CN114712366A

CN114712366A - Medicine for treating acute myeloid leukemia and application thereof

Info

Publication number: CN114712366A
Application number: CN202210337887.8A
Authority: CN
Inventors: 曾辉; 姜欣雅; 杜鹃; 刘芳舒; 华蕾; 孙晓凡
Original assignee: First Affiliated Hospital of Jinan University
Current assignee: First Affiliated Hospital of Jinan University
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-07-08

Abstract

The invention relates to a medicament for treating acute myeloid leukemia and application thereof, wherein the medicament for treating acute myeloid leukemia comprises NHE1 inhibitor HMA. The invention creatively discovers that the NHE1 inhibitor HMA can obviously inhibit the progress of acute myeloid leukemia, is used as a medicine for treating the acute myeloid leukemia, and enriches the treatment strategy of the acute myeloid leukemia. The invention proves that the HMA and the chemotherapeutic medicament are combined, on one hand, the killing effect of the chemotherapeutic medicament on AML cells can be enhanced; on the other hand, the dosage of the chemotherapeutic drug can be reduced, and the toxic and side effects of the chemotherapeutic drug on human bodies can be alleviated.

Description

Medicine for treating acute myeloid leukemia and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new medicinal application of an NHE1 inhibitor HMA, and particularly relates to a medicament for treating acute myeloid leukemia and application thereof.

Background

Acute Myeloid Leukemia (AML) is a group of highly heterogeneous hematological malignancies caused by clonal proliferation of immature myeloid progenitor cells, and currently there are limited treatments for AML, and induction chemotherapy is still the standard treatment, although some patients respond to induction chemotherapy, refractory disease and relapse are the major causes of treatment failure. In addition, the treatment of older patients is less effective, with a median survival of only 5-10 months. Since 2017, several new drugs for AML treatment have been approved by the FDA, including the BCL 2-specific inhibitor Venetocick (VEN). However, clinically acquired resistance to venetocks is a problem. Therefore, it is crucial to explore new therapeutic approaches to obtain more effective treatment of AML.

CN110678201A discloses anti-CD 19 antibodies and venetox for the treatment of non-hodgkin's lymphoma, chronic lymphocytic leukemia and/or small lymphocytic lymphoma. anti-CD 19 antibodies, in particular MOR00208 and venetock, are administered to patients with non-hodgkin's lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL) and/or Small Lymphocytic Lymphoma (SLL) to alleviate treatment-related tumor lysis syndrome, depending on the particular mode of treatment.

CN112533604A discloses a method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a plurality of AZD2811 nanoparticles and vinatork, wherein the preparation of AZD2811 nanoparticles is disclosed in international application publication No. WO 2015/036792. Studies have shown that the combination of AZD2811 nanoparticles and venetocks shows significantly enhanced combined efficacy compared to either agent alone.

Intracellular pH homeostasis is an important factor in tumor development. Unlike normal tissue, tumor cells exhibit an inverse pH gradient, i.e., intracellular pH (phi) is more basic to favor invasion and metastasis. Cytosolic alkalinization relies on sodium-hydrogen exchanger (NHEs) plasma transporters. NHE1 is the most widely expressed member of the NHE family, and elevated NHE1 has been a hallmark of tumorigenesis and prognosis in solid tumors such as gliomas and breast cancer. Therefore, induction of cellular acidification becomes a new approach for tumor therapy. The first widely used NHE1 inhibitor was a derivative of amiloride, known as a pyrazinylguanidine-type inhibitor. Among them, 5-N, N (hexamethylene) amide (HMA) has the strongest effect. Studies have demonstrated the effectiveness of HMA in treating breast cancer, however, the role of HMA in AML remains to be studied due to the apparent heterogeneity of solid and hematologic tumors.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new application of a NHE1 inhibitor HMA medicine, and particularly relates to a medicine for treating acute myeloid leukemia and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a medicament for treating acute myeloid leukemia, wherein the medicament for treating acute myeloid leukemia comprises NHE1 inhibitor HMA, and the chemical formula of HMA is as follows:

the invention creatively discovers that the NHE1 inhibitor HMA can obviously inhibit the progress of Acute Myeloid Leukemia (AML), and is used as a medicine for treating the acute myeloid leukemia.

Preferably, the medicament for treating acute myeloid leukemia further comprises a pharmaceutical adjuvant, wherein the pharmaceutical adjuvant comprises any one or a combination of at least two of carrier materials, diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners or pigments.

The combination of the at least two components, such as the combination of carrier material, filler, binder, lubricant, stabilizer and flavoring agent, the combination of excipient, filler and humectant, etc., can be selected in any combination manner, and will not be described in detail herein.

Preferably, the dosage form of the medicine for treating acute myeloid leukemia comprises tablets, capsules, granules, oral liquid, sustained-release preparations, nano preparations or injections.

In a second aspect, the invention provides the use of the NHE1 inhibitor HMA in the manufacture of a medicament for the treatment of acute myeloid leukaemia.

In a third aspect, the invention provides an application of an NHE1 inhibitor HMA in preparing a chemotherapeutic drug sensitizer.

The invention also provides the use of the NHE1 inhibitor HMA for the manufacture of a product for the treatment of patients with acute myeloid leukemia who are not susceptible to chemotherapeutic agents; also provides application of the NHE1 inhibitor HMA in preparing a product for improving the sensitivity of acute myeloid leukemia patients to chemotherapeutic drugs; also provides application of the NHE1 inhibitor HMA in preparing a product for improving the sensitivity of the acute myeloid leukemia cell line to chemotherapeutic drugs.

In the above application, the chemotherapeutic drug comprises vinatock, cytarabine or decitabine.

In a fourth aspect, the present invention provides a pharmaceutical composition for treating acute myeloid leukemia, wherein the pharmaceutical composition for treating acute myeloid leukemia comprises NHE1 inhibitor HMA and chemotherapeutic drugs, and the chemical formula of HMA is as follows:

the invention proves that the HMA and the chemotherapeutic drug are combined, on one hand, the killing effect of the chemotherapeutic drug on AML cells can be enhanced; on the other hand, the dosage of the chemotherapeutic drug can be reduced, and the toxic and side effects of the chemotherapeutic drug on human bodies can be alleviated. The test result shows that the HMA combined with the chemotherapeutic drug acts on the AML cell strain, the killing effect on the AML cell is obviously enhanced compared with that of the single chemotherapeutic drug or the HMA, namely the HMA and the chemotherapeutic drug have synergistic effect in combination, and the sensitivity of the AML cell to the chemotherapeutic drug can be obviously enhanced; the combined application of HMA and chemotherapeutic drugs can inhibit the proliferation of primary cells of AML patients and also has synergistic effect; by establishing a CDX mouse model and carrying out single drug and combined treatment of chemotherapeutic drugs and HMA, the combination of the two drugs can more effectively inhibit the progress of AML in vivo.

Preferably, the pharmaceutical composition is a combination of two separate formulations.

Preferably, the two separate preparations are independently selected from tablets, capsules, granules, oral liquids, sustained release preparations, nano preparations or injections.

The two separate formulations are administered simultaneously or sequentially at intervals or alternately in sequence.

Preferably, the chemotherapeutic agent comprises vinetocker, cytarabine or decitabine.

Preferably, the pharmaceutical composition further comprises a pharmaceutical excipient, wherein the pharmaceutical excipient comprises any one or a combination of at least two of carrier materials, diluents, excipients, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweeteners or pigments.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers that the NHE1 inhibitor HMA can obviously inhibit the progress of Acute Myeloid Leukemia (AML) and is used as a medicine for treating the acute myeloid leukemia, and the invention takes AML cell strains, mouse experiments and clinical samples as research objects, verifies that the NHE1 inhibitor HMA can inhibit the proliferation of cells and promote the apoptosis of the cells in the AML cell strains, has killing effect on primary cells of AML patients, and enriches the treatment strategy of the acute myeloid leukemia.

The invention proves that the combined use of HMA and chemotherapeutic drugs can enhance the killing effect of the chemotherapeutic drugs on AML cells on one hand; on the other hand, the dosage of the chemotherapeutic drug can be reduced, and the toxic and side effects of the chemotherapeutic drug on human bodies can be alleviated. The test result shows that the HMA combined with the chemotherapeutic drug acts on the AML cell strain, the killing effect on the AML cell is obviously enhanced compared with that of the single chemotherapeutic drug or the HMA, namely the HMA and the chemotherapeutic drug have synergistic effect in combination, and the sensitivity of the AML cell to the chemotherapeutic drug can be obviously enhanced; the combined application of HMA and chemotherapeutic drugs can inhibit the proliferation of primary cells of AML patients and also has synergistic effect; by establishing a CDX mouse model and carrying out single drug and combined treatment of chemotherapeutic drugs and HMA, the combination of the two drugs can more effectively inhibit the progress of AML in vivo.

Drawings

FIG. 1 is a graph showing the results of the effect of HMA on intracellular pH of MV 4-11;

FIG. 2 is a graph of the results of HMA effect on cell viability of MV4-11 cells;

FIG. 3 is a graph showing the results of the effect of HMA on apoptosis of MV4-11 cells;

FIG. 4 is a graph showing the effect of HMA on Caspase-3/7 activity in MV4-11 cells;

fig. 5 is a graph of the results of HMA effect on cell viability of AML primary cells;

fig. 6 is a graph of the results of HMA effect on apoptosis of AML primary cells;

FIG. 7 is a graph showing the effect of the combination of Venetok and HMA on the cell viability of MV4-11 cells;

FIG. 8 is a graph showing the effect of von Willebrand in combination with HMA on apoptosis of MV4-11 cells;

FIG. 9 is a graph of results of a Bliss model analysis of the synergy of HMA and Venetukast on MV4-11 cells;

FIG. 10 is a graph showing the effect of Winnetock and HMA on survival in mice;

FIG. 11 is a statistical plot of the results of a combination of Venetok and HMA on the AML infiltration rate of bone marrow, spleen and peripheral blood in mice;

FIG. 12 is a graph showing the results of an immunohistochemical assay on mouse bone marrow and spleen using a combination of Venetok and HMA;

fig. 13 is a graph of the results of the effect of a combination of venetocks and HMA on cell viability of AML primary cells;

FIG. 14 is a graph of the effect of von Willebrand in combination with HMA on apoptosis of AML primary cells;

FIG. 15 is a graph of the results of a Bliss model analysis of the synergy of Venetukast and HMA on AML primary cells;

FIG. 16 is a graph showing the effect of cytarabine in combination with HMA on the cell viability of MV4-11 cells;

figure 17 is a graph of the results of the effect of decitabine and HMA in combination on the cell viability of MV4-11 cells.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

The following examples relate to NHE1 inhibitor HMA available from gosha biotechnology limited, shanghai (MCE) as model No. HY-128067; the medicine Venetian Toxol is purchased from MCE and has model number HY-15531; cytarabine, a drug, was purchased from MCE and was type HY-13605; the drug decitabine was purchased from MCE and was model No. HY-a 0004.

The following examples relate to fresh bone marrow samples from AML patients sourced from the first hospital hematological department affiliated with river-south university.

The NCG mice referred to in the following examples were purchased from guangdong collectivium biotechnology limited, mice aged 6-8 weeks.

Example 1

NHE1 inhibitor HMA proliferation inhibition and apoptosis promotion effects on leukemia cells:

will be 4X 10⁶Inoculating MV4-11 cells in a six-well plate, adding NHE1 inhibitors with different concentrations to make the final concentrations respectively 0,5 μ M,10 μ M and 20 μ M; uniformly blowing, putting into an incubator for culturing for 48h, and then carrying out the following experiments:

(1) and (3) detecting the intracellular pH:

making a standard curve: cells were washed twice with PBS, added with BCECF-AM probe at a final concentration of 5. mu.M, and stained for 30 minutes at room temperature in the dark. Washing with PBS once, resuspending with calibration Buffer with different pH values, adding 10 μmol of Nigericin and Valinomycin, and incubating for 10 min to make the intracellular pH values reach 4.5,5.5,6.5 and 7.5 respectively; and (3) detecting the fluorescence intensity of the excitation light at 490nm and 440nm and the emission light at 535nm by using a microplate reader, calculating the fluorescence ratio of 490nm/440nm, and making a standard curve of the relation between the pH value and the fluorescence ratio.

Intracellular pH assay: the cells were collected, washed twice with PBS, added with BCECF-AM probe at a final concentration of 5. mu.M, and stained for 30 minutes at room temperature in the dark. The fluorescence intensity of excitation light at 490nm and 440nm and emission light at 535nm was measured using a microplate reader, the 490nm/440nm fluorescence ratio was calculated, and the intracellular pH was calculated from the standard curve.

The results are shown in fig. 1, which shows that as HMA concentration increases, intracellular pH decreases, indicating that HMA can lead to intracellular acidification.

(2) And (3) cell viability detection:

MV4-11 cells were plated at 5X 10⁴Inoculating the cells with the inoculation density of one/mL into a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively adding HMA to make the final concentration respectively 0,5 mu M,10 mu M and 20 mu M, slightly shaking and uniformly mixing, putting the 96-well plate into a cell culture box, and respectively culturing for 24h,48h and 72 h; and adding 10 mul of CCK8 working solution into each hole, mixing uniformly, continuously culturing for 3 hours in an incubator in the dark, and detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 2, and the results in fig. 2 indicate that HMA significantly inhibits AML cell viability, with concentration-dependent and time-dependent characteristics.

(3) And (3) detecting cell apoptosis:

MV4-11 cells were treated with HMA at the corresponding concentration for 48h, collected in a flow tube, washed once with PBS, and resuspended by adding Binding Buffer. Add 5. mu.l Annexin V and 10. mu.l PI for staining, while setting up the Annexin V and PI single stain controls and the negative control. Mix gently and incubate for 15 minutes at room temperature in the dark. Data were detected and collected using a BD FACS Canto flow cytometer. The results are shown in fig. 3, and the results in fig. 3 show that HMA promotes apoptosis.

(4) Detecting the activity of Caspase-3/7:

MV4-11 cells are treated with HMA with corresponding concentration for 48h, and then Caspase-3/7 substrate Z-DEVD-Rh 110-DVED-Z working solution is added to the cells, and the cells are placed in an incubator to be incubated for 2 h. Cells were collected in flow tubes, washed once with PBS, FITC channel fluorescence intensity was measured with a BD FACS Canto flow cytometer and data was collected. The results are shown in FIG. 4, which shows that HMA activates Caspase-3/7.

Example 2

Proliferation inhibition and apoptosis promotion effects of HMA on primary cells of AML patients:

(1) isolation and culture of bone marrow mononuclear cells:

collecting 5mL of fresh bone marrow sample of AML patient into a heparan violaceum anticoagulation tube, and uniformly mixing the fresh bone marrow sample with PBS (precooled at 4 ℃) with the same volume; and taking a clean 15mL centrifuge tube, adding 3mL human lymphocyte separation liquid into the tube, sucking the diluted bone marrow sample, inclining and fixing the centrifuge tube for 45 degrees to avoid shaking, and slowly adding the bone marrow sample onto the lymphocyte separation liquid along the tube wall of the centrifuge tube to ensure that the separation liquid and the liquid level of the bone marrow liquid are obviously layered. The tube was placed in a centrifuge and centrifuged at 2000rpm for 20 minutes. And (4) taking out the centrifuge tube gently to avoid shaking, adding another 15mL sterile centrifuge tube into 2mL PBS, carefully sucking the second mononuclear cell layer into the tube, blowing, beating and mixing uniformly. Placing the centrifuge tube in a centrifuge, setting the parameter to be 1000rpm, centrifuging for 5 minutes, and discarding the supernatant; the erythrocyte lysate is kept still and cracked for 7 minutes, and then the erythrocyte lysate is washed repeatedly by PBS solution precooled at 4 ℃ and centrifuged for 2 times for standby.

Primary cell cultures were prepared at a rate of 79% (v/v) α MEM + 20% (v/v) FBS + 1% (v/v) double antibodyAnd added with SCF (50ng/ml), IL3(10ng/ml), FLT3(50ng/ml), IL6(20ng/ml) and TPO (25 ng/ml). The primary cells isolated above were plated at 1X 10⁶The density of cells/mL was placed in an incubator.

(2) And (3) cell viability detection:

the primary cells were plated at 4X 10⁵Inoculating the cells with the density of one/mL in a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively treating with HMA (0, 5. mu.M, 10. mu.M, 20. mu.M and 40. mu.M) with different concentrations, slightly shaking and mixing uniformly, putting the 96-well plate into a cell culture box, and culturing for 48 h; and adding 10 mul of CCK8 working solution into each hole, mixing uniformly, continuously culturing for 3 hours in an incubator in the dark, and detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 5 (figure # represents different sample numbers), and fig. 5 shows that HMA significantly inhibits AML primary cell viability and is concentration gradient dependent.

(3) And (3) detecting cell apoptosis:

the primary cells were plated at 4X 10⁵The cells were seeded in six-well plates at individual/mL densities, treated with different concentrations of HMA (0, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M) for 48h, collected in flow tubes, washed once with PBS, and resuspended in cells by adding Binding Buffer. Add 5. mu.l Annexin V and 10. mu.l PI for staining, while setting up the Annexin V and PI single stain controls and the negative control. Mix gently and incubate for 15 minutes at room temperature in the dark. Data were detected and collected using a BD FACS Canto flow cytometer. The results are shown in fig. 6 (numbers # in the figure represent different sample numbers), and fig. 6 shows that HMA promotes apoptosis of AML primary cells.

Example 3

The combined application of HMA and Venitox has the functions of inhibiting the proliferation and promoting the apoptosis of leukemia cells:

will be 4X 10⁶Inoculating MV4-11 cells in a six-hole plate, and respectively adding no medicine, 0.1 mu M of Venetian Torke, 10 mu M of HMA and the combination of the two medicines; uniformly blowing, putting into an incubator for culturing for 48h, and then carrying out the following experiments:

(1) and (3) cell viability detection:

MV4-11 cells were plated at 5X 10⁴Cell inoculation of one/mLInoculating in 96-well plate at density, setting 3-4 auxiliary wells for different treated cells, and inoculating at volume of 100 μ L per well; respectively carrying out the treatments, slightly shaking and uniformly mixing, and then putting the 96-well plate into a cell culture box for culturing for 48 hours; and adding 10 mul of CCK8 working solution into each hole, mixing uniformly, continuously culturing for 3 hours in an incubator in the dark, and detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 7, and fig. 7 shows that both of venetocks and HMA can significantly inhibit AML cell viability, and that the combined use of both drugs can play a more significant role.

(2) And (3) detecting cell apoptosis:

MV4-11 cells were treated as above for 48h, collected in a flow tube, washed once with PBS, and resuspended by adding Binding Buffer. 5. mu.l of Annexin V and 10. mu.l of PI were added for staining, while Annexin V and PI single stain controls and negative controls were set up. Mix gently and incubate for 15 minutes at room temperature in the dark. Data were detected and collected using a BD FACS Canto flow cytometer. The results are shown in fig. 8, and fig. 8 shows that both of venetocel and HMA promote apoptosis, and the pro-apoptotic effect of the combination of both drugs is more significant.

(3) Calculating a combined index and constructing a Bliss model:

MV4-11 cells were plated at 5X 10⁴Inoculating the cells with the inoculation density of one/mL into a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively administering different concentrations of veronick, different concentrations of HMA and the combination of the two medicines, wherein the specific concentrations are as follows; uniformly blowing, and putting into an incubator for culturing for 48 hours; and adding 10 mul of CCK8 working solution into each hole, mixing uniformly, continuously culturing for 3 hours in an incubator in the dark, and detecting the absorbance at the wavelength of 450nm by using a microplate reader. Obtaining cell activity data, and calculating a combined index by utilizing Compuyn software; the synergy Fimder website (http:// www.synergyfinder.org /) was used to construct the Bliss model.

The results are shown in fig. 9, which shows that fig. 9 shows a combination index of 0.34-0.56 for both venetocks and HMA in synergy (a); and the combination of HMA and Venetork at different concentrations shows a synergistic effect (b); by constructing the Bliss model, the combined effect of HMA and venetock can also be judged as synergy (c).

Example 4

Therapeutic effects of HMA in combination with venetox on AML mice:

(1) constructing a CDX mouse model:

will be 1 × 10⁶Individual MV4-11 cells were injected tail vein into NCG mice to induce AML. On the fourth day of modeling, mice were randomly divided into four groups, a control group, a verniton group, an HMA group, and a combination group; the administration is started from the fifth day, the solvent is administered by intragastric administration to the control group, 50mg/kg of vernetokk is administered by intragastric administration to the vernetokk group, 20mg/kg of HMA is administered by intragastric administration to the HMA group, and two medicaments are administered by intragastric administration at the same time in the combination group five times per week. 3 mice were sacrificed in each group 14 days after administration, peripheral blood, bone marrow and spleen were collected, hCD45 ratio was examined, and survival time was recorded for the remaining mice in each group for survival analysis. The results are shown in FIG. 10, and the results in FIG. 10 show that: the survival time of the single vitamin E and vitamin E plus the single HMA is prolonged, and the two medicines are combined to play a more obvious role in prolonging the survival time.

(2) Detecting the AML infiltration proportion:

after washing the collected bone marrow, spleen was ground and peripheral blood was lysed for erythrocytes, washed once with PBS and filtered with a 45 μm filter, resuspended with buffer (PBS + 2% FBS), 200 μ l was taken in a flow tube and stained with the following antibodies: APC-Cy7-hCD45, PE-hCD33 and PI. The cells were stained in ice for 20 minutes in the dark, washed once with 2ml buffer, resuspended in 300. mu.l buffer, and then examined and data collected on a BD FACS Canto flow cytometer. The results are shown in fig. 11, which shows that hCD45 in bone marrow, spleen and peripheral blood of the two-drug combination group is significantly lower than that of the single-drug combination group, indicating that the two-drug combination group can achieve better AML killing effect.

(3) Fixation and immunohistochemistry of femur, spleen:

and (3) putting the collected femur and spleen of the mouse into 4% paraformaldehyde fixing solution to denature and coagulate the protein of the cell, so as to keep the original shape and structure of the cell and perform an immunohistochemical experiment. The results are shown in FIG. 12, which indicates that spleen and bone marrow in the combination group had a lower positive ratio of hCD45 than those in the two single groups.

Example 5

The combined application of HMA and Venetork has proliferation inhibiting and apoptosis promoting effects on primary cells of AML patients:

(1) isolation and culture of bone marrow mononuclear cells:

Primary cell culture media were prepared at a rate of 79% (v/v) α MEM + 20% (v/v) FBS + 1% (v/v) diabody, and SCF (50ng/ml), IL3(10ng/ml), FLT3(50ng/ml), IL6(20ng/ml) and TPO (25ng/ml) were added. The primary cells isolated above were plated at 1X 10⁶The density of cells/mL was placed in an incubator.

(2) And (3) cell viability detection:

the primary cells were plated at 4X 10⁵Inoculating the cells with the density of one/mL in a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively adding DMSO (0.1%), Venetuok (0.1 mu M), HMA (10 mu M) and the two medicines for combination, slightly shaking and uniformly mixing, putting a 96-well plate into a cell culture box, and culturing for 48 h; adding 10 to each wellAnd (3) mixing the micro-liter CCK8 working solution, continuously culturing for 3 hours in an incubator in a dark place, and detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 13 (numbers # in the figure represent different sample numbers), and show that HMA in combination with venetocks significantly inhibited AML primary cell viability compared to the single group.

(3) And (3) detecting cell apoptosis:

the primary cells were plated at 4X 10⁵Each/mL of the cells was inoculated into a six-well plate, treated with DMSO (0.1%), Vernitol (0.1. mu.M), HMA (10. mu.M) and a combination of the two for 48h, collected in a flow tube, washed once with PBS, and resuspended in a Binding Buffer. Add 5. mu.l Annexin V and 10. mu.l PI for staining, while setting up the Annexin V and PI single stain controls and the negative control. Mix gently and incubate for 15 minutes at room temperature in the dark. Data were detected and collected using a BD FACS Canto flow cytometer. The results are shown in fig. 14 (numbers # in the figure represent different sample numbers), and show that HMA in combination with venetox promotes apoptosis of AML primary cells.

(4) Constructing a Bliss model:

the primary cells were plated at 4X 10⁵Inoculating the cells with the density of one/mL in a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively administering different concentrations of veronick, different concentrations of HMA and two medicines for combined use, wherein the specific concentrations are shown as follows; uniformly blowing, and putting into an incubator for culturing for 48 hours; and adding 10 mu l of CCK8 working solution into each hole, mixing uniformly, then continuously culturing for 3 hours in an incubator in the dark, and then detecting the absorbance at the wavelength of 450nm by using a microplate reader. Cell activity data were obtained and the synergy Fimder website (http:// www.synergyfinder.org /) was used to construct the Bliss model.

The results are shown in FIG. 15 (in the figure, # indicates different sample numbers): the effect of HMA in combination with venetocks in AML primary cells can be judged as a synergistic effect.

Example 6

The proliferation inhibition effect of the combined application of HMA and cytarabine on leukemia cells is as follows:

MV4-11 cells were plated at 5X 10⁴Inoculating the cells with the inoculation density of one/mL into a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively administering 0.5 mu M cytarabine (Ara-C), 10 mu M HMA and the two medicines without adding medicines, slightly shaking and uniformly mixing, putting a 96-well plate into a cell culture box, and culturing for 48 h; and adding 10 mu l of CCK8 working solution into each hole, mixing uniformly, then continuously culturing for 3 hours in an incubator in the dark, and then detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 16, and fig. 16 shows that cytarabine and HMA both can significantly inhibit AML cell viability, and the combined application of the two drugs can play a more significant role.

Example 7

The combined application of HMA and decitabine has the proliferation inhibition effect on leukemia cells:

MV4-11 cells were plated at 5X 10⁴Inoculating the cells with the inoculation density of one/mL into a 96-well plate, setting 3-4 auxiliary wells for the cells with different treatments, and inoculating according to the volume of 100 mu L per well; respectively adding 5 mu M Decitabine (DAC), 10 mu M HMA and the two medicines without adding medicines, slightly shaking and uniformly mixing, putting a 96-well plate into a cell culture box, and culturing for 48 h; and adding 10 mu l of CCK8 working solution into each hole, mixing uniformly, then continuously culturing for 3 hours in an incubator in the dark, and then detecting the absorbance at the wavelength of 450nm by using a microplate reader. The results are shown in fig. 17, and fig. 17 shows that cytarabine and HMA both can significantly inhibit AML cell viability, and the combined use of the two drugs can play a more significant role.

The applicant states that the invention is described by the above examples to describe the drug for treating acute myeloid leukemia and the application thereof, but the invention is not limited to the above examples, i.e. it does not mean that the invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. The medicine for treating acute myeloid leukemia is characterized by comprising an NHE1 inhibitor HMA, wherein the HMA has the chemical formula as shown in the specification:

2. the medicament for treating acute myeloid leukemia according to claim 1, further comprising a pharmaceutical adjuvant, wherein the pharmaceutical adjuvant comprises any one or a combination of at least two of carrier material, diluent, excipient, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant, stabilizer, flavoring agent, sweetener or pigment.

3. The drug for treating acute myeloid leukemia according to claim 1 or 2, wherein the dosage form of the drug for treating acute myeloid leukemia comprises tablet, capsule, granule, oral liquid, sustained release preparation, nano preparation or injection.

Use of the NHE1 inhibitor HMA for the preparation of a medicament for the treatment of acute myeloid leukemia.

Application of an NHE1 inhibitor HMA in preparing a chemotherapeutic drug sensitizer.

6. The use of claim 5, wherein the chemotherapeutic agent comprises Venetock, Cytarabine, or Decitabine.

7. The pharmaceutical composition for treating acute myeloid leukemia is characterized by comprising an NHE1 inhibitor HMA and a chemotherapeutic drug, wherein the HMA has the following chemical formula:

8. the pharmaceutical composition for treating acute myeloid leukemia according to claim 7, wherein said pharmaceutical composition is a combination of two separate preparations;

9. The pharmaceutical composition of claim 7, wherein the chemotherapeutic agent comprises vinetork, cytarabine, or decitabine.

10. The pharmaceutical composition for treating acute myeloid leukemia according to any one of claims 7-9, wherein the pharmaceutical composition further comprises a pharmaceutical adjuvant, and the pharmaceutical adjuvant comprises any one or a combination of at least two of carrier material, diluent, excipient, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant, stabilizer, flavoring agent, sweetener or pigment.