Pharmaceutical compositions and their applications

Abstract

The present invention provides a pharmaceutical composition comprising panobinostat and tazemetostat. The inventors of the present application creatively discovered that when tazemetostat, an EZH2 inhibitor, is used in combination with panobinostat, pembrolizumab and ipilimumab to treat patients with end-stage diffuse midline glioma with H3K27 alteration accompanied by leptomeningeal / meningeal dissemination in clinical practice, and patients with diffuse midline glioma with primary, recurrent or progressive H3K27 alteration, the therapeutic effect is significant, tumor growth is significantly inhibited, tumor volume is reduced, the quality of life of patients is improved, and the survival time of patients is extended.

Description

[Technical field]

[0001] The present invention relates to the pharmaceutical technical field, in particular to pharmaceutical compositions and their applications. [Background technology]

[0002] Diffuse midline glioma (H3 K27-altered), named after the World Health Organization Classification of Tumors of the Central Nervous System, 5th Edition, 2021, is a pediatric diffuse high-grade glioma that predominates in the midline of the central nervous system, often affecting areas such as the thalamus, pons, and spinal cord. Surgical resection is extremely difficult, and biopsy is the main method for most patients. Until now, regardless of traditional treatment, targeted therapy, or immunotherapy, the overall survival of patients has not been extended. There are no global treatment guidelines, so effective treatment plans need to be explored as soon as possible. Patients with diffuse midline glioma with H3K27 alterations have a very poor prognosis, with a 2-year survival rate of less than 10%. In particular, it is noteworthy that diffuse midline glioma with H3K27 alterations, which originates in the spinal cord and is accompanied by leptomeningeal / meningeal dissemination, is an extremely rare case. Previous literature has shown that patients with high-grade gliomas with leptomeningeal / meningeal dissemination have a poorer prognosis and therefore have always been excluded from clinical studies, with a median survival time of only 1.6 to 3.8 months. Summary of the Invention

[0003] The present invention has been made in view of the above circumstances, and aims to provide a pharmaceutical composition and its application. The pharmaceutical composition provided in the present application can be used to treat diffuse midline glioma with H3K27 alteration, and can inhibit tumor growth, reduce tumor volume, significantly improve the quality of life of patients, and extend the survival time of patients.

[0004] The present invention provides a pharmaceutical composition comprising an HDAC inhibitor and an EZH2 inhibitor.

[0005] The present inventors creatively discovered that the combined use of a histone deacetylase inhibitor (HDAC) and an EZH2 inhibitor has a significant clinical effect in treating tumors, particularly diffuse midline gliomas with H3K27 alterations. In some specific embodiments, the mass ratio of the HDAC inhibitor to the EZH2 inhibitor is 100-200:1000-3000, preferably 100-200:1200-2800, more preferably 100-200:1500-2500.

[0006] In some specific embodiments, the HDAC inhibitor includes, but is not limited to, one or more of Panobinostat, Vorinostat, Romidepsin, Belinostat and Tucidinostat (also known as Chidamide).Preferably, the HDAC inhibitor is Panobinostat.Panobinostat was approved by the US Food and Drug Administration in 2015 as a treatment for multiple myeloma.Research reveals that Panobinostat can cross the blood-brain barrier and is a promising drug for the treatment of diffuse midline glioma with H3K27 alteration, but for recurrent glioma and anaplastic glioma, it cannot significantly extend the 6-month progression-free survival of patients.

[0007] In some specific embodiments, the EZH2 inhibitor is, but is not limited to, tazemetostat. Tazemetostat is an EZH2 small molecule inhibitor that was approved by the US Food and Drug Administration in 2020 as a treatment for epithelioid sarcoma, but no clinical studies have been conducted in glioma patients to date. The inventors of the present application have creatively discovered that EZH2 inhibitors can be used as a type of epigenetic drug to treat diffuse midline glioma patients with H3K27 alterations in clinical practice, and in particular, when tazemetostat is used in combination with panobinostat to treat end-stage diffuse midline glioma patients with H3K27 alterations with leptomeningeal / spinal membrane seeding, and diffuse midline glioma patients with primary, recurrent or progressive H3K27 alterations, the therapeutic effect is significant, tumor growth can be significantly inhibited, tumor volume can be reduced, the quality of life of patients can be improved, and the survival time of patients can be extended.

[0008] In some specific embodiments, the composition comprises panobinostat and tazemetostat, the mass ratio of the panobinostat to the tazemetostat being 100-200:1000-3000, preferably 100-200:1200-2800, more preferably 100-200:1500-2500.

[0009] In some specific embodiments, the pharmaceutical composition further comprises one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a VEGF inhibitor.

[0010] In some particular embodiments, the PD-1 inhibitor is one or more selected from the group consisting of Pembrolizumab, Nivolumab, Tislelizumab, Toripalimab, Sintilimab, Camrelizumab, Zimberelimab, Penpulimab, Serplulimab, and Pucotenlimab, preferably Pembrolizumab.

[0011] In some particular embodiments, the PD-L1 inhibitor is one or more selected from the group consisting of Atezolizumab, Durvalumab, Envafolimab, Sugemalimab, and Avelumab.

[0012] In some specific embodiments, the CTLA-4 inhibitor is one or more selected from the group consisting of Ipilimumab and Tremelimumab, preferably Ipilimumab.

[0013] In some specific embodiments, the VEGF inhibitor is one or more selected from the group consisting of Bevacizumab, Ramucirumab, Ranibizumab (trade name: Lucentis), and Aflibercept, preferably Bevacizumab.

[0014] In some specific embodiments, the pharmaceutical composition further contains pembrolizumab, which is a PD-1 inhibitor among immune checkpoint inhibitors. The mass ratio of the panobinostat to the pembrolizumab is 100-200:50-400, preferably 100-200:100-350, and more preferably 100-200:150-300.

[0015] In some specific embodiments, the pharmaceutical composition further comprises ipilimumab, a CTLA-4 inhibitor among immune checkpoint inhibitors. The present inventors have discovered that when pembrolizumab and ipilimumab are used in combination with panobinostat and tazemetostat to treat diffuse midline gliomas with H3K27 alterations, they have a significant therapeutic effect, inhibit tumor growth, reduce tumor volume, significantly improve the quality of life of patients, and extend the survival time of patients. The mass ratio of the panobinostat to the ipilimumab is 100-200:10-100, preferably 100-200:20-80.

[0016] In some specific embodiments, the pharmaceutical composition further comprises bevacizumab, a recombinant humanized monoclonal antibody targeting VEGF. In some specific embodiments, the mass ratio of the panobinostat to the bevacizumab is 100-200:300-1000, preferably 100-200:350-800, and more preferably 100-200:400-700.

[0017] In some particular embodiments, the pharmaceutical composition comprises panobinostat, tazemetostat, and pembrolizumab. In some particular embodiments, the pharmaceutical composition comprises panobinostat, tazemetostat, and ipilimumab. In some particular embodiments, the pharmaceutical composition comprises panobinostat, tazemetostat, pembrolizumab, and ipilimumab. In some particular embodiments, the pharmaceutical composition comprises panobinostat, tazemetostat, pembrolizumab, and ipilimumab. In some particular embodiments, the pharmaceutical composition comprises panobinostat, tazemetostat, pembrolizumab, ipilimumab, and bevacizumab.

[0018] In some specific embodiments, the pharmaceutical composition comprises an EZH2 inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor. In the pharmaceutical composition, the mass ratio of the EZH2 inhibitor to the PD-1 inhibitor or the PD-L1 inhibitor is 1000-3000:50-400. In some specific embodiments, the pharmaceutical composition comprises an EZH2 inhibitor and a CTLA-4 inhibitor, and the mass ratio of the EZH2 inhibitor to the CTLA-4 inhibitor is 1000-3000:10-100. In some specific embodiments, the pharmaceutical composition comprises an EZH2 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor, and a CTLA-4 inhibitor, and the mass ratio of the EZH2 inhibitor to the PD-1 inhibitor or the PD-L1 inhibitor to the CTLA-4 inhibitor is 1000-3000:50-400:10-100. In some possible embodiments, the pharmaceutical composition comprises one or more of a PD-1 inhibitor or a PD-L1 inhibitor, an EZH2 inhibitor, a CTLA-4 inhibitor, and a VEGF inhibitor, wherein the mass ratio of the EZH2 inhibitor, the PD-1 inhibitor or the PD-L1 inhibitor, the CTLA-4 inhibitor, and the VEGF inhibitor is 1000-3000:50-400:10-100:300-1000. In some specific embodiments, the pharmaceutical composition comprises tazemetostat and pembrolizumab. In some specific embodiments, the pharmaceutical composition comprises tazemetostat and ipilimumab. In some specific embodiments, the pharmaceutical composition comprises tazemetostat, pembrolizumab, and ipilimumab. In some specific embodiments, the pharmaceutical composition comprises tazemetostat, pembrolizumab, ipilimumab, and bevacizumab.

[0019] In some specific embodiments, the pharmaceutical composition comprises an HDAC inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor, and the mass ratio of the HDAC inhibitor to the PD-1 inhibitor or the PD-L1 inhibitor is 100-200:50-400. In some specific embodiments, the pharmaceutical composition comprises an HDAC inhibitor and a CTLA-4 inhibitor, and the mass ratio of the HDAC inhibitor to the CTLA-4 inhibitor is 100-200:10-100. In some specific embodiments, the pharmaceutical composition comprises an HDAC inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor, and a CTLA-4 inhibitor, and the mass ratio of the HDAC inhibitor to the PD-1 inhibitor or the PD-L1 inhibitor to the CTLA-4 inhibitor is 100-200:50-400:10-100. In some possible embodiments, the pharmaceutical composition comprises one or more of a PD-1 inhibitor or PD-L1 inhibitor, an HDAC inhibitor, a CTLA-4 inhibitor, and a VEGF inhibitor, wherein the mass ratio of the HDAC inhibitor, the PD-1 inhibitor or PD-L1 inhibitor, the CTLA-4 inhibitor, and the VEGF inhibitor is 100-200:50-400:10-100:300-1000. In some specific embodiments, the pharmaceutical composition comprises panobinostat and pembrolizumab. In some specific embodiments, the pharmaceutical composition comprises panobinostat and ipilimumab. In some specific embodiments, the pharmaceutical composition comprises panobinostat, pembrolizumab, and ipilimumab. In some specific embodiments, the pharmaceutical composition comprises panobinostat, pembrolizumab, and ipilimumab. In some specific embodiments, the pharmaceutical composition comprises panobinostat, pembrolizumab, ipilimumab, and bevacizumab.

[0020] The present invention also provides a use of the pharmaceutical composition in the manufacture of an antitumor medicament. In some particular embodiments, the tumor is a central nervous system tumor. In some particular embodiments, the tumor is a glioma. In some particular embodiments, the tumor is a pediatric diffuse high-grade glioma. In some particular embodiments, the tumor is a diffuse midline glioma with H3K27 alterations. The names of these tumors are derived from the 2021 5th edition of the World Health Organization Classification of Tumors of the Central Nervous System.

[0021] The most common H3K27 alteration, H3K27M (i.e., a lysine to methionine mutation at position 27 of histone H3), is a typical "oncogenic histone" and can drive epigenetic reprogramming in diffuse midline gliomas with H3K27 alterations. The chromatin landscape in diffuse midline gliomas with H3K27 alterations is characterized by widespread loss of trimethylation and upregulation of acetylation at H3K27 sites. Acetylation at H3K27 sites is located in intergenic regions and can promote the expression of endogenous retroviral elements, which can be further upregulated by epigenetic drugs. Such "viral mimicry" may activate innate immune responses and promote immune activation. Therefore, the inventors have demonstrated that epigenetic immunotherapy, particularly the combination of epigenetic agents with immune checkpoint inhibitors, can provide survival benefit to glioma patients by treating diffuse midline gliomas with H3K27 alterations.

[0022] The results of the study showed that the recurrent thoracic spinal cord lesions were safely resected as much as possible for diffuse midline gliomas with H3K27 alterations in the thoracic spinal cord, and a treatment regimen of panobinostat, tazemetostat, pembrolizumab, ipilimumab, and bevacizumab was used. Prior to the data cutoff date (day 371), the volume of all tumor lesions in the patient sustained a decrease after treatment, with the volume of the suprasellar region (T1) and dorsal medulla oblongata (T2) lesions decreasing by 137 mm, respectively. 3 and 14mm 3 The results showed that the leptomeningeal / spinal cord and periventricular enhancement signals were no longer detectable by imaging in the enhanced MRI image on the 367th day. During the entire treatment course, the patient's quality of life was significantly improved, and he returned to daily life at the 11th course.

[0023] Cerebrospinal fluid (CSF) samples were collected from the patients on the 26th, 72nd, 169th, 321st, and 340th days of treatment, and proteome analysis was performed using mass spectrometry. A total of 1812 proteins were identified through the analysis. Among them, 1118 proteins were differentially expressed at different time points during treatment, and were classified into 20 clusters based on their expression patterns. Four clusters of proteins that showed the same change trend were studied intensively. As a result, the expression levels of proteins related to tumor angiogenesis, metastasis, and proliferation, such as VEGFA, collagen family, and laminin family, were significantly decreased from the 26th day of treatment. Proteins related to tumor invasion, metastasis, epithelial-mesenchymal transition, and stemness, such as MMP9, VIM, and NES, showed a stable decrease trend from the 72nd day of treatment. The protein expression levels of tumor-associated M2-like macrophage marker MRC1, cytotoxic T cell activation inhibitory regulatory protein VISG4, and immunosuppressive marker PLAUR in gliomas showed a marked downward trend after day 26 of treatment. Proteins ICAM1 and ICAM2 associated with immune cell migration and activation were markedly elevated after day 72 of treatment. The expression levels of proteins associated with immune activation and immune enhancement, such as SELL, LYZ, and ATRN, were also markedly elevated after day 169 of treatment and continued to increase until the final sampling time point.

[0024] After the data cut-off date, the patient's tumor progressed to two new sites. The patient's overall survival was 20 months, and her survival after receiving epigenetic drug immunotherapy was 16 months. This therapy was also applied to three other patients with diffuse midline gliomas with H3K27 alterations, all of which achieved significant clinical responses. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows the pathological diagnosis results for Patient 1. [Diagram 2] FIG. 2: Treatment regimens and tumor response to the treatment regimens. [Diagram 3]FIG. 3: Response of patient-derived tumor cells to epigenetic therapy. [Figure 4] FIG. 4. Molecular response of patient-derived tumor cells to epigenetic therapy. [Diagram 5] FIG. 5 shows intracranial LM imaging changes (sagittal plane). [Figure 6] FIG. 6 shows intracranial LM imaging changes (axial slice). [Figure 7] FIG. 7 shows intracranial LM imaging changes (coronal plane). [Figure 8] FIG. 8 shows the imaging changes (sagittal plane) of a thoracic spinal cord tumor. [Figure 9] FIG. 9 shows the neurocognitive function scores. [Figure 10] FIG. 10 shows the clustering tendency of differentially expressed proteins in cerebrospinal fluid. [Figure 11] FIG. 11 is the complete sequence of differentially expressed proteins in cerebrospinal fluid. [Figure 12] FIG. 12 shows the clinical outcome for patient 2. [Figure 13] FIG. 13 shows the clinical outcome for patient 3. [Figure 14] FIG. 14 shows the clinical effect of Patient 4. [Figure 15] FIG. 15 shows the clinical outcome of patient 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides pharmaceutical compositions and their uses, and those skilled in the art can refer to the contents of the present invention and improve the process parameters accordingly to achieve the present invention.All similar replacements and modifications are obvious to those skilled in the art, and are considered to be included in the present invention.The method and use of the present invention have been described in the preferred embodiments, but it will be obvious to those skilled in the art to make modifications to the method and use of the present invention, or to make suitable modifications and combinations to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention. EXAMPLES

[0027] Example 1 Materials and Methods Patient 1 was a 28-year-old male patient who was finally diagnosed with diffuse midline glioma with H3K27 alterations (see Table 1 for details) originating from the thoracic spinal cord on September 13, 2020. After tumor resection, no adjuvant treatment was administered, and the patient recovered well after surgery, with only minor neurological function damage. Three months after surgery, the patient developed symptoms of weakness in both lower limbs, hiccups, lethargy, and progressive deterioration. On January 11, 2021 (day 1), the patient was admitted to Beijing Tsinghua Chang Gung Hospital due to confusion and sudden loss of consciousness, with a Glasgow Coma Scale score of 8 / 15 at admission. Magnetic resonance imaging (MRI) showed that the primary thoracic spinal cord tumor had recurred, with leptomeningeal / meningeal seeding of the brain and spinal cord. A subtotal resection was performed for the recurrent thoracic spinal lesion, and a biopsy (on the fifth day) was performed for the dorsal medulla oblongata lesion. The pathology of both lesions was diagnosed as diffuse midline glioma (WHO level 4) with H3K27 changes. Referring to Figure 1, Figure 1 shows the pathological diagnosis results of Patient 1. Figure 1 shows representative staining results from the pathological diagnosis of the thoracic spinal recurrent tumor (TT, top) and the dorsal medulla oblongata leptomeningeal metastasis (LM, bottom). HE staining showed angiogenesis, hemorrhage around the tumor cells, and necrosis. The Ki-67 scales of both lesions were relatively high, with positive staining of tumor cell H3K27M and negative staining of H3K27me3 and IDH1. In Figure 1 , TT is thoracic tumor, LM is leptomeningeal metastasis tumor, HE is hematoxylin and eosin, H3K27me3 is trimethylation of H3K27, and IDH1 is isocitrate dehydrogenase 1.

[0028] Patient details [Table 1]

[0029] Treatment regimen The Ethics Committee of Beijing Tsinghua Chang Gung Hospital approved the patient's treatment regimen for the purpose of compassionate medication. The patient and his family signed a written informed consent. There was no business sponsorship or participation in the entire treatment process. The treatment regimen is shown in Figure 2. Figure 2(a) shows the ITK-SNAP 15 The changes in tumor volume were measured in MRI at three locations: the thoracic spinal cord, suprasellar region, and medulla oblongata. Figure 2(b) shows the timeline of treatment, including epigenetic drugs (panobinostat and tazemetostat), the VEGF inhibitor bevacizumab, the immune checkpoint inhibitors pembrolizumab and ipilimumab, radiation therapy, and tumor treatment field therapy. The timeline shows the time of first onset of treatment-related adverse events (see Table 2 for details), cerebrospinal fluid analysis, and MRI evaluation simultaneously. The timeline spans from January 11, 2021 (day 1), 4 months after diagnosis, to January 16, 2022 (day 371), and included 17 treatment cycles of 21 or 28 days. For ease of reference, the cerebrospinal fluid samples are labeled 15A‐E (Figure 2b) in order of collection time. Here, Pan is panobinostat, Taz is tazemetostat, Bev is bevacizumab, Pem is pembrolizumab, Ipi is ipilimumab, RT is radiation therapy, TTF is tumor treating field therapy, AE is adverse event, CSF is cerebrospinal fluid, MRI is magnetic resonance imaging, LM is leptomeningeal metastasis, VEGFA is vascular endothelial growth factor A, and ICB is immune checkpoint inhibitor.

[0030] Specifically, the treatment course consisted of a 3-week cycle, in which panobinostat was orally administered 3 times / week (days 1, 3, 5, 8, 10, and 12) at 20 mg / dose in the first and second weeks, and bevacizumab 10 mg / kg and pembrolizumab 200 mg were intravenously administered in the third week. Because the patient's condition was severe at the time of admission, bevacizumab treatment was administered once every 2 weeks in the first to fourth cycles of treatment. In the third to fifth cycles, the patient received whole-brain radiotherapy (41.4 Gy, 23 fractions) and localized enhanced radiotherapy in the sellar region (10 Gy, 5 fractions). The patient experienced grade 3 adverse events, including peripheral leukopenia, neutropenia, and weight loss, so oral administration of panobinostat (Figure 2(b) and Table 2) was temporarily suspended in the fifth cycle. In vitro tumor cell activity and invasion assays revealed that THG15 cells (thoracic spinal tumor from patient 1) were more sensitive to the combination of panobinostat and tazemetostat than either drug alone (see Figures 3 and 4). From cycle 8, tazemetostat 400 mg was orally administered, and tumor-treating field therapy was performed in parallel with panobinostat. In cycle 11, a satisfactory therapeutic effect was obtained and peripheral blood alkaline phosphatase was elevated, so the oral dose of panobinostat was reduced to 10 mg (Figure 2(a), day 199, Tables 2 and 5), but due to late progression of LM, the oral dose of panobinostat was returned to 20 mg (Figure 2, day 277, Table 5) in cycle 12. Ipilimumab (50 mg, intravenous administration) was incorporated into the treatment regimen in cycle 12. 1. EZH2 inhibitors may enhance the efficacy of anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors and help reverse resistance to immune checkpoint inhibitors; 2. Periventricular LM progressed slightly in cycle 11; 3. All lesions significantly shrank after adjusting the oral medication (Figure 2 and Table 5); 4. The combination of programmed cell death protein 1 (PD-1) and CTLA-4 inhibitors has been reported to increase immune-related adverse events, which may lead to the suspension or discontinuation of treatment. Due to patient fatigue (CTCAE2 level time), the start time of cycle 13 was postponed by 1 week, during which no drug treatment was administered, and tazemetostat was reduced to 200 mg from cycle 13.The 3-week treatment regimen was extended to 4 weeks from cycle 17. Data collection deadline was day 371 (April 12, 2022).

[0031] Safety and Adverse Events Ten grade 1 to 3 adverse events potentially related to treatment are listed in Table 2. Grade 3 adverse events included anemia, leukopenia, neutropenia, and weight loss. All of these adverse events were alleviated after transfusion or discontinuation of panobinostat. Low-grade adverse events included diarrhea, fatigue, thrombocytopenia, and elevated transaminases and alkaline phosphatases. There were no grade 4 adverse events or treatment discontinuations due to adverse events throughout the course.

[0032] Treatment-related adverse events [Table 2]

[0033] Pathological diagnosis Formalin-fixed, paraffin-embedded pathological tissue was sectioned and then subjected to immunohistochemical staining using hematoxylin and eosin (HE). For specific procedures, refer to Yao, J., Wang, L., Ge, H., Yin, H. & Piao, Y. Diffuse midline glioma with H3 K27M mutation of the spinal cord: A series of 33 cases. Neuropathology 41, 183-190, doi:10.1111 / neup.12714 (2021). For sources of anti-Ki-67, anti-H3K27M, anti-H3K27me3, anti-isocitrate dehydrogenase 1 (IDH1) R132H mutant and secondary antibodies, see Yao, J., Wang, L., Ge, H., Yin, H. & Piao, Y. Diffuse midline glioma with H3 K27M mutation of the spinal cord: A series of 33 cases. Neuropathology 41, 183-190, doi:10.1111 / neup.12714(2021). The immune response was detected using a Leica Bond automated stainer.

[0034] Culturing patient-derived tumor cells After obtaining permission from the Ethics Committee of Beijing Tsinghua Chang Gung Hospital and consent from the patient and family, patient-derived tumor cells were collected. The surgical specimen was derived from the thoracic spinal tumor of patient 1 and named THG15 (Tsinghua Glioma 15). The specific procedure was as follows: Zhang, W. et al. Combination of oncolytic herpes simplex viruses armed with angiostatin and IL-12 enhances antitumor efficacy in human glioblastoma models. Neoplasia 15, 591-599, doi:10.1593 / neo.13158(2013) and Chen, WJ et al. The different role of YKL-40 in glioblastoma is a function of MGMT promoter methylation status. Cell Death Dis 11, 668, doi:10.1038 / s41419-020-02909-9(2020). Briefly, tissues were mechanically cut and digested with 0.1% trypsin (Hyclone) and 10 U / ml DNasel (Roche) at 37°C for 45 min to lyse red blood cells. After several washes, the tissues were passed through a 100 μm cell filter, and the cells that passed through the filter were collected and added to EF medium. The specific medium formulation was Neurobasal medium, 3 mM L-glutamine, 1× B27 supplement, 0.5× N2 supplement, 2 μg / mL heparin, 20 ng / ml recombinant human EGF (Sigma-Aldrich), 20 ng / ml recombinant human FGF2 (Sigma-Aldrich), and 0.5× double antibody complex. Fresh EF medium was replenished every 3 days during cell culture. Tumor cell spheroids were dissociated and passaged using the NeuroCult Chemical Dissociation Kit (StemCell).

[0035] Cell viability test Panobinostat (LBH589) and tazemetostat (EPZ-6438) were purchased from Selleck Chemicals and dissolved in 0.1% DMSO before use. Approximately 5000 THG15 cells were seeded in each well of a 96-well plate and incubated in EF medium for 24 h before pharmaceutical intervention, followed by incubation with different concentrations of panobinostat, tazemetostat, or both drugs for 72 h, resulting in a complex number of three wells. Cell viability was measured using a CCK-8 kit (GK10001) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a SPARK detector (TECAN). To examine the inhibitory effect of each intervention, standard dose-response curves with a Hill slope of -1.0 were constructed after comparing drug-intervention cell viability with that of unintervention cells, and the corresponding half maximal inhibitory concentration (IC50) was calculated on Prism 9 (GraphPad) based on the results of two separate experiments.

[0036] Inspection of the Transwell migration chamber Approximately 5×10 5 THG15 cells were resuspended in 100 μL serum-free DMEM medium and inoculated into the upper chamber of each Transwell of a 24-well plate, and DMEM + 20% FBS (Gibco) was added to the lower chamber. At the same time, 1 μM panobinostat, 50 μM tazemetostat, or a combination of both drugs were administered as intervention groups in triplicates. After 48 h of culture, cells were fixed with 4% paraformaldehyde (Solarbio) and stained with 1% crystal violet (Solarbio). The upper surface of the upper chamber of the Transwell was gently rubbed with a cotton swab to remove cells that did not pass through the membrane. The remaining cells were observed under an inverted fluorescence microscope (Ts2R-FL, Nikon). The average of three random views was taken and the relative area of ​​stained cells was quantified with ImageJ.

[0037] Western Blot Test To analyze epigenetic changes in tumor cells following pharmaceutical intervention, 5 × 10 5THG15 cells were inoculated and incubated with 1 μM panobinostat or 50 μM tazemetostat, or both, for 48 h in triplicate in 6-well plates. The status of cells after pharmacological intervention was observed in the bright field under an inverted fluorescence microscope (Ts2R-FL, Nikon), and representative images were taken with NIS Elements (Nikon) using autoexposure and autowhite balance. Cells were lysed in RIPA buffer (Solarbio), and samples were prepared by diluting 5× SDS-PAGE sample loading buffer (Beyotime) with PBS (Solarbio) 4:1. Total protein levels of samples were semiquantified on SurePAGE gels® (Genscript, Nanjing, China) using eStainTML1 protein staining system (Genscript). After adjusting the sample volume according to the total protein level, electrophoresis was performed on 20% Bis-Tris gels (Genscript). The isolated proteins were transferred onto Immobilon®-P PVDF membranes (Millipore) using the eBlot® L1 Rapid Wet Transfer System (Genscript). Blocking was performed with 5% nonfat dry milk prepared in TBST consisting of 1× TBS (Lablead) and 0.1% TWEEN20 (VWR), followed by incubation with anti-H3K27Me3, anti-H3K27Ac, ​​anti-H3K27M or anti-H3 antibodies. After completion of secondary antibody incubation, the blots were detected with the SuperSignal™ West Pico PLUS chemiluminescence system (Thermo Fisher Scientific) and imaged with an ImageQuant LAS 4000 (GE). The relative intensity of the blots was quantified by ImageJ densitometry.

[0038] Magnetic resonance imaging and tumor volume measurement Brain and spinal cord images were acquired with a 3.0 Tesla MRI and included T1-weighted, T2-weighted, and gadolinium-injected T1-weighted sequences. Tumor contours were manually sketched on the enhanced T1-weighted MRI images by two neuroimaging specialists independently, and tumor volumes were calculated using ITK-SNAP. The calculation method was adapted from Yushkevich, PA et al. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage 31, 1116-1128, doi:10.1016 / j.neuroimage.2006.01.015 (2006).

[0039] Assessment of health-related quality of life Patients' quality of life was assessed according to two quality of life questionnaires (QLQ) developed by the European Organization for Research and Treatment of Cancer. The core QLQ-C30 includes a five-item functioning scale, one general health and quality of life scale, three multi-item symptom scales, five single-item symptom scales, and one subjective scale of economic burden of disease treatment. The brain tumor-specific index, QLQ-BN20, includes four multi-item scales and seven single-item scales. To facilitate interpretation, the original scales and measurement scores were linearly converted to a standard scale spanning from 0 to 100, in which higher scores correspond to higher health functioning or levels. All scales and indices, except for the physiological functioning scale, were scaled based on the average health status and quality of life in the week prior to data collection. The results are shown in Tables 3 and 4. Table 3 shows the results of the QLQ-C30 evaluation. Table 4 shows the results of the QLQ-BN20 evaluation.

[0040] QLQ-C30 evaluation results [Table 3]

[0041] QLQ-BN20 evaluation results [Table 4]

[0042] Analysis of the cerebrospinal fluid proteome After collecting cerebrospinal fluid samples, high abundance proteins were first removed using High Select® Top14 High Abundance Protein Consuming Resin (Thermo Fisher Scientific). The purified proteins were separated using a Thermo Scientific UltiMate® 3000 RSLCnano system and mass analyzed using FAIMS PulseDIA and Orbitrap Exploris 480. For FAIMS experiments, optimal compensation voltages (CV) were -48V and -68V, respectively, with a cycle time of 1s. MS1 resolution was 60000, with a normalized AGC target of 300%. Mass range was 390-1010Th. Normalized AGC target was 200% with 12 windows of resolution 30000 after one full scan. For the first partial method, mass ranges were 520-630Th at -68V and 630-750Th at -48V. In the second partial method, the mass range was 400-520Th at -68V and 750-1000Th at -48V. The normalized HCD collision energy was 32%. The background was a human FASTA file downloaded from the UniProt proteome dataset on January 26, 2020. Database searches were performed using DIA-NN (version 1.7.8). The original file was saved in iProX:IPX0005197001.

[0043] statistical analysis One-way analysis of variance (ANOVA) was employed to determine the statistical differences between protein levels at different times, and the Benjamini & Hochberg method was employed to adjust the p-values. Differentially expressed proteins (ANOVA, adjusted p<0.05) during the treatment process were grouped into 20 groups according to their expression profiles using the Mfuzz package.

[0044] During the first cycle of treatment, the patient had a temporary return to drowsiness after surgical resection of the primary tumor recurrence in the thoracic spinal cord, but 10 days later, the patient reported that the volume of metastatic disease in the sellar region had decreased from baseline to 1905 mm 3 From 2606mm 3 The patient's electrolyte and fluid imbalance was completely corrected after two cycles of treatment. From the third cycle of treatment, the patient's consciousness was fully restored, and her neurocognitive function was also fully improved (see Figure 9 and Table 4). At the end of the sixth cycle of radiation therapy, the volume of all the patient's lesions had significantly decreased, with T1 and T2 decreasing to 1905 mm from the baseline volume, respectively. 3 and 829mm 3 From 1615mm on the 122nd day of treatment 3 and 265mm 3 The volume of periventricular enhancement was reduced to 100%. Interestingly, in cycle 11 (day 227), a decrease in the dose of panobinostat in the same cycle caused a slight increase in the periventricular enhancement foci on the images without any new neurological dysfunction, but an increase in the dose in cycle 12 (day 256) reduced the volume of the increased foci (see Figures 2a, 5-7, and Table 5). As the treatment progressed, the proteins in the cerebrospinal fluid were classified into 20 clusters (cluster features are shown in Figure 10) according to their differential expression trends. The heat map shows proteins that showed a tendency of changing in the same direction in the internal expression levels in the cerebrospinal fluid samples taken at different time points of treatment.

[0045] The specific results are as follows: Figure 3 shows the response of patient-derived tumor cells to epigenetic therapy. Figure 3(a) shows dose-response curves showing the change in tumor cell viability after intervention with panobinostat, tazemetostat or a combination of both for 72 hours. Results are shown as the mean ± standard deviation (n=6) of cell viability from two independent measurements. Drug vehicle 0.1% DMSO was not found to affect cell viability (data not shown). Comparison of IC50 values ​​between drugs was performed using an additive sum of squares F test. * indicates p<0.05, *** indicates p<0.001. The left side of Figure 3(a) shows the microscopic tumor cell viability after intervention with 1 μM panobinostat, 50 μM tazemetostat (close to IC50 estimates for both) or a combination of both for 48 hours. Figure 3(b) shows the effect of panobinostat and tazemetostat on tumor cell migration in Transwell. Tumor cells were treated with 1 μM panobinostat, 50 μM tazemetostat, or a combination of both for 48 hours, after which the tumor cells that had migrated through the transmembrane were stained and observed under a microscope. The area of ​​stained cells relative to the field of view was quantified using ImageJ, and the average of three random views was taken. The bar graph results are expressed as the mean ± standard deviation (n = 6). For unpaired t-tests, *** indicates p < 0.001. DMSO is dimethyl sulfoxide, Pan is panobinostat, Taz is tazemetostat, and IC50 is the half maximal inhibitory concentration.

[0046] Figure 4 shows the molecular response of patient-derived tumor cells to epigenetic therapy. Figure 4(a) shows the expression of H3K27me3 and H3K27ac in patient-derived tumor cells after intervention with DMSO, 1 μM panobinostat, 50 μM tazemetostat, or a combination of both for 48 h. The expression levels of tumor cells H3K27me3 and H3K27ac after drug intervention relative to the DMSO-treated reference group are shown in Figures 4(b) and 4(c), respectively. The t-test showed that the combined intervention of panobinostat and tazemetostat significantly decreased the expression of tumor cells H3K27me3 and significantly increased the expression of H3K27ac (n=4; NS stands for non-significant; ** stands for p<0.01). The expression levels of H3 and H3K27M in tumor cells were measured by Western blot (a). DMSO stands for dimethyl sulfoxide, Pan stands for panobinostat, Taz stands for tazemetostat, H3K27me3 stands for H3K27 trimethylation, and H3K27ac stands for H3K27 acetylation.

[0047] Figure 5 shows intracranial LM imaging changes (sagittal plane). T1-weighted sagittal plane MRI images are shown in the figure, and the dates of image acquisition are indicated by the labels in the upper left of the figure. Arrows indicate multiple leptomeningeal linear enhancing foci. T1 and T2 show the suprasellar region LM and medullary tumor at baseline (day 2). Axial and coronal images are shown in Figures 6 and 7.

[0048] Figure 6 shows intracranial LM imaging changes (axial slices). T1-weighted axial MRI images are shown in the figures, and the dates of image acquisition are indicated by labels in the upper left corner of the figures. Arrows indicate multiple leptomeningeal linear enhancing foci. T1 represents the suprasellar region LM at baseline (day 2). Sagittal and coronal images are shown in Figures 5 and 7.

[0049] Figure 7 shows intracranial LM imaging changes (coronal plane). T1-weighted coronal MRI images are shown in the figure, and the dates of image acquisition are indicated by the labels in the upper left of the figure. The arrows on the image at baseline (day 2) indicate multiple leptomeningeal linear enhancing foci and periventricular enhancing foci. Sagittal and axial images are shown in Figures 5 and 6.

[0050] Figure 8 shows the radiological changes of thoracic spinal cord tumors (sagittal plane). T1-weighted and T2-weighted MRI images are shown as above, and the dates of image acquisition are indicated by the labels in the upper left corner of the figure. T3 shows the recurrent thoracic spinal cord tumor lesions at baseline (day 2). Radiological tumor volume measurement data are shown in Table 5.

[0051] Imaging tumor volume measurement data (mm 3 ) [Table 5]

[0052] From Table 5, by the data cutoff date (day 371), the volume of all tumor lesions in the patient sustained a reduction after treatment, with the suprasellar region (T1) and dorsal medulla oblongata (T2) lesions each shrinking by 137 mm 3 and 14mm 3 The volume was found to have decreased by more than 94% compared to baseline. Enhanced MRI images on day 367 suggested that the leptomeningeal / spinal cord and periventricular enhancement signals were no longer detectable by imaging (Figures 5-8). Throughout the course of treatment, the patient's quality of life improved significantly, and he returned to daily life (Figure 9 and Table 4) after the 11th course.

[0053] Figure 9 shows the scores of neurocognitive function. The neurocognitive function of the patients was evaluated by the Mini-Mental State Examination (MMSE). The score range was 0 to 30 points, and a score of 27 points or more was evaluated as normal.

[0054] Figure 10 shows the cluster trend of differentially expressed proteins in cerebrospinal fluid. After removing high abundance proteins, Mfuzz was used to classify the differentially expressed proteins in cerebrospinal fluid during the treatment process into 20 clusters (ANOVA, corrected p<0.05) according to their relative expression patterns. The change trend of the relative expression of proteins in all 20 clusters is shown in Figure 10.

[0055] Figure 11 shows the complete sequence of differentially expressed proteins in CSF. Figure 11a-d shows heat maps of relative protein expression in CSF samples at various time points during the treatment course (sampling times are shown in Figure 2(b)). Differentially expressed proteins during the treatment course were classified into 20 clusters (ANOVA, adjusted p<0.05) according to their expression profiles (cluster features are shown in Figure 9) using Mfuzz.

[0056] Cerebrospinal fluid (CSF) samples were collected from the patients on the 26th, 72nd, 169th, 321st, and 340th days of treatment, and proteome analysis was performed using mass spectrometry. A total of 1812 proteins were identified through the analysis. Among them, 1118 proteins were differentially expressed at different time points of treatment, and were classified into 20 clusters (Figures 10 and 11) according to their expression patterns. Four clusters of proteins with the same change tendency were studied in particular. The results showed that the expression levels of proteins related to tumor angiogenesis, metastasis, and proliferation, such as VEGFA, collagen family, and laminin family, were significantly decreased from the 26th day of treatment. Proteins related to tumor invasion, metastasis, epithelial-mesenchymal transition, and stemness, such as MMP9, VIM, and NES, showed a stable decreasing trend from the 72nd day of treatment. Interestingly, the expression level of the protease inhibitor PZP increased significantly after the 26th day of treatment, while the expression levels of proteasome subunits such as PSMA3, PSMB3, and PSMB8 decreased significantly after the 72nd day of treatment. This result suggests that the remarkable therapeutic effect of the patient may be related to the protease inhibition effect that was not achieved by the use of the proteasome inhibitor Marizomib. Furthermore, the expression levels of the tumor-associated M2-like macrophage marker MRC1, the cytotoxic T cell activation inhibitory regulator protein VISG4, and the immunosuppressive marker PLAUR in gliomas showed a tendency to decrease significantly after the 26th day of treatment. The proteins ICAM1 and ICAM2, which are related to the migration and activation of immune cells, increased significantly after the 72nd day of treatment. The expression levels of proteins related to immune activation and immune enhancement, such as SELL, LYZ, and ATRN, also increased significantly after the 169th day of treatment and continued to increase until the final sampling time point.

[0057] After the data cut-off date, the patient's tumor progressed to two new sites.The patient's overall survival was 20 months, and her survival after receiving epigenetic agent immunotherapy was 16 months. Example 2

[0058] Patient 2 was a 53-year-old man with sudden paraplegia, paresthesia, and urinary incontinence as the main clinical manifestations. The findings of the tumor on T1-weighted and T2-weighted MRI images are shown in Figure 12. Figure 12 shows the clinical outcome of Patient 2. Please refer to the labels above the images for the dates of image acquisition. Diffuse midline glioma with H3K27 alterations in the cervical spinal cord was confirmed after tumor biopsy at another hospital. The patient was admitted to Beijing Tsinghua Chang Gung Hospital (on day -5, arrows indicate the tumor focus on T1-weighted images and the extent of the focus and surrounding edema on T2-weighted images) for the progression of tumor imaging and clinical symptoms. The patient received radiotherapy (45 Gy, 25 fractions) at Beijing Tsinghua Chang Gung Hospital, and was administered the VEGF inhibitor bevacizumab (10 mg / kg) as well as epigenetic immunotherapy with tazemetostat and panobinostat (panobinostat 20 mg and tazemetostat 400 mg were orally administered on Mondays, Wednesdays, and Fridays for 2 consecutive weeks, and ipilimumab 50 mg + pembrolizumab 100 mg were administered intravenously after discontinuation of panobinostat and tazemetostat on the third week) for 3 weeks. A discectomy was performed on the 71st day. After treatment, the patient's lower limb motor and sensory functions were significantly improved, and bladder control was restored. Corresponding to these improvements in clinical symptoms, a significant reduction in cervical spinal cord tumor volume was observed on imaging (day 415). The patient survived for more than 16 months after diagnosis and for more than 15 months after epigenetic drug immunotherapy, with last follow-up in October 2022. Example 3

[0059] Patient 3 was a 25-year-old woman with the main clinical manifestations of right-sided hemiplegia and aphasia. The findings of the tumor on T1-weighted MRI images are shown in Figure 13. Figure 13 shows the clinical response of Patient 3, and the dates of image acquisition are shown in the labels above the images. The patient's left thalamic diffuse midline glioma with H3K27 alterations was confirmed after tumor biopsy at another hospital (day -587, the primary tumor is indicated by a circle), and she received standard chemoradiotherapy at another hospital, but the patient's neurological condition continued to deteriorate, and she underwent radiation therapy again, but still without any clinical and imaging improvement. The baseline MRI image at Beijing Tsinghua Chang Gung Hospital shows the progression of the left thalamic tumor (day 1). Patient 3 was then administered the VEGF inhibitor bevacizumab (10 mg / kg) and an epigenetic drug immunotherapy with tazemetostat and panobinostat for 3 or 4 weeks (panobinostat 20 mg and tazemetostat 400 mg were orally administered on Mondays, Wednesdays, and Fridays for 2 consecutive weeks, and on the third week, panobinostat and tazemetostat were discontinued, followed by ipilimumab 50 mg + pembrolizumab 100 mg intravenously). After treatment, the patient's right-sided motor function improved significantly and he was able to walk independently. After treatment, his language ability improved significantly in terms of naming and fluency, and he enjoyed speaking. Corresponding to the improvement of clinical symptoms after treatment, a significant reduction in the tumor volume of the left thalamus was observed on images (day 157). Example 4

[0060] Patient 4 was a 44-year-old man with the main clinical manifestation being weakness of the left limbs. The findings of the tumor on T1-weighted MRI images are shown in Figure 14. Figure 14 shows the clinical response to treatment for Patient 4, and the dates of image acquisition are shown in the labels above the images. A diffuse midline glioma with right thalamic H3K27 alterations was diagnosed after tumor resection at another hospital (the primary tumor is indicated by a circle on the preoperative image on day -32, and the postoperative image on day -17). Patient 4 was transferred to Beijing Tsinghua Chang Gung Hospital (day -4) due to the deterioration of tumor imaging and clinical condition, and received radiotherapy (54 Gy, 27 fractions), tumor treatment field therapy, VEGF inhibitor bevacizumab (10 mg / kg), and epigenetic drug tazemetostat and panobinostat immunotherapy (panobinostat 20 mg and tazemetostat 400 mg were orally administered on Mondays, Wednesdays, and Fridays for 2 consecutive weeks, and pembrolizumab 200 mg was intravenously administered after discontinuing panobinostat and tazemetostat on the third week). After treatment, the patient's left motor function was significantly restored, his quality of life was significantly improved, and he was able to perform normal daily activities such as mountain climbing and various social activities. Corresponding to the improvement of these clinical symptoms, a significant reduction in tumor volume in the right thalamus was observed on imaging (day 228). Example 5

[0061] Patient 5 was a 25-year-old man with progressive weakness of the left upper limb and neck pain as clinical manifestations. The findings of T1-weighted and T2-weighted MRI images of the three tumors (T1-T3) are shown in Figure 15. Figure 15 shows the clinical treatment effect of Patient 5, and the dates of image acquisition are shown in the labels above the images. A diffuse midline glioma with H3K27 alterations in the cervical spinal cord was first diagnosed after tumor resection at a hospital in Germany in September 2017, and standard chemoradiotherapy was performed after surgery. As the tumor recurred, the patient underwent a second surgical treatment in Germany in February 2019, with the pathological diagnosis being the same as before. After the second surgery, the patient did not receive standard adjuvant therapy, and from May 2019 to March 2020, he participated in the US adult ONC201 clinical trial for recurrent H3K27M-mutated gliomas. In September 2020, patient 5 was admitted to Beijing Tsinghua Chang Gung Hospital with paraplegia, urinary and fecal incontinence, and right-sided facial paralysis. MRI showed recurrent cervical spinal cord tumor (T1) with multiple brain metastases (T2 and T3) (day -56). The patient underwent subtotal resection of cervical spinal cord tumor on day -38, but the intracranial metastases progressed significantly after the third operation, and the neurological symptoms also worsened (day 0). The patient received a 3-week course of combination treatment with panobinostat and bevacizumab (panobinostat 20 mg orally on Mondays, Wednesdays, and Fridays for 2 consecutive weeks, and bevacizumab 10 mg / kg intravenously after discontinuing panobinostat on the third week). After two courses, the T2 and T3 brain metastases had significantly shrunk (day 49), accompanied by a significant improvement in the neurological symptoms. However, brain metastases progressed after the fourth course. Patient 5's MRI scan on day 113, the last imaging study before death on day 163, showed increasing brain metastatic tumor burden. Patient 5 died 43 months after being diagnosed with diffuse midline glioma with H3K27 alterations, 5 months after treatment with panobinostat plus bevacizumab.The therapeutic effect of this patient suggests that the combination of panobinostat and bevacizumab has a certain therapeutic effect in the treatment of diffuse midline glioma with H3K27 alterations, but cannot significantly inhibit the intracranial metastasis of the patient's intramedullary tumor. Compared with the above patients 1 to 4, the clinical therapeutic effect of the combination of panobinostat, tazemetostat, pembrolizumab, ipilimumab and bevacizumab is significantly superior to that of the combination of panobinostat and bevacizumab.

[0062] Although the above are only preferred embodiments of the present invention, still, those skilled in the art may make some improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition comprising an HDAC inhibitor and an EZH2 inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that the mass ratio of the HDAC inhibitor to the EZH2 inhibitor is 100-200:1000-4000.

3. The HDAC inhibitor is one or more selected from the group consisting of panobinostat, vorinostat, romidepsin, belinostat, and tucidinostat; The EZH2 inhibitor is tazemetostat. The pharmaceutical composition according to claim 1 or 2.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a VEGF inhibitor.

5. The PD-1 inhibitor is one or more selected from the group consisting of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, camrelizumab, zimberelimab, penprimab, serplulimab, and pucotenlimab; The PD-L1 inhibitor is one or more selected from the group consisting of atezolizumab, durvalumab, embafolimab, sugemalimab, and avelumab; The VEGF inhibitor is one or more selected from the group consisting of bevacizumab, ramucirumab, ranibizumab, and aflibercept; The CTLA-4 inhibitor is one or more selected from the group consisting of ipilimumab and tremelimumab; The pharmaceutical composition according to claim 4 .

6. further comprising pembrolizumab, The pharmaceutical composition according to claim 5, wherein the mass ratio of the HDAC inhibitor to the pembrolizumab is 100-200:50-400.

7. Further comprising ipilimumab, The pharmaceutical composition according to claim 6, characterized in that the mass ratio of the HDAC inhibitor to the ipilimumab is 100-200:10-100.

8. further comprising bevacizumab, The pharmaceutical composition according to claim 7, characterized in that the mass ratio of said HDAC inhibitor to said bevacizumab is 100-200:300-1000.

9. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the manufacture of an antitumor medicament.

10. The use according to claim 9, characterized in that the tumor is a central nervous system tumor.

11. The use according to claim 10, characterized in that the tumor is a glioma.

12. The use according to claim 11, characterized in that the tumor is a pediatric diffuse high-grade glioma.

13. The use according to claim 12, characterized in that the tumor is a diffuse midline glioma with H3K27 alterations.

Images