Application of TRPML1-specific small molecule agonist ML-SA5 in the preparation of drugs for acute myeloid leukemia

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the use of a TRPML1-specific small molecule agonist ML-SA5 in the preparation of an acute myeloid leukemia drug. The ML-SA5 agonist of the present invention can reduce the infiltration of leukemia cells into mouse bone marrow, peripheral blood, liver, kidney and other tissues and organs, prolong the life span of leukemia mice, and comprehensively delay the progression of acute myeloid leukemia in mice, providing a new drug for the treatment of acute myeloid leukemia, and having good market value and clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of a TRPML1-specific small molecule agonist ML-SA5 in the preparation of an acute myeloid leukemia drug. Background Art

[0002] Acute myeloid leukemia (AML) is one of the most common and fatal acute leukemias in adults, with a low survival rate (about 5 years). The key hallmark of AML is the uncontrolled proliferation and blocked differentiation of immature bone marrow precursor cells, which leads to the uncontrolled clonal expansion of these immature bone marrow precursor cells and their entry into the peripheral blood. The clinical manifestations are malignant proliferation and enlargement of leukemia cells in hematopoietic organs such as the bone marrow, spleen, and liver, and infiltration into various tissues and organs throughout the body. In recent years, "differentiation therapy" that promotes the differentiation of AML immature precursor cells has attracted widespread attention. For example, by inducing leukemia cell differentiation, all-trans retinoic acid (ATRA) has greatly improved the clinical treatment effect of acute promyelocytic leukemia (APL), a unique subtype of AML. However, ATRA is ineffective for the clinical treatment of other subtypes of AML. Therefore, there is an urgent need to develop targeted drugs for the treatment of AML by inducing AML cell differentiation. Summary of the invention

[0003] In order to solve the above technical problems, the main purpose of the present invention is to provide the use of TRPML1-specific small molecule agonist ML-SA5 in the preparation of acute myeloid leukemia drugs to solve the problems in the related technology.

[0004] The agonist has been verified to be able to reduce the infiltration of leukemia cells into tissues and organs, prolong life, and comprehensively delay the progression of acute myeloid leukemia.

[0005] In the first aspect of the present invention, a TRPML1-specific small molecule agonist ML-SA5 is provided, and its chemical structure is as follows:

[0006]

[0007] The second aspect of the present invention provides the use of ML-SA5 in the preparation of drugs for acute myeloid leukemia.

[0008] Furthermore, in the preparation of acute myeloid leukemia drugs, the effective dose of the TRPML1-specific small molecule agonist ML-SA5 is 4 mg / kg.

[0009] Furthermore, the TRPML1-specific small molecule agonist ML-SA5 is used after acute myeloid leukemia.

[0010] Furthermore, the acute myeloid leukemia drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a TRPML1-specific small molecule agonist ML-SA5.

[0011] A pharmaceutical composition for treating acute myeloid leukemia comprises an effective therapeutic amount of a TRPML1-specific small molecule agonist ML-SA5 and pharmaceutically acceptable excipients.

[0012] Furthermore, the pharmaceutical composition is in the form of injection, tablet, capsule, granule, aerosol or oral liquid.

[0013] The beneficial effect of the present invention is that when the agonist ML-SA5 is used to prepare an acute myeloid leukemia drug, the ML-SA5 agonist of the present invention can reduce the infiltration of leukemia cells into mouse bone marrow, peripheral blood, liver, kidney and other tissues and organs, prolong the life span of leukemia mice, and comprehensively delay the progression of acute myeloid leukemia in mice, thereby providing a new drug for the treatment of acute myeloid leukemia and having good market value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The chemical structure diagram of the TRPML1-specific small molecule agonist ML-SA5 of the present invention;

[0015] Figure 2 The figure is the preparation and dosing time pattern of acute myeloid leukemia model mice;

[0016] Figure 3 The administration of ML-SA5 had a significant inducing differentiation effect on immature leukemia cells in the bone marrow and peripheral blood of acute myeloid leukemia mice;

[0017] Figure 4 Administration of ML-SA5 significantly improved the infiltration of leukemia cells into the bone marrow and peripheral blood of mice;

[0018] Figure 5 Administration of ML-SA5 significantly improved the infiltration of leukemia cells into the liver, kidney and other tissues of mice;

[0019] Figure 6 Administration of ML-SA5 significantly prolonged the lifespan of mice with acute myeloid leukemia;

[0020] Figure 7 Administration of ML-SA5 significantly improved the weight loss of acute myeloid leukemia mice;

[0021] Figure 8 Administration of ML-SA5 comprehensively delayed the progression of acute myeloid leukemia in mice. DETAILED DESCRIPTION

[0022] The new use of the TRPML1-specific small molecule agonist ML-SA5 discovered by the present invention is the use of the agonist in the preparation of a drug for treating acute myeloid leukemia. Specifically, when the ML-SA5 agonist is used to prepare a drug for treating acute myeloid leukemia, the drug containing the ML-SA5 agonist can reduce the infiltration of leukemia cells into mouse bone marrow, peripheral blood, liver, kidney and other tissues and organs, prolong the life span of leukemia mice, and comprehensively delay the progression of acute myeloid leukemia in mice.

[0023] When treating acute myeloid leukemia, the effective dose of the TRPML1-specific small molecule agonist ML-SA5 is 4 mg / kg.

[0024] Studies have shown that the lysosomal cation channel TRPML1 is a target for regulating the release of cations in lysosomes. By using TRPML1 agonists to activate the activity of the TRPML1 channel, the fusion process of lysosomes and autophagosomes in cells can be regulated, thereby inhibiting cell autophagy. Inhibiting leukemia cell autophagy can promote the differentiation of leukemia cells and reduce the malignant proliferation and expansion of immature leukemia cells. Therefore, the following is a method for confirming the specific small molecule agonist ML-SA5 of TRPML1 (its chemical structure is shown in Figure 1 ) Experiments showing that the drug has a therapeutic effect on acute myeloid leukemia by inducing differentiation of acute myeloid leukemia cells.

[0025] 1. Test methods

[0026] First, the human acute myeloid leukemia cell line THP-1-Luc linked to luciferase was injected into the blood system of NSG immunodeficient mice through the tail vein to form a systemic diffuse acute myeloid leukemia mouse model. After the model was successfully established, intervention was carried out and the mice were divided into two groups, namely the control group (Saline) and the ML-SA5 administration group. The model establishment and ML-SA5 intervention methods were as follows Figure 2 As shown:

[0027] Control group: After modeling, mice were intraperitoneally injected with normal saline;

[0028] ML-SA5 group: mice were intraperitoneally administered with ML-SA5 after model establishment.

[0029] Figure 2 Specifically, the xenograft NSG mice used for modeling were 5 to 8 weeks old, with an equal number of males and females. 5×10 6THP-1-Luc cells were injected into the tail vein of each NSG mouse. On the 7th day after transplantation, imaging was performed using an in vivo optical imaging system (IVIS Lumina S5, PerkinElmer), and the post-transplantation tumor burden was evaluated by detecting bioluminescence. Dosing began on the 2nd day after transplantation, and normal saline (10 mL / kg) and ML-SA5 (4 mg / kg) were given to the control group (number ≥ 6 mice) and the ML-SA5 administration group (number ≥ 6 mice) by intraperitoneal injection at the same time every day according to body weight. The administration lasted for 28 days from the start of administration. On the 28th day, peripheral blood, bone marrow, liver tissue, and kidney tissue of the mice were obtained after anesthesia with 1% sodium pentobarbital (40 mg / kg), and corresponding index detection and experimental operations were performed.

[0030] 2. ML-SA5 administration has a significant induction effect on the differentiation of immature leukemia cells in the bone marrow and peripheral blood of acute myeloid leukemia mice

[0031] 2.1 Measurement method

[0032] After 28 days of administration, the mice were anesthetized, and the whiskers on both sides were cut with ophthalmic scissors. The head of the mice was turned down, and the neck was gently pressed to make the eyeball protrude. The eyeballs were removed with curved forceps to obtain venous blood, which was placed on a glass slide to complete the preparation of a blood smear. After the mice were anesthetized, the femurs of the mice were completely removed, and PBS was slowly injected into the bone marrow cavity with a syringe, and the bone marrow was gradually pushed out. The bone marrow suspension was placed on a glass slide to complete the preparation of a bone marrow smear. The bone marrow and peripheral blood were stained with Giemsa blood pictures. Before staining, it was treated with protease and the like, and then stained with Giemsa stain. On the chromosomes, different shades of horizontal stripes can appear. Giemsa stain can dye the cell nucleus into purple-red or blue-purple, and the cytoplasm into pink, presenting a clear image of cells and chromosomes under a light microscope.

[0033] The results are as follows Figure 3 As shown: The bone marrow (BM) and peripheral blood (PB) of leukemia mice in the control group (Saline) are full of immature leukemia cells (indicated by arrows). Compared with the control group (Saline), ML-SA5 administration has a significant induction of differentiation of immature leukemia cells in the bone marrow and peripheral blood of acute myeloid leukemia mice, and the cells differentiate and mature into monocytes (indicated by arrows). The scale bar is 20 microns.

[0034] 3. ML-SA5 administration significantly improved leukemia cell infiltration in the bone marrow and peripheral blood of mice

[0035] 3.1 Measurement method

[0036] After the mice were anesthetized, the whole blood and bone marrow suspension of the mice were taken in the same way as in 2.1 and dripped into EDTA anticoagulation blood collection tubes (BD, Broken Bow, NE, USA), and the blood and EDTA were re-separated and combined by turning upside down. By adding red blood cell lysis buffer to the whole blood or bone marrow suspension of the venous blood, the white blood cells were separated and collected for subsequent flow detection experiments. The infiltration of leukemia cells in the peripheral blood and bone marrow of mice was detected using a flow cytometer (CytoFLEX SRT, Beckman Coulter), and the percentage of leukemia cells (GFP fluorescence positive) in the samples in the peripheral blood or bone marrow white blood cells was recorded. All data were statistically analyzed and statistical graphs were made using Graphpad 7.0 software.

[0037] The results are as follows Figure 4 As shown: Compared with the control group (Saline), the administration of ML-SA5 significantly reduced the infiltration of human leukemia cells THP-1 into the mouse bone marrow and peripheral blood.

[0038] 4. ML-SA5 administration has a significant improvement effect on leukemia cell infiltration in mouse liver, kidney and other tissues 4.1 Measurement method

[0039] After 28 days of modeling, mice in the control group (Saline) and the ML-SA5 administration group were anesthetized and perfused with PBS buffer by left ventricle puncture. The whole liver and kidney were collected, fixed in 4% paraformaldehyde at room temperature for 24 hours, embedded in paraffin, cut into 3 μm thick slices with a slicer, put into xylene for dewaxing, taken out after 30 minutes, and then transferred to 100%, 95%, 90%, 80%, and 70% ethanol solutions in sequence for 5 minutes each time, and finally transferred to distilled water and left for 5 minutes. The dewaxed slices were immersed in hematoxylin staining solution for 3 minutes, rinsed for 1 minute, transferred to 95% ethanol, and after 1 minute, immersed in eosin staining solution for 1 minute. After staining, the slices were transferred to 70%, 80%, 90%, 95%, and 100% ethanol solutions for dehydration, and then transferred to xylene for 5 minutes, and finally sealed with neutral glue for preservation. This pathological morphology experiment makes the cell structure and composition in the sample clearly visible, helping to analyze the leukemia infiltration in the sample. All data are statistically analyzed and statistical graphs are made using Graphpad 7.0 software.

[0040] The results are as follows Figure 5 As shown: Compared with the control group, ML-SA5 administration can alleviate the infiltration of human leukemia cells THP-1 in mouse liver (Liver) and kidney (Kidney). In the control group, the parts enclosed by the elliptical dotted lines are the leukemia tissues that invaded the liver and kidneys; the rectangular solid lines are the magnified tumor tissues. In the ML-SA5 administration group, the rectangular dotted lines are the circled tumor tissues, and the rectangular solid lines are the magnified tumor tissues. The scale is 200 microns.

[0041] 5. ML-SA5 administration significantly prolonged the lifespan of mice with acute myeloid leukemia

[0042] 5.1 Measurement method

[0043] After modeling, the survival days of mice in the control group (Saline) and the ML-SA5 administration group were recorded, and the survival curve was drawn using Graphpad 7.0 software.

[0044] The results are as follows Figure 6 As shown: Administration of ML-SA5 significantly prolonged the lifespan of acute myeloid leukemia mice compared with the control group.

[0045] 6. Administration of ML-SA5 significantly improved the weight loss of acute myeloid leukemia mice.

[0046] 6.1 Test methods

[0047] The body weight of mice was measured and recorded every day from day 1 to day 28 of modeling. The body weight of mice in the control group (Saline) and the ML-SA5 administration group was compared 28 days after modeling. All data were statistically analyzed and plotted using Graphpad 7.0 software.

[0048] The results are as follows Figure 7 As shown: Compared with the control group (Saline), ML-SA5 administration significantly improved the weight loss of acute myeloid leukemia mice.

[0049] 7. Administration of ML-SA5 delays the progression of acute myeloid leukemia in mice.

[0050] 7.1 Test methods

[0051] Mice carrying THP-Luc cells were anesthetized with isoflurane. 200 μl of substrate D-luciferin (VICMED) at a concentration of 150 mg / kg was intraperitoneally injected into the mice 12 minutes before imaging. Bioluminescence was then monitored using an in vivo optical imaging system (IVISLumina S5, PerkinElmer).

[0052] The results are as follows Figure 8 As shown: Compared with the control group (Saline), the administration of ML-SA5 delayed the invasion of the whole body of mice by human leukemia cells THP-1 carrying luciferase.

[0053] In summary, the present invention found that ML-SA5 intervention can reduce the infiltration of leukemia cells into mouse bone marrow, peripheral blood, liver, kidney and other tissues and organs, prolong the lifespan of leukemia mice, and comprehensively delay the progression of acute myeloid leukemia in mice.

[0054] The above experimental results prove that the ML-SA5 small molecule agonist can comprehensively improve the course of acute myeloid leukemia invasion in model mice. The present invention provides a new drug for the treatment of acute myeloid leukemia, which has good market value and clinical application prospects.

Claims

1. Application of TRPML1 specific small molecule agonist ML-SA5 in the preparation of acute myeloid leukemia drugs, characterized in that: The chemical structural formula of the TRPML1-specific small molecule agonist ML-SA5 is shown below:

2. The use according to claim 1, characterized in that: In the preparation of acute myeloid leukemia drugs, the effective dose of the TRPML1-specific small molecule agonist ML-SA5 is 4 mg / kg.

3. The use according to claim 1, characterized in that: The TRPML1-specific small molecule agonist ML-SA5 was used after acute myeloid leukemia.

4. The use according to claim 1, characterized in that: The acute myeloid leukemia drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a TRPML1-specific small molecule agonist ML-SA5.

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