Medicinal composition and medicine for treating acute myelogenous leukemia by using Vesonalar

Abstract

The invention discloses a pharmaceutical composition and a medicine for treating acute myelogenous leukemia in cooperation with vinca. The pharmaceutical composition is prepared from AGX51, vinca and a proper amount of solvent. In the pharmaceutical composition, the concentration ratio of the AGX51 to the vinca is 200: (1-40); the AGX51 in the pharmaceutical composition can improve the proliferation inhibition capability of a single Venoclara drug by 2.2 times or more; the AGX51 in the pharmaceutical composition can improve the apoptosis induction capability of a single drug of Venoclara by 3.6 times or more.

Description

Technical Field

[0001] The invention relates to the technical field of drug research and development, and in particular to a drug composition for treating acute myeloid leukemia in combination with venetoclax. Background Art

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous blood system malignancy caused by gene mutations, characterized by dysregulated clonal proliferation, blocked differentiation, and suppressed apoptosis of hematopoietic stem / progenitor cells. At present, the clinical treatment of adult AML (non-acute promyelocytic leukemia) is mainly intensive chemotherapy, and medium- and high-risk patients need hematopoietic stem cell transplantation after remission, supplemented by immunotherapy, targeted drug therapy, etc.

[0003] Venetoclax (also known as ABT-199) is a new targeted drug for the treatment of AML that was approved by the U.S. FDA in 2018 and approved for marketing by the Chinese FDA at the end of 2020. In 2021, my country's latest diagnosis and treatment guidelines added venetoclax combined with demethylating drugs or low-dose cytarabine to low-intensity treatment for patients aged 75 years and above with first-time or unfit AML, as well as relapsed and refractory AML. It has become a breakthrough new therapy that has profoundly changed the treatment landscape of AML and has broad application prospects. However, in clinical practice, 30%-40% of patients develop treatment resistance when they first receive venetoclax treatment, and most patients develop secondary resistance and relapse within months to years after treatment remission. Once resistance occurs, patients will lack effective treatment drugs and their survival time will be shortened. The mechanism of action of venetoclax is to target the B-cell lymphoma 2 (BCL2) protein and activate the mitochondrial apoptosis pathway. Resistance to venetoclax is an important cause of ultimate treatment failure and patient death. Researching highly effective drug combinations to enhance the sensitivity of venetoclax's anticancer activity is crucial to improving the cure rate of AML. Summary of the invention

[0004] A pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax comprises AGX51, venetoclax and an appropriate amount of solvent.

[0005] In the improved scheme, the concentration ratio of AGX51 and venetoclax in the pharmaceutical composition is: 200:(1-40).

[0006] In the improved scheme, AGX51 in the drug composition can increase the proliferation inhibition ability of venetoclax alone by more than 2.2 times.

[0007] In the improved scheme, AGX51 in the drug composition can increase the apoptosis-inducing ability of venetoclax alone by more than 3.6 times.

[0008] In an improved solution, the concentration of AGX51 is 6 μM-10 μM, and the concentration of venetoclax is 0.1 μM-0.4 μM. In a more preferred solution, the concentration of AGX51 is 6 μM, and the concentration of venetoclax is 0.1 μM.

[0009] In the improved scheme, the concentration of AGX51 in the pharmaceutical composition is 2-10 μM; the concentration of venetoclax in the pharmaceutical composition is 0.05-0.4 μM.

[0010] In the improved scheme, the solubility of venetoclax in DMSO is 115.14 mM.

[0011] In the improved scheme, the solubility of AGX51 in DMSO was 220.15 mM;

[0012] The present invention also discloses a medicine, including the pharmaceutical composition; the medicine is a liquid preparation or a solid preparation. When used, conventional amounts of auxiliary materials, such as diluents, starch, saccharin, etc., need to be added, which can be conventionally selected by those skilled in the art according to needs.

[0013] 1. The present invention combines venetoclax with AGX51. On the basis of the study of the molecular function of ID1, the ID1-specific small molecule inhibitor AGX51 is innovatively used in combination with venetoclax. AGX51 can significantly synergistically enhance the proliferation inhibition and apoptosis induction of AML cells by venetoclax, and has concentration dependence. The combination has the advantages of low drug toxicity and side effects and low raw material cost. Through two experiments, it was verified that 0.1 μM venetoclax and 6 μM AGX51 are the optimal drug combination concentrations for inhibiting proliferation of the AML cell line MV4-11, and at this concentration, the effect of venetoclax alone can be increased by 2-3 times. Compared with the same drugs, the effect of drug efficacy can be doubled.

[0014] 2. The pharmaceutical composition of venetoclax and AGX51 provided by the present invention can provide a new treatment plan with better effects and lower toxic side effects for AML patients, especially elderly patients with poor tolerance, and has high scientific research and production application value.

[0015] 3. The DNA binding inhibitor 1 of the present invention belongs to the highly conserved transcriptional regulator ID protein family and is a key gene for regulating cell proliferation and differentiation. Overexpression of ID1 in various cancers is related to tumor occurrence and development and drug treatment. It is a negative regulator of anticancer drug-induced cell apoptosis and a potential tumor treatment target and biomarker. The present invention combines the novel small molecule antagonist AGX51 of ID1 with the effect and dosage of venetoclax, thereby enhancing the anti-AML activity of venetoclax and improving the clinical application effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the MV4-11 cell STR identification report;

[0017] Figure 2 Schematic diagram of the molecular structure of AGX51;

[0018] Figure 3 It is a curve diagram of drug concentration and cell growth inhibition rate;

[0019] Figure 4 It is a schematic diagram of the synergistic effect at 48 h;

[0020] Figure 5 It is a schematic diagram of the synergistic effect at 72 h;

[0021] Figure 6 Schematic diagram of apoptosis induction results; DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1: AGX51 synergistically enhances the cell-killing effect of venetoclax

[0024] (1) Experimental Materials

[0025] ① Human AML cell line MV4-11; introduced and preserved by the Department of Hematology, People's Hospital Affiliated to Jiangsu University, collected by the American Type Culture Collection (ATCC) cell bank, coded as CRL-9591, and correctly identified by STR of Wuhan Pronocell Life Science Co., Ltd. (such as Figure 1 shown).

[0026] ② Venetoclax, purchased from Selleck, USA, item number S8048, chemical substance CAS number 1257044-40-8, molecular formula C45H50ClN7O7S, molecular weight 868.44, purity 99.97%, insoluble in water, solubility in DMSO is 115.14mM. The preparation method is: under the aseptic conditions of the clean bench, dissolve in DMSO to a 20mM mother solution and store at -80℃. For example, 5mg = 20mM × 287.9μl × 868.44, that is, 5mg Venetoclax powder is dissolved in 287.9μl DMSO solution.

[0027] ③AGX51 was purchased from TargetMol, USA, with the product number T10268, the chemical substance CAS number is 330834-54-3, the molecular formula is C27H29NO4, the molecular weight is 431.52, the purity is 99.85%, it is insoluble in water, and the solubility in DMSO is 220.15mM (such as Figure 2 The preparation method is: dissolve in DMSO to 10mM stock solution under sterile conditions in a clean bench and store at -80℃. For example, 5mg = 10mM × 1.16ml × 431.52, that is, 5mg AGX51 powder is dissolved in 1.16ml DMSO solution, ultrasonic dissolution, ultrasonic power 240W, 5min, completely dissolved.

[0028] ④Other reagents: culture medium (Vicente, 350-006-CL), fetal bovine serum (Vicente, 086-150), cell culture grade PBS (Vicente, 311-010-CL), double antibody (Biyuntian, C0222), CCK8 (TargetMol, C0005).

[0029] (2) MV4-11 cell culture conditions

[0030] MV4-11 is a suspension cell, routinely cultured in RPMI-1640 medium (Vicente, 350-006-CL) containing 10% fetal bovine serum and 1% double antibody in a TC-free 25 cm2 permeable cell culture flask, placed in a 37°C 5% CO2 incubator, and maintained at a cell density of no more than 80%. The medium was changed or passaged as needed, and the centrifugation condition was 1000 rpm at 4°C for 5 min (Eppendorf refrigerated centrifuge, model Centrifuge 5810R).

[0031] (3) Cell proliferation assay to detect the half inhibitory concentration (IC50) of AML cells to venetoclax and AGX51

[0032] ① Prepare a suspension of MV4-11 cells in good condition in the logarithmic growth phase, 90 μl per well of a 96-well plate, and inoculate 20,000 cells per well. To reduce errors, add sterile PBS to the outermost circle of wells on the culture plate and place it in a 37°C 5% CO2 incubator for 2 hours to allow the cells to recover to a good growth state before adding drugs.

[0033] ②Add each group of treatments to the cell culture wells, set up zero wells, blank control groups and different concentrations of drug treatment groups, dilute the mother solution of the drug with RPMI-1640 medium as the working solution, add 10 μl to each well to make the final volume 100 μl, and set up 3 replicates for each group. The concentration gradient of venetoclax is: 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 μM, and the concentration gradient of AGX51 is: 0.001, 0.01, 0.1, 1, 10, 15, 20 μM.

[0034] ③ After 48 hours of drug treatment, CCK8 detection was performed. 10 μl CCK8 solution was added to each well, and the cells were incubated in the incubator for another 2 hours. The absorbance A value of each well was detected at a wavelength of 450 nm using an ELISA reader to ensure that the reading value of the blank control group was between 0.8 and 1.2. The calculation formula was: cell survival rate = (experimental group A value - zero adjustment group A value) / (control group A value - zero adjustment group A value) × 100%, proliferation inhibition rate (inhibition) = 1 - cell survival rate.

[0035] ④ The curve of drug concentration and cell growth inhibition rate was plotted using Graphpad Prism10 software, and the IC50 value was calculated. The 48h IC50 of MV4-11 for venetoclax was 0.46 μM, and that for AGX51 was 11.88 μM (e.g. Figure 3 shown).

[0036] (4) Cell proliferation assay to detect the ability of AGX51 to synergistically enhance the ability of venetoclax to inhibit AML cell proliferation

[0037] ① Prepare a suspension of MV4-11 cells in good condition in the logarithmic growth phase, add 90 μl to each well of a 96-well plate, and inoculate 20,000 cells per well. To reduce errors, add sterile PBS to the outermost circle of wells on the culture plate and place it in a 37°C 5% CO2 incubator for 2 hours to allow the cells to recover to a good growth state before adding drugs.

[0038] ②Add treatment to each group, set up zero wells, blank control group and different concentration drug treatment groups, dilute the mother solution drug with RPMI-1640 medium as working solution, add 10μl to each well to make the final volume 100μl, and set up 3 replicate wells for each group. According to the above IC50, select the appropriate drug concentration for the combination of two drugs. The concentration gradient of venetoclax is: 0.05, 0.1, 0.2, 0.4μM, the concentration gradient of AGX51 is: 2, 4, 6, 8, 10μM, and various concentration combinations of the two drugs.

[0039] ③ After 48h and 72h of drug treatment, CCK8 detection was performed. 10μl CCK8 solution was added to each well, and the cells were incubated in the incubator for 2h. The absorbance A value of each well was detected at a wavelength of 450nm using an ELISA reader to ensure that the reading value of the blank control group was between 0.8-1.2. The calculation formula was: cell survival rate = (experimental group A value - zero adjustment group A value) / (control group A value - zero adjustment group A value) × 100%, proliferation inhibition rate (inhibition) = 1-cell survival rate.

[0040] ④ Use the SynergyFinder tool to analyze the combined drug data and use the ZIP synergy model to evaluate the synergistic potential of the two drugs. Select the "matrix" analysis mode in SynergyFinder, enter the inhibition values ​​of each group, and download the analysis results (such as Figure 4 and Figure 5 shown).

[0041] ⑤Result analysis: ZIP<-10 indicates that the two drugs have antagonistic effects, -10<ZIP<10 indicates that the effects of the two drugs are additive, and ZIP>10 indicates that the two drugs have synergistic effects. The results of the combined action of venetoclax and AGX51 for 48h and 72h were consistent, with ZIP values ​​of 29.8 and 27.9, respectively, indicating that the two drugs have a strong synergistic effect, that is, AGX51 can significantly promote the ability of venetoclax to inhibit the proliferation of AML cells. Figure 4 and Figure 5 The white box in the middle shows the area with the best synergistic effect, and the point corresponding to the small left corner of the box indicates the lowest drug concentration and the best effect, indicating that 0.1 μM venetoclax and 6 μM AGX51 are the best drug combination concentrations for inhibiting proliferation, which increases the proliferation inhibition ability of venetoclax alone by 2.2 times.

[0042] from Figure 4 and Figure 5 It can be seen that at some concentration ratios, the effect of the combination is lower than the sum of the effects of the individual substances (such as 0.1 μM venetoclax and 4 μM AGX51). At some concentrations (such as 0.1 μM venetoclax and 6 μM AGX51), the synergistic effect of the two drugs far exceeds that at other concentrations.

[0043] Example 2: AGX51 synergistically promotes venetoclax-induced apoptosis

[0044] (1) Experimental Materials

[0045] ①Annexin V-PE 7-AAD Apoptossi kit (Novozyme, A213), other reagents are the same as described in Example 1.

[0046] (2) Flow cytometry detection of AGX51 synergistically promoting apoptosis induction by venetoclax

[0047] ① Prepare a suspension of MV4-11 cells in good condition in the logarithmic growth phase and inoculate 1.8×10 cells per well of a 12-well plate. 5 Each group of cells had 3 replicate wells.

[0048] ② The concentrations of venetoclax are 0.1 and 0.2 μM, the concentrations of AGX51 are 4 and 6 μM, and various concentration combinations of the two drugs in combination.

[0049] ③ After 48 hours of drug treatment, apoptosis detection was performed: the cells to be detected were collected into a 1.5 ml EP tube, washed once with pre-cooled PBS, and resuspended in 100 μl 1×Binding buffer to adjust the concentration to 1×10 6 / ml, add 5μl PE Annexin V and 5μl 7-AAD (Novozyme, A213), blow gently, incubate at room temperature in the dark for 10min, add 400μl 1×Bindingbuffer to each well, mix gently, and complete the detection of the stained samples by flow cytometry within 1h. Set up a blank control group, PEAnnexin V and 7-AAD single staining group and double staining group. The excitation wavelength of the flow cytometer is 488nm, PE fluorescence is detected in the FL2 channel, 7-AAD fluorescence is detected in the FL3 channel, and 10,000 cells are collected for each sample.

[0050] ④ FlowJo software was used to analyze the results, and the data of three independent repetitions were combined and statistically drawn. The calculation formula was: apoptosis rate (%) = early apoptosis rate (Annexin V + / PE - )+late apoptosis rate (Annexin V + / PE + ). The experimental results are as follows Figure 6 As shown, the combination of venetoclax and AGX51 induced apoptosis in MV4-11 cells more effectively than venetoclax alone, and 0.1 μM venetoclax and 6 μM AGX51 were the optimal drug combination concentrations for inducing apoptosis, which increased the apoptosis-inducing ability of venetoclax alone by 3.6 times.

[0051] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition of venetoclax for treating acute myeloid leukemia, characterized in that: It comprises AGX51, Venetoclax and a proper amount of solvent, wherein AGX51 and Venetoclax are dissolved in the solvent.

2. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 1, characterized in that: The concentration ratio of AGX51 and venetoclax in the pharmaceutical composition is 200:(1-40).

3. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 2, characterized in that: AGX51 in the drug composition can increase the proliferation inhibition ability of venetoclax alone by more than 2.2 times.

4. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 3, characterized in that: AGX51 in the drug composition can enhance the apoptosis-inducing ability of venetoclax alone by more than 3.6 times.

5. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 1, characterized in that: The concentration of AGX51 in the pharmaceutical composition is 2-10 μM; the concentration of venetoclax in the pharmaceutical composition is 0.05-0.4 μM.

6. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 3, characterized in that: The solubility of venetoclax in DMSO is 115.14 mM.

7. The pharmaceutical composition for treating acute myeloid leukemia in combination with venetoclax according to claim 4, characterized in that: The solubility of AGX51 in DMSO is 220.15 mM.

8. A drug, characterized in that include: The pharmaceutical composition and excipients according to any one of claims 1 to 7.

9. The drug according to claim 8, characterized in that The medicine is a liquid preparation or a solid preparation.

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