Application of ALK / LTK inhibitor in preparation of product for treating and / or preventing chronic lymphocytic leukemia

Abstract

The invention discloses an application of an ALK / LTK inhibitor in preparation of a product for treating and / or preventing chronic lymphocytic leukemia. Cell tests prove that the ALK / LTK inhibitor can inhibit an AKT signal channel at the downstream of LTK, so that the cell cycle of tumor cells is stopped, cell apoptosis is promoted, and finally the inhibiting effect on CLL cell proliferation is achieved; in a mouse model, the ALK / LTK inhibitor is proved to be capable of remarkably prolonging the survival time of tumor-bearing mice; the invention provides more drug choices for clinical treatment of CLL.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of ALK / LTK inhibitors in the preparation of products for treating and / or preventing chronic lymphocytic leukemia. Background Art

[0002] Chronic lymphocytic leukemia (CLL) is a blood tumor with clonal proliferation of B lymphocytes. It is highly heterogeneous clinically, and most cases progress slowly with a median survival of 5 to 10 years. The pathogenesis of CLL is still unclear. Many studies have shown that CLL is highly heterogeneous, and its occurrence and development are related to multiple factors, such as abnormal expression of multiple pathway factors or abnormal activation of multiple signaling pathways. Current clinical treatment options for CLL include chemoimmunotherapy, targeted therapy, and sometimes combined with radiotherapy.

[0003] Despite the recent introduction of several highly effective drugs in the clinical treatment of CLL, such as ibrutinib and venetoclax, CLL remains an incurable disease for patients who have not received allogeneic stem cell transplantation, and the development of alternative and additional treatment options is therefore needed. New combination therapy approaches for CLL will continue to be an important research direction. In addition, the increased use of targeted therapies has triggered the emergence of new mutations that have been shown to confer drug resistance to tumor cells. Therefore, it is very important to explore drugs with novel mechanisms of action. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of ALK / LTK inhibitors in the preparation of products for treating and / or preventing chronic lymphocytic leukemia. The present invention confirms the inhibitory effect of ALK / LTK inhibitors on CLL cell proliferation through in vivo and in vitro experiments, and can significantly prolong the survival time of tumor-bearing mice, providing more options for the treatment of CLL.

[0005] The invention also provides a pharmaceutical composition.

[0006] The present invention also provides an application of the ALK / LTK inhibitor.

[0007] According to a first aspect of the present invention, the use of an ALK / LTK inhibitor in the preparation of a product for treating and / or preventing chronic lymphocytic leukemia is proposed.

[0008] In some embodiments of the present invention, the ALK / LTK inhibitor includes at least one of alectinib, AZD1480, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ribotinib, efonacol, ensartinib, iluacinib, anectinib and pharmaceutically acceptable salts thereof.

[0009] In some preferred embodiments of the present invention, the ALK / LTK inhibitor is lorlatinib.

[0010] According to a second aspect of the present invention, a pharmaceutical composition is provided, which comprises an ALK / LTK inhibitor.

[0011] In some embodiments of the present invention, the ALK / LTK inhibitor includes at least one of alectinib, AZD1480, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ribotinib, efonacol, ensartinib, iluacinib, anectinib and pharmaceutically acceptable salts thereof.

[0012] In some preferred embodiments of the present invention, the ALK / LTK inhibitor is lorlatinib.

[0013] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

[0015] In some embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone.

[0016] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or alginic acid.

[0017] In some embodiments of the present invention, the lubricant is selected from at least one of micro-powder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel.

[0018] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, cellulose acetate phthalate, polyvinyl phthalate, ethyl cellulose, and cellulose acetate.

[0019] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0020] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate or glyceryl monostearate.

[0021] In some embodiments of the present invention, the administration methods of the drug include but are not limited to oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retro-ocular, retinal, choroidal, and intrathecal injection.

[0022] In some embodiments of the present invention, the dosage form of the drug includes but is not limited to tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensoids, creams, jellies, and sprays.

[0023] In some embodiments of the present invention, the concentration of the ALK / LTK inhibitor in the pharmaceutical composition is 0.5 to 150 μM.

[0024] According to the third aspect of the present invention, the use of ALK / LTK inhibitors in the preparation of chronic lymphocytic leukemia cell line proliferation inhibitors, chronic lymphocytic leukemia cell line apoptosis promoters or chronic lymphocytic leukemia cell line cell cycle arresters is proposed.

[0025] In some embodiments of the present invention, the chronic lymphocytic leukemia cell line is a cell line that induces chronic lymphocytic leukemia.

[0026] In some embodiments of the present invention, the chronic lymphocytic leukemia cell line includes MEC-1 cells and / or HG-3 cells.

[0027] In some embodiments of the present invention, the ALK / LTK inhibitor includes at least one of alectinib, AZD1480, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ribotinib, efonacol, ensartinib, iluacinib, anectinib and pharmaceutically acceptable salts thereof.

[0028] In some preferred embodiments of the present invention, the ALK / LTK inhibitor is lorlatinib.

[0029] In some embodiments of the present invention, the half effective concentration of alectinib when treating MEC-1 cells is 5 to 6 μM.

[0030] In some embodiments of the present invention, the half effective concentration of alectinib when treating HG-3 cells is 6 to 7 μM.

[0031] In some embodiments of the present invention, the median effective concentration of AZD1480 when treating MEC-1 cells is 2-3 μM.

[0032] In some embodiments of the present invention, the median effective concentration of AZD1480 when treating HG-3 cells is 6-7 μM.

[0033] In some embodiments of the present invention, the half effective concentration of brigatinib when treating MEC-1 cells is 2 to 3 μM.

[0034] In some embodiments of the present invention, the half effective concentration of brigatinib when treating HG-3 cells is 5 to 6 μM.

[0035] In some embodiments of the present invention, the half effective concentration of ceritinib when treating MEC-1 cells is 5 to 6 μM.

[0036] In some embodiments of the present invention, the half effective concentration of ceritinib when treating HG-3 cells is 2-3 μM.

[0037] In some embodiments of the present invention, the median effective concentration of crizotinib when treating MEC-1 cells is 2-3 μM.

[0038] In some embodiments of the present invention, the median effective concentration of crizotinib when treating HG-3 cells is 1 to 2 μM.

[0039] In some embodiments of the present invention, the half effective concentration of entrectinib when treating MEC-1 cells is 3 to 4 μM.

[0040] In some embodiments of the present invention, the half effective concentration of entrectinib when treating HG-3 cells is 3 to 4 μM.

[0041] In some embodiments of the present invention, the half effective concentration of lorlatinib when treating MEC-1 cells is 9 to 10 μM.

[0042] In some embodiments of the present invention, the half effective concentration of lorlatinib when treating HG-3 cells is 1 to 2 μM.

[0043] In some embodiments of the present invention, the median effective concentration of ribotinib when treating MEC-1 cells is 1 to 2 μM.

[0044] In some embodiments of the present invention, the median effective concentration of ribotinib when treating HG-3 cells is 2 to 3 μM.

[0045] In some embodiments of the present invention, the median effective concentration of Ivonecarb when treating MEC-1 cells is 2-3 μM.

[0046] In some embodiments of the present invention, the median effective concentration of Ivonecarb when treating HG-3 cells is 0.5-1 μM.

[0047] In some embodiments of the present invention, the half effective concentration of Ensartinib when treating MEC-1 cells is 2 to 3 μM.

[0048] In some embodiments of the present invention, the half effective concentration of Ensartinib when treating HG-3 cells is 1 to 2 μM.

[0049] In some embodiments of the present invention, the median effective concentration of Iluac when treating MEC-1 cells is 2 to 3 μM.

[0050] In some embodiments of the present invention, the median effective concentration of Iluac when treating HG-3 cells is 3 to 4 μM.

[0051] In some embodiments of the present invention, the half effective concentration of Anectinib when treating MEC-1 cells is 1 to 2 μM.

[0052] In some embodiments of the present invention, the half effective concentration of Anectinib when treating HG-3 cells is 0.5-1 μM.

[0053] The present invention has at least the following beneficial effects:

[0054] The present invention provides the use of ALK / LTK inhibitors in the preparation of products for treating and / or preventing chronic lymphocytic leukemia. Cell experiments have confirmed that ALK / LTK inhibitors can inhibit the AKT signaling pathway downstream of LTK, thereby arresting the cell cycle of tumor cells and promoting cell apoptosis, and ultimately exerting an inhibitory effect on CLL cell proliferation; and in a mouse model, it has been confirmed that ALK / LTK inhibitors can significantly prolong the survival time of tumor-bearing mice; the present invention provides more drug options for the clinical treatment of CLL. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0056] Figure 1This is a cell viability curve diagram of CLL cell lines treated with different ALK / LTK inhibitors in Example 1 of the present invention; wherein A and B are MEC-1 cells, and C and D are HG-3 cells;

[0057] Figure 2 This is a cell viability curve of PBMCs of CLL patients treated with different ALK / LTK inhibitors in Example 1 of the present invention;

[0058] Figure 3 This is a cell viability curve of PBMCs of CLL patients treated with the ALK / LTK inhibitor Lorlatinib in Example 1 of the present invention;

[0059] Figure 4 This is a cell proliferation curve of MEC-1 cells treated with different ALK / LTK inhibitors in Example 2 of the present invention; wherein, "*" represents p less than 0.05, "**" represents p less than 0.01, "***" represents p less than 0.001, and "****" represents p less than 0.0001;

[0060] Figure 5 This is a graph showing the protein detection results after the CLL cell lines were treated with different ALK / LTK inhibitors in Example 3 of the present invention;

[0061] Figure 6 This is a graph showing the protein detection results after HG-3 was treated with different ALK / LTK inhibitors in Example 3 of the present invention;

[0062] Figure 7 The figure is a cell cycle detection result diagram after MEC-1 cells are treated with the ALK / LTK inhibitor Lorlatinib in Example 4 of the present invention; wherein A to C are flow cytometry result diagrams, and D is a statistical diagram of the flow cytometry result; wherein, "*" represents p less than 0.05, "**" represents p less than 0.01, and "***" represents p less than 0.001;

[0063] Figure 8 The results of apoptosis detection after MEC-1 cells were treated with the ALK / LTK inhibitor Lorlatinib in Example 5 of the present invention are shown in Figure 5; A to C are flow cytometry results, and D is a statistical graph of flow cytometry results; "***" represents p less than 0.001;

[0064] Fig. 9 This is a survival curve diagram of the CLL mouse model after administration of the ALK / LTK inhibitor Alectinibc in Example 6 of the present invention. DETAILED DESCRIPTION

[0065] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0066] Example 1 EC50 of different ALK / LTK inhibitors in treating CLL cells

[0067] This example detects the half-maximal effect concentration (EC50) of different ALK / LTK inhibitors in treating CLL cell lines (MEC-1 cells and HG-3 cells), peripheral blood mononuclear cells (PBMCs) of CLL patients, and PBMCs of healthy subjects. The specific test methods and results are as follows:

[0068] 1. EC50 of different ALK / LTK inhibitors in treating CLL cell lines

[0069] After counting the number of MEC-1 cells and HG-3 cells, the cells were resuspended to 1×10 in IMDM medium and RPMI 1640 medium containing 10% serum, respectively. 5 / mL, 100 μL were inoculated into 96-well plates, and ALK / LTK inhibitors Alectinib, AZD1480, Brigatinib, Ceritinib, Crizotinib, Entrictinib, Lorlatinib, Repotrectinib, Envonalkib, Ensartinib, and ibdihydrochloride, Iruplinakib and Unecritinib were added into the culture system in a concentration gradient (0nM, 100nM, 200nM, 500nM, 1μM, 5μM and 10μM) and treated for 24h. The cell viability was detected by CCK8 kit (MCE, HY-K0301). The curve of drug concentration and cell activity inhibition rate was drawn. The data were fitted by nonlinear regression analysis and EC50 (half-maximal effect concentration) was calculated. The results are shown in Figure 1 shown.

[0070] Depend on Figure 1 It can be seen that:

[0071] Alectinib has a half-effective effect at a concentration of 6354nM for MEC-1 cells and a half-effective effect at a concentration of 7240nM for HG-3 cells.

[0072] AZD1480 has a half-effective effect on MEC-1 cells at a concentration of 2852 nM; and has a half-effective effect on HG-3 cells at a concentration of 6445 nM.

[0073] Brigatinib has a half-effective effect at a concentration of 2016nM for MEC-1 cells and a half-effective effect at a concentration of 5296nM for HG-3 cells.

[0074] Ceritinib has a half-effective effect at a concentration of 5394nM for MEC-1 cells and a half-effective effect at a concentration of 2820nM for HG-3 cells.

[0075] Crizotinib has a half-effective effect at a concentration of 2256nM for MEC-1 cells and a half-effective effect at a concentration of 1831nM for HG-3 cells.

[0076] Entrictinib has a half-effective effect at a concentration of 3550nM for MEC-1 cells and a half-effective effect at a concentration of 3618nM for HG-3 cells.

[0077] Lorlatinib has a half-effective effect at a concentration of 9105nM for MEC-1 cells and 9186nM for HG-3 cells.

[0078] Repotrectinib has a half-effective effect at a concentration of 1911nM for MEC-1 cells and a half-effective effect at a concentration of 2235nM for HG-3 cells.

[0079] Envonalkib has a half-effective effect on MEC-1 cells at a concentration of 2549 nM; for HG-3 cells, it has a half-effective effect at a concentration of 824.1 nM;

[0080] Ensartinib dihydrochloride has a half-effective effect on MEC-1 cells at a concentration of 2421nM; and has a half-effective effect on HG-3 cells at a concentration of 1621nM.

[0081] Iruplinakib has a half-effective effect at a concentration of 2612 nM for MEC-1 cells and a half-effective effect at a concentration of 3334 nM for HG-3 cells.

[0082] Unecritinib has a half-effective effect at a concentration of 1507 nM for MEC-1 cells and a half-effective effect at a concentration of 785.7 nM for HG-3 cells.

[0083] The above results indicate that ALK / LTK inhibitors can effectively inhibit the cell viability of CLL cell lines in vitro and that ALK / LTK inhibitors have therapeutic potential for CLL.

[0084] 2. EC50 of different ALK / LTK inhibitors in treating PBMCs of CLL patients

[0085] 3-5 mL of peripheral blood samples were collected from CLL patients with informed consent and added to the upper layer of an equal volume of peripheral blood mononuclear cell separation solution (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.). The cells were collected after centrifugation at 1800 rpm for 20 min, resuspended with red blood cell lysis buffer and placed at room temperature for 3 min, centrifuged and discarded the supernatant, washed twice with PBS, and resuspended PBMCs of CLL patients in RPMI1640 medium containing 10% serum and counted. 1×10 6 100 μL of cell suspension was inoculated into a 96-well plate at a density of 0.1% / mL. ALK / LTK inhibitors Alectinib, AZD1480, Envonalkib and Lorlatinib were added into the culture system according to concentration gradients (0 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM and 10 μM) and treated for 24 h. Cell viability was detected, and a curve of drug concentration and cell activity inhibition rate was drawn. Nonlinear regression analysis was used to fit the data and EC50 was calculated. The results are shown in Figure 3. Figure 2 shown.

[0086] Depend on Figure 2 It can be seen that Alectinib has a half-efficacy for CLL patients' PBMCs at a concentration of 8408-12046nM; AZD1480 has a half-efficacy for CLL patients' PBMCs at a concentration of 635-3903nM; Envonalkib has a half-efficacy for CLL patients' PBMCa at a concentration of 702.6-2170nM; Lorlatinib has a half-efficacy for CLL patients' PBMCs at a concentration of 4.826μM; the above results indicate that ALK / LTK inhibitors can effectively inhibit the cell viability of CLL patients' PBMCs in vitro, and ALK / LTK inhibitors have therapeutic potential for CLL.

[0087] 3. EC50 of ALK / LTK inhibitors in healthy human PBMCs

[0088] In order to verify the safety of ALK / LTK inhibitors, Lorlatinib was used to treat healthy human PBMCs in this example. The specific steps were the same as those for treating CLL patient PBMCs. The EC50 results were as follows: Figure 3 shown.

[0089] Depend on Figure 3 It can be seen that the EC50 of Lorlatinib for healthy human PBMCs is greater than 200μM, indicating that ALK / LTK inhibitors have no obvious toxicity to normal cells at half the effective concentration, and are therefore suitable for the clinical treatment of CLL.

[0090] Example 2 Growth curves of CLL cells treated with different ALK / LTK inhibitors

[0091] This example detects the growth curves of MEC-1 cells after being treated with different ALK / LTK inhibitors. The specific test methods and results are as follows:

[0092] The MEC-1 cells were counted and the density was readjusted to 1×10 5 / mL, inoculated into 12-well plates, and added ALK / LTK inhibitors AZD1480 (0μM, 1μM, 2μM, 5μM, 10μM), Iruplinakib (0μM, 1μM, 2μM, 5μM, 10μM), Ensartinib dihydrochloride (0μM, 1μM, 2μM, 5μM, 10μM), Unecritinib (0μM, 1μM, 2μM, 4μM), Envonalkib (0μM, 1μM, 2μM, 4μM) and Lorlatinib (10μM, 20μM) into the culture system according to concentration gradient, and counted the cells on the first, third, fifth and seventh days of culture, or added CCK-8 (purchased from MCE, HY-K0301) to detect the absorbance value at a wavelength of 450nM, and plotted the growth curve. The results are shown in the following table. Figure 4 shown.

[0093] Depend on Figure 4It can be seen that AZD1480 at a dosage of 2 μM can significantly inhibit the proliferation of MEC-1 cells, Iruplinakib at a dosage of 5 μM can significantly inhibit the proliferation of MEC-1 cells, Ensartinib dihydrochloride at a dosage of 1 μM can significantly inhibit the proliferation of MEC-1 cells, Unecritinib at a dosage of 1 μM can significantly inhibit the proliferation of MEC-1 cells, Envonalkib at a dosage of 1 μM can significantly inhibit the proliferation of MEC-1 cells, and Lorlatinib at a dosage of 10 μM can significantly inhibit the proliferation of MEC-1 cells; the above results indicate that ALK / LTK inhibitors can significantly inhibit the proliferation of MEC-1 cells.

[0094] Example 3 Activation of AKT signaling pathway after treatment of CLL cells with different ALK / LTK inhibitors

[0095] In this example, western blot was used to detect the activation of the AKT signaling pathway after CLL cell lines were treated with different ALK / LTK inhibitors. The specific test methods and results are as follows:

[0096] After CLL cell lines were treated with Alectinib, AZD1480, Lorlatinib, Crizotinib, Envonalkib and Unecritinib for 48 hours, the expression levels of the target protein LTK (antibody purchased from Abcam, ab129155) and downstream proteins p-AKT (antibody purchased from CST, 4060S) and AKT (antibody purchased from CST, 4691S) were detected by sample preparation, protein quantification, SDS-PAGE electrophoresis, membrane transfer, blocking, primary and secondary antibody incubation, washing, signal detection, imaging and analysis before and after CLL cells were treated with ALK / LTK inhibitors. The results are shown in the figure. Figure 5 and Figure 6 shown.

[0097] Depend on Figure 5 and Figure 6 It can be seen that treating CLL cell lines with ALK / LTK inhibitors can significantly inhibit the expression of LTK protein and downregulate the expression of its downstream p-AKT and AKT proteins.

[0098] Example 4 Cell cycle status of CLL cells after treatment with ALK / LTK inhibitors

[0099] MEC-1 cells were intervened with DMSO, 20 μM Lorlatinib and 40 μM Lorlatinib for 48 hours and the cell cycle was detected by flow cytometry, which specifically included the following steps:

[0100] 1) Preparation of fixation buffer:

[0101] Solution 1 (2% formaldehyde): 20 μL formaldehyde plus 980 μL PBS;

[0102] Solution 2: 850 μL of anhydrous ethanol plus 400 μL of PBS;

[0103] Solution 3 (1% formaldehyde): 989 μL PBS plus 10 μL formaldehyde and 1 μL Tween-20;

[0104] Solution 4 (washing solution): 1 mL PBS plus 1 μL Tween-20;

[0105] Solution 5 (1% BSA): 1 mL PBS plus 1 μL Tween-20 and 0.01 g BSA;

[0106] FACS buffer (PBS containing 2% serum): 1 mL PBS plus 20 μL fetal bovine serum.

[0107] 2) MEC-1 cells were treated with DMSO, 20 μM Lorlatinib and 40 μM Lorlatinib respectively. After 48 hours of treatment, bromodeoxyuridine (BrdU) solution (BrdU kit purchased from Becton Dickinson Medical Devices Co., Ltd., Cat. No. 559619) was added to the culture system. For every 1 mL of culture system, 3 μL of BrdU solution with a reserve concentration of 10 mg / mL was added. The cells were incubated at 37°C in the dark for 3 hours.

[0108] 3) Collect cells into 1.5 mL EP tube, centrifuge at 500 g for 3 minutes, and discard the supernatant;

[0109] 4) Wash the cells twice with PBS and discard the supernatant;

[0110] 5) Add 1 mL of solution 1, fix at room temperature for 10 minutes, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0111] 6) Add 1 mL of solution 2, fix on ice for half an hour, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0112] 7) Add 1 mL of solution 3, fix on ice for more than 1 hour, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0113] 8) Add 100 μL of 300 μg / mL DNase solution, gently pipette to mix, incubate at 37°C for 1 hour, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0114] 9) Add 1 mL of solution 4 for washing, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0115] 10) Add 100 μL of solution 5 to resuspend the pellet, add 2 μL of anti-BrdU, mix by pipetting, incubate on ice in the dark for 30 minutes, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0116] 11) Add 100 μL FACS buffer to resuspend the pellet, add 1 μL 1 mg / mL DAPI solution (final concentration 10 μg / mL), add 0.5 μL RNase A, pipette to mix, and incubate at room temperature in the dark for 30 minutes;

[0117] 12) Flow cytometry was used to detect the levels of BrdU and DAPI in the cell samples; the results were as follows: Figure 7 shown.

[0118] Depend on Figure 7 It can be seen that after MEC-1 cells were intervened with DMSO, 20μM Lorlatinib and 40μM Lorlatinib for 48 hours, the proportion of cells in the G0-G1 phase increased and the proportion of cells in the S phase decreased with the increase of Lorlatinib concentration. Lorlatinib significantly blocked the cycle process of CLL cell line MEC-1 and stagnated in the G0-G1 phase.

[0119] Example 5: Apoptosis of CLL cells after treatment with ALK / LTK inhibitors

[0120] After MEC-1 cells were treated with DMSO, 20 μM Lorlatinib and 40 μM Lorlatinib for 48 hours, the cells were collected and washed twice with PBS, resuspended with 100 μL 1× Binding Buffer, and 1 μL PI and 1 μL Annexin V-Alexa Fluor 647 antibody (Annexin V-Alexa Fluor 647 / PI apoptosis detection kit purchased from admas, C8078) were added, mixed by pipetting, incubated on ice in the dark for 15 minutes, and cell apoptosis was detected by flow cytometry. The results are shown in the figure. Figure 8 shown.

[0121] Depend on Figure 8 It can be seen that after intervening MEC-1 cells with DMSO, 20μM Lorlatinib and 40μM Lorlatinib for 48 hours, the proportion of apoptotic cells increased with the increase of Lorlatinib concentration, indicating that Lorlatinib can promote cell apoptosis of CLL cell line MEC-1.

[0122] Example 6 Effect of ALK / LTK inhibitors on the survival of tumor-bearing immunodeficient mice

[0123] NOG mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were injected with MEC-1 cells (5×10 5 cells) and HG-3 (each corresponding to 3×10 5 After the injection of 40 mg / kg Alectinib, the control group was gavaged with a compound solvent (15% PEG400, 15% 2-hydroxypropyl-β-cyclodextrin, 10% Cremophor EL, 10% DMSO, 0.02M HCl) without adding drugs every day. The survival time of the mice was recorded and the survival curve was drawn. The results are shown in the figure. Fig. 9 shown.

[0124] Depend on Fig. 9 It can be seen that compared with the control group, administration of Alectinib can significantly prolong the survival time of CLL tumor-bearing mice, indicating that ALK / LTK inhibitors can be used in the clinical treatment of CLL.

[0125] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of ALK / LTK inhibitors in the preparation of products for treating and / or preventing chronic lymphocytic leukemia.

2. The use according to claim 1, characterized in that: The ALK / LTK inhibitors include at least one of alectinib, AZD1480, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ribotinib, efonacol, ensartinib, iluacol, anectinib and pharmaceutically acceptable salts thereof.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition includes an ALK / LTK inhibitor.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

5. The pharmaceutical composition according to claim 3, characterized in that The administration method of the pharmaceutical composition includes at least one of oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retro-ocular administration, retinal administration, choroidal administration and intrathecal injection.

6. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition includes, but is not limited to, at least one of tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensoids, creams, jellies and sprays.

7. The pharmaceutical composition according to claim 3, characterized in that The concentration of the ALK / LTK inhibitor in the pharmaceutical composition is 0.5 to 150 μM.

8. Use of ALK / LTK inhibitors in the preparation of chronic lymphocytic leukemia cell line proliferation inhibitors, chronic lymphocytic leukemia cell line apoptosis promoters or chronic lymphocytic leukemia cell line cell cycle inhibitors.

9. The use according to claim 8, characterized in that: The chronic lymphocytic leukemia cell line is a cell line that induces chronic lymphocytic leukemia.

10. The use according to claim 8, characterized in that: The chronic lymphocytic leukemia cell line includes MEC-1 cells and / or HG-3 cells.

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