A pharmaceutical composition comprising a Bcl-2 inhibitor and a STAT inhibitor

Abstract

The present invention relates to a pharmaceutical composition for treating leukemia. The active ingredients in the pharmaceutical composition include a BCL-2 inhibitor and a STAT inhibitor, wherein the molar ratio of the BCL-2 inhibitor to the STAT inhibitor is from 1:1 to 1:100. The BCL-2 inhibitor is selected from venetoclax, ABT-263 or ABT-737, and the STAT inhibitor is selected from SH-4-54, nifuroxazide or pimozide. The composition of the BCL-2 inhibitor and the STAT inhibitor of the present invention has a synergistic killing effect on AML cell lines and patient primary cells, and can synergistically inhibit cell proliferation and promote cell apoptosis. Therefore, this composition provides a treatment plan with better effects and low toxic side effects for AML patients, especially elderly patients with poor tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and particularly relates to a pharmaceutical composition for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a hematological malignancy characterized by the differentiation and maturation disorder of bone marrow hematopoietic cells, malignant clonal proliferation, and inhibition of normal hematopoietic function. It has characteristics such as high heterogeneity and rapid progression.

[0003] The chemotherapy regimen based on anthracyclines and cytarabine ("3 + 7" regimen) is the current standard treatment for AML and has not changed much in the past 40 years. However, the cure rate of patients using the existing treatment regimen is low, the overall prognosis is poor, and the 5-year overall survival (OS) is less than 20%. In addition, due to the high toxicity of chemotherapy drugs, elderly patients can hardly tolerate them, and there are few alternative treatment options. Therefore, exploring therapeutic drugs and new regimens with good effects and low toxic and side effects is the main research direction at present.

[0004] With the popularization of next-generation sequencing technology, it has been found that more than 97% of AML patients have gene mutations related to various cytogenetic characteristics in their bodies, such as FLT3-ITD, IDH1 / 2, PML-RARA, DNMT3A, TET2, etc. Small molecule drugs targeting these mutations provide new options for the treatment of AML, making the treatment of AML more precise and individualized. However, these targeted drugs also have certain limitations. They are only effective for patients with corresponding gene mutations, and the efficacy of single-drug treatment is limited and prone to drug resistance. Therefore, the improvement of treatment regimens targeting multiple targets, such as the combined use of targeted drugs, traditional Chinese medicine monomers, chemotherapy drugs, etc., is the new direction for optimizing the current AML treatment regimen.

[0005] B-cell lymphoma 2 (BCL-2) is a key regulator of the apoptosis process. In the cellular stress response, the pro-apoptotic proteins BAX and BAK translocate to the mitochondria and induce the activation of caspases 9 and caspases cascade reaction by releasing cytochrome c (CytC) to induce mitochondrial outer membrane permeability, thereby inducing apoptosis. The mitochondrial pathway is regulated by pro-apoptotic BH3-only proteins, which activate BAX and inhibit BCL-2 and MCL-1. Among them, BCL-2 is the genetic material that inhibits apoptosis depending on the mitochondrial apoptosis pathway. By blocking the expression of apoptotic proteins, AML cells can escape apoptosis, and the overexpression of BCL-2 is also related to drug resistance and poor prognosis.

[0006] Venetoclax (ABT-199) is a highly effective and highly selective oral inhibitor of BCL-2. It can bind to the BCL-2 protein, inhibit the formation of the BCL-2 and BIM protein complex, promote the activation of BAX and BAK, and ultimately induce apoptosis. Since June 2011, Venetoclax has been used in clinical trials for various hematological malignancies, showing good anti-tumor activity. In 2016, the US FDA approved Venetoclax for the treatment of AML. Currently, the combination of Venetoclax with low-dose cytarabine, azacitidine or decitabine has also been approved by the US FDA as a first-line treatment regimen for elderly or chemotherapy-ineligible AML patients, with an overall remission rate (complete remission, CR) of 67%. However, 30% of AML patients still have poor efficacy and cannot achieve complete remission through combination therapy or relapse within a short period of time.

[0007] STAT3 / 5 are signal transducers and activators of transcription. As a convergence point of multiple oncogenic signaling pathways, they play an important role in regulating anti-tumor immune responses. In various cancers, the constitutive activation of STAT3, and to a lesser extent STAT5, increases tumor proliferation, survival, angiogenesis and metastasis, while also inhibiting anti-tumor immunity. The JAK-STAT signal in the hematopoietic system also coordinates the survival, self-renewal, hematopoiesis and proliferation of hematopoietic stem cells (HSCs). STAT3 / 5 are the main oncogenic transcription factors of the JAK-STAT pathway and are constitutively activated in 44%-76% of AML patients. Studies have found that the disease-free survival of those with constitutive activation is shorter. Therefore, abnormal JAK-STAT signals are also closely related to the occurrence of hematopoietic malignancies.

[0008] SH-4-54 is a highly effective STAT3 / 5 inhibitor. Studies have shown that SH-4-54 can effectively kill glioblastoma brain cancer stem cells (BTSCs), effectively inhibit STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations, and show no discernible off-target effects at therapeutic doses.

[0009] Currently, there are no relevant literature reports on the combination of BCL-2 inhibitors and STAT inhibitors for the treatment of leukemia. Summary of the Invention

[0010] To solve the above technical problems, the present invention includes the following aspects:

[0011] The first aspect of the present invention provides a pharmaceutical composition for the treatment of leukemia, wherein the active ingredients include a BCL-2 inhibitor and a STAT inhibitor.

[0012] Preferably, the molar ratio of the BCL-2 inhibitor to the STAT inhibitor in the active ingredient is from 1:1 to 1:100. More preferably, the molar ratio of the BCL-2 inhibitor to the STAT inhibitor in the active ingredient is from 1:1 to 1:80. Further preferably, the molar ratio of the BCL-2 inhibitor to the STAT inhibitor in the active ingredient is from 1:5 to 1:50. Most preferably, the molar ratio of the BCL-2 inhibitor to the STAT inhibitor in the active ingredient is from 1:5 to 1:10.

[0013] Preferably, the STAT inhibitor is a STAT3 / 5 inhibitor.

[0014] Preferably, the BCL-2 inhibitor is selected from Venetoclax (ABT-199), ABT-263 or ABT-737.

[0015] Preferably, the STAT inhibitor is selected from SH-4-54, Nifuroxazide or Pimozide.

[0016] Preferably, the active ingredient of the pharmaceutical composition comprises Venetoclax and SH-4-54.

[0017] More preferably, the active ingredient of the pharmaceutical composition consists of Venetoclax and SH-4-54.

[0018] Preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is from 1:1 to 1:100. More preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is from 1:1 to 1:80. Further preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is from 1:5 to 1:50. Most preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is from 1:5 to 1:10.

[0019] Preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is 1:9, and the leukemia is acute myeloid leukemia of the MV4-11 cell line.

[0020] Preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is 1:50, and the leukemia is acute myeloid leukemia of primary cells of AML patients.

[0021] Preferably, the molar ratio of Venetoclax to SH-4-54 in the active ingredient is 1:6.7, and the leukemia is acute myeloid leukemia of primary cells of AML patients.

[0022] Preferably, the pharmaceutical composition is an oral preparation.

[0023] More preferably, the oral preparation is a tablet, capsule, granule, dripping pill, sustained-release or controlled-release preparation, etc.

[0024] Preferably, the leukemia is selected from acute myeloid leukemia, acute lymphoblastic leukemia or multiple myeloma.

[0025] More preferably, the leukemia is acute myeloid leukemia.

[0026] Even more preferably, the acute myeloid leukemia is MV4-11 cell line or primary cells of AML patients.

[0027] The second aspect of the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating leukemia.

[0028] Preferably, the leukemia is selected from acute myeloid leukemia, acute lymphoblastic leukemia or multiple myeloma.

[0029] More preferably, the leukemia is acute myeloid leukemia.

[0030] Even more preferably, the acute myeloid leukemia is MV4-11 cell line or primary cells of AML patients.

[0031] Advantages of the present invention:

[0032] Both the BCL-2 inhibitor venetoclax and SH-4-54 are oral drugs with anti-tumor and low toxic side effects. The present invention unexpectedly finds that the combination of a BCL-2 inhibitor and a STAT inhibitor, especially the combination of venetoclax and SH-4-54, has a synergistic killing effect on AML cell lines and primary patient cells, and can synergistically inhibit cell proliferation and promote apoptosis. Therefore, the combination scheme of the BCL-2 inhibitor and the STAT inhibitor (preferably venetoclax and SH-4-54) provided by the present invention provides a new treatment scheme with better efficacy and lower toxic side effects for AML patients, especially elderly patients with poor tolerance, and has important significance for realizing individualized precision treatment of AML. Description of the Drawings

[0033] Figure 1 shows the results of the synergistic killing effect of venetoclax combined with SH-4-54 on AML cell lines;

[0034] Figure 2 shows the results of the synergistic killing effect of venetoclax combined with SH-4-54 on primary cells of AML patients;

[0035] Figure 3 shows the results of the inhibitory effect of venetoclax combined with SH-4-54 on the proliferation of AML cells;

[0036] Figure 4It is the result of the synergistic effect of Venetoclax combined with SH-4-54 in promoting apoptosis of AML cells. Specific Embodiments

[0037] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] Experimental Example 1: Synergistic Effect of Venetoclax Combined with SH-4-54 on AML Cells

[0039] 1. Experimental Method

[0040] 1.1 Synergistic Killing Effect of Drug Combination on AML Cell Lines and Primary Cells

[0041] Collect AML cell lines (MV4-11) and primary AML cells (AML#1, AML#2, AML#3) in the logarithmic growth phase. After centrifugation, resuspend them in fresh medium. According to the density of 2×10 5 / mL for AML cell lines and 1x10 6 / mL for primary AML cells, plate them at 100 μL per well in a 96-well plate. Each sample is plated in three replicates. Add 1 μL of the test concentration of Venetoclax solution, SH-4-54 solution, and a mixed solution of Venetoclax and SH-4-54 (0.5 μL each of Venetoclax and SH-4-54 solutions in the mixed solution). The drugs are all dissolved in DMSO. Set a blank control group and surround it with PBS, 100 μL / well. After incubating in an incubator at 37°C and 5% CO2 concentration for 24 hours, add 10 μL of MTS working solution to each well, and continue to incubate in the incubator for 4 hours. Then, measure the optical density (OD value) at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader. The OD value of each well is positively correlated with the number of its cells. Calculate the inhibition rate of the drug on cells and the single-drug IC 50 , use the software GraphPad Prism to draw a cell proliferation graph, and use the Calcusyn software to calculate the combination index CI. When the CI value > 1, it is judged as antagonistic; when the CI value = 1, it is judged as additive; when the CI value < 1, it is judged as synergistic.

[0042] 1.2 Synergistic Inhibitory Effect of Drug Combination on AML Cell Proliferation

[0043] Collect AML cell lines (MV4-11) in the logarithmic growth phase. After centrifugation, resuspend them in fresh medium. According to the density of 1×10 5Plated at a density of / mL, inoculated 100 μL per well into a 96-well plate, with three replicates for each sample. Respectively added 1 μL of venetoclax solution, SH-4-54 solution, and a mixed solution of venetoclax and SH-4-54 (0.5 μL each of venetoclax and SH-4-54 solution in the mixed solution). The drugs were all dissolved in DMSO. A blank control group was set, with PBS plated around, 100 μL / well. After incubating in an incubator at 37 °C and 5% CO2 concentration for 0, 24, 48, 72, and 96 hours respectively, 20 μL of MTS working solution was added to each well, and then continued to incubate in the incubator for 4 h. The optical density (OD value) at a wavelength of 490 nm was measured with an enzyme-linked immunosorbent assay reader. The OD value of each well was positively correlated with its cell number, and software GraphPad Prism was used to draw the cell proliferation graph.

[0044] 1.3, Synergistic pro-apoptotic effect of drug combination on AML cells

[0045] Collect AML cell line (MV4-11) in the logarithmic growth phase, resuspend it in fresh medium after centrifugation, and plate it at a density of AML cell line 2×10 5 / mL, inoculated 1 ml per well into a 24-well plate. Respectively added 10 μL of venetoclax solution, SH-4-54 solution, and a mixed solution of venetoclax and SH-4-54 (5 μL each of venetoclax and SH-4-54 solution in the mixed solution). The drugs were all dissolved in DMSO. A blank control group was set. After centrifuging to collect AML (MV4-11) cells treated with drugs at 1000 rpm for 5 min, washed 3 times with PBS, transferred to a flow cytometry tube, and 300 μL of 1×buffer, 6 μL of PI, and 3 μL of AnnexinV-FITC were added to each tube in turn. After incubating in the dark at room temperature for 10 min, it was detected by machine.

[0046] 2. Experimental results

[0047] 2.1, Synergistic killing effect of drug combination on AML cell line and primary cells

[0048] To explore the synergistic killing effect of venetoclax combined with SH-4-54 in AML, this experimental example conducted a drug sensitivity test on the AML cell line MV4-11. The experimental results are as Figure 1 shown in Figures A and 1B. The IC 50 values of venetoclax and SH-4-54 were 162.2 nM and 1164 nM respectively.

[0049] In this experimental example, the killing effects of the venetoclax monotherapy group (dose gradients: 60 nM, 80 nM, 100 nM, 120 nM), the SH-4-54 monotherapy group (dose gradients: 540 nM, 720 nM, 900 nM, 1080 nM), and the combination group of the two drugs (dose gradients: 60 nM + 540 nM, 80 nM + 720 nM, 100 nM + 900 nM, 120 nM + 1080 nM) on MV4-11 cells were investigated respectively.

[0050] The experimental results showed that the combination of venetoclax and SH-4-54 could synergistically inhibit the proliferation of MV4-11 cells ( Figure 1 C). The drug combination curve was plotted and the combination index CI at 50%, 75% and 90% effective rates (ED50, ED75, ED90) of the drug combination was calculated. It was found that the combination index CI of the two drugs after combination was < 1, indicating that the combination of venetoclax and SH-4-54 had a significant synergistic effect ( Figure 1 D).

[0051] To further confirm the synergistic killing effect of the combination of venetoclax and SH-4-54 on AML cells, this experimental example conducted a proliferation test using primary cells from AML patients. The experimental results showed that the venetoclax monotherapy group (dose gradients: 30 nM, 150 nM, 180 nM, 240 nM), the SH-4-54 monotherapy group (dose gradients: 0.2 μM, 1 μM, 1.2 μM, 1.6 μM), and the combination drug group (dose gradients of venetoclax + SH-4-54: 30 nM + 0.2 μM, 150 nM + 1 μM, 180 nM + 1.2 μM, 240 nM + 1.6 μM) could all inhibit the proliferation of primary AML cells AML#1. The venetoclax monotherapy group (dose gradients: 2 nM, 4 nM, 8 nM, 16 nM, 32 nM), the SH-4-54 monotherapy group (dose gradients: 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM), and the combination drug group (dose gradients of venetoclax + SH-4-54: 2 nM + 0.1 μM, 4 nM + 0.2 μM, 8 nM + 0.4 μM, 16 nM + 0.8 μM, 32 nM + 1.6 μM) could all inhibit the proliferation of primary AML cells AML#2 and AML#3, and the inhibitory effect of the combination drug group was stronger than that of the monotherapy groups of the two drugs ( Figure 2 ). The combination index of venetoclax and SH-4-54 with different concentration ratios acting synergistically on primary AML cells (AML#1, AML#2, AML#3) is shown in Table 1.

[0052] Table 1 Combination index CI of the combination of venetoclax and SH-4-54 acting on AML cells

[0053] Primary cell ED50 ED75 ED90 AML#1 0.38906 0.29082 0.21934 AML#2 0.47894 0.49005 0.63197 AML#3 0.40180 0.48930 0.59947

[0054] 2.2 Synergistic inhibitory effect of drug combination on the proliferation of AML cells

[0055] To explore the effect of venetoclax combined with SH-4-54 on the proliferation of AML cells, in this experimental example, the MTS method was used to detect the effects of the control group (Control), the venetoclax single-drug group (dose 100 nM), the SH-4-54 single-drug group (dose 900 nM), and the combined drug group (venetoclax dose 100 nM + SH-4-54 dose 900 nM) on cell growth. The experimental results showed that the combined drug group had a significant inhibitory effect on the proliferation of MV4-11 cells ( Figure 3 ).

[0056] 2.3 Synergistic pro-apoptotic effect of drug combination on AML cells

[0057] To explore the effect of venetoclax combined with SH-4-54 on the apoptosis of AML cells, flow cytometry was used to detect the proportion of apoptosis in the control group (Control), the venetoclax single-drug group (dose 100 nM), the SH-4-54 single-drug group (dose 900 nM), and the combined drug group (venetoclax dose 100 nM + SH-4-54 dose 900 nM) after acting on AML cells for 24 h. Venetoclax and SH-4-54 single drugs could induce less apoptosis of AML cells, while the combined drug group could significantly promote the apoptosis of AML cells compared with the single drugs ( Figure 4 ).

[0058] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that, The active ingredients of the pharmaceutical composition are venetoclax and SH-4-54, and the molar ratio of venetoclax to SH-4-54 in the active ingredients is from 1:5 to 1:

50.

2. The pharmaceutical composition according to claim 1, wherein The molar ratio of venetoclax to SH-4-54 in the active ingredients is from 1:5 to 1:

10.

3. The pharmaceutical composition according to claim 1, wherein The molar ratio of venetoclax to SH-4-54 in the active ingredients is from 1:10 to 1:

50.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The pharmaceutical composition is an oral preparation.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating leukemia, wherein the leukemia is acute myeloid leukemia.

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