Use of sr-b1 inhibitors in the treatment of acute myeloid leukemia

Abstract

The application relates to the field of biological medicine, and particularly discloses application of an SR-B1 inhibitor BLT-1 in preparation of a drug for treating acute myeloid leukemia. The application aims to provide a new treatment method for treating acute myeloid leukemia. The application finds that the SR-B1 inhibitor BLT-1 has the effects of inhibiting proliferation of acute myeloid leukemia cells, promoting apoptosis of the acute myeloid leukemia cells and improving individual level, can provide a treatment drug for the acute myeloid leukemia drug, and provides a new thought for further research on the mechanism of the acute myeloid leukemia and development of the acute myeloid leukemia drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of an SR-B1 inhibitor in treatment of acute myeloid leukemia. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in large numbers in bone marrow and other hematopoietic tissues due to uncontrolled proliferation, differentiation disorders, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs while inhibiting normal hematopoiesis. AML is the most common acute leukemia in adults and is also a newly diagnosed malignant tumor. The treatment methods for AML include intensive induction therapy, non-intensive therapy, hematopoietic cell transplantation, and immunotherapy, which have improved the complete remission (CR) and 5-year disease-free survival (DFS) of AML patients. However, drug resistance and high recurrence rate are still serious problems faced by patients. In recent years, although new immunotherapy has achieved certain results, it still faces challenges such as clear treatment targets and lack of specific markers for AML mutations, and it is still a difficult point that needs to be broken through in the future scientific field.

[0003] The scavenger receptor (SR) family is a kind of glycoprotein mainly located on the surface of macrophages, and is an important pattern recognition receptor in innate immunity. Among them, SR-B1 is a high-density lipoprotein receptor, which mediates the selective uptake or flow of high-density lipoprotein cholesterol (HDL-C) into cells and tissues, promotes the efflux of cholesterol from peripheral tissues (including macrophages) back to the liver, and promotes the uptake of fat-soluble vitamins and viral entry into host cells. The role of SR-B1 in various diseases has been studied, such as colon cancer, breast cancer, prostate cancer, ovarian cancer, leukemia, etc. Studies have shown that SR-B1 is highly expressed in hematological malignant tumor cells, can recruit cholesterol needed for cell division, and promotes the proliferation of tumor cells and the development of cancer. So far, there has been no report on the role of SR-B1 gene in acute myeloid leukemia. SUMMARY

[0004] The application aims to provide the application of SR-B1 gene in the treatment of acute myeloid leukemia, and to provide a therapeutic drug for acute myeloid leukemia drugs to solve the technical problem that the existing drugs for treating acute myeloid leukemia need to be further developed.

[0005] In order to achieve the above object, the present application adopts the following technical scheme:

[0006] Use of SR-B1 inhibitor BLT-1 in the preparation of a drug for treating acute myeloid leukemia.

[0007] The principle and advantages of the present technical solution are that:

[0008] The inventors have carried out a large number of experimental verification to explore the effect of SR-B1 inhibitor BLT-1 in treating acute myeloid leukemia, as follows:

[0009] (1) Effect of SR-B1 inhibitor BLT-1 on the proliferation of acute myeloid leukemia: different concentrations of SR-B1 inhibitor BLT-1 were incubated with acute myeloid leukemia cells for 48 h, and the proliferation ability of the cells was detected by using a cell counting kit (CCK-8).

[0010] (2) Effect of SR-B1 inhibitor BLT-1 on the apoptosis of acute myeloid leukemia cells: the apoptosis of acute myeloid leukemia cells after incubation with SR-B1 inhibitor BLT-1 was detected by flow cytometry (FCM).

[0011] (3) Effect of SR-B1 inhibitor BLT-1 on acute myeloid leukemia model mice: first, the changes of various cells in the blood of mice were detected by blood routine test. Second, the number of leukemia cells and the proportion of blast cells in the blood and bone marrow of mice were observed by blood smear and bone marrow smear; the body weight change and survival period of mice were counted. Finally, the viscera ratio of mice was calculated and the differences in the viscera of mice were observed by H&E staining.

[0012] Compared with the prior art, the present technical solution has the following beneficial effects.

[0013] The present application first reports that SR-B1 inhibitor BLT-1 can inhibit the in vitro proliferation of acute myeloid leukemia cells by promoting the apoptosis of acute myeloid leukemia cells. In addition, the present application first reports that SR-B1 inhibitor BLT-1 can reduce the proportion of blast cells in leukemia mice, prolong the survival period of leukemia mice, and effectively improve the organ lesions of leukemia mice. The above findings can provide a therapeutic drug for acute myeloid leukemia drugs, and provide a new idea for further studying the pathogenesis of acute myeloid leukemia; at the same time, SR-B1 inhibitor BLT-1 can be applied to the screening and testing of new anti-acute myeloid leukemia drugs, and provides guidance for the development of new drugs for treating acute myeloid leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1Chemical structural formula of SR-B1 inhibitor BLT-1;

[0015] Figure 2 Figure of anti-proliferation results of CCK-8 test for different acute myeloid leukemia cells in Example 1 after incubation with SR-B1 inhibitor BLT-1 for 48 h;

[0016] Figure 3 Figure of apoptosis results of flow cytometry for acute myeloid leukemia cells C1498 in Example 2 after incubation with different concentrations of SR-B1 inhibitor BLT-1 for 48 h;

[0017] Figure 4 Statistical figure of blood routine test results of blank group, model group and BLT-1 group mice in Example 3;

[0018] Figure 5 Results and statistical figure of blood smear and bone marrow smear of blank group, model group and BLT-1 group mice in Example 3;

[0019] Figure 6 Statistical figure of body weight change curve and survival time curve of blank group, model group and BLT-1 group mice in Example 3;

[0020] Figure 7 Pictures of organs, statistical results of organ / body ratio and H&E staining results of organs of blank group, model group and BLT-1 group mice in Example 3. DETAILED DESCRIPTION

[0021] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described below in combination with the drawings and specific examples. In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0022] Example 1: Effect of SR-B1 inhibitor on proliferation of acute myeloid leukemia cells

[0023] 1. Experimental methods

[0024] 1.1 Cell culture

[0025] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2. Acute myeloid leukemia cells HL-60 were donated by the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, and cultured in 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2. Acute myeloid leukemia cells KG-1a were purchased from Shanghai Fuheng Biotechnology Co., Ltd. and cultured in IMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2.

[0026] 1.2 CCK-8 assay for cell proliferation

[0027] The effect of SR-B1 inhibitor BLT-1 on the proliferation of acute myeloid leukemia cells C1498, HL-60 and KG-1a was analyzed by CCK-8 method. Cells were seeded in 96-well plates, 200 μL of different concentrations of drug-containing medium were added to each well, and the cells were incubated, and the 96-well plates were incubated in a 37°C cell incubator containing 5% CO2 for 48 h. 20 μL of CCK-8 solution was added to each well, and incubated in a 37°C, 5% CO2 incubator for 4 h. The absorbance at 450 nm was measured by a microplate reader and the IC 50 .

[0028] 1.3 Statistical analysis

[0029] All experiments were from three independent experiments, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism software, independent sample t test was used for comparison between groups, and p<0.05 was considered statistically significant.

[0030] 2 Experimental results

[0031] As shown in the description accompanying Figure 2 (A), SR-B1 inhibitor BLT-1 can effectively inhibit the proliferation of acute myeloid leukemia cells. As shown in the description accompanying Figure 2 (B), the inhibitory effect on acute myeloid leukemia cells is enhanced with the increase of the concentration of SR-B1 inhibitor BLT-1. As shown in the description accompanying Figure 2 (C), the IC 50 of acute myeloid leukemia cells C1498, HL-60 and KG-1a after 48 h incubation with SR-B1 inhibitor BLT-1 were 2.92 ± 0.33, 5.38 ± 0.26 and 4.34 ± 0.43 μM, respectively.

[0032] Example 2: Effect of SR-B1 inhibitor on apoptosis of acute myeloid leukemia cells

[0033] 1. Experimental methods

[0034] 1.1 Cell culture

[0035] Acute myeloid leukemia cells C1498 were purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2.

[0036] 1.2 Apoptosis experiment

[0037] (1) Experimental process:

[0038] After co-incubating acute myeloid leukemia cells C1498 with different concentrations (0, 2, 4, 8 μM) of SR-B1 inhibitor BLT-1 in a 6-well plate for 24 h, the cells and culture medium were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was removed, washed with PBS for 3 times, and the supernatant was removed; 500 μL of 1x binding buffer was added to resuspend the cells, centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was removed, and the step was repeated; 500 μL of 1x binding buffer was added to resuspend the cells, and the concentration was adjusted to 1-5x10 6 / mL, 100 μL of cell suspension was taken in a 5 mL flow tube, 5 μL of Annexin V / FITC was added and mixed, and then incubated at room temperature for 5 min in the dark; 5 μL of propidium iodide solution (PI) was added, and 400 μL of PBS was added, and flow cytometry or fluorescence microscopy detection was immediately performed.

[0039] (2) Experimental design:

[0040] ① Blank tube: negative control group cells, without Annexin V / FITC and PI. Used to adjust the voltage. ② Single dye tube: positive control group cells, only with Annexin V / FITC or only with PI. Used to adjust the compensation. ③ Detection tube: treated cells, with Annexin V / FITC and PI. After adjusting the voltage and compensation with the blank tube and single dye tube, the required flow cytometry data was obtained.

[0041] 1.3 Statistical analysis

[0042] All experiments were from 3 independent experiments, and the experimental results were expressed as mean ± standard deviation (Mean ± SD). Statistical processing and analysis were performed using GraphPad Prism software, independent sample t-test was used for comparison between groups, and p<0.05 was considered statistically significant.

[0043] 2 Experimental Results

[0044] The results of the apoptosis experiment are shown in the attached instruction manual. Figure 3 As shown in (A)(B)(C)(D), the SR-B1 inhibitor BLT-1 can promote apoptosis in acute myeloid leukemia (AML) cells. The apoptosis rate of AML cells C1498 increases with increasing concentration of the SR-B1 inhibitor BLT-1. (See attached instructions) Figure 3 (E) shows that the apoptosis rates of acute myeloid leukemia cells C1498 at concentrations of 0, 2, 4, and 8 μM SR-B1 inhibitor BLT-1 were 6.80±0.99%, 8.60±1.07%, 12.5±1.27%, and 23.35±1.95%, respectively.

[0045] Example 3: Effects of SR-B1 inhibitors on a mouse model of acute myeloid leukemia

[0046] 1 Experimental Methods

[0047] 1.1 Establishment of a mouse leukemia model

[0048] Collect C1498 acute myeloid leukemia cells in good growth condition at a quantity of 2 × 10⁻⁶. 6 C57BL / 6 mice were injected intravenously via tail vein and randomly divided into a blank control group (tail vein injection of physiological saline), a model group, and a BLT-1 group (25 mg / kg / day). After 24 hours, mice were treated daily with an equal volume of solvent and BLT-1 (25 mg / kg) for 14 consecutive days. Peripheral blood smears and complete blood counts were performed at 0, 10, 20, and 40 days to observe changes in leukocytes and immature cells. Additionally, mice were weighed every 3 days to record survival and mortality, and survival time was statistically analyzed. When mice were near death, their spleen and liver were photographed and weighed, and eosin staining was performed on the spleen and liver for microscopic observation of leukemia cell infiltration. Simultaneously, bone marrow smears were collected for microscopic examination, and the proportion of immature cells in the bone marrow cells was statistically analyzed.

[0049] 1.2 Statistical Analysis

[0050] All experiments were conducted in triplicate, and results are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism software. Independent samples t-tests were used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0051] 2 Experimental Results

[0052] Included in the instruction manual Figure 4It can be seen that compared with the blank group, the number of mononuclear cells in the model group increased significantly (p<0.01), and the number of mononuclear cells in the BLT-1 group had no significant change (p>0.05); in addition, compared with the model group, the number of mononuclear cells in the BLT-1 group decreased significantly (p<0.01). The specification is attached Figure 5 (C) The results show that compared with the control group, the number of white blood cells in the model group increased significantly (p<0.05), and there was no significant difference in the BLT-1 group (p>0.05); compared with the model group, the number of white blood cells in the BLT-1 group decreased significantly (p<0.05). The specification is attached Figure 5 (D) The results show that compared with the control group, the number of immature cells in the model group and the BLT-1 group increased significantly (p<0.001), but the increase in the BLT-1 group was less than that in the model group; compared with the model group, the number of immature cells in the BLT-1 group decreased significantly (p<0.05). The specification is attached Figure 6 From the survival curve results in the specification attached Figure 7 (A) (B) The results show that compared with the blank group, the liver and spleen of the model group mice were enlarged and there were a large number of white nodules in the liver, and the BLT-1 group had no obvious enlargement and organ lesions. The specification is attached Figure 7 (C) The H&E staining results show that compared with the blank group, there are a large number of white blood cell infiltrations in the lungs, liver, spleen and bone marrow of the model group mice, and the lung structure and liver lobular structure are destroyed, while the BLT-1 group has no obvious white blood cell infiltration and organ structure destruction.

Claims

1. Use of an SR-B1 inhibitor for the manufacture of a medicament for the treatment of acute myeloid leukemia, characterized in that, The SR-B1 inhibitor is BLT-1, which is a compound with the following structure:

2. Use of a SR-B1 inhibitor according to claim 1 for the manufacture of a medicament for the treatment of acute myeloid leukemia, characterized in that, The compound has an inhibitory effect on the proliferation of acute myeloid leukemia cells.

3. Use of a SR-B1 inhibitor according to claim 2 for the manufacture of a medicament for the treatment of acute myeloid leukemia, characterized in that, The compound can promote the apoptosis of acute myeloid leukemia cells.

4. The application of the SR-B1 inhibitor according to claim 2 in the preparation of drugs for treating acute myeloid leukemia, characterized in that, The cells include at least one of the following: HL-60, KG-1a, C1498, and other acute myeloid leukemia cells.

5. The use according to any one of claims 1-4, wherein the drug is BLT-1 or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable carrier.

6. The use according to any one of claims 5, wherein the drug is administered orally in the form of tablets, capsules, pills, granules, powders, oral solutions, suspensions. The tablets are sugar-coated tablets, film-coated tablets, enteric-coated tablets, effervescent tablets, sublingual tablets. The capsules are hard capsules, soft capsules, microcapsules. The pills are drop pills.

7. The use according to any one of claims 5, wherein the drug is administered by inhalation in the form of aerosols.

8. The use according to any one of claims 5, wherein the drug is administered intravenously in the form of injections. The injections are lyophilized powder injections or injection emulsions.

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