Pharmaceutical use of sp / klf transcription factor inhibitors in combination with deacetylase inhibitors

Abstract

The present invention relates to the pharmaceutical use of a combination of an SP / KLF transcription factor inhibitor, phorboxazole or a variant thereof, and a sirtuin inhibitor. The present invention provides the use of a sirtuin inhibitor and phorboxazole or a variant thereof for the manufacture of a medicament for the prevention and / or treatment of glioma. The present invention also provides a pharmaceutical composition for the prevention and / or treatment of glioma, comprising a sirtuin inhibitor and phorboxazole or a variant thereof, and optionally a pharmaceutically acceptable adjuvant. The glioma is adult glioma or pediatric glioma. The inventors of the present invention have found that the combination of a sirtuin inhibitor and phorboxazole or a variant thereof can significantly improve the therapeutic effect on glioma and reduce the side effects of single treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drugs for preventing and / or treating tumors. Specifically, the present application relates to the use of SP / KLF transcription factor inhibitor phorboxazole or its variant in combination with a deacetylase inhibitor for the preparation of a drug for preventing and / or treating glioma, and a pharmaceutical composition comprising SP / KLF transcription factor inhibitor phorboxazole or its variant and a deacetylase inhibitor. BACKGROUND

[0002] Glioma is the most common primary tumor in the central nervous system, with a high incidence in adults, up to 8 per 100,000 per year. Patients often have symptoms such as dizziness and headache, nausea and vomiting, blurred vision or consciousness, aphasia, mental abnormalities, etc., which seriously affect the normal life and work of patients, and if not treated in time, it may lead to aggravation of the disease and even threaten life.

[0003] At the same time, the incidence of glioma in children is also high. For example, diffuse intrinsic pontine glioma (DIPG) is a highly malignant brainstem tumor in children that originates in the pons, usually histopathologically diagnosed as high-grade astrocytic glioblastoma, and is almost only seen in children, with a median age of 6-7 years at diagnosis. The prognosis of DIPG is very poor, with a median survival of less than 9 months after diagnosis, a 2-year survival rate of less than 10%, and a 5-year survival rate of less than 1%, making it one of the most deadly malignant tumors in children [1-3] . Due to its special location of onset and highly invasive characteristics, it cannot be resected by surgical means [1，2，4，5] .

[0004] Although for decades, more and more researchers have focused on the pathogenesis of DIPG and provided diverse treatment strategies. Clinical trials have also evaluated various DIPG treatment strategies, including various combinations of radiotherapy and chemotherapy drugs, but there is still no particularly effective treatment method. Radiotherapy is still the current standard treatment strategy, but this is only a palliative option that can only temporarily relieve symptoms and cannot change the poor prognosis [4，6] .

[0005] Chinese patent application for invention with publication number CN115120722A discloses the use of SP / KLF transcription factor inhibitors, which proves that SP / KLF transcription factor inhibitors phorboxazole and its variants EC-8042, etc. can effectively inhibit the occurrence and development of DIPG when used alone.

[0006] However, it is still a technical problem to be solved at present to provide a more feasible combined drug regimen for the clinical treatment of DIPG in order to improve the therapeutic effect and reduce the side effects of single treatment. SUMMARY

[0007] The technical solution of the present application is based on the following discoveries and researches:

[0008] In clinical practice, single administration can interfere with the normal function of unintended targets or activate anti-protection and compensation mechanisms, leading to unexpected off-target toxicity or drug resistance. However, combination therapy of two or more drugs can improve therapeutic effect and reduce side effects of single treatment [7，8] On this basis, the inventors found and confirmed through a large number of experimental screening and research that the combination of SP / KLF transcription factor inhibitor mithramycin or its variant and sirtuin inhibitor in the clinical treatment of DIPG is effective, providing a more feasible new combination therapy for the clinical treatment of DIPG.

[0009] Therefore, the purpose of the present application is to overcome the shortcomings of the prior art, and to provide the use of sirtuin inhibitors and mithramycin or its variant in the preparation of a drug for preventing and / or treating childhood diffuse intrinsic pontine glioma (DIPG). The present application also provides a pharmaceutical composition for preventing and / or treating childhood diffuse intrinsic pontine glioma (DIPG).

[0010] The purpose of the present application is achieved by the following technical solutions:

[0011] In one aspect, the present application provides the use of sirtuin inhibitors and mithramycin or its variant in the preparation of a drug for preventing and / or treating glioma.

[0012] Preferably, the glioma is adult glioma or childhood glioma.

[0013] More preferably, the childhood glioma is childhood high-grade glioma (pHGG) or childhood diffuse intrinsic pontine glioma (DIPG).

[0014] In the present application, mithramycin (Mithramycin), also known as plicamycin, has the following structure:

[0015] .

[0016] According to some embodiments of the application, wherein the deacetylase inhibitor is selected from one or more of PCI-34051 (CAS number 950762-95-5), Vorinostat (CAS number 149647-78-9), Droxinostat (CAS number 99873-43-5), Danusertib (CAS number 404951-53-7), Pracinostat (CAS number 404950-80-7), Tubastatin A HC1 (CAS number 1310693-92-5), ITSA-1 (CAS number 200626-61-5), Fimepinostat (CUDC-907) (CAS number 1339928-25-4), and Chidamide (CAS number 1616493-44-7). Wherein, CAS (Chemical Abstract Service) number is the American Chemical Abstracts Society registration number.

[0017] Preferably, the deacetylase inhibitor is selected from one or more of PCI-34051, Vorinostat, Droxinostat, Danusertib, Pracinostat, and Chidamide; more preferably, the deacetylase inhibitor is Vorinostat and / or Chidamide.

[0018] According to some embodiments of the application, wherein the variant of the Photofrin (EC-8042) has the structure shown in the following formula:

[0019] .

[0020] According to some embodiments of the application, wherein the ratio of the amount of the deacetylase inhibitor and the Photofrin or variant thereof is (10 nM ~ 750 µM):(2.5 nM ~ 250 nM);

[0021] Preferably, the ratio of the amount of the deacetylase inhibitor and the Photofrin is (10 nM ~ 750 µM):(5 nM ~ 250 nM);

[0022] More preferably, the amount ratio of Volitinostat and Plicamycin is (0.2 µM~10 µM):(5 nM~250 nM); or, the amount ratio of Droxinostat and Plicamycin is (15 µM~750 µM):(5 nM~250 nM); or, the amount ratio of PCI-34051 and Plicamycin is (10 µM~250 µM):(5 nM~125 nM); or, the amount ratio of Danothostat and Plicamycin is (10 nM~500 nM):(5 nM~250 nM); or, the amount ratio of Paribulin and Plicamycin is (10 nM~500 nM):(5 nM~250 nM); or, the amount ratio of Chidamide and Plicamycin is (0.4 µM~20 µM):(5 nM~250 nM);

[0023] Further preferably, the amount ratio of Volitinostat and Plicamycin is (0.4 µM~0.8 µM):(10 nM~20 nM); or, the amount ratio of Chidamide and Plicamycin is (0.8 µM~1.6 µM):(10 nM~20 nM);

[0024] More further preferably, the molar ratio of Volitinostat and Plicamycin is (40~80): 1; or, the molar ratio of Droxinostat and Plicamycin is 3000: 1; or, the molar ratio of PCI-34051 and Plicamycin is 2000: 1; or, the molar ratio of Danothostat and Plicamycin is 2: 1; or, the molar ratio of Paribulin and Plicamycin is 2: 1; or, the amount ratio of Chidamide and Plicamycin is (80~160):1;

[0025] Most preferably, the molar ratio of Volitinostat and Plicamycin is 40:1; or, the amount ratio of Chidamide and Plicamycin is 160:1;

[0026] or,

[0027] Preferably, the amount ratio of the Deacetylase Inhibitor and the variant of Plicamycin is (10 nM~375 µM):(2.5 nM~125 nM);

[0028] More preferably, the amount ratio of vorinostat and the variant of mithramycin is (0.2 µM~10 µM):(2.5 nM~125 nM); or, the amount ratio of Droxinostat and the variant of mithramycin is (15 µM~375 µM):(2.5 nM~62.5 nM); or, the amount ratio of PCI-34051 and the variant of mithramycin is (10 µM~120 µM):(2.5 nM~30 nM); or, the amount ratio of Damocinostat and the variant of mithramycin is (10 nM~500 nM):(2.5 nM~125 nM); or, the amount ratio of Paribulin and the variant of mithramycin is (10 nM~500 nM):(2.5 nM~125 nM); or, the amount ratio of Chidamide and the variant of mithramycin is (0.4 µM~10 µM):(5 nM~125 nM);

[0029] Further preferably, the amount ratio of vorinostat and the variant of mithramycin is (0.4 µM~0.8 µM):(10 nM~20 nM); or, the amount ratio of Chidamide and the variant of mithramycin is (0.8 µM~1.6 µM):(10 nM~20 nM).

[0030] More further preferably, the molar ratio of vorinostat and the variant of mithramycin is (40~80): 1; or, the molar ratio of Droxinostat and the variant of mithramycin is 6000: 1; or, the molar ratio of PCI-34051 and the variant of mithramycin is 4000: 1; or, the molar ratio of Damocinostat and the variant of mithramycin is 4: 1; or, the molar ratio of Paribulin and the variant of mithramycin is 4: 1; or, the molar ratio of Chidamide and the variant of mithramycin is (80~160):1.

[0031] Most preferably, the molar ratio of vorinostat and the variant of mithramycin is 80:1; or, the molar ratio of Chidamide and the variant of mithramycin is 160:1.

[0032] In another aspect, the present application provides a pharmaceutical composition for preventing and / or treating glioma, comprising a sirtuin inhibitor and mithramycin or a variant thereof, and optionally a pharmaceutically acceptable adjuvant.

[0033] Preferably, the glioma is adult glioma or pediatric glioma.

[0034] More preferably, the pediatric glioma is pediatric high-grade glioma (pHGG) or pediatric diffuse intrinsic pontine glioma (DIPG).

[0035] According to some embodiments of the application, wherein the deacetylase inhibitor is selected from one or more of PCI-34051 (CAS No. 950762-95-5), Vorinostat (CAS No. 149647-78-9), Droxinostat (CAS No. 99873-43-5), Danusertib (CAS No. 404951-53-7), Pracinostat (CAS No. 404950-80-7), Tubastatin A HC1 (CAS No. 1310693-92-5), ITSA-1 (CAS No. 200626-61-5), Fiamepinostat (CUDC-907) (CAS No. 1339928-25-4), and Chidamide (CAS No. 1616493-44-7). Wherein, CAS (Chemical Abstract Service) No. is the American Chemical Abstracts Society registration number.

[0036] Preferably, the deacetylase inhibitor is selected from one or more of PCI-34051, Vorinostat, Droxinostat, Danusertib, Pracinostat, and Chidamide; more preferably, the deacetylase inhibitor is Vorinostat and / or Chidamide.

[0037] According to some embodiments of the application, wherein the variant of the Photorhabdus luminescens (EC-8042) has the structure shown in the following formula:

[0038] .

[0039] According to some embodiments of the application, wherein the ratio of the amount of the deacetylase inhibitor and the amount of the Photorhabdus luminescens or its variant is (10 nM ~ 750 µM):(2.5 nM ~ 250 nM);

[0040] Preferably, the ratio of the amount of the deacetylase inhibitor and the amount of the Photorhabdus luminescens is (10 nM ~ 750 µM):(5 nM ~ 250 nM);

[0041] More preferably, the amount ratio of Volitinostat and Plicamycin is (0.2 µM~10 µM):(5 nM~250 nM); or, the amount ratio of Droxinostat and Plicamycin is (15 µM~750 µM):(5 nM~250 nM); or, the amount ratio of PCI-34051 and Plicamycin is (10 µM~250 µM):(5 nM~125 nM); or, the amount ratio of Danothostat and Plicamycin is (10 nM~500 nM):(5 nM~250 nM); or, the amount ratio of Paribulin and Plicamycin is (10 nM~500 nM):(5 nM~250 nM); or, the amount ratio of Chidamide and Plicamycin is (0.4 µM~20 µM):(5 nM~250 nM);

[0042] Further preferably, the amount ratio of Volitinostat and Plicamycin is (0.4 µM~0.8 µM):(10 nM~20 nM); or, the amount ratio of Chidamide and Plicamycin is (0.8 µM~1.6 µM):(10 nM~20 nM);

[0043] More further preferably, the molar ratio of Volitinostat and Plicamycin is (40~80): 1; or, the molar ratio of Droxinostat and Plicamycin is 3000: 1; or, the molar ratio of PCI-34051 and Plicamycin is 2000:1; or, the molar ratio of Danothostat and Plicamycin is 2: 1; or, the molar ratio of Paribulin and Plicamycin is 2: 1; or, the amount ratio of Chidamide and Plicamycin is (80~160):1;

[0044] Most preferably, the molar ratio of Volitinostat and Plicamycin is 40:1; or, the amount ratio of Chidamide and Plicamycin is 160:1;

[0045] or,

[0046] Preferably, the amount ratio of the Deacetylase Inhibitor and the variant of Plicamycin is (10 nM~375 µM):(2.5 nM~125 nM);

[0047] More preferably, the ratio of the amounts of vorinostat and the variant of mithramycin is (0.2 µM~10 µM):(2.5 nM~125 nM); or, the ratio of the amounts of Droxinostat and the variant of mithramycin is (15 µM~375 µM):(2.5 nM~62.5 nM); or, the ratio of the amounts of PCI-34051 and the variant of mithramycin is (10 µM~120 µM):(2.5 nM~30 nM); or, the ratio of the amounts of Damocinostat and the variant of mithramycin is (10 nM~500 nM):(2.5 nM~125 nM); or, the ratio of the amounts of Paribulin and the variant of mithramycin is (10 nM~500 nM):(2.5 nM~125 nM); or, the ratio of the amounts of Chidamide and the variant of mithramycin is (0.4 µM~10 µM):(5 nM~125 nM);

[0048] Further preferably, the ratio of the amounts of vorinostat and the variant of mithramycin is (0.4 µM~0.8 µM):(10 nM~20 nM); or, the ratio of the amounts of Chidamide and the variant of mithramycin is (0.8 µM~1.6 µM):(10 nM~20 nM).

[0049] More further preferably, the molar ratio of vorinostat and the variant of mithramycin is (40~80): 1; or, the molar ratio of Droxinostat and the variant of mithramycin is 6000: 1; or, the molar ratio of PCI-34051 and the variant of mithramycin is 4000: 1; or, the molar ratio of Damocinostat and the variant of mithramycin is 4: 1; or, the molar ratio of Paribulin and the variant of mithramycin is 4: 1; or, the molar ratio of Chidamide and the variant of mithramycin is (80~160):1.

[0050] Most preferably, the molar ratio of vorinostat and the variant of mithramycin is 80:1; or, the molar ratio of Chidamide and the variant of mithramycin is 160:1.

[0051] According to some embodiments of the application, wherein the pharmaceutical composition comprises 2.5~250 nM of mithramycin or a variant thereof;

[0052] Preferably, the pharmaceutical composition comprises 5~250 nM of mithramycin or 2.5~125 nM of a variant of mithramycin.

[0053] According to some embodiments of the application, wherein the pharmaceutical composition comprises 10 nM~750 µM of a deacetylase inhibitor;

[0054] Preferably, the pharmaceutical composition comprises 0.2-10 µM of vorinostat, or 15-750 µM of Droxinostat, or 10-250 µM of PCI-34051, or 10-500 nM of Danusertib, or 10-500 nM of Pridinostat, or 0.4-10 µM of Chidamide.

[0055] According to a preferred embodiment of the present application, the pharmaceutical composition comprises 0.2-10 µM of vorinostat and 5-250 nM of mithramycin, and pharmaceutically acceptable excipients. More preferably, the molar ratio of vorinostat and mithramycin is 40: 1. For example, the pharmaceutical composition comprises 0.2 µM of vorinostat and 5 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 0.4 µM of vorinostat and 10 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 0.8 µM of vorinostat and 20 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 1.2 µM of vorinostat and 30 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 1.6 µM of vorinostat and 40 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 2.4 µM of vorinostat and 60 nM of mithramycin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 3.2 µM of vorinostat and 80 nM of mithramycin, and pharmaceutically acceptable excipients.

[0056] According to another preferred embodiment of the present application, the pharmaceutical composition comprises 0.2-10 µM of vorinostat and 2.5-125 nM of a variant of calcinacin, and pharmaceutically acceptable excipients. More preferably, the molar ratio of vorinostat and the variant of calcinacin is 80: 1. For example, the pharmaceutical composition comprises 0.2 µM of vorinostat and 2.5 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 0.4 µM of vorinostat and 5 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 0.8 µM of vorinostat and 10 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 1.2 µM of vorinostat and 15 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 1.6 µM of vorinostat and 20 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 2.4 µM of vorinostat and 30 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 3.2 µM of vorinostat and 40 nM of a variant of calcinacin, and pharmaceutically acceptable excipients.

[0057] According to another preferred embodiment of the present application, the pharmaceutical composition comprises 0.4-10 µM of chidamide and 5-125 nM of a variant of calcinacin, and pharmaceutically acceptable excipients. More preferably, the molar ratio of chidamide and the variant of calcinacin is 80: 1. For example, the pharmaceutical composition comprises 0.4 µM of chidamide and 5 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 0.8 µM of chidamide and 10 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 1.6 µM of chidamide and 20 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 2.4 µM of chidamide and 30 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 3.2 µM of chidamide and 40 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 4.8 µM of chidamide and 60 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 6.4 µM of chidamide and 80 nM of a variant of calcinacin, and pharmaceutically acceptable excipients; or, the pharmaceutical composition comprises 10 µM of chidamide and 125 nM of a variant of calcinacin, and pharmaceutically acceptable excipients.

[0058] According to another preferred embodiment of the present application, the pharmaceutical composition comprises 0.4-10 µM of chidamide and 5-125 nM of calicheamicin, and pharmaceutically acceptable adjuvants. More preferably, the molar ratio of chidamide and calicheamicin is 80: 1. For example, the pharmaceutical composition comprises 0.4 µM of chidamide and 5 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 0.8 µM of chidamide and 10 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 1.6 µM of chidamide and 20 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 2.4 µM of chidamide and 30 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 3.2 µM of chidamide and 40 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 4.8 µM of chidamide and 60 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 6.4 µM of chidamide and 80 nM of calicheamicin, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 10 µM of chidamide and 125 nM of calicheamicin, and pharmaceutically acceptable adjuvants.

[0059] According to another preferred embodiment of the present application, the pharmaceutical composition comprises 0.4-10 µM of chidamide and 5-125 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants. More preferably, the molar ratio of chidamide and calicheamicin variant is 160: 1. For example, the pharmaceutical composition comprises 0.4 µM of chidamide and 2.5 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 0.8 µM of chidamide and 5 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 1.6 µM of chidamide and 10 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 2.4 µM of chidamide and 15 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 3.2 µM of chidamide and 20 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 4.8 µM of chidamide and 30 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants; or, the pharmaceutical composition comprises 6.4 µM of chidamide and 40 nM of calicheamicin variant, and pharmaceutically acceptable adjuvants.

[0060] Compared with the prior art, the present application has the following beneficial effects:

[0061] The in vitro experiment of the present application proves that the combination of the deacetylase inhibitor and the phorboxazole and the combination of the deacetylase inhibitor and the variant of the phorboxazole have more significant inhibitory effect on DIPG tumor cells and can more effectively inhibit cell division and proliferation compared with the single drug. In addition, the use of the deacetylase inhibitor alone promotes the hypoxia adaptation and infiltration ability of the DIPG tumor cells, thereby producing drug resistance. However, the combination of the deacetylase inhibitor and the phorboxazole or the variant thereof can reverse the drug resistance of the tumor cells caused by the use of the deacetylase inhibitor alone.

[0062] The animal experiment of the present application proves that the combination of the deacetylase inhibitor and the phorboxazole or the variant thereof can inhibit the occurrence and development of DIPG in vivo and make the expression profile of the tumor cells close to the expression characteristics of the normal glial progenitor cells and the longer survival genes.

[0063] In particular, among the deacetylase inhibitors, vorinostat can pass through the blood-brain barrier and directly enter the brain tumor cells to play a role. In the in vitro combination experiment (see Figures 1 to 4 ), the combination of vorinostat and the phorboxazole has the best effect. Although Figure 2 the ranking of PCI-34051 is higher than that of vorinostat, it is further found that the half-inhibitory concentration of PCI-34051 is higher and the amount of PCI-34051 passing through the blood-brain barrier into the tumor lesion is limited, so the clinical effect is second.

[0064] In addition, chidamide is a new type of antitumor drug used for the clinical treatment of various malignant tumors, and chidamide is also a histone deacetylase (HDAC) inhibitor. The in vitro experiment and the animal experiment of the present application prove that the combination of chidamide and the variant of the phorboxazole can achieve a combination effect obviously better than the single drug group.

[0065] In addition, the in vitro experiment of the present application also proves that the combination of the present application is also effective for adult glioma, such as U87 cells.

[0066] The inventors have found that Sirtuin inhibitors alone upregulate the acetylation level of chromatin in cells, active gene transcription process, and are accompanied by excessive opening of SP / KLF transcription factor binding sites. Through functional analysis of these abnormally active genes, the inventors have further found that they are related to hypoxic adaptation and tumor metastasis / infiltration, and other indications that promote tumor drug resistance. Therefore, the phorboxazole or its variant EC-8042 can inhibit the expression of related genes by inhibiting the binding of SP / KLF transcription factors, thereby reducing the drug resistance of tumors. Not only that, EC-8042 can also synergize with sirtuin inhibitors to inhibit the growth and proliferation of tumor cells. Therefore, the combined use of the two drugs can achieve a more safe and effective clinical treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0067] The embodiments of the present application will be described in detail below with reference to the accompanying drawings, in which:

[0068] Figure 1 The treatment scheme for DIPG cell lines in Example 1 of the present application is shown.

[0069] Figure 2 The screening results of combined use of phorboxazole in Example 1 of the present application are shown.

[0070] Figure 3 The effects of combined use of sirtuin inhibitors and phorboxazole in Example 1 of the present application are shown, in which A, B and C respectively represent the cell lines SU-DIPG-IV, SU-DIPG-XIII and SU-DIPG-XVII.

[0071] Figure 4 The effects of combined use of sirtuin inhibitors and phorboxazole variants in Example 1 of the present application are shown, in which A, B and C respectively represent the cell lines SU-DIPG-IV, SU-DIPG-XIII and SU-DIPG-XVII.

[0072] Figure 5 The clustering analysis results of DIPG cell transcriptome sequencing of the vorinostat alone group and the vorinostat and EC-8042 combined use group in Example 1 of the present application are shown.

[0073] Figure 6 The results of functional analysis of the first group of genes in Example 1 of the present application are shown.

[0074] Figure 7 The RT-qPCR results of the EC-8042 alone group, the vorinostat alone group, and the combined use group in Example 1 of the present application are shown.

[0075] Figure 8Results of cell proliferation experiments in Example 1 of the present application are shown, wherein A shows the results of MTS detection of cell proliferation; B, C and D show the results of experiments using EdU incorporation method, for cell lines SU-DIPG-IV and SU-DIPG-XVII.

[0076] Figure 9 Deacetylase inhibitor alone promotes hypoxia adaptation and infiltration ability of tumor cells, wherein A and B show the results of experiments using real-time quantitative PCR to detect changes in expression of hypoxia adaptation and tumor metastasis related genes, for cell lines SU-DIPG-IV, SU-DIPG-XIII, SU-DIPG-XVII and SHSMU-DIPG-PDX; C shows the results of experiments using transwell chamber to detect cell metastasis and invasion ability, for cell lines SU-DIPG-IV, SU-DIPG-XIII, SU-DIPG-XVII and SHSMU-DIPG-PDX.

[0077] Figure 10 EC-8042 and deacetylase inhibitor combination can reverse tumor cell resistance caused by deacetylase inhibitor alone, wherein A shows that according to high-throughput sequencing results of SU-DIPG-XVII cell RNA, genes are divided into 6 groups, and the genes in group 1 are increased in expression when deacetylase inhibitor alone is used, and the expression is decreased again after EC-8042 is added; B shows the results of GO enrichment analysis of the genes in group 1 in A; C shows the results of KEGG enrichment analysis of the genes in group 1 in A; D shows the results of experiments using real-time quantitative PCR to detect changes in expression of hypoxia adaptation and tumor metastasis related genes, for cell lines SU-DIPG-IV and SU-DIPG-XVII; E shows the results of experiments using transwell chamber to detect cell metastasis and invasion ability, for cell lines SU-DIPG-IV and SU-DIPG-XVII.

[0078] Figure 11 A scheme of implanting tumor cells and administering drugs in Example 2 of the present application is shown.

[0079] Figure 12 Results of quantitatively analyzing tumor size in mice by detecting spontaneous light intensity of tumor cells in Example 2 of the present application are shown, wherein A is a photograph of a mouse implanted with SU-DIPG-XVII cells, B is a statistical analysis result of light intensity of the mouse implanted with SU-DIPG-XVII cells, C is a photograph of a mouse implanted with SHSMU-DIPG-PDX cells, and D is a statistical analysis result of light intensity of the mouse implanted with SHSMU-DIPG-PDX cells.

[0080] Figure 13 The survival probability and survival days of each experimental group after tumor implantation in mice in Example 2 of the present application are shown, wherein A shows the survival probability and survival days of each experimental group after implantation of SU-DIPG-XVII cells, and B shows the survival probability and survival days of each experimental group after implantation of SHSMU-DIPG-PDX cells.

[0081] Figure 14 The results of the immunohistochemical experiment in Example 2 of the present application are shown, wherein A is the staining diagram of each experimental group implanted with SU-DIPG-XVII cells, B is the quantitative statistical diagram of each experimental group implanted with SU-DIPG-XVII cells, C is the staining diagram of each experimental group implanted with SHSMU-DIPG-PDX cells, and D is the quantitative statistical diagram of each experimental group implanted with SHSMU-DIPG-PDX cells.

[0082] Figure 15 The results of analyzing the expression profile data and survival period data of DIPG clinical patients are shown, wherein A shows the results of GSEA analysis between the control group and the combined drug group using the top 100 genes specifically up-regulated in DIPG tumors screened from the expression profile of DIPG tumors and normal cells in the article published by Manav Pathania et al. in 2017 [9] ; B shows the results of GSEA analysis between the control group and the combined drug group using the top 100 genes specifically down-regulated in DIPG tumors screened from the expression profile of DIPG tumors and normal cells in the article published by Manav Pathania et al. in 2017 [9] ; C shows the results of GSEA analysis between the control group and the combined drug group using the characteristic gene set related to longer lifespan in DIPG patients defined in the article published by Jamie N. Anastas et al. in 2019

[10] ; wherein A, B, and C are all SU-DIPG-IV and SU-DIPG-XVII cell lines.

[0083] Figure 16The effect of EC-8042 combined with chidamide is shown; wherein, A shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-IV; B shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-IV; C shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-XVII; D shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-XVII.

[0084] Figure 17 The effect of EC-8042 combined with chidamide is shown; wherein, A shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-IV; B shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-IV; C shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-XVII; D shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-XVII.

[0085] Figure 18 The effect of EC-8042 combined with chidamide is shown; wherein, A shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-IV; B shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-IV; C shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-XVII; D shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-XVII.

[0086] Figure 19 The effect of EC-8042 combined with chidamide is shown; wherein, A shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-IV; B shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-IV; C shows the relative cell number of EC-8042, chidamide and the combined use on cell line SU-DIPG-XVII; D shows the combination index (CI) of EC-8042 combined with chidamide on cell line SU-DIPG-XVII.

[0087] Figure 20The effect of EC-8042 combined with chidamide is shown; wherein, A shows the relative cell number of EC-8042, chidamide and combined drug on cell line U87; B shows the combined drug index (CI) of EC-8042 combined with chidamide on cell line U87; C shows the relative cell number of EC-8042, chidamide and combined drug on cell line CT-2A; D shows the combined drug index (CI) of EC-8042 combined with chidamide on cell line CT-2A.

[0088] Figure 21 The combination of EC-8042 and chidamide significantly down-regulates the proliferation rate of adult glioblastoma cell line (U87) and mouse glioma cell line (CT-2A); wherein, A shows the effect of EC-8042, chidamide and combined drug on the proliferation rate of adult glioblastoma cell line (U87); B shows the effect of EC-8042, chidamide and combined drug on the proliferation rate of mouse glioma cell line (CT-2A). DETAILED DESCRIPTION

[0089] Unless otherwise specified, the reagents used in the following examples are analytical grade reagents and can be commercially available from regular channels.

[0090] Cell line sources:

[0091] Child diffuse intrinsic pontine glioma primary cells SU-DIPG-IV (H3.1K27M), SU-DIPG-XIII (H3.3K27M), SU-DIPG-XVII (H3.3K27M) were gifted by Michelle Monje lab at Stanford University and Tang Yujie lab at Shanghai Jiaotong University, SHSMU-DIPG-PDX cells were gifted by Tang Yujie lab at Shanghai Jiaotong University, U87 cells were purchased from ATCC (Cat. No. HTB-14), CT-2A cells were gifted by Kang Chunsheng lab at Tianjin Medical University.

[0092] Drug sources:

[0093] Mithramycin (brand MCE, Cat. No. HY-A0122); Mithramycin variant EC-8042 was gifted by EntreChem, Spain.

[0094] Experimental instruments:

[0095] Biosafety cabinet (brand Thermo Fisher, model 1300IIA2); carbon dioxide cell incubator (brand Thermo Fisher, model 311); cell imaging microplate reader (brand BIOTEK, model Cytation5); inverted microscope (brand Leica, model DFC295); flow cytometry (brand BD, model Fortessa); fluorescent quantitative PCR instrument (brand Roche, model LightCycler® 480).

[0096] Example 1: In vitro experiment

[0097] 1.1 Experimental method

[0098] (1) Cell proliferation ability detection (MTS method)

[0099] The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (promega, item number G5430) kit detection method was used. First, 1000 cells / 100 μl of cell culture medium were plated in each well of a 96-well plate, with 6 replicate wells for each cell. For median lethal dose (IC50) and combination efficacy (CI) detection experiments, the number of cells was detected after 3 days of culture in each drug group; for cell proliferation ability detection experiments, 0 days, 1 day and 3 days were set as the detection time points. Before detection, the culture medium in the culture well was removed, 100 μl of MTS buffer (prepared in the ratio of DMEM medium:MTS:PMS=100:20:1) was added to each well, the 96-well plate was wrapped with tin foil, and incubation was carried out at 37°C for 2 hours (note: the incubation time must be the same each time), then detection was carried out using a microplate reader, with an absorbance value of 490 nm.

[0100] (2) Combination index (CI) calculation

[0101] The relative number of cells under each concentration gradient of drug alone and drug combination conditions compared with the control group without drug use was detected, and the CalcuSyn software (Biosoft, Cambridge, UK) was introduced for combination index calculation.

[0102] (3) Cell migration and invasion ability detection

[0103] For cell invasion assay, first melt the matrigel on ice, then dilute with 8 times volume of pre-cooled medium without supplements, ready for use. Add the medium with chemoattractant to the 24-well plate, carefully put the transwell (purchased from corning, item number 3422) on the medium, make sure no air bubble is under the transwell, add 50 μl prepared matrigel to each transwell. Incubate at 37℃ for 30 minutes, aspirate the liquid. For cell migration assay, omit the above step, directly prepare the transwell. Resuspend 5 x 10 4 -1 x 10 5 cells in 100-150 μl medium without chemoattractant, inoculate into the transwell, incubate for 6-8 hours, wipe off the residual cells and matrigel in the upper transwell with a cotton swab, fix with 10% formaldehyde for 10 minutes, wash once with PBS, then stain with 20% crystal violet for 15 minutes, wash with PBS until no floating color, then observe and take pictures under an inverted microscope.

[0104] (4) RNA extraction, reverse transcription and real-time quantitative PCR

[0105] A. Extraction of cell RNA

[0106] Digest and centrifuge to collect the cell precipitate of each drug group, wash once with PBS, add 1 ml TRIzol solution (brand life technology, item number 15596018), vortex to mix. Incubate at room temperature for 10 minutes, add 200 μl chloroform, shake vigorously for 15 seconds, incubate at room temperature for 3 minutes, centrifuge at 12000 rpm at 4℃ for 10 minutes. At this time, the sample is divided into three layers, carefully aspirate 400 μl of the uppermost aqueous phase into a new centrifuge tube, add 400 μl of isopropanol, mix well by inverting, incubate at room temperature for 10 minutes. Centrifuge at 12000 rpm at 4℃ for 10 minutes, at this time a small amount of white precipitate can be seen at the bottom of the tube, discard the upper waste liquid. Add 1 ml of freshly prepared 75% ethanol, invert to mix, wash the RNA precipitate. Centrifuge at 7500 rpm at 4℃ for 5 minutes, discard the upper waste liquid. Air dry the precipitate to a translucent state. Add 20 μl of RNase-free ddH2O to dissolve, determine the concentration, and proceed to the next step or store at -80℃.

[0107] B. Transcriptome high-throughput sequencing

[0108] RNA library construction and high-throughput sequencing were completed by Beijing Novogene Bioinformatics Technology Co., Ltd.

[0109] C. Reverse transcription of RNA

[0110] Reverse transcription kit (brand life technology, item number K1622) experimental method. First remove the RNA extracted in the last step may be residual genomic DNA. Reaction system as follows:

[0111]

[0112] Put in PCR instrument, 37℃ reaction 30 minutes.

[0113] Add 1 μl 50mM EDTA, 65℃ incubation 10 minutes to stop the reaction.

[0114] Add 1 μl Oligo dT(18), 65℃ incubation 10 minutes, after the reaction is placed on ice.

[0115] Reverse transcription reaction, system as follows:

[0116]

[0117] Mix and centrifuge, 42℃ incubation 60 minutes, 70℃ incubation 5 minutes to stop the reaction.

[0118] D. Real-time quantitative PCR (RT-qPCR)

[0119] With reverse transcription product cDNA as template, RT-qPCR. Reaction system as follows:

[0120]

[0121] The above reaction system is placed in PCR amplification instrument for reaction, reaction program as follows:

[0122]

[0123] (5) EdU staining method to detect cell proliferation

[0124] BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071) kit detection method. First EdU incorporation into cell culture medium, culture 24 hours after digestion into cell suspension, 4% paraformaldehyde fixation 30 minutes, 0.3% Triton X-100 permeabilization 15 minutes. Wash twice after PBS, add the kit in the dark incubation for 30 minutes. After washing, flow cytometry for detection.

[0125] (6) data analysis

[0126] Reads were mapped to the human genome reference sequence hg38 using hisat2 version 2.1.0. Full-length transcript assembly and quantification were performed for each gene using StringTie v2.1.1. KEGG and GO enrichment analysis was done using R package cluster Profiler.

[0127] 1.2 Experimental results

[0128] (1) Sirtuin inhibitors and SP / KLF transcription factor family inhibitors, such as the drug blebbistatin or its variant EC-8042, have good combined effects in DIPG

[0129] In order to find a combination therapy strategy, the inventors used 182 small molecule drugs targeting epigenetic modification proteins (purchased from Selleck China, product name: Epigenetic Compound Library, product number: L1900, which contains inhibitors of epigenetic enzymes, including histone deacetylases (HDACs), SIRTs, lysine demethylases, histone acetyltransferases (HATs), DNA methyltransferases (Dnmts) and SIRTs activators, etc.) to treat DIPG cell lines in a concentration gradient, and at the same time, dimethyl sulfoxide (DMSO) or blebbistatin treatment, as shown in Figure 1 .

[0130] For these 182 small molecule drugs, 130 inhibitors with high IC50 values were first excluded, and among the remaining 52 drugs, 18 drugs had good combined effects with blebbistatin, with log2 (single drug group / combination group IC50) fold greater than 1 considered to have good combined effects, and the combined effects decreased from top to bottom, of which 8 were inhibitors of deacetylases, as shown in Figure 2 .

[0131] The inventors further verified the combined effects of the top five deacetylase inhibitors (PCI-34051, vorinostat, droxinostat, danusertib and pracinostat) and blebbistatin on the inhibition of DIPG cell proliferation in vitro. The results were calculated by the mixed drug analysis software CalcuSyn 2.0, as shown in Figure 3 , wherein CI less than 1 represents the combined effect of the two drugs, and CI less than 0.7 represents a significant combined effect of the two drugs, and the lower dotted line in the figure represents CI is 0.7. The inventors found that deacetylase inhibitors and blebbistatin had good combined effects within a certain concentration range, and the concentrations of various deacetylase inhibitors and blebbistatin were as shown in Figure 3 .

[0132] The inventors also verified the combined effect of the deacetylase inhibitor and the variant of photofrin, EC-8042, and the results are shown in Figure 4 Likewise, the deacetylase inhibitor and EC-8042 also have a good combined effect in a certain concentration range, and the concentrations of various deacetylase inhibitors and EC-8042 are shown in Figure 4

[0133] (2) Deacetylase inhibitors and EC-8042 can synergistically down-regulate the proliferation ability of DIPG cells

[0134] The inventors performed transcriptome sequencing on DIPG cells in the deacetylase inhibitor vorinostat alone group (referred to as the vorinostat alone group) and the deacetylase inhibitor vorinostat combined with EC-8042 group (referred to as the combined group, in which the concentration of vorinostat is 0.8 µM, the concentration of EC-8042 is 10 nM, and the molar ratio of vorinostat to EC-8042 is 80: 1). Cluster analysis was performed, and the results are shown in Figure 5 As can be seen from Figure 5 , the genes in the first cluster are down-regulated compared with the control group without drug treatment in both the vorinostat alone group and the combined group, and the down-regulation amplitude in the combined group is greater than that in the vorinostat alone group. Functional analysis of this cluster of genes showed that this cluster of genes was mainly enriched in proliferation-related signaling pathways such as cell replication and cell cycle, as shown in Figure 6 .

[0135] The transcription levels of cell cycle-related genes (CDK1, CDK2, CDK4, CCNA2, CCNB1, CCNB2, CCND1, and CCNE2) in the EC-8042 alone group, the vorinostat alone group, and the vorinostat combined with EC-8042 group (referred to as the combined group, in which the concentration of vorinostat is 0.8 µM, the concentration of EC-8042 is 10 nM, and the molar ratio of vorinostat to EC-8042 is 80: 1) were studied by RT-qPCR, and a group without drug treatment was set as the control group. The results of RT-qPCR are shown in Figure 7 . Figure 7 The results show that the transcription levels of cell cycle-related genes are significantly down-regulated in the single use group and the control group, and the transcription levels of cell cycle-related genes in the combined group also show a significant down-regulation trend compared with the single use group.

[0136] The experimental results of cell proliferation are shown in Figure 8 From Figure 8 ​As can be seen, the proliferation rate of cells in the EC-8042 alone group, the vorinostat alone group and the control group was significantly down-regulated; the proliferation rate of cells in the combination group (in which the concentration of vorinostat was 0.8 µM, the concentration of EC-8042 was 10 nM, and the molar ratio of vorinostat to EC-8042 was 80: 1) showed a significant downward trend compared with the EC-8042 alone group and the vorinostat alone group.

[0137] (3) EC-8042 combined with deacetylase inhibitors can block the drug resistance of DIPG cells to deacetylase inhibitors

[0138] The inventors also found that deacetylase inhibitors alone can promote the hypoxic adaptation and infiltration ability of tumor cells, leading to drug resistance of tumors to such drugs. As shown in Figure 9 , in which Figure 9 Real-time quantitative PCR experiments of A showed that under hypoxic conditions, the downstream genes VEGFA and GLUT1 of the hypoxia-related signaling pathway in DIPG cells were significantly activated; Figure 9 Real-time quantitative PCR experiments of B showed that after adding deacetylase inhibitors vorinostat, pracinostat or danusertib, the hypoxia-responsive genes VEGFA and GLUT1 were further activated, at the same time, the expressions of tumor metastasis and infiltration-related genes CDH2 and TWIST1 were significantly up-regulated, while the expression of CDH1 gene which characterized the low infiltration ability of tumors was significantly decreased; Figure 9 The multi-well chamber experiment of C also showed that deacetylase inhibitors vorinostat and danusertib can promote tumor metastasis under hypoxic conditions, and deacetylase inhibitors vorinostat, pracinostat and danusertib can promote tumor cell invasion under hypoxic conditions.

[0139] EC-8042 combined with deacetylase inhibitors (in which the concentration of vorinostat was 0.8 µM, the concentration of EC-8042 was 10 nM, and the molar ratio of vorinostat to EC-8042 was 80: 1) not only can synergistically inhibit cell proliferation, but also can reverse the above-mentioned side effects caused by deacetylase inhibitors alone. As shown in Figure 10 , according to the transcriptome high-throughput sequencing data, the genes were clustered and analyzed as shown in Figure 10 A, the results showed that the expression of the first group of genes was significantly increased when vorinostat was used alone (left), and the expression was significantly decreased after combination with EC-8042 (right), indicating that this group of genes was activated by deacetylase vorinostat and inhibited by EC8042; the GO analysis of the first group of genes shown in Figure 10 A, the results are shown in Figure 10 B, which enriched the functions related to hypoxic adaptation (red words) and tumor metastasis and infiltration (blue words); the GO analysis of the second group of genes shown in Figure 10KEGG analysis was performed on the genes of group A shown in Figure 1, and the results are as follows: Figure 10 As shown in C, signaling pathways related to hypoxia adaptation (red text) and tumor metastasis and invasion (blue text) were also enriched; real-time quantitative PCR experiments were performed as follows: Figure 10 As shown in D, hypoxia response-related genes VEGFA and GLUT1 and tumor metastasis-invasive genes CDH2 and TWIST1 were activated in the deacetylase vorinostat monotherapy group and were significantly downregulated in the EC-8042 and vorinostat combination group, while the expression changes of CDH1 gene, which characterizes low tumor invasiveness, showed the opposite trend. Figure 10 The multi-compartment experiment of EC-8042 also demonstrated that the deacetylase inhibitor vorinostat can promote tumor metastasis and invasion under hypoxic conditions, while the ability of tumor metastasis and invasion is inhibited when EC-8042 and vorinostat are used in combination.

[0140] (4) The combined use of EC-8042 and deacetylase inhibitors significantly downregulates the proliferation rate of glioma cells.

[0141] The inventors tested the combined effect of EC-8042 and the deacetylase inhibitor chidamide in DIPG and found that chidamide and EC-8042 also had a good combined effect within a certain concentration range. The concentrations of chidamide and EC-8042 were as follows: Figure 16 As shown.

[0142] Compared with the EC-8042 monotherapy group, the chidamide monotherapy group, and the control group, SU-DIPG-IV cells ( Figure 17 A) and SU-DIPG-XVII cells ( Figure 17 B) The proliferation rate of SU-DIPG-IV cells was significantly downregulated; the EC-8042 and chidamide combination group (where the concentration of chidamide was 1.6 µM, the concentration of EC-8042 was 10 nM, and the molar ratio of chidamide to EC-8042 was 160:1) Figure 17 A) and SU-DIPG-XVII cells ( Figure 17 The proliferation rate of B) showed a significant downward trend compared to the EC-8042 monotherapy group and the damide monotherapy group.

[0143] The inventors also tested the combined effects of the deacetylase inhibitor vorinostat and chidamide in the adult glioblastoma cell line (U87) and the mouse glioma cell line (CT-2A), and the results are as follows: Figures 18 to 21As shown in FIG. 6A, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 18 As shown in FIG. 6B, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 20

[0144] As shown in FIG. 6C, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 19 As shown in FIG. 6D, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 19 A) and mouse glioma cell line CT-2A (B) in the combination group of EC-8042 and vorinostat (wherein the concentration of vorinostat is 0.8 µM, the concentration of EC-8042 is 10 nM, and the molar ratio of vorinostat to EC-8042 is 80: 1) showed a significant downward trend compared with the single use group of EC-8042 and the single use group of vorinostat. Figure 19

[0145] As shown in FIG. 7A, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 21 As shown in FIG. 7B, the combination of EC-8042 and vorinostat at a molar ratio of 1: 80 (EC-8042: vorinostat) showed a significant synergistic effect on the proliferation of human glioblastoma cell line U87 (A) and mouse glioma cell line CT-2A (B) in a certain concentration range. Figure 21 A) and mouse glioma cell line CT-2A (B) in the combination group of EC-8042 and vorinostat (wherein the concentration of vorinostat is 0.8 µM, the concentration of EC-8042 is 10 nM, and the molar ratio of vorinostat to EC-8042 is 80: 1) showed a significant downward trend compared with the single use group of EC-8042 and the single use group of vorinostat. Figure 21

[0146] Example 2: Animal experiment

[0147] 2.1 Experimental method

[0148] (1) In vivo animal experiment

[0149] In order to detect the anticancer effect of the drug in vivo, the inventors first established a mouse DIPG orthotopic tumor model, and 1 x 10 5 ​​​SU-DIPG-XVII-luciferase or SHSMU-DIPG-PDX-luciferase cells were injected into the position of the brainstem of 4-week-old female mice in situ. The survival time of the mice was recorded from the day of implantation. Two weeks after the implantation day, the mice were divided into four groups of six each and injected with the drug vorinostat or EC-8042 or a combination of the two drugs through the tail vein, three injections per day for four consecutive weeks. The injection dose of the vorinostat single-drug group was 200 mg (vorinostat) / kg (mouse weight) / time, the injection dose of the EC-8042 single-drug group was 20 mg (EC-8042) / kg (mouse weight) / time, and the injection dose of the combination of the two drugs was 200 mg (vorinostat) / kg (mouse weight) / time and 20 mg (EC-8042) / kg (mouse weight) / time. Because EC-8042 has poor brain entry effect, the concentration of EC-8042 used in the animal experiment was increased, and the small molecule nano-packaging technology was used (the packaging was completed by Sun Shaokai Laboratory of Tianjin Medical University). In the second week, the fourth week, and the sixth week after the implantation, the size of the tumor in the mouse body was quantitatively analyzed by detecting the spontaneous light intensity of the tumor cells using the IVIS spectral in vivo imaging system (PerkinElmer).

[0150] (2) Immunohistochemistry

[0151] A. Baking slices: select the slices that need to be dyed and place them in a 60°C oven for 3-6 hours;

[0152] B. Gradient rehydration: xylene I 15 min, xylene II 15 min, 100% ethanol I 5 min, 100% ethanol II 5 min, 95% ethanol I 5 min, 95% ethanol II 5 min, 80% ethanol 5 min, 70% ethanol 5 min, distilled water 5 min;

[0153] C. Antigen repair: take 5 ml of 50x sodium citrate antigen repair solution (pH=6) and dilute it to 1x with distilled water. Add the antigen repair solution to the antigen repair box containing the tissue slices. Heat in the microwave oven for 5-6 min until the liquid boils, then let it cool at room temperature for 6 min. Heat again for about 1 min until boiling, then let it cool at room temperature for 6 min. Repeat the boiling four times. Place the antigen repair box containing the tissue slices at room temperature for about 60 min to cool to room temperature;

[0154] D. Block endogenous peroxidase: Remove the sections from the antigen retrieval solution, drain as much liquid as possible, and add endogenous peroxidase blocking reagent (select rabbit / mouse origin according to the antibody to be stained) to the tissue, and incubate at room temperature for 10 minutes. Tilt the slide to drain the endogenous peroxidase blocking reagent, and place the sections in the antigen retrieval cassette and wash with PBS buffer for 3 times, 3-5 minutes each time;

[0155] E. Remove the sections from the liquid, add excess 1% BSA (prepared with PBS buffer) to the tissue, and place the sections in the wet box, and seal in the 37°C oven for 60 minutes;

[0156] F. Primary antibody incubation: Take the wet box out of the 37°C oven, remove the sections to drain the 1% BSA, add 20-50 μl of the antibody diluted with 1% BSA to the tissue, cover the tissue area with sealing film, and place the sections in the wet box and incubate in the 4°C refrigerator overnight. Take the wet box out, and place it at room temperature for 1-2 hours to warm up, and place the sections in the antigen retrieval cassette and wash with PBS buffer for 3 times, 3-5 minutes each time;

[0157] G. Add reaction enhancer: Remove the sections from the liquid, add reaction enhancer to the tissue, and place the sections in the wet box and incubate at room temperature for 20 minutes. Remove the sections to drain the liquid, and place the sections in the antigen retrieval cassette and wash with PBS buffer for 3 times, 3-5 minutes each time;

[0158] H. Secondary antibody incubation: Remove the sections from the liquid, add the corresponding enhanced goat anti-rabbit / mouse IgG polymer to the tissue, and place the sections in the wet box and incubate at room temperature for 20 minutes. Remove the sections to drain the liquid, and place the sections in the antigen retrieval cassette and wash with PBS buffer for 3 times, 3-5 minutes each time;

[0159] I. DAB color developing solution color development: Remove the sections from the liquid, add the diluted DAB color developing solution (prepared as needed) to the tissue, and incubate at room temperature for 5-10 minutes until color development. Remove the sections to drain the liquid, and place the sections in the antigen retrieval cassette and wash with PBS buffer for 3 times, 3-5 minutes each time;

[0160] J. Hematoxylin re-stain the cell nucleus: Remove the sections from the liquid, add hematoxylin staining solution to the tissue, and incubate at room temperature for 3-5 minutes until the cell nucleus is blue-violet. Remove the sections to drain the liquid, and place the sections in the antigen retrieval cassette and wash with distilled water to remove the hematoxylin staining solution;

[0161] K. Hydrochloric acid differentiation: Remove the sections from the liquid, and place the sections in the hydrochloric acid differentiation solution for about 20 seconds;

[0162] L. Ammonia blue recovery: Remove the sections from the liquid, and place the sections in the ammonia blue recovery solution for about 20 seconds;

[0163] M. Gradient dehydration, transparency: Distilled water 5 min, 70% ethanol 5 min, 80% ethanol 5 min, 95% ethanol II 5 min, 95% ethanol I 5 min, 100% ethanol II 5 min, 100% ethanol I 5 min, Xylene II 10 min, Xylene I 10 min;

[0164] N. Neutral gum sealing: 2 drops of neutral gum are added to the slice with a pipette, and a cover glass is covered on the tissue, and air-dried at room temperature (as much as possible without bubbles on the tissue, if there are bubbles, use forceps to gently press out or use xylene to wash off the cover glass and re-seal the slice);

[0165] O. Microscopy and photography.

[0166] 2.2 Experimental results

[0167] In vivo combination of HDAC inhibitors and EC-8042 inhibits tumor growth

[0168] In this example, tumor cells were transplanted in situ in mice, and single and double drug treatments were given, as shown in Figure 11 .

[0169] The results of the spontaneous light intensity of tumor cells are shown in Figure 12 , and the survival probability and survival days of mice after tumor implantation are shown in Figure 13 . From the results of Figure 12 and 13 , it can be seen that the combination of EC-8042 and vorinostat has better therapeutic effect compared with the EC-8042 single use group and the vorinostat single use group. The results of immunohistochemistry are shown in Figure 14 . From Figure 14 , it can be seen that the results of immunohistochemistry also show that the combination of the two drugs can significantly down-regulate the proliferation ability of tumor cells, and reverse the hypoxic adaptation ability and infiltration ability of tumor cells caused by the use of HDAC inhibitors alone, and reduce the drug resistance of tumor cells.

[0170] The results of analyzing the expression profile data and survival data of DIPG clinical patients disclosed in [9],

[10] are shown in Figure 15 . From the results of Figure 15 , it is found that the combination drug strategy can well correlate the expression profile of tumor cells to the gene expression pattern related to normal neural progenitor cells (as shown in Figure 15 A and Figure 15 B) and longer survival (as shown in Figure 15 C).

[0171] While the application has been described in some detail, it is apparent that modifications can be made without departing from the spirit and scope of the application. It is to be understood that the application is not limited to the described embodiments, but encompasses all equivalents falling within the scope of the claims.

[0172] References:

[0173] 1. Long, W., et al., Potential New Therapies for Pediatric Diffuse Intrinsic Pontine Glioma. Front Pharmacol, 2017. 8: p. 495.

[0174] 2. Freeman, C.R. and G. Perilongo, Chemotherapy for brain stem gliomas. Childs Nerv Syst, 1999. 15(10): p. 545-53.

[0175] 3. Sturm, D., et al., Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge. Nat Rev Cancer, 2014. 14(2): p. 92-107.

[0176] 4. Gallitto, M., et al., Role of Radiation Therapy in the Management of Diffuse Intrinsic Pontine Glioma: A Systematic Review. Adv Radiat Oncol, 2019. 4(3): p. 520-531.

[0177] 5. Kramm, C.M., et al., Thalamic high-grade gliomas in children: a distinct clinical subset? Neuro Oncol, 2011. 13(6): p. 680-9.

[0178] 6. Vanan, M.I. and D.D. Eisenstat, DIPG in Children - What Can We Learn from the Past? Front Oncol, 2015. 5: p. 237.

[0179] 7. Liu, H., et al., The recent progress of deep-learning-based in silico prediction of drug combination. Drug Discov Today, 2023. 28(7): p. 103625.

[0180] 8. Shtar, G., et al., CDCDB: A large and continuously updated drug combination database. Sci Data, 2022. 9(1): p. 263.

[0181] 9. Pathania, M., et al., H3.3 Cooperates with Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas. Cancer Cell, 2017. 32(5): p. 684-700.e9.

[0182] 10. Anastas, J.N., et al., Re-programing Chromatin with a Bifunctional LSD1 / HDAC Inhibitor Induces Therapeutic Differentiation in DIPG. Cancer Cell, 2019. 36(5): p. 528-544 e10.

Claims

1. Use of a HDAC inhibitor and a phorboxazole or a variant thereof in the manufacture of a medicament for the prevention and / or treatment of a glioma; wherein the variant of the phorboxazole has a structure shown in the following formula: wherein the HDAC inhibitor is selected from one or more of PCI-34051, Vorinostat, Droxinostat, Danusostat, Pracinostat and Chidamide, and the glioma is a childhood diffuse intrinsic pontine glioma; or the HDAC inhibitor is Vorinostat and / or Chidamide, and the glioma is an adult glioblastoma.

2. Use according to claim 1, characterized in that, The ratio of the amount of the HDAC inhibitor to the amount of the phorboxazole or the variant thereof is (10 nM ~ 750 µM):(2.5 nM ~ 250 nM).

3. Use according to claim 1, characterized in that, The ratio of the amount of the HDAC inhibitor to the amount of the phorboxazole is (10 nM ~ 750 µM):(5 nM ~ 250 nM).

4. Use according to claim 1, characterized in that, The ratio of the amount of Vorinostat to the amount of the phorboxazole is (0.2 µM ~ 10 µM):(5 nM ~ 250 nM); or the ratio of the amount of Droxinostat to the amount of the phorboxazole is (15 µM ~ 750 µM):(5 nM ~ 250 nM); or the ratio of the amount of PCI-34051 to the amount of the phorboxazole is (10 µM ~ 250 µM):(5 nM ~ 125 nM); or the ratio of the amount of Danusostat to the amount of the phorboxazole is (10 nM ~ 500 nM):(5 nM ~ 250 nM); or the ratio of the amount of Pracinostat to the amount of the phorboxazole is (10 nM ~ 500 nM):(5 nM ~ 250 nM); or the ratio of the amount of Chidamide to the amount of the phorboxazole is (0.4 µM ~ 20 µM):(5 nM ~ 250 nM).

5. Use according to claim 1, characterized in that, The molar ratio of Vorinostat to the phorboxazole is 40:1; or the molar ratio of Droxinostat to the phorboxazole is 3000:1; or the molar ratio of PCI-34051 to the phorboxazole is 2000:1; or the molar ratio of Danusostat to the phorboxazole is 2:1; or the molar ratio of Pracinostat to the phorboxazole is 2:1; or the ratio of the amount of Chidamide to the amount of the phorboxazole is (80 ~ 160):

1.

6. Use according to claim 1, characterized in that, The ratio of the amount of the HDAC inhibitor to the amount of the variant of the phorboxazole is (10 nM ~ 375 µM):(2.5 nM ~ 125 nM).

7. Use according to claim 1, characterized in that, The ratio of the amount of Droxinostat and the variant of Calicheamicin is (0.2 µM~10 µM):(2.5 nM~125 nM); or, the ratio of the amount of Droxinostat and the variant of Calicheamicin is (15 µM~375 µM):(2.5 nM~62.5 nM); or, the ratio of the amount of PCI-34051 and the variant of Calicheamicin is (10 µM~120 µM):(2.5 nM~30 nM); or, the ratio of the amount of Danoxtat and the variant of Calicheamicin is (10 nM~500 nM):(2.5 nM~125 nM); or, the ratio of the amount of Pariprost and the variant of Calicheamicin is (10 nM~500 nM):(2.5 nM~125 nM); or, the ratio of the amount of Sedarobin and the variant of Calicheamicin is (0.4 µM~20 µM):(5 nM~125 nM).

8. Use according to claim 1, characterized in that, The molar ratio of Droxinostat and the variant of Calicheamicin is 80: 1; or, the molar ratio of Droxinostat and the variant of Calicheamicin is 6000: 1; or, the molar ratio of PCI-34051 and the variant of Calicheamicin is 4000: 1; or, the molar ratio of Danoxtat and the variant of Calicheamicin is 4: 1; or, the molar ratio of Pariprost and the variant of Calicheamicin is 4: 1; or, the molar ratio of Sedarobin and the variant of Calicheamicin is (80~160): 1.

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