Pharmaceutical composition for treating acute myelogenous leukemia and application thereof

Abstract

The invention relates to a pharmaceutical composition for treating acute myelogenous leukemia. The pharmaceutical composition comprises lonidamine and a GPX4 inhibitor. The invention also relates to application of the pharmaceutical composition in preparation of drugs for resisting acute myelogenous leukemia cells. Aiming at an oxidation-antioxidation defense system of AML tumor cells, lonidamine and the GPX4 inhibitor are creatively combined for use, the inhibition rate of lonidamine and the GPX4 inhibitor on the AML cells is remarkably higher than that of independent medication, and the combination of lonidamine and the GPX4 inhibitor can synergistically inhibit the activity of the AML cells, induce mitochondria damage of the AML cells and promote ferroptosis of the AML cells, so that the purpose of treating the AML is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition for treating acute myeloid leukemia and an application of the pharmaceutical composition in preparing a drug for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem cells characterized by abnormal proliferation of primitive immature myeloid cells in the bone marrow and peripheral blood. In recent years, the incidence of AML has been increasing year by year. Although targeted therapies have been recently approved for patients with IDH1 or Flt3 mutations, the first-line drug treatment regimen for AML has remained basically unchanged for 20 years. The main treatments for AML are chemotherapy and hematopoietic stem cell transplantation. Cytarabine combined with anthracyclines is the standard treatment for AML. Although current treatments have improved the prognosis of young patients, relapse, adverse reactions to chemotherapy, and treatment effects in elderly patients remain severe challenges. Allogeneic hematopoietic stem cell transplantation, as a fundamental treatment, can effectively treat relapsed / refractory AML. However, stem cell transplantation is limited by many factors, such as the patient's physical condition, economic situation, and the source of matching hematopoietic stem cells. Therefore, safe, effective, and economical drug treatment regimens still need to be further developed.

[0003] Lonidamine (Lonidamine, LND) is a safe and well-tolerated mitochondrial targeted drug approved by the US FDA, which can open the mitochondrial permeability transition pore, leading to the production of intracellular ROS and causing cell apoptosis. However, when lonidamine is used alone, the clinical efficacy is poor.

[0004] Glutathione (GSH) is the most abundant antioxidant in cells. Glutathione peroxidase 4 (GPX4) can convert lipid peroxides into lipid alcohols with GSH as a cofactor, playing the role of the cornerstone of the cellular antioxidant system. Ras-selective lethal 3 (RSL3) is an inhibitor of GPX4. RSL3 inhibits GPX4 activity, which can induce the accumulation of membrane lipid peroxides, induce cell ferroptosis, and promote the process of oxidative cell death.

[0005] At present, studies have confirmed that combination therapy can enhance the activity of a single drug and reduce its toxicity, and is a relatively mature and reliable way to solve the problem of single-drug efficacy and drug resistance. However, to date, there have been no reports on the combined use of lonidamine and GPX4 inhibitors in the treatment of AML. Summary of the invention

[0006] The object of the present invention is to provide a pharmaceutical composition for treating acute myeloid leukemia and its application in the preparation of a drug for treating acute myeloid leukemia. The present invention combines lonidamine and a GPX4 inhibitor, effectively solving the problems of poor efficacy and drug resistance that occur in the treatment of acute myeloid leukemia patients in the prior art.

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a pharmaceutical composition for treating acute myeloid leukemia, and the pharmaceutical composition contains lonidamine and a GPX4 inhibitor.

[0009] Further, in the aforementioned pharmaceutical composition for treating acute myeloid leukemia, the GPX4 inhibitor is RSL3.

[0010] Further, in the aforementioned pharmaceutical composition for treating acute myeloid leukemia, the molar ratio of lonidamine to RSL3 can be 250 - 1000:1.

[0011] Further, in the aforementioned pharmaceutical composition for treating acute myeloid leukemia, the molar ratio of lonidamine to RSL3 is preferably 750 - 1000:1.

[0012] On the other hand, the present invention also provides the application of the aforementioned pharmaceutical composition in the preparation of a drug for anti - acute myeloid leukemia cells.

[0013] Further, in the aforementioned application, the drug contains lonidamine, RSL3 and a pharmaceutically acceptable excipient.

[0014] Further, in the aforementioned application, the dosage form of the drug is an oral tablet, granule, injection or capsule.

[0015] Further, in the aforementioned application, the acute myeloid leukemia cells are Kasumi - 1 or HL60 cells

[0016] Compared with the prior art, the above - mentioned technical solutions of the present invention have the following beneficial effects:

[0017] The present invention creatively combines lonidamine and a GPX4 inhibitor for the oxidation - antioxidant defense system of AML tumor cells. The inhibition rate of AML cells is significantly higher than that of single drug use. The combined use of the two drugs can synergistically inhibit the viability of AML cells, promote the ferroptosis of AML cells, and induce oxidative damage to AML cells, thereby achieving the purpose of treating AML. Description of the Drawings

[0018] Figure 1Show the cytotoxic effect of lonidamine at different concentrations on AML cell lines and determine the half-lethal concentration (IC50) of the drug;

[0019] Figure 2 Show the synergistic effect of the cross-combination of lonidamine and RSL3 at different concentrations on AML cell lines;

[0020] Figure 3 Show the synergistic killing effect of the combination of lonidamine at the optimal drug concentration and RSL3 on AML cell lines;

[0021] Figure 4 Show the killing effect diagram of the combination of lonidamine and RSL3 on AML cells detected by flow cytometry;

[0022] Figure 5 Show the effect of the combination of lonidamine and RSL3 on oxidative stress in AML cell lines detected by confocal laser microscopy;

[0023] Figure 6 Show the effect of the combination of lonidamine and RSL3 on the lipid peroxidation level in AML cell lines detected by flow cytometry;

[0024] Figure 7 Show the recovery effect of different death pathway inhibitors on cell viability after treatment with the combination of lonidamine and RSL3. Detailed implementation mode

[0025] The following further describes the present invention in detail in combination with embodiments and drawings, but it is not used as a basis for limiting the technical solution of the present invention.

[0026] The pharmaceutical composition for treating acute myeloid leukemia of the present invention comprises lonidamine and a GPX4 inhibitor, and can be used for preparing drugs against acute myeloid leukemia cells. Among them, the GPX4 inhibitor can specifically be RSL3. In the pharmaceutical composition, the molar ratio of lonidamine to RSL3 can be 250-1000:1, preferably 750-1000:1.

[0027] The following further illustrates the present invention in combination with experiments.

[0028] Experiment 1: Inhibitory effect of lonidamine on the activity of AML cell lines

[0029] Select Kasumi-1 cells in the logarithmic growth phase, resuspend and mix them with 2 mL of fresh medium after centrifugation, transfer 20 μL of the cell suspension to a cell counting plate, and adjust the cell concentration of the mother liquor to 8×10 according to the counting results of the cell counter 5 / mL, aspirate 50 μL of the cell suspension and inoculate it into a 96-well plate. According to the experimental protocol, it is divided into a blank group, a control group, and an experimental group. Each group has replicate wells, and the drug dosage is calculated. Using the method of serial dilution, RPMI-1640 basal medium is used as the dilution solvent, and different concentrations of lonidamine (0, 50, 100, 150, 200 μM) are added to the corresponding 96-well plates containing cells at a volume of 50 μL / well. Incubate in a cell culture incubator at 37 °C and 5% CO 2 2. Add 15 μL of CCK8 and react for 4 hours at 24 and 48 hours respectively to detect the cell viability. Read the absorbance (A450) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Define the A450 value of the cell viability of the control group as 1, calculate the relative cell viability of each concentration of the drug treatment group, use Graphpad Prism software to plot the toxicity curve and calculate the IC50. The IC50 values of lonidamine in Kasumi-1 cells are 115.5 μM (24 h) and 54.67 μM (48 h), which indicates that the antitumor effect of lonidamine in the AML cell line is dose- and time-dependent (see Figure 1 ).

[0030] Experiment 2: Synergistic effect of lonidamine and RSL3 on the inhibition of viability and killing effect of AML cell lines

[0031] Select HL-60 and Kasumi-1 cells in the logarithmic growth phase. After centrifugation, resuspend and mix them with 2 mL of fresh medium. Transfer 20 μL of the cell suspension to a cell counting chamber, and adjust the cell concentration of the mother liquor to 8×10 5 / mL, aspirate 50 μL of the cell suspension and inoculate it into a 96-well plate. Divide it into a blank group, a control group and an experimental group according to the 5×5 drug combination scheme. Each group has replicate wells. The blank group does not contain cells and only contains 100 μL of RPMI-1640 medium. The control group contains cells and 100 μL of RPMI-1640 medium. The experimental group contains cells and the required drugs. The specific drug usage plan is as follows: Treat Kasumi-1 cells with lonidamine (0, 50, 100, 200, 300 μM), RSL3 (0, 0.25, 0.5, 0.75, 1 μM) alone or in combination of each concentration of the two drugs; Treat HL-60 cells with lonidamine (0, 50, 100, 150, 200 μM), RSL3 (0, 0.05, 0.1, 0.15, 0.2 μM) alone or in combination of each concentration of the two drugs. Using the method of serial dilution, dilute lonidamine and RSL3 into the required concentrations with RPMI-1640 basal medium in 1.5 mL EP tubes according to the experimental protocol, and add them to the corresponding 96-well plates containing cells at a volume of 50 μL / well. After incubating the 96-well plate in a cell incubator for 12 h, add 15 μL of CCK-8 reagent to each well and continue to incubate in the incubator for 4 h. Read the absorbance (A450) at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Define the A450 value of the cell viability of the control group as 1, calculate the relative cell viability of each concentration of the drug treatment group, and after sorting according to the drug concentration order, use the SynergyFinder tool kit and the ZIP model to analyze and construct a synergistic scoring map of the drug combination. The results show that both lonidamine and RSL3 have cytotoxic effects on AML cells and show a dose-dependent manner. In addition, we calculated the drug ZIP synergy effect score with the SynergyFinder tool kit. Generally, it is considered that the ZIP synergy score > 10, which indicates that there is a synergistic effect between the combined drugs. The ZIP model results show that the ZIP synergy scores of the two drugs combined to treat Kasumi-1 cells and HL-60 cells are 13.23 and 16.866 respectively, indicating that lonidamine and RSL3 have a synergistic effect in the treatment of AML ( Figure 2 ). For Kasumi-1 cells, the combination of 100 μM lonidamine and 0.4 μM RSL3 showed the best synergistic effect. For HL-60 cells, the best synergistic effect was obtained when 100 μM lonidamine was combined with 0.1 μM RSL3 ( Figure 2 、 Figure 3 ).

[0032] In addition, 1.5 mL of Kasumi-1 cell suspension was seeded into each well of a 6-well culture plate and subjected to corresponding treatments according to the experimental groups: control group (Vehicle), lonidamine treatment group (100 μM), RSL3 treatment group (0.4 μM), and combined drug treatment group. The cells were cultured in an incubator for 12 h. The cells were collected into an EP tube, centrifuged, washed with PBS, and then resuspended gently in 195 μL of binding buffer. Then 5 μL of AnnexinV-FITC was added, and the mixture was gently vortexed and incubated in the dark at room temperature for 10 min. After centrifugation at 1000 g for 5 min, the supernatant was discarded, and the cells were gently resuspended in 190 μL of binding buffer. Then 10 μL of PI staining solution was added, and the mixture was gently vortexed. Immediately, flow cytometry was performed. The results showed that compared with single drug treatment, the proportion of dead cells (the proportion of cells positive for both AnnexinV and PI) in AML cells treated with lonidamine combined with RSL3 was significantly increased ( Figure 4 ).

[0033] Experiment 3: Effect of the combination of lonidamine and RSL3 on the oxidative stress level of AML cell lines

[0034] Kasumi-1 cells and HL-60 cells in good growth condition were collected. The experiment was divided into a control group, a lonidamine treatment group, an RSL3 treatment group, and a combined drug treatment group. The cells were resuspended in basal medium, counted, and the cell density was adjusted to 1.5×10 6 / mL. 500 μL of cell suspension was seeded into each well of a 12-well culture plate. 500 μL of lonidamine (100 μM) and RSL3 (0.4 μM) prepared with basal medium were added to the corresponding culture plates to treat Kasumi-1 cells alone or in combination; 500 μL of basal medium containing lonidamine (100 μM) and RSL3 (0.1 μM) were added to the corresponding culture plates to treat HL-60 cells alone or in combination. The cells were incubated in an incubator for 8 h. The cells were collected into an EP tube, centrifuged, washed with PBS, and then centrifuged again. 5 μM BODIPY C11 (a fluorescent probe for detecting intracellular lipid peroxides, which can specifically bind to intracellular lipid peroxides) was freshly prepared with basal medium. 500 μL of the staining solution was used to resuspend the cell pellet in the corresponding EP tube, and the cells were stained in a cell culture incubator for 30 min. After centrifugation, the cells were washed with PBS to remove the medium containing the fluorescent probe. The cells were resuspended in 500 μL of basal medium, and the intracellular lipid peroxide level was detected by flow cytometry. The results showed that compared with single drug treatment, the fluorescence intensity of BODIPY C11 in the AML cell line treated with lonidamine combined with RSL3 was significantly increased ( Figure 5 ), indicating that the combination of lonidamine and RSL3 drugs can significantly promote lipid peroxidation in AML cells.

[0035] Kasumi-1 cells and HL-60 cells were treated under the same experimental conditions for 6 h. After centrifugation and washing, 4 μM DCFH-DA (a fluorescent probe for detecting intracellular reactive oxygen species, which itself has no fluorescence and is hydrolyzed by esterase to generate DCFH after entering the cells. The latter is oxidized by intracellular reactive oxygen species to 2'-7'-dichlorofluorescein DCF, showing green fluorescence) was prepared. 500 μL of the staining solution was used to resuspend the cell pellet, and the cells were incubated in a cell culture incubator for 20 min. Then, 5 μg / ml Hoechst 33342 (a fluorescent probe for labeling cell nuclei) staining solution was added to each EP tube, and the incubation was continued in the dark for 10 min. After that, the cells were collected and centrifuged at low speed. 500 μL of the basal medium was used to resuspend the cell pellet in each tube and transferred to a polylysine-coated four-chamber glass-bottom dish, and the cells were left standing in the dark for 10 min. After the cells adhered to the bottom of the dish, the fluorescence intensity of the cells in each experimental group was observed using a laser confocal microscope in a dark room, and images were taken. The results showed that after the combined treatment of clonidamine and RSL3, the green fluorescence in the AML cells was significantly enhanced( Figure 6 ). It indicates that the combination of clonidamine and RSL3 drugs can significantly promote the oxidative stress of AML cells.

[0036] Experiment 4: The combination of clonidamine and RSL3 can induce ferroptosis in AML cells

[0037] In the cytotoxicity recovery experiment, HL-60 cells were treated with clonidamine (100 μM), RSL3 (0.1 μM), and a series of drug inhibitors for specific cell death modes. The drug inhibitors included iron ion chelator (DFO, 10 μM), ferroptosis inhibitor (Fer-1, 1 μM), autophagy inhibitor (BafA1, 20 nM), apoptosis inhibitor (Z-VAD, 2 μM), and necrosis inhibitor (Necro, 500 nM). The volume of the liquid in each well was 120 μL (each well contained 40 μL of the drugs clonidamine and RSL3, 40 μL of the drug inhibitor, and 40 μL of the cell suspension. The control group and the two-drug combination group were made up with the basal medium). After the 96-well plate was incubated in a cell culture incubator for 12 h, 15 μL of CCK-8 reagent was added to each well, and the plate was continued to be incubated in the incubator for 4 h. The absorbance (A450) at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay reader. The A450 value of the cell viability of the control group was defined as 1, and the relative cell viability of each concentration of the drug treatment group was calculated. The results showed that the ferroptosis inhibitor Fer-1 significantly reversed the inhibitory effect of clonidamine / RSL3 on the viability of AML cells, and the iron chelator DFO partially reversed this inhibitory effect, while the other pathway inhibitors did not show obvious recovery effects, indicating that ferroptosis plays a major role in the cell death induced by the combination of clonidamine and RSL3( Figure 7 ).

[0038] The above results indicate that the combined use of lonidamine and RSL3 can significantly inhibit the viability of AML cells, promote cellular oxidative stress, and induce ferroptosis. It is feasible to use a pharmaceutical composition containing lonidamine and a GPX4 inhibitor as a drug option and treatment regimen for the treatment of AML.

Claims

1. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that: The pharmaceutical composition comprises lonidamine and a GPX4 inhibitor.

2. The pharmaceutical composition for treating acute myeloid leukemia according to claim 1, characterized in that: The GPX4 inhibitor is RSL3.

3. The pharmaceutical composition for treating acute myeloid leukemia according to claim 2, characterized in that: In the pharmaceutical composition, the molar ratio of lonidamine to RSL3 is 250-1000:

1.

4. The pharmaceutical composition for treating acute myeloid leukemia according to claim 3, characterized in that: In the pharmaceutical composition, the molar ratio of lonidamine to RSL3 is 750-1000:

1.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of an anti-acute myeloid leukemia cell drug.

6. The use according to claim 5, characterized in that: The medicine comprises lonidamine, RSL3 and pharmaceutically acceptable excipients.

7. The use according to claim 5, characterized in that: The drug is in the form of oral tablets, granules, injections or capsules.

8. The use according to claim 5, characterized in that: The acute myeloid leukemia cells are Kasumi-1 or HL60 cells.

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