Pharmaceutical composition for treating leukemia and application thereof

Abstract

The invention relates to the field of medicines, in particular to a pharmaceutical composition for treating leukemia and application thereof. The invention finds that the c-Jun controls the expression of HDAC1 in leukemia tumor cells, and cell proliferation and CD33 expression can be effectively reduced by inhibiting the c-Jun. Meanwhile, the HDAC1 inhibitor panobinostat and the c-Jun inhibitor ailanthone are combined to be used for treating the AML, compared with single use of the HDAC1 inhibitor or the c-Jun inhibitor, the synergistic interaction effect is achieved, and the discovery provides a promising way for medicine combined treatment in leukemia treatment.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a drug combination for treating leukemia and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a common and fatal acute leukemia in adults, characterized by abnormal proliferation and differentiation of myeloid primitive cells in the bone marrow, resulting in the inhibition of normal hematopoiesis. AML patients usually present with symptoms such as anemia, bleeding, and infection, with rapid disease progression and poor prognosis.

[0003] At present, the main treatments for AML include chemotherapy, hematopoietic stem cell transplantation, etc. However, these treatments have some limitations. Although chemotherapy can relieve the symptoms of AML to a certain extent, chemotherapy drugs often have large toxic side effects and can cause serious damage to the patient's body. In addition, chemotherapy is prone to drug resistance, resulting in poor treatment effects. Hematopoietic stem cell transplantation is a relatively effective treatment method, but the transplantation process is complicated, the risk is high, and the donor source is limited, which limits its widespread application.

[0004] Due to the limitations of existing treatments, finding new therapeutic targets for AML is of great clinical significance. New therapeutic targets can provide a basis for developing safer and more effective treatments and improve the survival rate and quality of life of AML patients. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies of the prior art, the present invention provides a highly specific and effective AML treatment target to reduce the damage of existing chemotherapy drugs to normal cells and improve the survival rate and quality of life of AML patients.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In one aspect, the present invention provides a pharmaceutical composition comprising a histone deacetylase 1 (HDAC1) inhibitor and a c-JUN inhibitor.

[0010] In one embodiment, the HDAC1 inhibitor is selected from Vorinostat, Romidepsin, Belinostat, Panobinostat, Chidamide, or one or more of the pharmaceutically acceptable salts, crystal forms, and solvates of the above drugs.

[0011] In one embodiment, the c-JUN inhibitor is selected from one or more of SP600125, Ailanthone, Veratramine, SR11302, NY2267 or pharmaceutically acceptable salts, crystal forms, and solvates of the above drugs.

[0012] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-50):(1-50).

[0013] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-20):(1-20).

[0014] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-10):(1-10).

[0015] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-5):(1-5).

[0016] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-3):(1-3).

[0017] In one embodiment, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is 1:1.

[0018] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0019] In one aspect, the present invention provides a pharmaceutical preparation comprising the above-mentioned pharmaceutical composition.

[0020] In one embodiment, the pharmaceutical formulation comprises a solid dosage form, a semisolid dosage form, a liquid dosage form, or a gaseous dosage form.

[0021] In one embodiment, the solid dosage forms include powders, pills, tablets, and capsules; the semisolid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include lotions, mixtures, solutions, and injections; and the gaseous dosage forms include aerosols and sprays.

[0022] In one aspect, the present invention provides the use of the above-mentioned pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for preventing, inhibiting and / or treating leukemia.

[0023] In one embodiment, the above HDAC1 inhibitor and c-JUN inhibitor are used as main active ingredients in a pharmaceutical composition or pharmaceutical preparation.

[0024] In one embodiment, HDAC1 inhibitor and c-JUN inhibitor as main active ingredients means that HDAC1 inhibitor and c-JUN inhibitor account for more than 10% of the active ingredients; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0025] In one aspect, the present invention provides a method for treating a leukemia patient, comprising administering to the patient a therapeutically effective amount of an HDAC1 inhibitor and a c-JUN inhibitor.

[0026] In one embodiment, the leukemia is selected from acute myeloid leukemia (AML).

[0027] (III) Beneficial effects

[0028] The present invention provides a highly specific and effective AML therapeutic target, which has the following beneficial effects compared with the prior art:

[0029] The present invention finds that c-Jun controls the expression of HDAC1 in leukemia tumor cells. Inhibiting c-Jun can effectively reduce cell proliferation and CD33 expression. At the same time, the present invention combines the HDAC1 inhibitor panobinostat with the c-Jun inhibitor Ailanthone for the treatment of AML, which achieves a synergistic effect compared to the use of HDAC1 inhibitors or c-Jun inhibitors alone. These findings provide a promising approach for drug combination therapy in the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1Differential analysis of HDAC1 expression and pathway activity in hematopoietic stem-progenitor (HSC-Prog) cells. A. Violin plots visualizing the expression levels of HDAC1 gene in AML and HC samples within HSC-Prog cells. B. Evaluation of pathway activity differences between HDAC1+HSC-Prog and HDAC1-HSC-Prog cells by gene set variation analysis (GSVA), providing t-values ​​obtained from linear models. C. CellChat analysis for all cell types showing the number and strength of interactions. D. Bar graph depicting the strength of outgoing interactions in HDAC1+HSC-Prog and HDAC1-HSC-Prog cells. E. Incoming and outgoing signaling pathways for each cell type. F. Comparison of top-level activities of transcription factors between patients and healthy samples using the regulator specificity score (RSS). G. Comparison of top-level activities of transcription factors between HDAC1+HSC-Prog and HDAC1-HSC-Prog cells using the RSS. H. Western blotting was used to detect the expression of HDAC1 and c-JUN in MV4-11 cells treated with SP600125 for 6 hours and 18 hours. I. Statistical analysis of Western blotting results, depicting the expression levels of HDAC1 and c-JUN in MV4-11 cells treated with SP600125 at 6 hours and 18 hours.

[0032] Figure 2 Determination of the function of c-JUN in leukemic cells. A. Schematic diagram of the chemical structure and function of SP600125. B. Experimental flow chart of MV4-11 and THP-1 cells treated with SP600125 and analyzed for cell proliferation using a CFSE dye-based assay. CF. Representative flow cytometric images and bar graphs showing proliferation of MV4-11 (C, D) and THP-1 (E, F) cells after treatment with different concentrations of SP600125, as assessed by CFSE dilution and flow cytometry (n=3); the numbers in the figures indicate the frequency of cell proliferation. G. Overview of the co-culture system using MV4-11 cells treated with SP600125 and natural killer (NK) cells. HI. Determination of NK cell cytotoxicity against MV4-11 cells treated with different concentrations of SP600125, by flow cytometric staining of MV4-11 cells with a viability dye (n=3); the numbers in the figures indicate the frequency of cell lysis. JK. Flow cytometric staining and quantification of THP-1 cells treated with different concentrations of SP600125 using anti-CD33 antibody.

[0033] Figure 3Comprehensive evaluation of treatment efficacy in leukemia mouse models. A. Schematic diagram of the treatment process in leukemia mouse models. B, D. Final volume growth curves (B) (n=6) and weights (D) (n=4) of C1498 tumors in different treatment groups. C. Images of C1498 tumors in each C57BL / 6J mouse in different treatment groups (n=4). E. Survival curves of leukemia mouse models bearing C1498 cells treated with Ailanthone, panobinostat, or Ailanthone combined with panobinostat (n=10). F, G. In vivo bioluminescence imaging and quantification of C1498-GFP-Luc mouse models under different treatment conditions (n=3). H. Schematic diagram of the working principle of the iSMART system. IK. iSMART imaging and quantification of C1498-GFP-Luc mouse models under different therapeutic interventions (n=3). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The present invention provides a highly specific and effective AML treatment target to reduce the damage of existing chemotherapy drugs to normal cells and improve the survival rate and quality of life of AML patients.

[0036] definition:

[0037] The term "inhibit" means to reduce by a measurable amount or to prevent entirely.

[0038] The term "therapeutically effective amount" refers to the amount of a combination of an HDAC1 inhibitor and a c-JUN inhibitor that is effective in treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of an HDAC1 inhibitor combined with a c-JUN inhibitor can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably stop to a certain extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably stop to a certain extent) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent. To the extent that a drug can inhibit growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining a response rate (RR).

[0039] The term "patient" refers to a subject to whom the HDAC1 inhibitor of the present invention is administered in combination with a c-JUN inhibitor. Patients include, but are not limited to, humans, rats, mice, guinea pigs, non-human primates, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. Typically, the patient is a rat, mouse, dog, human, or non-human primate, more typically a human.

[0040] The term "treat or treatment" refers to therapeutic treatment and preventive treatment, wherein the purpose is to inhibit or slow down (mitigate) undesirable physiological changes or conditions, such as the development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of disease extent, stabilization of disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extension of survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from the disease or condition and those susceptible to the disease or condition.

[0041] In the context of cancer, the term "treat" includes any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or reducing the number of cancer cells; and ameliorating one or more symptoms of associated diseases.

[0042] As used herein, the phrase "a salt thereof" refers to the salt form of a compound.

[0043] As used herein, “HDAC1 inhibitor and c-JUN inhibitor as main active ingredients” means that the proportion of HDAC1 inhibitor and c-JUN inhibitor in the active ingredients is more than 10%; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0044] "Crystal form" refers to the arrangement of drug molecules in the crystal and the different specific structures brought about by different solid structures. Although drugs in different crystal forms have different lattice parameters, including the shape, size and space group of the unit cell, as long as they have the same biological effect of inhibiting HDAC1 or c-JUN, they are also within the scope of protection of the present invention.

[0045] "Solvate" refers to a form of matter formed when solvent molecules are incorporated into the crystal lattice during the crystallization process. Although the solvate is composed of solute (drug or compound) and solvent molecules, and there is more solvent in its crystal composition, as long as it has the same biological effect of inhibiting HDAC1 or c-JUN, it is also within the scope of protection of the present invention.

[0046] Histone deacetylase 1 (HDAC1) is an enzyme that plays a key role in the biological processes of cells. HDAC1 is a protein with a relative molecular mass of about 55kDa. It contains a catalytic domain that is responsible for catalyzing the deacetylation reaction of histones. In addition, HDAC1 also has some regulatory domains that can interact with other proteins and participate in regulating their activity and function.

[0047] c-Jun is a protein that is an important member of the AP-1 (activator protein 1) family of transcription factors. c-Jun contains a basic leucine zipper (bZIP) domain, which consists of a region rich in basic amino acids and a leucine repeat sequence. This domain enables c-Jun to form dimers with other AP-1 family members (such as c-Fos, etc.) or itself, thereby binding to specific DNA sequences and regulating gene transcription.

[0048] The following is a detailed description through specific embodiments.

[0049] Cell lines and cell culture

[0050] Human AML cell line MV4-11 and mouse AML cell line C1498 were both from Abiowell. MV4-11 cells were cultured in IMDM (AW-M005) containing 10% FBS (Gibco) and 1% penicillin-streptomycin, while C1498 cells were cultured in DMEM (Gibco) containing 10% FBS (Gibco) and 1% P / S.

[0051] C1498-GFP-Luc cell line was purchased from zqxzbio and cultured in DMEM (Gibco) containing 10% FBS (Gibco) and 1% P / S, and puromycin was added at a concentration of 1 μg / ml. Human AML cell line THP-1 was cultured in RPMI 1640 (Gibco) containing 10% FBS (Gibco) and 1% P / S.

[0052] Mouse samples

[0053] 6-week-old female C57BL / 6J mice were purchased from spfbiotech (Beijing). We used 6- to 8-week-old male or female mice for experiments and used sex- and age-matched littermates as controls.

[0054] Flow cytometry

[0055] The cultured THP-1 cells were centrifuged at 500 g for 5 min and collected. The total number of live cells was determined using a Bright-Line hemocytometer, and dead cells were excluded using trypan blue staining. For flow cytometry, 1 × 10 6 Single cells were incubated with 10ul mouse serum on ice for 20 minutes to block FcγR, and then labeled with specific monoclonal antibodies on ice for 30 minutes. Dead cells were labeled with 7-AAD (BioLegend). Anti-human CD 33 (PE, WM53) antibody was purchased from BioLegend. Flow cytometry was performed using the standard protocol of the CytoFLEX analyzer (Beckman Coulter) and analyzed using FlowJo software.

[0056] Cell proliferation assay

[0057] MV4-11 and THP-1 cells were collected by centrifugation at 500 g for 5 minutes, and cell viability was maintained. Resuspended in 1 ml 1× PBS and counted to ensure that the number of cells was at least 1×10 6 1 μl of CFSE (BioLegend) stock solution was added per ml to a final concentration of 5 μM and incubated at 37°C for 15-20 minutes. 5 volumes of cold medium containing serum were added to terminate the staining and incubated on ice for 5 minutes. The cells were washed three times with complete RPMI 1640 medium and analyzed immediately on a flow cytometer in the dark.

[0058] NK cell cytotoxicity assay

[0059] In KBM581 supplemented with 10% FBS (Gibco) and 1% P / S Cultured in culture medium. Activation of NK cells was induced by adding 200U / ml IL-2 (Proteintech). MV4-11 target cells were pre-labeled with CFSE before co-culture. To detect the cytotoxic capacity of human NK cells, AML cell line (MV4-11) was seeded in 96-well plates. Subsequently, the ratio of effector cells (NK cells) to target cells (MV4-11 cells) was 1:1. After co-culture at 37°C for 4 hours, each sample was supplemented with 1.5μl 7-AAD dye (BioLegend), mixed thoroughly for 5-10 minutes, and then analyzed using flow cytometry.

[0060] Western blot analysis

[0061] Tumor cells were lysed using radioimmunoprecipitation analysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, P0011). Protein samples (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (Bio-Rad, 1610173) and transferred to polyvinylidene difluoride membranes (Millipore, ISEQ00010). The membrane was blocked with 5% skim milk in 1×Tris buffered saline at room temperature. The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-HDAC1 (Proteintech, 10197-1-AP), anti-JUN (Proteintech, 66313-1-Ig). GAPDH was used as a loading control. After incubation with HRP-labeled anti-rabbit immunoglobulin G (IgG) (Proteintech, SA00001-2) or anti-mouse IgG (Proteintech, SA00001-1) antibodies for 1 h, the immunolabeled proteins were detected by chemiluminescence using a chemiluminescent HRP substrate (Millipore, WBKLS0500) and scanned using the Amersham Imager 600 system (Cytiva).

[0062] Subcutaneous tumor model

[0063] C1498 and C1498-GFP-Luc tumor cells were digested into single cells and washed three times in cold PBS. Then, 1 × 10 6 C1498 and 1×10 6 C1498-GFP-Luc tumor cells were injected subcutaneously into the right side of the mice. The tumor volume was measured on the 5th day using the formula (length × width × height) / 2. The tumor was monitored every 2 days. When the tumor reached 1000 mm 3 The mice were killed at 4 000 rpm and recorded as dead, and the survival was observed.

[0064] In vivo treatment

[0065] In the subcutaneous C1498 and C1498-GFP-Luc tumor models, when the tumor size reached approximately 50 mm 3 The mice were randomly divided into groups and treated on the 5th day of tumor bearing, and the treatment was continued once a day until the 15th day. Each mouse was intraperitoneally injected with 10 mg / kg of the drug in a total volume of 200 μl.

[0066] Bioluminescence imaging

[0067] Bioluminescence imaging was used to capture the photon flux emitted by subcutaneous C1498-GFP-Luc tumors for subsequent data analysis. Ten minutes before imaging, mice were injected intraperitoneally with 30 mg / ml D-luciferin (Gold Biotechnology) at a dose of 150 mg / kg body weight. Mice were placed in an IVIS imaging chamber (Caliper Life Sciences) after full anesthesia with isoflurane. Luciferase expression was imaged and calculated using Living Image software.

[0068] iSMAART

[0069] On the day of imaging, mice were anesthetized and injected with 150 mg / kg luciferin and then fixed in an animal holder. CT and bioluminescence imaging were performed sequentially. For CT imaging, the exposure time of the flat-panel detector was 0.124 seconds, and the rotating stage rotated at a speed of 8° per second, requiring 45 seconds to complete a full 360° acquisition. For bioluminescence imaging, the CCD camera had an exposure time of 60 seconds and used 4×4 pixel binning (52 mm pixel pitch) to capture bioluminescence signals at 0°, 90°, 180°, and 270° angles. Multi-projection data acquisition increased the number of measurements and reduced the ill-posedness of the reconstruction problem. The field of view was approximately 120°, and acquiring 4 views was sufficient to cover the entire animal surface around the reconstruction volume. The bioluminescence view was integrated into the CT-assisted bioluminescence tomography (BLT) reconstruction framework. The entire imaging process took approximately 6 minutes.

[0070] A pre-calibrated geometric mapping program integrates the 2D bioluminescence projections (bioluminescence imaging data) onto the surface contours obtained by CT. After image reconstruction, bioluminescence tomography is combined with CT for dual-modality visualization.

[0071] All animal experiments involved in the examples were in compliance with the regulations of the Animal Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China.

[0072] Example 1c-JUN has high regulatory activity in leukemia

[0073] The present invention found that HDAC1 expression was higher in hematopoietic stem cell-progenitor cell (HSC-Prog) samples from leukemia patients compared with healthy samples ( Figure 1 A). GSVA analysis of HDAC1+ and HDAC1- HSC-Prog cells also confirmed the role of HDAC1. HDAC1+ HSC-Prog cells showed high enrichment in cell proliferation-related pathways (DNA_REPAIR and G2M_CHECKPOINT), providing additional evidence for the involvement of HDAC1 ( Figure 1 B).

[0074] Given the changes in the composition of different cell types observed in leukemia samples, this is likely due to alterations in complex intercellular communication. In this study, we used the CellChat R software package, which is designed to quantitatively infer intercellular communication based on single-cell RNA sequencing (scRNA-seq) data, to determine intercellular communication and understand how intercellular crosstalk is associated with the expression of HDAC1 in HSC-Prog cells.

[0075] The present invention infers the number and strength of interactions between different cell populations in leukemia samples ( Figure 1 C). The most significant interaction was observed between HSC-Prog cells and proliferating T cells. When the interaction strength (represented by the interaction weight) was considered, HSC-Prog cells played a key role in the microenvironment. Figure 1 D details the inferred incoming and outgoing interaction strengths, where HDAC1+ HSC-Prog cells and HDAC1- HSC-Prog cells are the main cell types expressing ligands and receptors, actively participating in cell-to-cell interactions. Specifically, HDAC1+ HSC-Prog cells exhibit higher outgoing interaction strengths than HDAC1- HSC-Prog cells. These findings suggest that HDAC1+ HSC-Prog cells play a central role in leukemia. In order to determine the signaling pathways involved in complex intercellular communication, the present invention calculates the outgoing and incoming interaction strengths for each signaling pathway. It is noteworthy that OX40 signaling exhibits the most dominant activity in HDAC1+ HSC-Prog cells, with outgoing and incoming interaction strengths significantly greater than other signaling pathways. Therefore, the present invention believes that the expression of HDAC1 is associated with changes in a series of pathways related to OX40 signal transduction ( Figure 1 E) These findings have important implications for the development of novel therapeutic strategies for leukemia targeting multiple pathways.

[0076] The present invention further uses the SCENIC process to reveal the differences in transcription factors in HSC-Prog cells between disease samples and healthy samples. Based on the calculated regulator specificity score (RSS), the top-ranked transcription factors in the two groups are different, including JUN, JUND, MYB and JUNB ( Figure 1F). These transcription factors showed higher regulatory activity in leukemia. To further investigate whether the transcription factors with the highest Z scores regulate the expression of HDAC1, the present invention isolated HSC-Prog cells from patient samples and divided them into HDAC1+ HSC-Prog cells and HDAC1- HSC-Prog cells. After performing similar analyses, the present invention confirmed that JUN showed higher regulatory activity in HDAC1+ HSC-Prog cells ( Figure 1 G). Therefore, JUN may play a key role in the risk of leukemia caused by HDAC1. In addition, the inhibition of c-JUN inhibitor SP600125 significantly reduced the expression of HDAC1 at the protein level in leukemia cells MV4-11 ( Figure 1 H, I).

[0077] Example 2c-JUN can be used as a target for leukemia treatment

[0078] like Figure 2 As shown, SP600125 can enter the nucleus of leukemia cells and affect the expression of c-JUN ( Figure 2 A). Subsequently, the present invention treated two leukemia cell lines, MV4-11 and THP-1 cells, with SP600125 for 48 hours, adding the compound once every 24 hours. Flow cytometry was used to evaluate cell proliferation ( Figure 2 B), the results showed that with the increase of SP600125 concentration, the proliferation of leukemia cells was significantly reduced ( Figure 2 C-4F). To verify this phenotype, MV4-11 cells were incubated with SP600125, resulting in a significant increase in natural killer (NK) cell cytotoxicity ( Figure 2 GI). According to previous literature reports, CD33 is a member of the sialic acid-binding immunoglobulin-like lectin (siglec) family and is a diagnostic marker and therapeutic target for acute myeloid leukemia. Therefore, the present invention observed that the expression of CD33 in THP-1 cells treated with SP600125 was significantly reduced ( Figure 2 J, K).

[0079] Example 3 The combination of HDAC1 inhibitor and c-JUN inhibitor shows a strong anti-leukemia effect

[0080] In view of the potential role of c-JUN in leukemia cells, the present invention investigates whether the combination of Panobinostat, a drug targeting HDAC1, and Ailanthone, a drug targeting c-JUN, is a promising strategy for leukemia treatment.

[0081] The specific administration method is mainly as follows: mice were given the drug once a day from 5 days to 15 days after tumor bearing, and the administration volume was 200ul. Among them, Ailanthone and Panobinostat were each dissolved in 5% glucose, and the administration amount was 10mg / kg. Ailanthone and Panobinostat combined treatment group was 100ul of each drug administration volume, a total of 200ul, and the drug amount was 5mg / kg.

[0082] The combination of panobinostat and Ailanthone significantly slowed tumor growth and prolonged survival in leukemia mice compared with each drug alone ( Figure 3 AE), the treatment of AML has a synergistic effect. The same conclusion can be drawn by in vivo bioluminescence imaging ( Figure 3 F, G). ISMAART is capable of three-dimensional bioluminescence and / or fluorescence tomography, providing precise depth information of the disease site, which is not possible with traditional two-dimensional optical imaging. In order to more accurately quantify the difference in efficacy between the combination of panobinostat and Ailanthone and each drug alone, the present invention uses the iSMAART system for imaging ( Figure 3 H). The integration of X-ray CT and optical tomography in iSMAART provides a natural registration for better 3D reconstruction of tumor morphology and location. Analysis of tumor volumes calculated from 3D reconstructions after treatment of leukemic mice showed that the combination of panobinostat and Ailanthone had a significant therapeutic effect ( Figure 3 IK).

[0083] In summary, the above results show that c-Jun controls the expression of HDAC1 in leukemia tumor cells. Inhibition of c-Jun can effectively reduce cell proliferation and CD33 expression. At the same time, the present invention combines the HDAC1 inhibitor panobinostat with the c-Jun inhibitor Ailanthone for the treatment of AML, which achieves a synergistic effect compared to the use of HDAC1 inhibitors or c-Jun inhibitors alone. These findings provide a promising approach for drug combination therapy in the treatment of leukemia.

[0084] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises an HDAC1 inhibitor and a c-JUN inhibitor.

2. The pharmaceutical composition according to claim 1, It is characterized in that The HDAC1 inhibitor is selected from one or more of Vorinostat, Romidepsin, Belinostat, Panobinostat, Chidamide or pharmaceutically acceptable salts, crystal forms and solvates of the above drugs.

3. The pharmaceutical composition according to claim 1, It is characterized in that The c-JUN inhibitor is selected from one or more of SP600125, Ailanthone, Veratramine, SR11302, NY2267 or pharmaceutically acceptable salts, crystal forms, and solvates of the above drugs.

4. The pharmaceutical composition according to claim 1, It is characterized in that The mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-50):(1-50); preferably, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-20):(1-20); preferably, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-10):(1-10); preferably, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-5):(1-5); preferably, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is (1-3):(1-3); preferably, the mass ratio of the HDAC1 inhibitor to the c-JUN inhibitor is 1:

1.

5. The pharmaceutical composition according to any one of claims 1 to 4, It is characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

6. A pharmaceutical preparation, comprising the pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical preparation according to claim 6, It is characterized in that The pharmaceutical preparation includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gas dosage form.

8. The pharmaceutical preparation according to claim 7, It is characterized in that The solid dosage forms include powders, pills, tablets, and capsules; the semisolid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include lotions, mixtures, solutions, and injections; and the gaseous dosage forms include aerosols and sprays.

9. Use of the pharmaceutical composition according to any one of claims 1 to 5 or the pharmaceutical preparation according to any one of claims 6 to 8 in the preparation of a drug for preventing, inhibiting and / or treating leukemia.

10. The use according to claim 9, It is characterized in that The above leukemia is selected from acute myeloid leukemia (AML).

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