention are clearer and more complete, the technical solutions in the embodiments of this invention will be described in more detail below with reference to the accompanying drawings. Specific embodiments are further illustrations of this invention and are not intended to limit it. Based on the embodiments of this invention, embodiments obtained by those skilled in the art without inventive technological improvements will be considered to fall within the scope of protection of this invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0040] All data concerning "dosing regimen," "dosage," and "dosing frequency" (GSK-J4 10 mg / kg, TMZ 40 mg / kg, once every 3 days for 30 consecutive days) are strictly derived from in vivo experiments using the NOD-Scid mouse orthotopic xenograft model. This invention has completed preclinical animal experiments and patient-derived organoid model validation. Specific human dosage, route of administration, and treatment duration are not within the scope of this invention and need to be further determined in subsequent clinical trials. The scope of this patent covers reasonable modifications based on the effective animal experimental regimen applied to humans through conventional dosage conversion.

[0041] Example 1: Inhibitory effect of GSK-J4 on the in vitro proliferation of glioblastoma cells

[0042] I. Materials and Methods

[0043] (1) Experimental materials

[0044] The human glioblastoma cell line U87 was purchased from the China Center for Type Culture Collection (CCTCC), accession number: SCSP-5432.

[0045] GSK-J4 was prepared as a stock solution using DMSO. Before the experiment, it was diluted with DMEM medium containing 10% fetal bovine serum to the required working concentration. The final concentration of DMSO should not exceed 0.1%.

[0046] (2) Experimental methods

[0047] U87 cells in logarithmic growth phase were seeded at a density of 1 × 10³ cells per well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The following day, the cells were divided into the following treatment groups: solvent control group (medium containing an equal volume of DMSO); and GSK-J4 single-drug group, with four concentration gradients: 30 nM, 300 nM, 3 μM, and 30 μM.

[0048] Each group had 3 replicates. Drug treatment lasted for 8 days, with the culture medium containing fresh drug replaced every 2 days. After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for another 3 hours. The absorbance at 450 nm was then measured using a microplate reader, and the relative cell viability of each group was calculated with the absorbance of the control group as 100%.

[0049] II. Experimental Results

[0050] Figure 1 The results of CCK-8 assays on U87 cells treated with different concentrations of GSK-J4 (30 nM - 30 μM) for 8 days show that GSK-J4 inhibits U87 cell proliferation in a dose-dependent manner, with a significant inhibitory effect observed at a concentration of 300 nM.

[0051] Example 2: Therapeutic effect of GSK-J4 combined with temozolomide on a mouse model of glioblastoma orthotopic xenograft.

[0052] I. Experimental Methods

[0053] (1) Establishment of orthotopic xenograft model

[0054] Female NOD-Scid immunodeficient mice aged 4–6 weeks were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then fixed in a stereotaxic apparatus. Under aseptic conditions, the skull was drilled open 2 mm to the right of the anterior fontanelle and 1 mm anterior to the anterior fontanelle. A 10 μL microsyringe was used to inject sodium pentobarbital at a rate of 1 μL / min containing 2 × 10⁻⁶ mg / kg of sodium pentobarbital. 510 μL of serum-free DMEM suspension containing one U87 cell was injected into the right cerebral cortex to a depth of 3.5 mm. The needle was left in place for 5 minutes after injection, then slowly withdrawn, and the skin was sutured. The mouse was placed on a temperature-controlled mat to recover and then returned to the SPF-grade animal facility for routine care.

[0055] (2) Animal grouping and administration regimen

[0056] Mice with successfully established orthotopic tumors were randomly divided into four groups of 6–8 mice each. The treatments for each group were as follows: Control group: intraperitoneal injection of an equal volume of sterile saline every 3 days, continuously until mouse death or an ethical endpoint; Temozolomide monotherapy group: intraperitoneal injection of temozolomide every 3 days at a dose of 40 mg / kg body weight; GSK-J4 monotherapy group: intraperitoneal injection of GSK-J4 every 3 days at a dose of 10 mg / kg body weight; Combination therapy group: simultaneous intraperitoneal injection of GSK-J4 (10 mg / kg) and temozolomide (40 mg / kg) every 3 days. All drugs were prepared with sterile saline. GSK-J4 was first dissolved with a small amount of DMSO and then diluted with saline to a final concentration (DMSO final concentration ≤1%). Temozolomide was prepared directly with saline. All administrations began on day 3 post-tumor inoculation.

[0057] (3) Observation indicators and survival analysis

[0058] From the date of tumor inoculation, the general condition and neurobehavioral symptoms (including arched back posture, gait changes, lethargy, and weight loss) of mice were observed daily. Mice exhibiting severe neurological symptoms (such as persistent arched back, inability to feed independently, severe gait disturbance, or weight loss exceeding 20%) were considered at an ethical endpoint, euthanized, and their survival time was recorded. Kaplan-Meier survival curves were plotted, and the Log-rank test was used for statistical analysis of intergroup survival differences.

[0059] (4) Pathological and histological examination

[0060] On day 25 after tumor inoculation, one mouse was randomly selected from each group, sacrificed after deep anesthesia, and the intact brain tissue was removed. After fixation with 4% paraformaldehyde, paraffin embedding, sectioning, and hematoxylin-eosin (H&E) staining, the size of the brain tumor and the degree of invasion of tumor cells into the surrounding normal brain tissue were observed under an optical microscope.

[0061] II. Experimental Results

[0062] Figure 3This study investigated the effects of GSK-J4 and temozolomide on the survival of mice with orthotopic glioblastoma xenografts. Survival analysis showed that the median survival was approximately 45 days in the control group, 57 days in the temozolomide monotherapy group, and 63 days in the GSK-J4 monotherapy group. The median survival in the combined GSK-J4 and temozolomide group was significantly prolonged to 66 days. The combined therapy group was approximately 9 days longer than the temozolomide monotherapy group and approximately 3 days longer than the GSK-J4 monotherapy group. The Log-rank test showed that the differences were statistically significant (P < 0.001). Figure 2 The study investigated the inhibitory effects of GSK-J4 and temozolomide on orthotopic glioblastoma xenografts in mice. HE staining further confirmed that the combined treatment group showed the smallest tumor volume and the most limited tumor cell invasion in the brain, while the control group had the largest tumor volume and extensive infiltration of surrounding brain tissue. These results indicate that the combined use of GSK-J4 and temozolomide synergistically inhibits glioblastoma growth in vivo and significantly prolongs the survival of tumor-bearing mice. Figure 4 The results show the biosafety of GSK-J4 and temozolomide in mice with orthotopic glioblastoma xenografts. Staining of liver and kidney sections in mice indicated that the combined administration did not cause liver or kidney damage, demonstrating good biosafety. Figure 4 ).

[0063] Example 3: Inhibitory effect of GSK-J4 combined with temozolomide on patient-derived glioma organoids

[0064] I. Experimental Methods

[0065] (1) Establishment of organoids

[0066] After obtaining informed consent from the patients and approval from the hospital's ethics committee, surgically resected glioblastoma tumor tissue was collected to construct glioblastoma organoids without the H3K27M mutation (designated G36-O) and glioblastoma organoids carrying the H3K27M mutation (designated G33-O). The procedure was briefly described as follows: the tumor tissue was washed with pre-cooled D-Hank's solution, manually cut into small pieces approximately 1 mm³, and then inoculated into freshly prepared organoid culture medium (composition: 50% DMEM: F12 + 50% Neurobasal + 1 × Glutamax + 1 × NEAA + 1 × penicillin-streptomycin + 1 × B-27 Supplement (without vitamin A) + 1 × N-2 Supplement + 55 μM β-mercaptoethanol + 2.375 μg / mL human insulin), and cultured in suspension at 120 r / min in a 37 °C, 5% CO2 incubator. The successfully established organoids were stabilized through passage and used for drug sensitivity testing.

[0067] (2) Drug treatment methods

[0068] G33-O or G36-O organoids cultured to the 3rd generation were digested into small cell clusters and seeded into ultra-low adsorption plates at a density of approximately 20 organoid bodies per well, allowing them to recover overnight. The experiment was divided into four groups: a solvent control group (medium containing an equal volume of DMSO); a GSK-J4 monotherapy group with a final concentration of 1 μM; a temozolomide monotherapy group with a final concentration of 50 μM; and a combination therapy group, which simultaneously received GSK-J4 (1 μM) and temozolomide (50 μM).

[0069] Each group had 3 replicates. The drug treatment lasted for 14 days, and the culture medium containing fresh drug was changed every 3 days. During the treatment, the organoids in each well were photographed using a bright-field microscope on days 0, 7, and 14 to observe and record the morphological changes and growth of the organoids.

[0070] II. Experimental Results

[0071] Figure 5 This study investigated the inhibitory effects of GSK-J4 and temozolomide on organoids from patient-derived H3K27M mutant glioblastoma. In the H3K27M mutant organoid G33-O, the solvent control group showed continuous enlargement and intact morphology within 14 days; the GSK-J4 monotherapy group exhibited partial inhibition of organoid growth, with some organoids showing edge fragmentation; the temozolomide monotherapy group showed slower organoid volume growth, but most maintained intact structure; while the combined treatment group showed significant morphological disintegration and fragmentation, with most organoids showing significant volume reduction or disintegration, and severely inhibited growth. This indicates that the combination of GSK-J4 and temozolomide has a strong synergistic inhibitory effect on H3K27M mutant glioblastoma organoids. As a control, GSK-J4 monotherapy did not show significant efficacy in the H3K27M wild-type organoid G36-O, further suggesting that this combination regimen has relative specificity against the H3K27M mutant subtype. Figure 5 ).

[0072] in conclusion:

[0073] This invention is the first to propose a combination therapy of the KDM6B small molecule inhibitor GSK-J4 and temozolomide, a first-line chemotherapy drug for glioblastoma, for the treatment of glioblastoma. Using in vivo orthotopic xenograft mouse models and patient-derived H3K27M mutant glioblastoma organoid models, this invention demonstrates that the combination therapy has a significant synergistic anti-tumor effect, effectively inhibiting tumor growth and significantly prolonging the survival of tumor-bearing animals, with effects superior to either single agent.

Claims

1. A pharmaceutical composition for treating glioma, characterized in that, The pharmaceutical composition comprises GSK-J4 or a pharmaceutically acceptable salt thereof, and temozolomide or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of GSK-J4 to temozolomide in the pharmaceutical composition is 1:(4-24).

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

4. The pharmaceutical composition according to claim 1, characterized in that, The glioma is glioblastoma; preferably, the glioblastoma is H3K27M mutant glioblastoma.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1-4.

6. The pharmaceutical preparation according to claim 5, characterized in that, The dosage form of the formulation includes injectable dosage form, cavity dosage form, or skin dosage form.

7. Use of the pharmaceutical composition of any one of claims 1-4 or the pharmaceutical preparation of any one of claims 5-6 in the preparation of a medicament for treating glioma.

8. The application according to claim 7, characterized in that, The glioma is glioblastoma; preferably, the glioblastoma is H3K27M mutant glioblastoma.

9. A method for inhibiting glioma cell growth and promoting glioma cell apoptosis in vitro for non-therapeutic purposes, characterized in that, The method includes treating glioma cells with the pharmaceutical composition of any one of claims 1-4 or the pharmaceutical preparation of any one of claims 5-6.

10. The method according to claim 9, characterized in that, The glioma cells include H3K27M mutant glioblastoma cells and / or U87 glioblastoma cells.

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