Application of CERT inhibitors in the preparation of drugs for treating acute myeloid leukemia

Abstract

The present invention relates to the use of CERT inhibitors in the preparation of drugs for treating acute myeloid leukemia. The present invention creatively finds that CERT inhibitors represented by HPA-12 have obvious therapeutic effects on acute myeloid leukemia. The present invention is based on AML cell lines, mouse experiments and clinical samples as research objects, and verifies that CERT inhibitors can inhibit cell proliferation and promote apoptosis in AML cell lines, prolong the survival of AML mouse models, reduce the infiltration degree of AML cells and enhance the sensitivity of AML cells to FLT3 inhibitors, which provides a new strategy for the treatment of acute myeloid leukemia (AML).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, relates to a new strategy for treating acute myeloid leukemia, and specifically relates to the use of a CERT inhibitor in the preparation of a drug for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease of the blood system characterized by the blockage of differentiation of myeloid primitive cells in the hematopoietic system and the aggregation of immature precursor cells. With the continuous progress in the field of molecular biology, the emergence of specific targeted drugs represented by FMS-like Tyrosine Kinase-3 (FLT3) inhibitors such as gilteritinib has broken the treatment dilemma of AML and provided clinically with precise treatment weapons. About 1 / 3 of adult AML patients with FLT3 activating mutations have a new treatment option and have improved the disease remission rate to a certain extent. However, this is especially true for patients with relapsed / refractory (RR) AML. Some patients may develop primary resistance due to the bone marrow microenvironment or secondary resistance due to other mutations after medication. Therefore, exploring more efficient and reasonable drug combinations is expected to increase the sensitivity of AML to FLT3 inhibitors and prevent the occurrence of resistance.

[0003] Sphingomyelin (SM) is an important class of lipids involved in building cell membranes and regulating almost all cellular functions. The sphingomyelin metabolic network is centered on the ceramide-sphingomyelin axis. Ceramide (Cer) is recognized as a pro-apoptotic signal, while SM, as the most abundant type of phospholipid, is required for cell growth. Therefore, the balance between these two sphingolipids is crucial for the survival and function of cancer cells. Ceramide Transfer Protein (CERT) determines the ratio of Cer and SM in cells and is the only lipid transporter that specifically transfers Cer from the endoplasmic reticulum to the Golgi apparatus. In the Golgi apparatus, Cer serves as a substrate for SM synthesis. Cer is a bioactive sphingolipid that can be produced in response to treatment with various chemotherapeutic drugs, including FLT3 inhibitors. However, it is unclear whether simultaneous targeting of FLT3 signaling and Cer metabolism can be used to modulate AML treatment. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a new strategy for treating acute myeloid leukemia, specifically to provide the use of CERT inhibitors in the preparation of drugs for treating acute myeloid leukemia.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides use of a CERT inhibitor in the preparation of a medicament for treating acute myeloid leukemia.

[0007] The present invention creatively discovered that CERT inhibitors represented by HPA-12 have significant therapeutic effects on acute myeloid leukemia. The present invention is based on AML cell lines, mouse experiments and clinical samples as research objects, and verifies that CERT inhibitors can inhibit cell proliferation and promote apoptosis in AML cell lines, prolong the survival of AML mouse models, reduce the infiltration of AML cells and enhance the sensitivity of AML cells to FLT3 inhibitors, which provides a new strategy for the treatment of acute myeloid leukemia (AML).

[0008] Preferably, the CERT inhibitor comprises HPA-12.

[0009] HPA-12 is a ceramide transport inhibitor, which was first discovered and synthesized by Hanada and Kobayashi, and its chemical structure is as follows. HPA-12 has been used in various biological science studies as a CERT inhibitor. For example, HPA-12 has antiviral and antibacterial properties, and can resist the growth of hepatitis C virus (HCV) and host cell bacteria Chlamydia in cultured human cells. The present invention repositions the drug function of HPA-12, further expands its new application in the treatment of acute myeloid leukemia (AML), and provides potential treatment ideas and reference for clinical practice.

[0010]

[0011] Preferably, the medicine further contains pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0013] Preferably, the dosage form of the drug is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0014] In a second aspect, the present invention provides use of a CERT inhibitor in the preparation of a preparation for promoting apoptosis of acute myeloid leukemia cells.

[0015] Preferably, the CERT inhibitor comprises HPA-12.

[0016] According to the research results of the present invention, CERT inhibitors represented by HPA-12 can promote apoptosis of acute myeloid leukemia cell lines at the cellular level (in vitro level), that is, CERT inhibitors represented by HPA-12 can be made into a simple experimental preparation for exploring the physiological metabolic process of acute myeloid leukemia cell lines, that is, an application in the preparation of a preparation for promoting apoptosis of acute myeloid leukemia cells for non-therapeutic purposes.

[0017] In a third aspect, the present invention provides use of a CERT inhibitor in the preparation of a preparation for inhibiting proliferation of acute myeloid leukemia cells.

[0018] Preferably, the CERT inhibitor comprises HPA-12.

[0019] According to the research results of the present invention, CERT inhibitors represented by HPA-12 can inhibit the proliferation of acute myeloid leukemia cell lines at the cellular level (in vitro level), that is, CERT inhibitors represented by HPA-12 can be made into a simple experimental preparation for exploring the physiological metabolic process of acute myeloid leukemia cell lines, that is, an application in the preparation of a preparation for inhibiting the proliferation of acute myeloid leukemia cells for non-therapeutic purposes.

[0020] In a fourth aspect, the present invention provides use of a CERT inhibitor in the preparation of a sensitizer for a FLT3 inhibitor.

[0021] The present invention found that the FLT3 inhibitor represented by Cleranib alone has limited therapeutic effect on acute myeloid leukemia, but CERT inhibitors can promote the sensitivity of acute myeloid leukemia cells to FLT3 inhibitors, greatly improving the effect of treating acute myeloid leukemia.

[0022] Preferably, the CERT inhibitor comprises HPA-12.

[0023] Preferably, the FLT3 inhibitor comprises crellanib.

[0024] In a fifth aspect, the present invention provides a combination pharmaceutical composition for treating acute myeloid leukemia, wherein the combination pharmaceutical composition comprises a CERT inhibitor and a FLT3 inhibitor.

[0025] The present invention also creatively combines CERT inhibitors and FLT3 inhibitors as a drug for treating acute myeloid leukemia. The combination of the two can not only reduce the dosage of CERT inhibitors or FLT3 inhibitors and improve the safety of medication, but also has a more significant effect of treating acute myeloid leukemia than a single CERT inhibitor or a single FLT3 inhibitor, and plays a synergistic promotion effect. The present invention experiments have proved that the pharmaceutical composition can inhibit the proliferation of acute myeloid leukemia cell lines and promote their apoptosis, providing more effective protection for acute myeloid leukemia in vivo. The present invention provides an effective drug combination strategy for the treatment of acute myeloid leukemia, which is of great significance.

[0026] Preferably, the CERT inhibitor comprises HPA-12.

[0027] Preferably, the FLT3 inhibitor comprises crellanib.

[0028] Preferably, the combination pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0029] Preferably, the combined pharmaceutical composition is a combination of two separate preparations, and the two separate preparations are administered simultaneously or sequentially.

[0030] The combination pharmaceutical composition may be in the form of a single compound preparation or a combination of two separate preparations; when it is a combination of two separate preparations, the administration method may be simultaneous administration, cross administration or sequential administration.

[0031] Preferably, the preparation is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0032] Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0033] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention creatively discovered that CERT inhibitors represented by HPA-12 have significant therapeutic effects on acute myeloid leukemia. The present invention is based on AML cell lines, mouse experiments and clinical samples as research objects, and verifies that CERT inhibitors can inhibit cell proliferation and promote apoptosis in AML cell lines, prolong the survival of AML mouse models, reduce the infiltration of AML cells and enhance the sensitivity of AML cells to FLT3 inhibitors, which provides a new strategy for the treatment of acute myeloid leukemia (AML).

[0036] The present invention also creatively combines CERT inhibitors and FLT3 inhibitors as drugs for treating acute myeloid leukemia. The combination of the two can not only reduce the dosage of CERT inhibitors or FLT3 inhibitors and improve the safety of medication, but also has a more significant effect in treating acute myeloid leukemia than a single CERT inhibitor or a single FLT3 inhibitor, achieving a synergistic promotion effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a statistical graph showing the inhibition of HPA-12 on the viability of MV4-11, MOLM13, HL-60 and THP-1 cells;

[0038] Figure 2 This is a graph showing the results of HPA-12 inhibiting the proliferation of MV4-11 cells;

[0039] Figure 3 This is the flow cytometry result and statistical result diagram of HPA-12 promoting apoptosis of MV4-11 cells;

[0040] Figure 4 This is the flow cytometry result and statistical result diagram of HPA-12 promoting apoptosis of MOLM13 cells;

[0041] Figure 5 This is a graph showing the inhibition of caspase3 / 7 activity in MV4-11 cells by HPA-12;

[0042] Figure 6 This is the statistical result of the inhibition of the activity of MV4-11 and MOLM13 cells by the combination of HPA-12 and Creno;

[0043] Figure 7 This is the result of HPA-12 combined with Creno inhibiting the proliferation of MV4-11 cells;

[0044] Figure 8 This is the flow cytometry result and statistical result diagram of the apoptosis promotion of MV4-11 cells by the combination of HPA-12 and Creno;

[0045] Fig. 9This is the result of the inhibition of caspase3 / 7 activity in MV4-11 cells by the combination of HPA-12 and Creno;

[0046] Fig.10 It is the Bliss, Loewe, ZIP, and HSA model diagram constructed by combining HPA-12 and Creno;

[0047] Fig.11 These are the in vivo imaging images of each group of mice before and after medication in Example 3;

[0048] Fig.12 is a survival curve diagram of each group of mice in Example 3;

[0049] Fig.13 is a graph showing the hCD45 cell infiltration results in the bone marrow of each group of mice in Example 3;

[0050] Fig.14 is a diagram showing the hCD45 cell infiltration results in the spleen of each group of mice in Example 3;

[0051] Fig.15 is a graph showing the hCD45 cell infiltration results in the peripheral blood of each group of mice in Example 3;

[0052] Fig.16 is a statistical diagram of spleen and spleen weight of each group of mice in Example 3;

[0053] Fig.17 is the immunohistochemical staining of the bone marrow sections of each group of mice in Example 3;

[0054] Fig.18 is the immunohistochemical staining of spleen sections of each group of mice in Example 3;

[0055] Fig.19 This is the result of HPA-12 inhibiting the activity of primary cells from AML patients;

[0056] Fig. 20 This is a graph showing the results of the combined use of HPA-12 and Creno to inhibit the activity of primary cells from AML patients. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0058] The HPA-12 involved in the following examples is a product purchased from TCI with model number H1553-5MG, and Clarane is a product purchased from Selleck with model numbers S2730-zky and s2730 (the concentrations in the experiments were calculated based on the actual active ingredients in the drugs).

[0059] The MV4-11 (FLT3 / ITD), MOLM13 (FLT3 / ITD), HL-60 (FLT3 / WT), THP-1 (FLT3 / WT), and KG-1α (FLT3 / WT) cells involved in the following examples were derived from ATCC; B-NDG mice were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. (6 weeks old, female).

[0060] Example 1-1

[0061] Inhibitory effect of HPA-12 on AML cell viability:

[0062] MV4-11 (FLT3 / ITD), MOLM13 (FLT3 / ITD), HL-60 (FLT3 / WT) and THP-1 (FLT3 / WT) cells were used as the research objects. The cells were respectively cultured at 5×10 4 The cells were inoculated at a density of 100 μM / mL in a 96-well plate. Four duplicate wells were set for cells with different treatments, and each well was inoculated with a volume of 100 μL. HPA-12 was added to make the final concentrations of 0, 60 μM, 80 μM, and 100 μM, respectively. After slight shaking and mixing, the 96-well plate was placed in a cell culture incubator and cultured for a total of 48 hours. 10 μL of CCK8 working solution was added to each well, mixed, and cultured in a light-proof incubator for 3 hours, and then the absorbance at a wavelength of 450 nm was detected with an ELISA reader. The results are shown in Figure 2. Figure 1 As shown, the results showed that HPA-12 could significantly inhibit the viability of AML cells in a concentration-dependent manner.

[0063] Example 1-2

[0064] Inhibitory effect of HPA-12 on AML cell proliferation:

[0065] MV4-11 (FLT3 / ITD) cells were used as the research object. MV4-11 cells were cultured at 5×10 4Cells were seeded at a density of 100 μg / mL in a 96-well plate and treated with DMSO, 60 μM, 80 μM and 100 μM HPA-12, respectively, and cultured for 48 h. EdU working solution was added to a final concentration of 10 μM and incubated for 3 h. After EdU labeling of cells, the culture medium was removed and 1 mL of 4% paraformaldehyde was added to fix at 20 ° C for 15 min. Centrifuge at 1000 rpm for 5 min, remove the fixative, and wash 3 times with 1 ml of PBS, 5 min each time. Centrifuge to remove PBS, add 1 ml of PBS containing 0.5% Triton X-100, incubate at 20 ° C for 15 min, and repeat the washing step. Centrifuge to remove PBS, prepare the staining reaction solution according to the instructions, add 500 μL of the reaction solution to each sample, incubate at 20 ° C in the dark for 30 min, and repeat the washing step. Centrifuge to remove PBS, add DAPI to a final concentration of 2 μg / mL, incubate at 20 ° C in the dark for 10 min, and repeat the washing step. Smear the cells on a glass slide, and after they are completely air-dried, drop a little anti-fluorescence quenching mounting medium, cover with a coverslip, be careful to avoid bubbles, and seal all around with transparent nail polish. After they are completely dry, observe and take pictures under a fluorescent inverted microscope, and count the fluorescence intensity. The results are as follows: Figure 2 As shown, the results showed that HPA-12 could significantly inhibit the proliferation of AML cells in a concentration-dependent manner.

[0066] Examples 1-3

[0067] HPA-12 promotes apoptosis of AML cells:

[0068] MV4-11 (FLT3 / ITD) and MOLM13 (FLT3 / ITD) cells were used as the research objects. MV4-11 and MOLM13 cells were cultured at 5×10 4 Cells were seeded at a density of 100 / mL in a 96-well plate. After being treated with the corresponding concentration of HPA-12 for 48 hours, the cells were collected in a flow tube, washed once with PBS, and resuspended in Binding Buffer. 5 μL Annexin V and 10 μL PI were added for staining. Annexin V and PI single staining controls and negative controls were also set up. Gently mix and incubate at 20°C in the dark for 15 minutes. Detection and data collection were performed using a BD FACS Canto flow cytometer. The results are as follows: Figure 3 (MV4-11) and Figure 4 As shown in (MOLM13), the results showed that HPA-12 could promote apoptosis of AML cells in a concentration-dependent manner.

[0069] Examples 1-4

[0070] Effect of HPA-12 on Caspase-3 / 7 Activity:

[0071] MV4-11 (FLT3 / ITD) cells were used as the research object. After MV4-11 cells were treated with HPA-12 at the corresponding concentration for 48 hours, Caspase-3 / 7 substrate Z-DEVD-Rh 110-DVED-Z working solution was added and incubated in an incubator for 2 hours. The cells were collected in a flow tube, washed once with PBS, and the fluorescence intensity of the FITC channel was detected using a BD FACS Canto flow cytometer and the data was collected. The results are shown in Figure 2. Figure 5 As shown, the results showed that HPA-12 can activate Caspase-3 / 7.

[0072] Example 2-1

[0073] HPA-12 combined with Creno inhibits AML cell viability:

[0074] MV4-11 (FLT3 / ITD) and MOLM13 (FLT3 / ITD) cells were used as the research objects. The cells were respectively divided into 5×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. Four sub-wells were set for cells with different treatments, and the cells were inoculated at a volume of 100 μL per well. No drug (DMSO), 4 μM crelanib, 80 μM HPA-12, and a combination of the two drugs were given respectively. After slight shaking and mixing, the 96-well plate was placed in a cell culture incubator and cultured for 48 hours. 10 μL of CCK8 working solution was added to each well, and after mixing, the incubator was incubated in the dark for 3 hours, and then the absorbance at a wavelength of 450 nm was detected with an enzyme marker. The statistical results are as follows: Figure 6 As shown, the results showed that both Creno and HPA-12 could significantly inhibit the viability of AML cells, and the combined use of the two drugs could have a more significant effect.

[0075] Example 2-2

[0076] HPA-12 combined with Creno inhibits AML cell proliferation:

[0077] MV4-11 (FLT3 / ITD) cells were used as the research object. The cells were divided into 5×10 4Cells were seeded at a density of 100 μM / mL in a 96-well plate. Four wells were set up for cells with different treatments, and each well was seeded with a volume of 100 μL. No drug (DMSO), 4 μM Creno, 80 μM HPA-12, and a combination of the two drugs (4 μM Creno + 80 μM HPA-12) were given respectively; cultured for 48 hours. EdU working solution was added to a final concentration of 10 μM, and incubated for 3 hours. After EdU labeling of cells, the culture medium was removed, and 1 ml of 4% paraformaldehyde was added, and fixed at 20°C for 15 minutes. Centrifuge at 1000 rpm for 5 minutes, remove the fixative, and wash 3 times with 1 ml PBS, each time for 5 minutes. Centrifuge to remove PBS, add 1 ml of PBS containing 0.5% Triton X-100, incubate at 20°C for 15 minutes, and repeat the washing steps. Remove PBS by centrifugation, prepare staining reaction solution according to the instructions, add 500μl reaction solution to each sample, incubate at 20℃ in dark for 30min, and repeat the washing step. Remove PBS by centrifugation, add DAPI to a final concentration of 2μg / ml, incubate at 20℃ in dark for 10min, and repeat the washing step. Apply the cells on the slide, and after they are completely air-dried, drop a little anti-fluorescence quenching sealing agent, cover with a coverslip, be careful to avoid bubbles, seal all around with transparent nail polish, and after they are completely dry, observe and take pictures under a fluorescence inverted microscope, and calculate the fluorescence intensity. The results are as follows Figure 7 As shown, the results showed that the combination of CERT inhibitor HPA-12 and Creno more significantly inhibited the proliferation of AML (FLT3 / ITD) cells than single drug.

[0078] Example 2-3

[0079] HPA-12 combined with Creno promotes apoptosis of AML cells:

[0080] MV4-11 (FLT3 / ITD) cells were used as the research object. The cells were divided into 5×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. Four replicate wells were set up for cells with different treatments, and each well was inoculated with a volume of 100 μL. No drug (DMSO), 4 μM Creno, 80 μM HPA-12, and a combination of the two drugs (4 μM Creno + 80 μM HPA-12) were given respectively; cultured for 48 hours. Collected in a flow tube, washed once with PBS, and resuspended in Binding Buffer. Added 5 μl Annexin V and 10 μl PI for staining, and Annexin V and PI single staining controls and negative controls were set up at the same time. Gently mix and incubate at 20°C in the dark for 15 minutes. Detected and collected data using a BD FACS Canto flow cytometer. The results are shown in Figure 8As shown, the results showed that both Creno and HPA-12 can promote cell apoptosis, and the pro-apoptotic effect of the combined use of the two drugs is more significant.

[0081] Embodiment 2-4

[0082] Effect of HPA-12 combined with Creno on Caspase-3 / 7 activity:

[0083] MV4-11 (FLT3 / ITD) cells were used as the research object. The cells were divided into 5×10 4 The cells were seeded at a density of 100 μL / mL in a 96-well plate. Four replicate wells were set up for cells with different treatments, and the cells were seeded at a volume of 100 μL per well. No drug (DMSO), 4 μM Creno, 80 μM HPA-12, and a combination of the two drugs (4 μM Creno + 80 μM HPA-12) were given respectively; and cultured for 48 hours. Caspase-3 / 7 substrate Z-DEVD-Rh 110-DVED-Z working solution was added and incubated in an incubator for 2 hours. The cells were collected in a flow tube, washed once with PBS, and the fluorescence intensity of the FITC channel was detected using a BD FACS Canto flow cytometer and the data was collected. The results are shown in Fig. 9 As shown, the results showed that the single application of CERT inhibitors HPA-12 and Creno could increase the activation of Caspase-3 / 7 in the MV4-11 cell line, while the activation was more obvious in the combined application of HPA-12 and Creno.

[0084] Embodiment 2-5

[0085] HPA-12 and Creno combined index and synergistic prediction model:

[0086] MV4-11 (FLT3 / ITD) and KG-1α (FLT3 / WT) cells were used as the research objects. MV4-11 (FLT3 / ITD) and KG-1α (FLT3 / WT) cells were cultured at 5×10 4Cells were inoculated at a density of 100 μL / mL in a 96-well plate. Three sub-wells were set for cells with different treatments, and the volume of each well was 100 μL. Different concentrations of crelanib (4 μM, 6 μM, 8 μM), different concentrations of HPA-12 (60 μM, 80 μM, 100 μM) and the combination of the two drugs were given respectively. The mixture was blown evenly and placed in an incubator for 48 hours. 10 μl of CCK8 working solution was added to each well, mixed and cultured in an incubator in the dark for 3 hours, and then the absorbance at a wavelength of 450 nm was detected by an enzyme marker. The cell activity data were obtained, and the combination index was calculated using Compusyn software. The results are shown in Table 1 (MV4-11) and Table 2 (KG-1α). The Bliss, Loewe, ZIP, and HSA models were constructed using the SynergyFimder website (http: / / www.synergyfinder.org / ). Fig.10 The results showed that the combined index of crellanib and HPA-12 was 0.134-0.779, showing a synergistic effect; and the combination of HPA-12 and crellanib at different concentrations showed a synergistic effect; by constructing the Bliss, Loewe, ZIP, HSA model, it can also be determined that the combined effect of HPA-12 and crellanib is synergistic.

[0087] Table 1

[0088] Group Crylanil(μM) HPA-12(μM) CL 1 4 60 0.779 2 4 80 0.677 3 4 100 0.614 1 6 60 0.467 2 6 80 0.423 3 6 100 0.356 1 8 60 0.294 2 8 80 0.256 3 8 100 0.134

[0089] Table 2

[0090]

[0091]

[0092] Example 3

[0093] The therapeutic effect of HPA-12 combined with Creno on AML mice:

[0094] (1) Construction of CDX mouse model and grouping: 1×10 6 MV4-11 cells were injected into B-NDG mice via the tail vein to induce AML. On the fourth day of modeling, the mice were randomly divided into four groups: control group, crelani group, HPA-12 group and combination group, with 8 mice in each group;

[0095] (2) Administration: Administration began on the seventh day. The control group was intraperitoneally injected with normal saline, the crelanib group was intraperitoneally injected with 15 mg / kg crelanib, the HPA-12 group was subcutaneously injected with 4 mg / kg HPA-12, and the combination group was intraperitoneally and subcutaneously injected with both drugs five times a week. After three weeks of administration, three mice in each group were killed, and peripheral blood, bone marrow, femur and spleen were collected to detect the proportion of hCD45.

[0096] (3) Effect on the survival of mice: The remaining mice in each group were subjected to in vivo imaging on the 7th and 27th days. Fig.11 As shown. Record the survival time and perform survival analysis. The results are as follows Fig.12 As shown, the results showed that the survival time of patients treated with Cleranib or HPA-12 alone increased, and the combination of the two drugs played a more significant role in prolonging survival;

[0097] (4) Effect on AML infiltration ratio: After the collected bone marrow was flushed, the spleen was ground, and the peripheral blood was lysed, washed once with PBS and filtered with a 45μm filter, resuspended with buffer (PBS + 2% FBS), 200μl was taken into a flow tube, and stained with the following antibodies: APC-Cy7-hCD45, PE-hCD33 and PI. The staining was carried out on ice in the dark for 20 minutes, 2ml of buffer was added to wash once, and after resuspending with 300μl of buffer, the BD FACS Canto flow cytometer was used to detect and collect data. The results are as follows: Fig.13 (marrow), Fig.14 (spleen), Fig.15 (Peripheral blood) The results showed that the hCD45 in the bone marrow, spleen and peripheral blood of the two-drug combination group was significantly lower than that of the single-drug group, indicating that the combination of the two drugs can have a better killing effect on AML;

[0098] (5) Fixation and immunohistochemistry of femur and spleen: The collected mouse femur and spleen were placed in 4% paraformaldehyde fixative to denature and coagulate the cell proteins, thereby maintaining the original morphology and structure of the cells. The appearance and weight statistics of the spleen of each group of mice are shown in the figure below. Fig.16 The immunohistochemistry experiments were performed by Wuhan Saiweier Biotechnology Co., Ltd. Fig.17 (bone marrow BM) and Fig.18 (Spleen) As shown, the results showed that the hCD45 positive rate in spleen and bone marrow of the combination group was lower than that of the two single-use groups.

[0099] Example 4

[0100] Inhibitory effect of HPA-12 on primary cells from AML patients:

[0101] (1) Isolation and culture of bone marrow mononuclear cells:

[0102] Collect 5mL of fresh bone marrow samples from AML patients into a purple-headed heparin anticoagulant tube, and mix the fresh bone marrow samples with an equal volume of PBS (precooled at 4°C); take a clean 15mL centrifuge tube, add 3mL of human lymphocyte separation solution to the tube, absorb the diluted bone marrow sample, tilt the centrifuge tube to 45 degrees to avoid shaking, and slowly add the bone marrow sample to the lymphocyte separation solution along the wall of the centrifuge tube to make a clear stratification between the separation solution and the bone marrow fluid. Place the centrifuge tube in a centrifuge and centrifuge at 2000rpm for 20min. Gently remove the centrifuge tube to avoid shaking, take another 15mL sterile centrifuge tube and add 2mL PBS, carefully absorb the second layer of mononuclear cells and add it to the tube, and mix it by blowing. Place the centrifuge tube in a centrifuge, set the parameters to 1000rpm for 5min, and discard the supernatant; let the red blood cell lysis solution stand for 6min, then wash it repeatedly with 4°C precooled PBS solution, centrifuge it twice and set it aside.

[0103] The primary cell culture medium was prepared according to the ratio of 79% (v / v) αMEM + 20% (v / v) FBS + 1% (v / v) double antibody, and SCF (50 ng / ml), IL3 (10 ng / ml), FLT3 (50 ng / ml), IL6 (20 ng / ml) and TPO (25 ng / ml) were added. The primary cells separated above were cultured at 1×10 6 The density of cells / mL was placed in the incubator.

[0104] (2) Detection of cell viability:

[0105] Primary cells were cultured at 4 × 10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. Four sub-wells were set for cells with different treatments, and each well was inoculated with a volume of 100 μL. Different concentrations of HPA-12 were treated (0, 60 μM, 80 μM, 100 μM), and mononuclear cells (donor cells) from fresh bone marrow samples of healthy donors were used as controls. After slight shaking and mixing, the 96-well plate was placed in a cell culture incubator and cultured for 48 hours. 10 μl of CCK8 working solution was added to each well, mixed and cultured in a light-proof incubator for 3 hours, and then the absorbance at a wavelength of 450 nm was detected with an enzyme marker. The results are shown in the figure. Fig.19 As shown, the results showed that HPA-12 could significantly inhibit the viability of primary AML cells in a concentration gradient-dependent manner.

[0106] Example 5

[0107] The inhibitory effect of HPA-12 combined with Cleranib on primary cells of AML patients:

[0108] (1) Isolation and culture of bone marrow mononuclear cells:

[0109] Collect 5mL of fresh bone marrow samples from AML patients into a purple-headed heparin anticoagulant tube, and mix the fresh bone marrow samples with an equal volume of PBS (precooled at 4°C); take a clean 15mL centrifuge tube, add 3mL of human lymphocyte separation solution to the tube, absorb the diluted bone marrow sample, tilt the centrifuge tube to 45 degrees to avoid shaking, and slowly add the bone marrow sample to the lymphocyte separation solution along the wall of the centrifuge tube to make a clear stratification between the separation solution and the bone marrow fluid. Place the centrifuge tube in a centrifuge and centrifuge at 2000rpm for 20min. Gently remove the centrifuge tube to avoid shaking, take another 15mL sterile centrifuge tube and add 2mL PBS, carefully absorb the second layer of mononuclear cells and add it to the tube, and mix it by blowing. Place the centrifuge tube in a centrifuge, set the parameters to 1000rpm for 5min, and discard the supernatant; let the red blood cell lysis solution stand for 6min, then wash it repeatedly with 4°C precooled PBS solution, centrifuge it twice and set it aside.

[0110] The primary cell culture medium was prepared according to the ratio of 79% (v / v) αMEM + 20% (v / v) FBS + 1% (v / v) double antibody, and SCF (50 ng / ml), IL3 (10 ng / ml), FLT3 (50 ng / ml), IL6 (20 ng / ml) and TPO (25 ng / ml) were added. The primary cells separated above were cultured at 1×10 6 The density of cells / mL was placed in an incubator.

[0111] (2) Detection of cell viability:

[0112] Primary cells were cultured at 4 × 10 5 Cells were inoculated at a density of 100 μL / mL in a 96-well plate. Four wells were set for cells with different treatments, and each well was inoculated at a volume of 100 μL. DMSO (0.1%), 4 μM of crellaranib, 80 μM of HPA-12, and the combination of the two drugs were given respectively. After slight shaking and mixing, the 96-well plate was placed in a cell culture incubator and cultured for 48 h. 10 μl of CCK8 working solution was added to each well, and after mixing, the incubator was protected from light and cultured for 3 h. Then, the absorbance at a wavelength of 450 nm was detected with an ELISA reader. The results are shown in the figure. Fig. 20 The results showed that HPA-12 combined with clelanib could significantly inhibit the viability of AML primary cells compared with the single-use group.

[0113] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the application of the CERT inhibitor of the present invention in the preparation of drugs for treating acute myeloid leukemia, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Use of an active ingredient consisting of a CERT inhibitor HPA-12 and a FLT3 inhibitor crelanib in the preparation of a drug for treating acute myeloid leukemia, wherein the CERT inhibitor HPA-12 is used as a sensitizer of the FLT3 inhibitor crelanib; The chemical structure of CERT inhibitor HPA-12 is shown below: 。 2. A combined pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The active ingredients in the combined pharmaceutical composition are CERT inhibitor HPA-12 and FLT3 inhibitor Cleranib; the chemical structure of CERT inhibitor HPA-12 is as follows: 。 3. The combined pharmaceutical composition according to claim 2, characterized in that: The combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

4. The combined pharmaceutical composition according to claim 3, characterized in that: The combined pharmaceutical composition is a combination of two separate preparations, and the two separate preparations are administered simultaneously or sequentially.

5. The combined pharmaceutical composition according to claim 3, characterized in that: The preparation is in any pharmaceutically acceptable dosage form.

6. The combined pharmaceutical composition according to claim 2, characterized in that: The combined pharmaceutical composition also contains pharmaceutically acceptable excipients.

7. The combined pharmaceutical composition according to claim 6, characterized in that: The pharmaceutically acceptable excipients include any one of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

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