Pharmaceutical composition and application thereof

Abstract

The invention relates to a pharmaceutical composition, which comprises panobinostat and tazeostat. The EZH2 inhibitor tazeostat is combined with panobinostat, palbolizumab and ipimumab for use, is clinically used for treating patients with advanced diffuse glioma accompanied with H3K27 change and patients with primary, relapse or progression diffuse glioma accompanied with H3K27 change, has a remarkable curative effect, and can be used for treating patients with primary, relapse or progression diffuse glioma accompanied with H3K27 change. Tumor growth can be remarkably inhibited, the tumor size is reduced, the living quality of a patient is improved, and the lifetime of the patient is prolonged.

Description

Pharmaceutical composition and its application Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a pharmaceutical composition and application thereof. Background Art

[0002] Diffuse midline glioma with H3K27 alteration (Diffuse midline glioma, H3 K27-altered) is derived from the 2021 fifth edition of the World Health Organization Classification of Central Nervous System Tumors. It is a type of pediatric diffuse high-grade glioma that is prone to occur in the midline part of the central nervous system, often involving the thalamus, pons, spinal cord and other parts. Surgical resection is extremely difficult, and most patients undergo biopsy. So far, neither traditional treatment, targeted therapy nor immunotherapy has been able to prolong the patient's overall survival. Internationally, there are no treatment guidelines, so effective treatment options are urgently needed. The prognosis of patients with diffuse midline glioma with H3K27 alteration is extremely poor, with a 2-year survival rate of less than 10%. It is particularly noteworthy that cases of diffuse midline glioma with H3K27 alteration that originate in the spinal cord and are accompanied by leptomeningeal / meningeal dissemination are even rarer. Historically, patients with high-grade gliomas with leptomeningeal / meningeal spread were often excluded from clinical studies due to their worse prognosis, with a median survival of only 1.6-3.8 months.

[0003] Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a pharmaceutical composition and its application. The pharmaceutical composition provided in this application can be used to treat diffuse midline glioma with H3K27 changes, which can inhibit tumor growth, reduce tumor volume, significantly improve the patient's quality of life, and prolong the patient's survival.

[0005] The present invention provides a pharmaceutical composition comprising: an HDAC inhibitor and an EZH2 inhibitor.

[0006] The inventors of this application have creatively discovered that the combination of a histone deacetylase inhibitor (HDAC) and an EZH2 inhibitor has significant clinical efficacy in treating tumors, especially diffuse midline gliomas with H3K27 alterations. In some specific implementations, the mass ratio of the HDAC inhibitor to the EZH2 inhibitor is 100-200:1000-3000, preferably 100-200:1200-2800, and more preferably 100-200:1500-2500.

[0007] In some specific implementations, the HDAC inhibitor includes, but is not limited to, one or more of panobinostat, vorinostat, romidepsin, belinostat, and chidamide, preferably panobinostat. Panobinostat, also known as panobinostat in English, is a drug approved by the U.S. Food and Drug Administration in 2015 for the treatment of multiple myeloma. Studies have shown that panobinostat can penetrate the blood-brain barrier and is a promising drug for the treatment of diffuse midline gliomas with H3K27 alterations. However, panobinostat did not significantly prolong the six-month progression-free survival of patients with recurrent glioblastoma and anaplastic glioma.

[0008] In some specific implementation schemes, the EZH2 inhibitor includes but is not limited to tazemetostat. Tazemetostat, also known as Tazemetostat in English, is a small molecule EZH2 inhibitor. It was approved by the U.S. Food and Drug Administration in 2020 for the treatment of epithelioid sarcoma. No clinical studies have yet been conducted in glioma patients. The inventors of this application have creatively discovered that EZH2 inhibitors can be used as a type of epigenetic drug to treat patients with diffuse midline gliomas with H3K27 changes in clinical practice, especially tazemetostat in combination with panobinostat for the treatment of patients with advanced diffuse midline gliomas with H3K27 changes and associated leptomeningeal / meningeal dissemination, as well as primary, recurrent or progressive diffuse midline gliomas with H3K27 changes. It has significant therapeutic effects, can significantly inhibit tumor growth, reduce tumor volume, improve the quality of life of patients, and prolong the survival of patients.

[0009] In some specific implementations, the composition comprises panobinostat and tazemetostat in a mass ratio of 100-200:1000-3000, preferably 100-200:1200-2800, and more preferably 100-200:1500-2500.

[0010] In some specific implementations, 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.

[0011] In some specific implementations, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, camrelizumab, zimberelimab, penpulimab, serplulimab or pucotenlimab, preferably pembrolizumab.

[0012] In some specific implementations, the PD-L1 inhibitor is selected from one or more of Atezolizumab, Durvalumab, Envafolimab, Sugemalimab, or Avelumab.

[0013] In some specific implementations, the CTLA-4 inhibitor is selected from one or more of Ipilimumab and Tremelimumab, preferably Ipilimumab.

[0014] In some specific implementations, the VEGF inhibitor is selected from one or more of bevacizumab, ramucirumab, lucentis or aflibercept, preferably bevacizumab.

[0015] In some specific implementations, the pharmaceutical composition further comprises pembrolizumab, a PD-1 inhibitor among immune checkpoint inhibitors. The mass ratio of panobinostat to pembrolizumab is 100-200:50-400, preferably 100-200:100-350, and more preferably 100-200:150-300.

[0016] In some specific implementations, the pharmaceutical composition also includes ipilimumab, a CTLA-4 inhibitor among immune checkpoint inhibitors. The inventors of this application have discovered that combining pembrolizumab and ipilimumab with panobinostat and tazemetostat for the treatment of diffuse midline gliomas with H3K27 alterations has significant efficacy, inhibiting tumor growth, reducing tumor volume, significantly improving patient quality of life, and prolonging patient survival. The mass ratio of panobinostat to ipilimumab is 100-200:10-100, preferably 100-200:20-80.

[0017] In some specific implementations, the pharmaceutical composition further comprises bevacizumab, a recombinant humanized monoclonal antibody targeting VEGF. In some specific implementations, the mass ratio of panobinostat to bevacizumab is 100-200:300-1000, preferably 100-200:350-800, and more preferably 100-200:400-700.

[0018] In some specific implementations, the pharmaceutical composition includes panobinostat, tazemetostat, and pembrolizumab. In some specific implementations, the pharmaceutical composition includes panobinostat, tazemetostat, and ipilimumab. In some specific implementations, the pharmaceutical composition includes panobinostat, tazemetostat, pembrolizumab, and ipilimumab. In some specific implementations, the pharmaceutical composition includes panobinostat, tazemetostat, pembrolizumab, and ipilimumab. In some specific implementations, the pharmaceutical composition includes panobinostat, tazemetostat, pembrolizumab, ipilimumab, and bevacizumab.

[0019] In some specific implementations, the pharmaceutical composition comprises an EZH2 inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor, wherein 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 implementations, the pharmaceutical composition comprises an EZH2 inhibitor and a CTLA-4 inhibitor, wherein the mass ratio of the EZH2 inhibitor to the CTLA-4 inhibitor is 1000-3000:10-100. In some specific implementations, the pharmaceutical composition comprises an EZH2 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor, and a CTLA-4 inhibitor, wherein the mass ratio of the EZH2 inhibitor, the PD-1 inhibitor or the PD-L1 inhibitor, and the CTLA-4 inhibitor is 1000-3000:50-400:10-100. In some possible implementations, the pharmaceutical composition includes one or more of an EZH2 inhibitor, a PD-1 inhibitor or a PD-L1 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 implementations, the pharmaceutical composition includes tazemetostat and pembrolizumab. In some specific implementations, the pharmaceutical composition includes tazemetostat and ipilimumab. In some specific implementations, the pharmaceutical composition includes tazemetostat, pembrolizumab, and ipilimumab. In some specific implementations, the pharmaceutical composition includes tazemetostat, pembrolizumab, ipilimumab, and bevacizumab.

[0020] In some specific implementations, the pharmaceutical composition comprises an HDAC inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor, wherein 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 implementations, the pharmaceutical composition comprises an HDAC inhibitor and a CTLA-4 inhibitor, wherein the mass ratio of the HDAC inhibitor to the CTLA-4 inhibitor is 100-200:10-100. In some specific implementations, the pharmaceutical composition comprises an HDAC inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor, and a CTLA-4 inhibitor, wherein the mass ratio of the HDAC inhibitor, the PD-1 inhibitor or the PD-L1 inhibitor, and the CTLA-4 inhibitor is 100-200:50-400:10-100. In some possible implementations, the pharmaceutical composition includes one or more of an HDAC inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor, a CTLA-4 inhibitor, and a VEGF inhibitor, wherein the mass ratio of the HDAC inhibitor, the PD-1 inhibitor or the PD-L1 inhibitor, the CTLA-4 inhibitor, and the VEGF inhibitor is 100-200:50-400:10-100:300-1000. In some specific implementations, the pharmaceutical composition includes panobinostat and pembrolizumab. In some specific implementations, the pharmaceutical composition includes panobinostat and ipilimumab. In some specific implementations, the pharmaceutical composition includes panobinostat, pembrolizumab, and ipilimumab. In some specific implementations, the pharmaceutical composition includes panobinostat, pembrolizumab, ipilimumab, and bevacizumab.

[0021] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of an anti-tumor drug. In some specific implementations, the tumor is a central nervous system tumor. In some specific implementations, the tumor is a glioma. In some specific implementations, the tumor is a pediatric diffuse high-grade glioma. In some specific implementations, the tumor is a diffuse midline glioma with H3K27 alterations. These tumor names are derived from the 2021 fifth edition of the World Health Organization Classification of Tumors of the Central Nervous System.

[0022] H3K27M, the most common H3K27 change, is a mutation from lysine to methionine at position 27 of histone H3. It is a classic "oncogenic histone" that can drive epigenetic reprogramming in diffuse midline glioma with H3K27 changes. The chromatin landscape of diffuse midline glioma with H3K27 changes is characterized by extensive loss of trimethylation and upregulation of acetylation at the H3K27 site. Acetylation of the H3K27 site is often located in the intergenic region and can promote the expression of endogenous retroviral sequences (Endogenous retroviral element). Epigenetic drugs can further upregulate acetylation at the H3K27 site. This "viral mimicry" can activate the innate immune response and promote immune activation. Based on this, the inventors of the present application will use immunotherapy based on epigenetic drugs to treat diffuse midline glioma with H3K27 changes, especially the combination of epigenetic drugs and immune checkpoint inhibitors, which can bring survival benefits to glioma patients.

[0023] The experimental results showed that for primary diffuse midline glioma of the thoracic spinal cord with H3K27 alterations, the recurrent thoracic spinal cord lesions were safely resected to the greatest extent possible and treated with a regimen of panobinostat, tazemetostat, pembrolizumab, ipilimumab, and bevacizumab. As of the data cutoff date (Day 371), the volume of all tumor lesions in the patients continued to shrink after treatment, with the lesions in the suprasellar region (T1) and dorsal medullary region (T2) being 137 mm, respectively. 3 and 14mm 3 , with a volume reduction of over 94% compared to baseline. Enhanced MRI imaging on day 367 demonstrated that the enhancement signals in the leptomeninges / meninges and periventricular space were no longer detectable. Throughout treatment, the patient's quality of life improved significantly, and he returned to his daily life by the 11th cycle.

[0024] Cerebrospinal fluid (CSF) samples were collected from the patient on days 26, 72, 169, 321, and 340 of treatment, and proteomic analysis was performed using mass spectrometry. A total of 1,812 proteins were identified, of which 1,118 showed differential expression across treatment time points. These proteins were clustered into 20 groups based on their expression patterns, and four groups with similar expression patterns were specifically analyzed. The results showed that starting from day 26 of treatment, the expression levels of proteins associated with tumor angiogenesis, metastasis, and proliferation, such as VEGFA, the collagen family, and the laminin family, decreased significantly. Proteins associated with tumor invasion, migration, epithelial-mesenchymal transition, and stemness, such as MMP9, VIM, and NES, showed a steady downward trend starting from day 72 of treatment. In addition, protein expression levels of MRC1, a marker of M2-like tumor-associated macrophages; VISG4, a negative regulator of cytotoxic T cell activation; and PLAUR, an immunosuppressive marker in gliomas, all showed significant decreases after treatment day 26. Immune cell migration and activation-related proteins ICAM1 and ICAM2 were significantly upregulated after treatment day 72. The expression levels of immune activation and immune enhancement-related proteins such as SELL, LYZ, and ATRN also showed a significant increase after treatment day 169, and this increase persisted until the last sampling time point.

[0025] After the data cutoff, the patient's tumor progressed at two new sites. His overall survival was 20 months, compared to 16 months after receiving epigenetic immunotherapy. This therapy was further applied to three additional patients with diffuse midline gliomas and H3K27 alterations, all of whom achieved significant clinical responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the pathological diagnosis of patient 1;

[0027] Figure 2 shows the treatment regimen and the tumor's response to the treatment regimen;

[0028] Figure 3 shows the response of patient-derived tumor cells to epigenetic therapy;

[0029] Figure 4 shows the molecular response of patient-derived tumor cells to epigenetic therapy;

[0030] Figure 5 shows the imaging changes of intracranial LM (sagittal view);

[0031] Figure 6 shows the imaging changes of intracranial LM (axial view);

[0032] Figure 7 shows the imaging changes of intracranial LM (coronal view);

[0033] Figure 8 shows the imaging changes of thoracic spinal cord tumors (sagittal view);

[0034] Figure 9 shows the neurocognitive function scores;

[0035] Figure 10 shows the clustering trend of differentially expressed proteins in cerebrospinal fluid;

[0036] Figure 11 is a complete list of differentially expressed proteins in cerebrospinal fluid;

[0037] Figure 12 shows the clinical efficacy of patient 2;

[0038] Figure 13 shows the clinical efficacy of patient 3;

[0039] Figure 14 shows the clinical efficacy of patient 4;

[0040] Figure 15 shows the clinical efficacy of patient 5. DETAILED DESCRIPTION

[0041] The present invention provides a pharmaceutical composition and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] Example 1

[0043] Materials and Methods

[0044] Patient No. 1 is a 28-year-old male patient who was diagnosed with a primary diffuse midline glioma of the thoracic spinal cord with H3K27 changes on September 13, 2020 (see Table 1 for details). After tumor resection, no adjuvant treatment was received, and the patient recovered well after surgery with only mild neurological impairment. Three months after the operation, the patient developed weakness in both lower limbs, hiccups, and drowsiness, and these symptoms progressively worsened. On January 11, 2021 (day 1), the patient was admitted to Beijing Tsinghua Chang Gung Memorial Hospital due to confusion and sudden coma. The Glasgow Coma Scale was 8 / 15 points at admission. Magnetic resonance imaging (MRI) showed recurrence of the primary tumor of the thoracic spinal cord, accompanied by leptomeningeal / meningeal dissemination to the brain and spinal cord. The recurrent thoracic spinal cord lesion was subtotally resected, and a biopsy was performed on the dorsal medulla lesion (day 5). The pathological diagnosis of both lesions was diffuse midline glioma with H3K27 alterations (WHO grade 4). See Figure 1, which shows the pathological diagnosis of patient No. 1. Figure 1 shows representative staining results of the pathological diagnosis of the recurrent tumor from the thoracic spinal cord (TT, top) and the dorsal medullary leptomeningeal metastasis (LM, bottom). HE staining showed angiogenesis and hemorrhage and necrosis around the tumor cells. The Ki-67 scores of both lesions were high, and the tumor cells stained positive for H3K27M and negative for H3K27me3 and IDH1. In Figure 1, TT is a thoracic tumor; LM is a leptomeningeal metastasis; HE is hematoxylin and eosin; H3K27me3 is H3K27 trimethylation; IDH1 is isocitrate dehydrogenase 1.

[0045] Table 1 Detailed information of patients

[0046]

[0047] Treatment options

[0048] The Ethics Committee of Beijing Tsinghua Chang Gung Memorial Hospital approved the patient's treatment plan for compassionate use. The patient and his family provided written informed consent. No commercial sponsorship or involvement was involved in the treatment. The treatment plan is shown in Figure 2, which shows the treatment plan and the tumor's response to the treatment plan. Figure 2(a) shows the tumor response to the treatment plan. 15Changes in tumor volume in the thoracic spinal cord, suprasellar region, and medulla oblongata were measured on MRI. Figure 2(b) shows the treatment timeline, including epigenetic drugs (panobinostat and tazemetostat), VEGF inhibitor bevacizumab, immune checkpoint inhibitors pembrolizumab and ipilimumab, radiotherapy, and tumor electric field treatment. The timeline also shows the first occurrence of treatment-related adverse events (see Table 2 for details), cerebrospinal fluid analysis, and MRI assessment. The timeline is from January 11, 2021 (Day 1), 4 months after diagnosis, to January 16, 2022 (Day 371), including 17 21- or 28-day treatment cycles. For ease of reference, cerebrospinal fluid samples are labeled 15A-E in order of sampling time (Figure 2b). Among them, Pan is panobinostat; Taz is tazemetostat; Bev is bevacizumab; Pem is pembrolizumab; Ipi is ipilimumab; RT is radiotherapy; TTF is tumor treating fields; AE is adverse event; CSF is cerebrospinal fluid; MRI is magnetic resonance imaging; LM is leptomeningeal metastasis; VEGFA is vascular endothelial growth factor A; ICB is immune checkpoint inhibitor.

[0049] Specifically, the treatment course consisted of a 3-week cycle, with panobinostat 20 mg administered orally three times weekly during weeks 1 and 2 (i.e., on days 1, 3, 5, 8, 10, and 12). Bevacizumab 10 mg / kg and pembrolizumab 200 mg were administered intravenously during week 3. Due to the patient's severe condition at admission, bevacizumab was administered every two weeks during cycles 1-4. During cycles 3-5, the patient received whole-brain radiotherapy (41.4 Gy, 23 fractions) and a localized boost to the sellar region (10 Gy, 5 fractions). Due to grade 3 adverse reactions, including leukopenia, neutropenia, and weight loss, oral panobinostat was suspended during cycle 5 (Figure 2(b) and Table 2). In vitro tumor cell viability and invasion assays demonstrated that THG15 cells (derived from a thoracic spinal cord tumor in Patient 1) were more sensitive to the combination of panobinostat and tazepinestat compared to either agent alone (see Figures 3 and 4). Starting in cycle 8, tazepinestat was administered orally at a dose of 400 mg, with tumor electric field therapy administered concurrently with panobinostat. In cycle 11, due to satisfactory efficacy and elevated peripheral blood alkaline phosphatase, the oral dose of panobinostat was reduced to 10 mg (Figure 2(a), day 199; Tables 2 and 5). However, due to the progression of LM in the later stages, the oral dose of panobinostat was restored to 20 mg in cycle 12 (Figure 2, day 277; Table 5). Ipilimumab (50 mg intravenously) was added to the treatment regimen during cycle 12 for the following reasons: 1. EZH2 inhibition may enhance the efficacy of anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors and help reverse resistance to immune checkpoint inhibitors; 2. Periventricular LMs showed mild progression during cycle 11; 3. All lesions significantly decreased after adjustment of oral medications (Figure 2 and Table 5); and 4. The combination of programmed cell death protein 1 (PD-1) and CTLA-4 inhibitors has been reported to increase the incidence of immune-related adverse events, which may lead to treatment suspension or discontinuation. Due to fatigue (CTCAE grade 2 event), the start of cycle 13 was delayed by one week. No medical intervention was administered during this period, and the tazemetostat dose was reduced to 200 mg starting in cycle 13. Starting in cycle 17, the 3-week treatment regimen was extended to 4 weeks. Data collection was cutoff on day 371 (April 12, 2022).

[0050] Safety and adverse events

[0051] Ten grade 1-3 adverse events potentially related to treatment are shown in Table 2. Grade 3 adverse events included anemia, leukopenia, neutropenia, and weight loss, all of which resolved after blood transfusion or discontinuation of panobinostat. Low-grade adverse events included diarrhea, fatigue, thrombocytopenia, and elevated transaminases and alkaline phosphatase. No grade 4 adverse events occurred during the entire course of treatment, nor did any adverse events lead to treatment discontinuation.

[0052] Table 2 Treatment-related adverse events

[0053]

[0054] Pathological diagnosis

[0055] Formalin-fixed, paraffin-embedded pathological tissues were sectioned and subjected to hematoxylin and eosin (HE) and immunohistochemical staining. The specific steps were referred 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). Anti-Ki-67 antibody, anti-H3K27M antibody, anti-H3K27me3 antibody, anti-isocitrate dehydrogenase 1 (IDH1) R132H mutant antibody and secondary antibody sources are 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). Immunoreactivity was detected by Leica Bond automatic staining instrument.

[0056] Patient-derived tumor cell culture

[0057] Patient-derived tumor cells were obtained with the approval of the Ethics Committee of Beijing Tsinghua Chang Gung Memorial Hospital and informed consent from the patient and family. The surgical specimen was derived from a thoracic spinal cord tumor in patient 1 and designated THG15 (Tsinghua Glioma 15). The detailed protocol was based on 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, W. J. 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, tissue was mechanically dissected and digested with 0.1% trypsin (Hyclone) and 10 U / ml DNase1 (Roche) at 37°C for 45 minutes. Red blood cells were lysed and washed multiple times before being passed through a 100 μm cell strainer. Cells that passed through the strainer were collected and added to EF medium containing the following ingredients: 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 the cell culture period. Tumor cell spheres were isolated and passaged using the NeuroCult Chemical Dissociation Kit (StemCell).

[0058] Cell viability assay

[0059] Panobinostat (LBH589) and tazepam (EPZ-6438) were purchased from Selleck Chemicals and dissolved in 0.1% DMSO before use. Approximately 5,000 THG15 cells were seeded into each well of a 96-well plate and cultured in EF medium for 24 hours before drug intervention. They were then incubated with varying concentrations of panobinostat, tazepam, or both drugs for 72 hours, with a replicate of 3. Cell viability was determined using a CCK-8 kit (GK10001) according to the manufacturer's instructions, and absorbance was measured at 450 nm using a SPARK detector (TECAN). To determine the inhibitory effect of each intervention, a standard dose-response curve with a Hill slope of -1.0 was generated by comparing the viability of drug-treated cells with that of untreated cells. The corresponding half-maximal inhibitory concentration (IC50) was calculated using Prism 9 (GraphPad) based on the results of two independent experiments.

[0060] Transwell migration chamber assay

[0061] About 5×10 5 THG15 cells were resuspended in 100 μL of serum-free DMEM medium and seeded into the upper chamber of each transwell in a 24-well plate. DMEM + 20% FBS (Gibco) was added to the lower chamber. 1 μM panobinostat, 50 μM tazemetostat, or a combination of the two drugs were set as intervention groups, and repeated three times. After 48 hours of culture, the cells were fixed with 4% paraformaldehyde (Solarbio) and stained with 1% crystal violet (Solarbio). The upper surface of the transwell upper chamber was gently wiped with a cotton swab to remove cells that failed to migrate across the membrane. Residual cells were observed under an inverted fluorescence microscope (Ts2R-FL, Nikon). The relative area of ​​stained cells was quantified on ImageJ, and the average of three random views was taken.

[0062] Western blotting assay

[0063] In order to analyze the epigenetic changes of tumor cells after drug intervention, 5×10 5 THG15 cells were seeded into each well of a 6-well plate and incubated with 1 μM panobinostat, 50 μM tazemetostat, or both for 48 hours, with three replicates in the 6-well plate. The cell status after drug intervention was observed in the bright field using an inverted fluorescence microscope (Ts2R-FL, Nikon), and representative images were captured using auto-exposure and auto-white balance on NIS Elements (Nikon). Cells were lysed in RIPA buffer (Solarbio), and samples were prepared using 5× SDS-PAGE sample loading buffer (Beyotime) diluted 4:1 with PBS (Solarbio). eStain was used. TML1 protein staining system (Genscript) in SurePAGE TM The total protein level of the samples was semi-quantitated on gels (Genscript, Nanjing, China). The sample volume was adjusted according to the total protein level and then electrophoresed on 20% Bis-Tris gels (Genscript). The separated proteins were used L1 Rapid Wet Transfer System (Genscript) transfers to -P PVDF membrane (Millipore). Blocking was performed with 5% skim milk in TBST consisting of 1× TBS (Lablead) and 0.1% TWEEN 20 (VWR). The membrane was then incubated with anti-H3K27Me3, anti-H3K27Ac, ​​anti-H3K27M, or anti-H3 antibodies. Following incubation with the corresponding secondary antibodies, the blot was detected using the SuperSignal™ West Pico PLUS chemiluminescence system (Thermo Fisher Scientific) and imaged on an ImageQuant LAS 4000 (GE). Relative intensities of the blots were quantified using ImageJ densitometry analysis.

[0064] Magnetic resonance imaging and tumor volume measurement

[0065] Brain and spinal cord scans were performed on a 3.0 Tesla magnetic resonance imaging instrument, including T1-weighted, T2-weighted, and post-gadolinium T1-weighted sequences. Tumor contours were manually delineated on enhanced T1-weighted MRI images by two neuroradiologists independently, and tumor volumes were calculated using ITK-SNAP, according to 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).

[0066] Health-related quality of life assessment

[0067] Patients' quality of life was assessed using two quality-of-life questionnaires (QLQs) developed by the European Organization for Research and Treatment of Cancer. The core QLQ-C30 consists of five functional scores, one global health and quality of life score, three multi-item symptom scores, five single-item symptom scores, and a subjective rating of the economic burden of disease treatment. The brain tumor-specific QLQ-BN20 includes four multi-feature scales and seven single-item symptom scales. For ease of interpretation, the original scales and measurement scores were linearly converted to standardized scores ranging from 0 to 100, with higher scores corresponding to higher levels of health functioning. With the exception of the physical function score, all scales and indicators were based on average health status and quality of life scores during the week prior to data collection. The results are shown in Tables 3 and 4. Table 3 shows the QLQ-C30 evaluation results, and Table 4 shows the QLQ-BN20 evaluation results.

[0068] Table 3 QLQ-C30 evaluation results

[0069]

[0070] Table 4 QLQ-BN20 evaluation results

[0071]

[0072] Cerebrospinal fluid proteomic analysis

[0073] After collecting the cerebrospinal fluid sample, first use high select TM Top14 Protein Depletion Resin (Thermo Fisher Scientific) was used to remove high-abundance proteins. TMThe purified proteins were separated on a 3000 RSLCnano system and analyzed by mass spectrometry using a FAIMS PulseDIA and an Orbitrap Exploris 480. The optimal compensation voltages (CVs) for the FAIMS experiments were -48 V and -68 V, respectively, with a cycle time of 1 s. The MS1 resolution was set to 60,000, and the normalized AGC target was 300%. The mass range was set to 390-1010 Th. Twelve windows with a resolution of 30,000 and a normalized AGC target of 200% were used after a full scan. For the first part of the method, the mass range was set to 520-630 Th at -68 V and 630-750 Th at -48 V. For the second part of the method, the mass range was set to 400-520 Th at -68 V and 750-1000 Th at -48 V. 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 search was performed using DIA-NN (version 1.7.8). The original file has been deposited in iProX: IPX0005197001.

[0074] Statistical analysis

[0075] One-way analysis of variance (ANOVA) was used to determine the statistical differences in protein levels at different time points, and the Benjamini & Hochberg method was used to adjust the p-values. The Mfuzz package was used to group differentially expressed proteins (ANOVA, corrected p < 0.05) during treatment into 20 clusters based on their expression profiles.

[0076] In the first cycle of treatment, the patient's consciousness disorder transiently recovered to drowsiness after surgical resection of the recurrent lesion of the primary thoracic spinal cord tumor. However, 10 days later, the volume of the sellar metastasis increased from the baseline of 1905 mm 3 Increased to 2606mm 3 , the patient developed diabetes insipidus and hyponatremia (see Figure 2a and Table 5 for details). The metastatic lesions in the leptomeninges and periventricular space were also progressing. After 2 cycles of treatment, the electrolyte and fluid balance disorders were completely corrected. Since the third cycle of treatment, the patient's consciousness has fully recovered and his neurocognitive function has also been comprehensively improved (see Figure 9 and Table 4 for details). At the end of radiotherapy in the sixth cycle of treatment, the volume of all lesions of the patient was significantly reduced, with T1 and T2 decreasing from their baseline volumes of 1905 mm and 1906 mm, respectively. 3 and 829mm 3 , reduced to 1615 mm on the 122nd day of treatment 3 and 265mm 3Interestingly, in cycle 11 (day 227), the periventricular enhancement lesions slightly increased on imaging in the absence of any new neurological deficits due to a dose reduction of ipabinostat during that cycle. However, after increasing the dose in cycle 12 (day 256), the volume of the increased lesions decreased (see Figures 2a, 5-7 and Table 5 for details). Over the course of treatment, proteins in the cerebrospinal fluid were divided into 20 clusters based on their differential expression trends (cluster characteristics are shown in Figure 10). The heat map shows proteins whose expression levels showed the same trend of change in the cerebrospinal fluid samples collected at different time points of treatment.

[0077] The specific results are as follows:

[0078] Figure 3 shows the response of patient-derived tumor cells to epigenetic therapy. Figure 3(a) shows dose-response curves demonstrating changes in tumor cell viability after 72 hours of treatment with panobinostat, tazepinestat, or their combination. Results are presented as mean ± standard deviation of two independent cell viability measurements (n = 6). The drug solvent, 0.1% DMSO, did not affect cell viability (data not shown). Inter-drug IC50 values ​​were compared using an additional sum-of-squares F-test: *, p < 0.05; ***, p < 0.001. Figure 3(a) (left) shows microscopic observation of tumor cell viability after 48 hours of treatment with 1 μM panobinostat, 50 μM tazepinestat (approximately their estimated IC50 values), or their combination. Figure 3(b) shows the effects of panobinostat and tazepinestat on tumor cell migration in transwells. Tumor cells migrating across the transwell membrane were stained and observed under a microscope after 48 hours of treatment with 1 μM panobinostat, 50 μM tazepinestat, or their combination. The results are shown on the left. The area of ​​stained cells relative to the field of view was quantified using ImageJ. The average of three randomly selected views was calculated, and the bar graphs are presented as mean ± standard deviation (n = 6). Unpaired t-test: ***, p < 0.001. DMSO, dimethyl sulfoxide; Pan, panobinostat; Taz, tazemetostat; IC50, half-maximal inhibitory concentration.

[0079] 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 treated with DMSO, 1 μM panobinostat, 50 μM tazemetostat, or both for 48 hours. The expression levels of H3K27me3 and H3K27ac in tumor cells after drug treatment relative to the DMSO-treated control group are shown in Figures 4(b) and 4(c), respectively. A t-test showed that H3K27me3 expression was significantly decreased, while H3K27ac expression was significantly increased, in tumor cells treated with panobinostat and tazemetostat combined (n = 4; NS, not significant; **, p < 0.01). Western blotting was used to simultaneously detect the expression levels of H3 and H3K27M in tumor cells (a). DMSO, dimethyl sulfoxide; Pan, panobinostat; Taz, tazemestat; H3K27me3, H3K27 trimethylation; H3K27ac, H3K27 acetylation.

[0080] Figure 5 shows the intracranial LM imaging changes (sagittal view). A T1 contrast-enhanced sagittal MRI image is shown. The acquisition date is indicated in the upper left corner of the image. Arrows indicate multiple linear lesions of leptomeningeal enhancement. T1 and T2 represent the suprasellar LM and medullary tumor at baseline (day 2). Axial and coronal images are shown in Figures 6 and 7.

[0081] Figure 6 shows the intracranial LM imaging changes (axial). A T1-enhanced axial MRI image is shown. The acquisition date is indicated in the upper left corner of the image. Arrows indicate multiple linear lesions of leptomeningeal enhancement. T1 represents the suprasellar LM at baseline (day 2). Sagittal and coronal images are shown in Figures 5 and 7.

[0082] Figure 7 shows the intracranial LM imaging changes (coronal view). A T1-enhanced coronal MRI image is shown. The acquisition date is indicated in the upper left corner of the image. Arrows on the baseline (day 2) image indicate multiple linear leptomeningeal and periventricular enhancement foci. Sagittal and axial images are shown in Figures 5 and 6.

[0083] Figure 8 shows the radiographic changes of a thoracic spinal cord tumor (sagittal view). T1-enhanced and T2-weighted MRI images are shown above. Image acquisition dates are indicated in the upper left corner of the figure. T3 represents the recurrent thoracic spinal cord tumor lesion at baseline (day 2). Radiographic tumor volume measurements are shown in Table 5.

[0084] Table 5 Imaging tumor volume measurement data (mm 3 )

[0085] Time (Cycle) Thoracic spinal cord (T3) Suprasellar region (T1) Medulla oblongata (T2) 2 (C1) 9057190582918 (C1) 2538260684045 (C3) 2842255760371 (C4) 2476217444694 (C5) 18962591637122 (C6) 1394161526 5169(C9)459107877199(C10)45470761227(C11)44686466256(C12)4428035 9277(C13)43550843320(C15)43327021341(C16)34617718367(C17)32413714

[0086] As shown in Table 5, before the data cutoff date (Day 371), the volume of all tumor lesions in the patients continued to shrink after treatment, with the lesions in the suprasellar region (T1) and dorsal medulla oblongata (T2) being 137 mm and 26 mm, respectively. 3 and 14mm 3 The volume of the cerebral ischemia-reperfusion injury (CSRI) was reduced by over 94% compared to baseline. Enhanced MRI imaging on day 367 demonstrated that the enhancement signal in the leptomeninges / meninges and periventricular space was no longer detectable (Figures 5-8). Throughout treatment, the patient's quality of life improved significantly, and he returned to his daily life by the 11th cycle (Figure 9 and Table 4).

[0087] Figure 9 shows the neurocognitive function scores. The Mini-Mental State Examination (MMSE) was used to assess the patients' neurocognitive function, with scores ranging from 0 to 30, with 27 or above indicating normal cognition.

[0088] Figure 10 shows the clustering trends of differentially expressed proteins in CSF. After removing highly abundant proteins, Mfuzz was used to group differentially expressed proteins in CSF during treatment into 20 clusters based on their relative expression patterns (ANOVA, corrected p < 0.05). The trends in relative expression of proteins in all 20 clusters are shown in Figure 10.

[0089] Figure 11 shows a complete list of differentially expressed proteins in CSF. Figures 11a-d show heat maps of relative protein expression in CSF samples at various time points during treatment (see Figure 2(b) for sampling time). Mfuzz was used to categorize differentially expressed proteins during treatment into 20 clusters based on their expression profiles (clustering features shown in Figure 9) (ANOVA, adjusted p < 0.05).

[0090] Cerebrospinal fluid (CSF) samples were collected from the patient on days 26, 72, 169, 321, and 340 of treatment, and proteomic analysis was performed using mass spectrometry. A total of 1,812 proteins were identified, of which 1,118 showed differential expression over the course of treatment and were clustered into 20 groups based on their expression patterns (Figures 10 and 11). We focused on four groups of proteins with similar trends. The results showed that starting from day 26 of treatment, the expression levels of proteins associated with tumor angiogenesis, metastasis, and proliferation, such as VEGFA, the collagen family, and the laminin family, were significantly reduced. Proteins associated with tumor invasion, migration, epithelial-mesenchymal transition, and stemness, such as MMP9, VIM, and NES, showed a steady downward trend starting from day 72 of treatment. Interestingly, the expression of the protease inhibitor PZP increased significantly after the 26th day of treatment, while the expression of proteasome subunits such as PSMA3, PSMB3, and PSMB8 decreased significantly after the 72nd day of treatment, suggesting that the significant therapeutic effect of the patients may be related to the inhibition of proteasomes, and this effect is not achieved by the application of the proteasome inhibitor Marizomib. In addition, the protein expression levels of MRC1, a marker of M2-like tumor-associated macrophages, VISG4, a negative regulator of cytotoxic T cell activation, and PLAUR, an immunosuppressive marker in glioma, all showed a significant decrease after the 26th day of treatment. Immune cell migration and activation-related proteins ICAM1 and ICAM2 were significantly upregulated after the 72nd day of treatment; the expression levels of immune activation and immune enhancement-related proteins such as SELL, LYZ, and ATRN also showed a significant increase after the 169th day of treatment and continued until the last sampling time point.

[0091] After the data cutoff, the patient's tumor progressed at two new sites. His overall survival was 20 months, compared with 16 months with epigenetic-based immunotherapy.

[0092] Example 2

[0093] Patient 2 was a 53-year-old male with sudden onset of paraplegia, paresthesia, and urinary incontinence as primary clinical manifestations. Figure 12 shows the tumor findings on T1-enhanced and T2-weighted MRI images. Figure 12 shows the clinical outcome of Patient 2. The dates of image acquisition are indicated above the images. Diffuse midline glioma of the cervical spinal cord with H3K27 alterations was diagnosed after a tumor biopsy performed at another hospital. The patient was admitted to Beijing Tsinghua Chang Gung Memorial Hospital due to progression of tumor imaging and clinical symptoms (day -5; arrows indicate the tumor lesion on T1-weighted images and the area of ​​edema surrounding the lesion on T2-weighted images). The patient received radiotherapy (45 Gy, 25 fractions) and the VEGF inhibitor bevacizumab (10 mg / kg) at Beijing Tsinghua Chang Gung Memorial Hospital. Simultaneously, they received a 3-week course of epigenetic immunotherapy with the drugs tazemetostat and panobinostat (20 mg panobinostat and 400 mg tazemetostat, taken orally on Mondays, Wednesdays, and Fridays for 2 consecutive weeks. Panobinostat and tazemetostat were discontinued on the third week, and ipilimumab 50 mg and pembrolizumab 100 mg were given intravenously). A laminectomy was performed on day 71. Following treatment, the patient experienced significant improvement in lower limb motor and sensory function, and bladder control was restored. Parallel to these clinical improvements was a significant reduction in cervical spinal cord tumor volume on imaging (day 415). The patient has survived for more than 16 months since diagnosis and more than 15 months after epigenetic immunotherapy. The last follow-up was in October 2022.

[0094] Example 3

[0095] Patient 3 was a 25-year-old female with clinical manifestations of right hemiplegia and aphasia. The tumor was seen on T1 enhanced MRI images as shown in Figure 13. Figure 13 shows the clinical efficacy of patient 3. The image acquisition date is indicated above the image. The patient was diagnosed with a diffuse midline glioma of the left thalamus with H3K27 changes after a tumor biopsy at an outside hospital (day -587, the primary tumor is circled). She had received standard chemoradiotherapy at the outside hospital, but her neurological symptoms continued to worsen. Repeated radiotherapy did not improve her clinical or imaging. Baseline MRI images at Beijing Tsinghua Chang Gung Memorial Hospital showed progression of the left thalamic tumor (day 1). Later, patient 3 received immunotherapy based on the epigenetic drugs tazemetostat and panobinostat in cycles of 3 or 4 weeks (panobinostat 20 mg, tazemetostat 400 mg, taken orally on Mondays, Wednesdays, and Fridays for 2 consecutive weeks. Panobinostat and tazemetostat were discontinued on the third week, and ipilimumab 50 mg + pembrolizumab 100 mg were given by intravenous infusion). The VEGF inhibitor bevacizumab (10 mg / kg) was also used. After treatment, the patient's right motor function improved significantly and he could walk independently. His language ability in terms of naming and fluency was significantly improved, and he was willing to talk. Parallel to these clinical improvements was the significant reduction in the volume of the left thalamic tumor on imaging (day 157).

[0096] Example 4

[0097] Patient 4 was a 44-year-old male with clinical symptoms primarily presenting with left limb weakness. The tumor findings on T1 contrast-enhanced MRI are shown in Figure 14 , which also illustrates the clinical outcome of Patient 4. Image acquisition dates are indicated above the images. A diffuse midline glioma of the right thalamus with H3K27 alterations was diagnosed following tumor resection at an outside hospital (preoperative image on day -32, with the primary tumor circled; postoperative image on day -17). Patient 4 was referred to Beijing Tsinghua Chang Gung Memorial Hospital (Day -4) due to worsening tumor radiographic and clinical symptoms. Patient 4 received radiotherapy (54 Gy in 27 fractions), tumor treating fields, the VEGF inhibitor bevacizumab (10 mg / kg), and immunotherapy with the epigenetic agents tazepinestat and panobinostat, administered orally on Mondays, Wednesdays, and Fridays for two consecutive weeks, with panobinostat and tazepinestat discontinued on the third week and pembrolizumab 200 mg administered intravenously. Following treatment, the patient experienced significant recovery of left-sided motor function, a marked improvement in quality of life, and the ability to perform normal daily activities, including mountain climbing and various social activities. Parallel to these clinical improvements was a significant reduction in the right thalamic tumor volume on imaging (Day 228).

[0098] Example 5

[0099] Patient 5 was a 25-year-old male who presented with progressive left upper limb weakness and neck pain. Three tumors (T1-T3) were visualized on T1-enhanced and T2-weighted MRI images, as shown in Figure 15 . Figure 15 shows the clinical outcome of Patient 5. The dates of image acquisition are indicated above the images. Patient 5 was diagnosed with a diffuse midline cervical spinal cord glioma with H3K27 alterations after initial tumor resection at a German hospital in September 2017. He underwent conventional postoperative chemoradiotherapy. Due to tumor recurrence, he underwent a second surgical procedure in Germany in February 2019, with the same pathological diagnosis. No conventional adjuvant therapy was administered after the second surgery, and he participated in the ONC201 clinical trial in the United States for adults with recurrent H3K27M-mutant gliomas from May 2019 to March 2020. In September 2020, Patient 5 developed paraplegia, accompanied by incontinence and right-sided facial palsy, and was subsequently treated at Tsinghua Chang Gung Memorial Hospital in Beijing. MRI showed recurrence of the cervical spinal tumor (T1) with multiple brain metastases (T2 and T3) (day -56). Patient 5 underwent subtotal resection of the cervical spinal tumor on day -38, but the intracranial metastases progressed significantly after the third surgery, and neurological symptoms worsened (day 0). The patient received a 3-week course of panobinostat and bevacizumab combination therapy (panobinostat 20 mg, taken orally on Mondays, Wednesdays, and Fridays for 2 consecutive weeks, with panobinostat discontinued on the third week and bevacizumab 10 mg / kg intravenous infusion given). After two courses of treatment, the T2 and T3 brain metastases were significantly reduced (day 49), accompanied by significant improvement in neurological symptoms. However, the brain metastases progressed after the fourth course of treatment. The MRI scan of patient 5 on day 113, the last imaging examination before his death on day 163, showed an increase in the tumor burden of brain metastases. Patient 5 died 43 months after diagnosis of diffuse midline glioma with H3K27 alterations and 5 months after treatment with panobinostat combined with bevacizumab. This patient's efficacy suggests that panobinostat combined with bevacizumab has some efficacy in treating diffuse midline glioma with H3K27 alterations, but it did not significantly inhibit intracranial metastasis of the patient's intramedullary tumor. Compared with the aforementioned patients 1-4, the combination of panobinostat, tazemetostat, pembrolizumab, ipilimumab, and bevacizumab showed significantly better clinical efficacy than the combination of panobinostat and bevacizumab.

[0100] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition comprising: HDAC inhibitors and EZH2 inhibitors.

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 pharmaceutical composition according to claim 1 or 2, characterized in that The HDAC inhibitor is selected from one or more of panobinostat, vorinostat, romidepsin, belinostat and chidamide; The EZH2 inhibitor is selected from tazemetostat.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that It also includes one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor and a VEGF inhibitor.

5. The pharmaceutical composition according to claim 4, characterized in that The PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, carrelizumab, sepalimab, penampalimab, slulizumab or putalimab; The PD-L1 inhibitor is selected from one or more of atezolizumab, durvalumab, envomalimab, sugemalimab or avelumab; The VEGF inhibitor is selected from one or more of bevacizumab, ramucirumab, ranibizumab or aflibercept; The CTLA-4 inhibitor is selected from one or more of ipilimumab and tremelimumab.

7. The pharmaceutical composition according to claim 5, characterized in that It also includes pembrolizumab, and the mass ratio of the HDAC inhibitor to pembrolizumab is 100-200:50-400.

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

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

9. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

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 was a diffuse midline glioma with H3K27 alterations.

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