Drug combination for treating chronic myeloid leukemia and its application

Abstract

The present invention discloses a drug combination and application for treating chronic myeloid leukemia, belonging to the field of medical technology. The present invention uses chronic myeloid leukemia cells KBM5, KU-812, and primary cells from clinical patients as research subjects. Disulfiram and TKIs are administered alone or in combination through in vitro drug addition experiments, and cell viability and cell proliferation are measured. The results show that DSF or TKIs alone have certain anti-tumor effects, and the combination of disulfiram and TKIs can significantly enhance the anti-tumor effect of TKIs. Based on this, we speculate that the combination of disulfiram and TKIs can be applied to the clinical treatment of chronic myeloid leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a drug combination for treating chronic myeloid leukemia and its application. Background Art

[0002] As essential tools for the prevention and treatment of diseases, pharmaceuticals have always been a core component of the medical field. In recent years, the exploration and invention of new drugs has been ongoing, focusing on emerging diseases or those for which specific treatments are lacking. However, with the increasing requirements for new drug development, the increasing difficulty of research and development, and the stringent and complex regulatory procedures for new drugs, there is a need to explore new therapeutic uses for existing drugs. This involves identifying new uses for existing drugs based on their modes of action and manageable side effects. This can be a shortcut to drug development, shortening the timeline from laboratory research to clinical application.

[0003] Chronic myelogenous leukemia (CML) accounts for 0.3% of all cancers and 20% of adult leukemia. In the general population, approximately 1 to 2 people per 100,000 suffer from the disease. [1] . CML is an abnormal proliferative tumor of myeloid cells that originates from pluripotent stem cells and is characterized by the BCR-ABL fusion gene and the Philadelphia chromosome (Ph); clinically, the main manifestations are a significant increase in the number of immature granulocytes and splenomegaly. Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein, such as imatinib (IM, trade name Gleevec), can competitively inhibit the binding of ATP to the catalytic center of the BCR-ABL fusion protein, prevent the activation of tyrosine kinase, and thus exert a targeted therapeutic effect. In 1994, the results of the clinical trial of Gleevec for the treatment of CML were announced, which brought about major changes in the treatment of CML and made its long-term survival possible. [2] TKI treatment has many problems, such as abl gene mutation-induced resistance and secondary resistance, as well as the inability to inhibit leukemia stem cells (LSCs). Although the use of second-generation and third-generation TKIs has continuously improved the efficacy, TKI resistance, blast crisis, and relapse caused by minimal residual lesions are still difficult problems in CML treatment. [3]Because the immunophenotype of CML LSCs is very similar to that of normal hematopoietic stem cells, and CML LSCs do not completely rely on BCR-ABL for survival, TKIs are essentially ineffective against these cells. CML LSCs are the root cause of CML relapse. Therefore, in order to move from treating the symptoms to treating the root cause, in addition to targeting and inhibiting more mature leukemia cells, effectively inhibiting and killing CML LSCs and other drug-resistant cells may be one of the important means of curing CML.

[0004] Disulfiram (DSF), also known as disulfiram, was approved by the U.S. Food and Drug Administration (FDA) in 1951 as a drug for the treatment of alcoholism. [4,5] The specific mechanism is: Disulfiram can inhibit the activity of acetaldehyde dehydrogenase (ALDH) in the body, preventing acetaldehyde from being converted into acetic acid, causing acetaldehyde to accumulate in the body and causing discomfort, such as decreased blood pressure, dry mouth, and sweating (also known as disulfiram-like reaction). As a drug for quitting alcohol, it has the characteristics of good safety and mild adverse reactions. [6] In 1977, Johns Hopkins Hospital used DSF to treat a 35-year-old breast cancer patient, but unexpectedly eliminated the tumor. This was the first report that DSF had anti-cancer activity. [7] In recent years, DSF has been reported to induce apoptosis in a wide range of human cancer cells, such as prostate cancer cells. [8] , lung cancer cells [9] , melanoma cells

[10] Pharmacokinetics show that after oral administration, DSF is mainly absorbed in the gastrointestinal tract. DSF is reduced to diethyldithiocarbamic acid (DDC) in the liver. As a divalent metal ion chelator, it can chelate with copper ions in the body to form copper diethyldithiocarbamate (CuET). [11,12] . It has been documented that disulfiram combined with copper has an effect on B-cell lymphoma.

[13] , liver cancer

[14] , colorectal cancer

[15] , multiple myeloma

[16] , acute myeloid leukemia [17,18] Studies have also found that DSF can be combined with other chemotherapy drugs to treat different types of cancer. In breast cancer cells, it was found that the combination of DSF and cisplatin can induce more cell apoptosis, which is significantly higher than that of the single drug group.

[19] In human colorectal cancer cells, DSF enhances 5-FU-induced apoptosis by inhibiting the activity of the NF-κB pathway

[20] Similar reports have been reported in pancreatic cancer cells

[21] ; The combined use of the above drugs can reduce the resistance of cancer cells to certain chemotherapy drugs and has good clinical application prospects.

[0005] In summary, based on the shortcomings of existing CML clinical treatments such as drug resistance and relapse, it is necessary to study new options to improve its treatment.

[0006] References:

[0007] [1]Jabbour E,Kantarjian H.Chronic myeloid leukemia:2020 update ondiagnosis,therapy and monitoring.Am J Hematol,2020,95:691-709

[0008] [2]Hehlmann R, Lauseker M, Saussele S, Pfirrmann M, Krause S, Kolb HJ, Neubauer A, Hossfeld DK, Nerl C, Gratwohl A, Baerlocher GM, Heim D, Brummendorf TH, Fabarius A, Haferlach C, Schlegelberger B, Muller MC, Jeromin S, Proetel U, Kohlbrenner K, Voskanyan A, Rinaldetti S, Seifarth W, Spiess B, Balleisen L, Goebeler MC, Hanel M, Ho A, Dengler J, Falge C, Kanz L, Kremers S, Burchert A, KnebaM, Stegelmann F, Kohne CA, Lindemann HW, Waller CF, Pfreundschuh M, Spiekermann K, Berdel WE, Muller L, Edinger M, Mayer J, Beelen DW, Bentz M, Link H, Hertenstein B, Fuchs R, Wernli M, Schlegel F, Schlag R, de Wit M, Trumper L, Hebart H, Hahn M, Thomalla J, Scheid C, Schafhausen P, Verbeek W, Eckart MJ, Gassmann W, Pezzutto A, Schenk M, Brossart P, Geer T, Bildat S, Schafer E, Hochhaus A, Hasford J. Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized cml study iv and impact of non-cml determinants. Leukemia, 2017, 31: 2398-2406

[0009] [3]Li Y,Lin C,Schmidt CA.New insights into antigen specificimmunotherapy for chronic myeloid leukemia.Cancer Cell Int,2012,12:52

[0010] [4]Wright C,Moore RD.Disulfiram treatment of alcoholism.Am J Med,1990,88:647-655

[0011] [5]Suh JJ,Pettinati HM,Kampman KM,O'Brien CP.The status ofdisulfiram:A half of a century later.J Clin Psychopharmacol,2006,26:290-302

[0012] [6]Johansson B.A review of the pharmacokinetics and pharmacodynamicsof disulfiram and its metabolites.Acta Psychiatr Scand Suppl,1992,369:15-26

[0013] [7]Lewison EF.Spontaneous regression of breast cancer.Prog Clin BiolRes,1977,12:47-53

[0014] [8]Schweizer MT,Lin J,Blackford A,Bardia A,King S,Armstrong AJ,RudekMA,Yegnasubramanian S,Carducci MA.Pharmacodynamic study of disulfiram in menwith non-metastatic recurrent prostate cancer.Prostate Cancer Prostatic Dis,2013,16:357-361

[0015] [9]Butcher K,Kannappan V,Kilari RS,Morris MR,McConville C,ArmesillaAL,Wang W.Investigation of the key chemical structures involved in theanticancer activity of disulfiram in a549 non-small cell lung cancer cellline.BMC Cancer,2018,18:753

[0016]

[10] Cen D,Gonzalez RI,Buckmeier JA,Kahlon RS,Tohidian NB,Meyskens FL,Jr.Disulfiram induces apoptosis in human melanoma cells:Aredox-relatedprocess.Mol Cancer Ther,2002,1:197-204

[0017]

[11] Chen D,Cui QC,Yang H,Dou QP.Disulfiram,a clinically used anti-alcoholism drug and copper-binding agent,induces apoptotic cell death inbreast cancer cultures and xenografts via inhibition of the proteasomeactivity.Cancer Res,2006,66:10425-10433

[0018]

[12] Li H,Wang J,Wu C,Wang L,Chen ZS,Cui W.The combination ofdisulfiram and copper for cancer treatment.Drug Discov Today,2020,25:1099-1108

[0019]

[13] Yang Y,Li M,Sun X,Zhou C,Wang Y,Wang L,Chen L,Liang Z,Zhu L,YangH.The selective cytotoxicity of dsf-cu attributes to the biomechanicalproperties and cytoskeleton rearrangements in the normal and cancerousnasopharyngeal epithelial cells.Int J Biochem Cell Biol,2017,84:96-108

[0020]

[14] Ren X,Li Y,Zhou Y,Hu W,Yang C,Jing Q,Zhou C,Wang X,Hu J,Wang L,Yang J,Wang H,Xu H,Li H,Tong X,Wang Y,Du J.Overcoming the compensatoryelevation of nrf2 renders hepatocellular carcinoma cells more vulnerable todisulfiram / copper-induced ferroptosis.Redox Biol,2021,46:102122

[0021]

[15] Hu Y,Qian Y,Wei J,Jin T,Kong X,Cao H,Ding K.The disulfiram / coppercomplex induces autophagic cell death in colorectal cancer by targetingulk1.Front Pharmacol,2021,12:752825

[0022]

[16] Xu Y,Zhou Q,Feng X,Dai Y,Jiang Y,Jiang W,Liu X,Xing X,Wang Y,NiY,Zheng C.Disulfiram / copper markedly induced myeloma cell apoptosis throughactivation of jnk and intrinsic and extrinsic apoptosis pathways.BiomedPharmacother,2020,126:110048

[0023]

[17] Xu B,Wang S,Li R,Chen K,He L,Deng M,Kannappan V,Zha J,Dong H,WangW.Disulfiram / copper selectively eradicates aml leukemia stem cells in vitroand in vivo by simultaneous induction of ros-jnk and inhibition of nf-kappaband nrf2.Cell Death Dis,2017,8:e2797

[0024]

[18] Hassani S,Ghaffari P,Chahardouli B,Alimoghaddam K,Ghavamzadeh A,Alizadeh S,Ghaffari SH.Disulfiram / copper causes ros levels alteration,cellcycle inhibition,and apoptosis in acute myeloid leukaemia cell lines withmodulation in the expression of related genes.Biomed Pharmacother,2018,99:561-569

[0025]

[19] Yang Z, Guo F, Albers AE, Sehouli J, Kaufmann AM. Disulfiram modulatesros accumulation and overcomes synergistically cisplatin resistance in breastcancer cell lines. Biomed Pharmacother, 2019,113:108727

[0026]

[20] Wang W, McLeod HL, Cassidy J. Disulfiram-mediated inhibition of nf-kappab activity enhances cytotoxicity of 5-fluorouracil in human colorectalcancer cell lines. Int J Cancer, 2003,104:504-511

[0027]

[21] Cong J, Wang Y, Zhang Lett,2017,409:9-19. Summary of the Invention

[0028] To solve the related problems, the primary purpose of the present invention is to provide the use of disulfiram in the preparation of drugs for treating chronic myeloid leukemia (CML).

[0029] Another object of the present invention is to provide the use of disulfiram combined with imatinib in the preparation of a drug for treating chronic myeloid leukemia.

[0030] Another object of the present invention is to provide a drug combination for treating chronic myeloid leukemia.

[0031] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0032] The application of disulfiram in the preparation of drugs for treating chronic myeloid leukemia (CML).

[0033] Furthermore, in the application, the effective dose of disulfiram is 0.01 to 1 μM.

[0034] Application of disulfiram combined with imatinib in the preparation of drugs for the treatment of chronic myeloid leukemia.

[0035] Furthermore, in the application, the effective dose of disulfiram is 0.01 to 1 μM, and the effective dose of imatinib is 0.01 to 1 μM.

[0036] Furthermore, in the application, the concentration ratio of disulfiram to imatinib is 0.25-1 μM:0.25-1 μM.

[0037] Furthermore, the drug for treating chronic myeloid leukemia is a drug that has the functions of promoting apoptosis, inhibiting proliferation, and reducing activity of cells in patients with chronic myeloid leukemia.

[0038] Furthermore, the cells are peripheral blood mononuclear cells and / or CD34+ cells.

[0039] A drug combination for treating chronic myeloid leukemia, comprising disulfiram and imatinib.

[0040] Furthermore, the disulfiram and imatinib are mixed in a concentration ratio of 0.25-1 μM:0.25-1 μM.

[0041] Furthermore, the drug combination also contains pharmaceutically acceptable excipients.

[0042] Furthermore, the pharmaceutically acceptable excipient is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

[0043] The present invention has the following advantages and effects compared to the prior art:

[0044] (1) Disulfiram is a hangover remedy that has been used clinically for many years. It has a strong pharmacological effect in inhibiting aldehyde dehydrogenase in the human body and is also highly safe and has mild adverse reactions.

[0045] (2) The present invention uses chronic myeloid leukemia cells KBM5, KU-812, K562, and primary cells from clinical patients as research subjects. Disulfiram and TKIs are administered alone or in combination through in vitro drug addition and other experimental procedures, and cell viability and cell death are measured. The results show that DSF or TKIs alone have certain anti-tumor effects, and the combination of disulfiram and TKIs can significantly enhance the anti-tumor effect of TKIs. Based on this, we speculate that the combination of disulfiram and TKIs can be applied to the clinical treatment of chronic myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The results of DSF concentration gradient treatment of KBM5 cells are shown in Figure 2; (a) and (b) respectively show its effects on Annexin V+ positive cells and cell activity using flow cytometry and clone formation experiments.

[0047] Figure 2 The figures show the results of DSF concentration gradient treatment of CML, HI PBMCs and CML LSCs cells; among them, (a) shows the effect of DSF concentration gradient treatment of CML, HI PBMCs on proliferation using CCK8, (b) shows the effect of DSF concentration gradient treatment of CML PBMCs on Annexin V+ positive cells using flow cytometry, and (c) shows the effect of DSF concentration gradient treatment of CML on Annexin V+ positive cells using flow cytometry.

[0048] Figure 3 The results of treating KBM5 cells with DSF and IM alone and in combination are shown in Figure 2. (a) shows the effect of CCK8 on cell proliferation and the calculation of the synergy coefficient. (b) and (c) show the effects on Annexin V+ positive cells and cell activity by flow cytometry and clone formation assay, respectively. (d) shows the effect of combined treatment of KU-812 and K562 cells with DSF and IM, and the effect of CCK8 on their proliferation.

[0049] Figure 4 The results of treating CML, HI PBMCs and CML LSCs with DSF and IM alone or in combination are shown in Figure 2; (a) and (b) / (c) show their effects on Annexin V+ positive cells and cell activity using CCK8 and flow cytometry, respectively. DETAILED DESCRIPTION

[0050] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0051] The experimental materials involved in the following examples are:

[0052] (1) Chronic myeloid leukemia cells KBM5, KU-812, and K562 cells were purchased from ATCC.

[0053] (2) Clinical specimens: Peripheral blood specimens from normal subjects were obtained from Guangzhou Overseas Chinese Hospital; bone marrow specimens from CML patients were obtained from Guangzhou Overseas Chinese Hospital. All patients were newly diagnosed. After signing a written informed consent form, bone marrow specimens were obtained from patients when necessary for examination.

[0054] (3) Preparation of DSF and IM solutions: Dissolve each in DMSO and prepare a 10 mM stock solution, then store at -80°C after aliquoting.

[0055] Example 1

[0056] (1) Cell culture

[0057] ① KBM5 cells: The complete culture medium is 90% IMDM medium containing 10% Gibco fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL); culture in a 5% CO2, 37°C incubator. The cells are suspension cells and the density is maintained at 1-2 × 10 6 / mL to ensure that the cells are in the logarithmic growth phase, and the cells are passaged every 2-3 days on average.

[0058] ②KU-812 cells: The complete culture medium is 90% 1640 medium containing 10% Gibco fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL); culture in a 5% CO2, 37°C incubator. The cells are suspension cells and the density is maintained at 1-2×10 6 / mL to ensure that the cells are in the logarithmic growth phase, and the cells are passaged every 2-3 days on average.

[0059] ③ K562 cells: The complete culture medium is 90% 1640 medium containing 10% Gibco fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL); culture in a 5% CO2, 37°C incubator. The cells are suspension cells and the density is maintained at 1-2 × 10 6 / mL to ensure that the cells are in the logarithmic growth phase, and the cells are passaged every 2-3 days on average.

[0060] ④ Primary PBMC cells: The complete culture medium is 90% IMDM medium containing 10% Gibco fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL); culture in a 5% CO2 concentration, 37°C constant temperature incubator.

[0061] ⑤ CD34 in newly diagnosed patients +Cells (LSCs): Freshly sorted CD34 + Cells were seeded in a cell culture plate and supplemented with IMDM medium and cytokines. Culture was performed in a 5% CO2, 37°C incubator. 50 μL each of 100 mg / mL of GSF and CSF was added, along with 10 μL each of 20 mg / mL of IL-6 and IL-3.

[0062] (2) Extraction of mononuclear cells (PBMCs):

[0063] ① Bring Ficoll (human peripheral blood lymphocyte separation medium) from a 4°C refrigerator to room temperature and return to temperature for at least 30 minutes.

[0064] ② Dilute the blood sample containing heparin / EDTA anticoagulant with sterile 1× PBS at a volume ratio of 1:1.

[0065] ③ Remove a 15ml sterile centrifuge tube and add Ficoll separation solution to the bottom of the tube (the volume of Ficoll added should be 1:1 with the volume of the diluted blood specimen). Then, use a Pasteur pipette to draw up the blood dilution solution from the previous step and add it dropwise, ensuring that the blood specimen is above the Ficoll layer and remains separated. Carefully place the centrifuge tube in a centrifuge at 1500 rpm at room temperature for 15-20 minutes.

[0066] ④ After centrifugation, the liquid in the centrifuge tube is divided into 4 layers, from top to bottom: plasma layer, mononuclear cell layer, Ficoll layer, red blood cells and granulocyte layer; use a Pasteur pipette to carefully extract the mononuclear cell layer and transfer it to a new centrifuge tube.

[0067] ⑤ Add 5-6 ml of sterile 1× PBS to the centrifuge tube containing mononuclear cells, mix well with a Pasteur pipette, then centrifuge at room temperature at 350g for 10 minutes, and repeat washing twice.

[0068] ⑥ Discard the supernatant and add 2-4 ml of red blood cell lysis buffer, depending on the volume of the cell pellet. Lyse the cells in the dark at room temperature for 15 minutes. Centrifuge at 350g for 5 minutes at room temperature. If the cell pellet remains red after centrifugation, repeat this step.

[0069] ⑦ Wash the cells twice with 6-8 ml of sterile 1× PBS and centrifuge at 350 g for 5 minutes at room temperature. If LSCs are not being isolated, resuspend the cell pellet in 90% IMDM medium supplemented with 10% Gibco fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 mg / mL). Count the mononuclear cell viability and cell number using trypan blue staining. Proceed with the next experimental step or store in a -80°C freezer in cell freezing medium.

[0070] (3)CD34+ Isolation of LSCs

[0071] CD34 + Cells were sorted using the Magnet-Activated Cell Sorting (MACS) system.

[0072] ① After washing with 1×PBS, remove the supernatant from the PBMCs and resuspend them in the corresponding volume of MACS buffer (1×10 8 The cells were resuspended in 300 μL of PBS, and 100 μL of blocking reagent and 100 μL of CD34-coupled magnetic bead antibody (CD34 MultiSortMicroBeads) were added to the suspension. After thorough mixing, the suspension was incubated in a refrigerator at 4°C for 30 min, with mixing performed every 10 min.

[0073] ② After incubation, resuspend the cells in 5-6 ml of MACS buffer and centrifuge at 300g for 10 minutes at room temperature. After centrifugation, resuspend the cells in 3 ml of MACS buffer.

[0074] ③ Install the appropriate adsorption column in the magnetic field. Rinse the MACS column with 3 ml of MACS buffer. Add the cell suspension to the rinsed MACS column and repeat the wash process with 3 ml of MACS buffer at least three times. (The next wash can only be performed when the previous buffer in the column is empty; avoid creating bubbles during the wash process.)

[0075] ④ Since cells that cannot bind to the magnetic beads will flow out from the bottom of the adsorption column, CD34 + The cells will be adsorbed in the adsorption column. Therefore, after three washes, make sure that the effluent is cell-free. Finally, remove the adsorption column from the magnetic field and place it on a suitable collection tube. Add 3-5 ml of MACS buffer to the adsorption column. Push the plunger firmly into the column to immediately flush out the magnetically labeled cells (CD34 + cell).

[0076] ⑤ After sorting, CD34 + Cell purity analysis: extract some of the cells obtained after the above sorting, wash them twice with 1×PBS, and then use CD34 + Resuspend the antibody (CD34 Antibody, anti-human) in 1× PBS (100:1). Incubate at room temperature in the dark for 15 minutes. Wash two to three times with 1× PBS after incubation. Analyze on a flow cytometer. Analyze the results using FlowJo software.

[0077] (4) Flow cytometry detection of apoptosis

[0078] KBM5 cells:

[0079] ① Collect KBM5 cells in the logarithmic growth phase into a sterile centrifuge tube and centrifuge at 350g for 5 minutes at room temperature. Remove the supernatant and wash once with sterile 1% PBS. After washing, resuspend the cell pellet in complete medium depending on the size of the cell pellet and count the cells using a counting chamber.

[0080] ② Take a 12-well plate with a final volume of 2 ml of complete medium as an example. There are two major groups: one is the DSF concentration gradient group (divided into four groups: 0, 0.25, 0.5, and 1 μM); the other is the DSF and IM combination group (divided into four groups: control group (Ctrl), DSF group (0.25 μM), IM group (0.25 μM), and DSF + IM combination group (DSF and IM each at 0.25 μM)). There are a total of eight wells, and the number of cells in each well is approximately 1-1.5×10 5 cells.

[0081] ③ Based on the final concentration of the drug, the concentration of the mother solution, and the final volume of the culture medium, use a pipette to aspirate the corresponding volume of the drug to dilute it to the corresponding final concentration, then use a pipette to thoroughly mix the drug and cells and seed them into the corresponding position of the well plate.

[0082] ④ After 48 hours, harvest the cells and wash twice with sterile 1× PBS. According to the instructions for the apoptosis kit, resuspend each well in 100 μL of 1× binding buffer and add 0.5 μL of Annexin V-APC antibody solution and 1 μL of Propidium-PE antibody solution. Gently pipette the cells to mix thoroughly and incubate at room temperature in the dark for 5 minutes.

[0083] ⑤ Detect on flow cytometer and the experimental results can be analyzed using FlowJo software.

[0084] Primary PBMC cells / LSCs cells from clinical specimens:

[0085] For primary cells, take 12-well plates as an example, the number of cells in each well is 5-10×10 5 Cells were cultured in a final volume of 2 mL of complete medium. The cells were divided into two groups: a DSF concentration gradient group (divided into four groups: 0, 0.25, 0.5, and 1 μM); and a DSF and IM combination group (divided into four groups: control group (Ctrl), DSF group (1 μM), IM group (1 μM), and DSF+IM combination group (DSF and IM each at 1 μM). The remaining experimental procedures were the same as for KBM5 cells.

[0086] (5) CCK8 detection of cell viability

[0087] KBM5, KU-812, K562 cells in logarithmic growth phase and primary PBMC cells / LSCs cells from fresh clinical specimens were seeded into 96-well culture plates, with 10,000 to 20,000 KBM5, KU-812, and K562 cells per well and 5 to 10×10 PBMC cells / LSCs cells per well. 4 / well; the final volume of culture medium was 100 μL / well. The drug addition procedure was the same as in Experimental Method 4 (Flow Cytometry Apoptosis Detection). A Blank group (only 100 μL of the corresponding culture medium was added), and three replicates were set up for each intervention in each group. Finally, the 96-well plate was mixed in an "eight" pattern to thoroughly mix the cell suspension and drug culture medium. The cells were then cultured in a 5% CO2, 37°C incubator. After 48 hours, 10 μL of CCK8 reagent was added to each well (be careful not to create bubbles in the wells, as this will affect the OD reading). The culture plate was incubated at 37°C for another 4-6 hours. The A450 value was measured at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader, which indirectly reflects the number of viable cells. Cell viability was calculated according to the following formula: Cell viability (%) = [A(drug added) - A(blank group)] / [A(solvent control group) - A(blank group)] × 100%.

[0088] (6) Methylcellulose colony formation assay

[0089] ① Take KBM5 cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, wash twice with sterile 1× PBS and count.

[0090] ② Set up different treatment groups: one with a gradient of DSF concentrations (divided into four groups: 0, 0.25, 0.5, and 1 μM); and one with a combination of DSF and IM (divided into four groups: Ctrl, DSF (1 μM), IM (1 μM), and DSF + IM (1 μM each for DSF and IM). Eight wells in total were used. Cell density was adjusted to 2,000–5,000 cells / well using a gradient dilution method, ensuring that the number of cells in each well remained consistent at the beginning.

[0091] ③ Dilute the complete culture medium corresponding to the cells with methylcellulose at a volume ratio of 1:1 and shake to mix.

[0092] ④ Mix the diluted methylcellulose with the cells and drug and add to a 12-well plate. Add sterile 1x PBS to the surrounding wells. Incubate the plates in a 5% CO2, 37°C incubator for 10-14 days. During this time, observe under an inverted microscope. If clear colonies emerge, stain and photograph the colony morphology and calculate the colony formation rate (colony formation rate = number of colonies formed / number of cells seeded).

[0093] (7) Compusyn calculates the synergy index of combined drug use

[0094] Following the CCK8 protocol in step 5, three treatments were set up in KBM5 cells: an IM monotherapy with a concentration gradient (0, 0.25, 0.5, 1 μM), an IM concentration gradient (0, 0.25, 0.5, 1 μM) plus 0.25 μM DSF, and an IM concentration gradient (0, 0.25, 0.5, 1 μM) plus 0.5 μM DSF. The inhibition rates of the latter two combinations were input into Compusyn, and the combination index (CI) was calculated. A CI > 1 indicates an antagonistic effect between the two drugs; a CI = 1 indicates an additive effect; and a CI < 1 indicates a synergistic effect.

[0095] (8) Statistical analysis

[0096] All data are expressed as the mean ± SD of three independent experiments and were statistically analyzed using GraphPad Prism 8.0 software. Statistical significance was analyzed using the rank sum test and one-way analysis of variance, and p values ​​were used (p values ​​< 0.05 were considered significant. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001).

[0097] Experimental results:

[0098] 1. After treating KBM5 cells with DSF (0, 0.25, 0.5, 1 μM) for 48 hours, flow cytometry was used to detect apoptosis in each experimental group ( Figure 1 (a)). We found that compared with the control group, the apoptosis rate of cells in the other groups increased with the increase of DSF concentration. At the same time, the methylcellulose colony formation experiment was performed on these four groups ( Figure 1 In (b), cell colony formation was observed 10-14 days after drug treatment. The results showed that with increasing DSF concentration, fewer cell colonies were formed and their growth was slower. This fully demonstrates that DSF can inhibit the proliferation of KBM5 cells and promote cell apoptosis.

[0099] 2. The cell viability of mononuclear cells (PBMCs) from normal subjects (HI) and newly diagnosed chronic myeloid leukemia (CML) treated with DSF (0, 0.25, 0.5, 1 μM) was detected by CCK8 assay ( Figure 2In (a), the results showed that for normal human PBMCs, cell viability did not change much after 48 hours of DSF treatment compared to before. This shows that DSF has the characteristics of good safety and low toxicity. However, for PBMCs of newly diagnosed chronic myeloid leukemia, cell viability continued to decline with increasing concentration after 48 hours of DSF treatment, and the change range was large. At the same time, we also used flow cytometry to detect the apoptosis of PBMCs of newly diagnosed chronic myeloid leukemia ( Figure 2 (b)), the results are consistent with those of CCK8. We also treated the sorted LSCs for 48 hours and found that DSF can effectively kill LSCs ( Figure 2 (c)) effectively solves the problem of TKI resistance and insensitivity. This fully demonstrates that DSF can inhibit the proliferation of PBMCs and LSCs in CML and promote cell apoptosis.

[0100] 3. The CCK8 assay was used to examine the effects of three treatments on cell proliferation: IM monotherapy (0, 0.25, 0.5, 1 μM), IM concentration gradient (0, 0.25, 0.5, 1 μM) plus 0.25 μM DSF, and IM concentration gradient (0, 0.25, 0.5, 1 μM) plus 0.5 μM DSF. The synergistic coefficients of the latter two combinations were calculated. The CI for the combination of 0.25 μM IM and 0.25 μM DSF was 0.38197. Since the CI < 1, the two treatments exhibited a synergistic effect ( Figure 3 (a)). Four groups were used to treat KBM5 cells, namely the control group, DSF group, IM group and combined drug group. Flow cytometry was used to detect apoptosis in each experimental group for 48 hours. ( Figure 3 (b)). We found that when DSF was combined with IM, the cell apoptosis rate was higher than that of the control group and the drug-only group. At the same time, the methylcellulose colony formation experiment was performed on these four groups ( Figure 3 In (c), we observed the formation of cell colonies 10-14 days after drug treatment. The results showed that the combined group had fewer cell colonies and grew slower than the single-drug group, and the two single-drug groups were also the same relative to the control group. We also used CCK8 to detect KU-812 and K562 cells ( Figure 3 In (d), the cell viability after the combination of the two drugs was found to be lower than that of the control group and the single drug. This fully demonstrates that DSF combined with IM can synergistically inhibit the proliferation of CML cell lines and promote cell apoptosis.

[0101] 4. The cell viability of the control group, DSF group, IM group and combined drug group in normal subjects (HI) and newly diagnosed chronic myeloid leukemia (CML) mononuclear cells (PBMCs) was detected by CCK8 method ( Figure 4In (a), the results showed that for normal human PBMCs, the cell viability of single drug and combined drug treatment for 48 hours did not change much compared with the control group. This also shows that DSF has the characteristics of good safety and low toxicity. For PBMCs of newly diagnosed chronic myeloid leukemia, the cell viability after the combination of the two drugs was lower than that of the control group and the single drug. At the same time, we also used flow cytometry to detect the apoptosis of PBMCs of newly diagnosed chronic myeloid leukemia ( Figure 4 (b)), the results are consistent with those of CCK8. We also treated the sorted LSCs for 48 hours ( Figure 4 In (c), the combination of DSF and IM was found to be effective in killing LSCs. This fully demonstrates that the combination of DSF and IM can synergistically inhibit the proliferation of PBMCs and LSCs in CML and promote cell apoptosis.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of disulfiram combined with imatinib in the preparation of a drug for treating chronic myeloid leukemia, characterized in that: The chronic myeloid leukemia is drug-resistant chronic myeloid leukemia In the application, the concentration ratio of disulfiram to imatinib is 0.25-1 μM:0.25-1 μM.

2. The use according to claim 1, characterized in that: The medicine further contains pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

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