CN116531395A - Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia - Google Patents
Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia Download PDFInfo
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- CN116531395A CN116531395A CN202210085636.5A CN202210085636A CN116531395A CN 116531395 A CN116531395 A CN 116531395A CN 202210085636 A CN202210085636 A CN 202210085636A CN 116531395 A CN116531395 A CN 116531395A
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- Prior art keywords
- cytarabine
- artesunate
- venetoclax
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical class C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 title claims abstract description 16
- 208000032839 leukemia Diseases 0.000 title claims abstract description 7
- FIHJKUPKCHIPAT-AHIGJZGOSA-N artesunate Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@@H](OC(=O)CCC(O)=O)[C@@H]4C FIHJKUPKCHIPAT-AHIGJZGOSA-N 0.000 claims abstract description 165
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/50—Pyridazines; Hydrogenated pyridazines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of biological medicine, and in particular relates to a composition containing artemisinin derivatives and application of the composition in preparation of medicines for treating leukemia. The composition of the invention is prepared by mixing the component A, the component B and the component C according to the proportion of 8-40:1-2:1-80; wherein, the A component is one or more of artesunate and analogues thereof; the analogues can be artemisinin, dihydroartemisinin, artemether, arteether, etc.; the component B is one or more of venetoclax and analogues thereof; the analog may be navitocrax, etc.; the component C is one or more of cytarabine and analogues thereof; the analog may be azacitidine, decitabine, gemcitabine, and the like; the invention overcomes the drug resistance generated by the feedback upregulation of other anti-apoptosis proteins in the combined scheme of artesunate and cytarabine and the combined scheme of venetoclax and cytarabine in the prior art, and obviously improves the treatment effect of the combined scheme of venetoclax and cytarabine on AML.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a pharmaceutical composition containing artesunate, venetoclax and cytarabine and application thereof in preparing medicines for treating acute myelogenous leukemia.
Background
Acute Myelogenous Leukemia (AML) is one of the most aggressive hematological malignancies, older AML patients are mainly treated with either demethylating drugs (HMAS) or low-dose cytarabine (LDAC), with very poor prognosis and overall survival rates of no more than 10% for 5 years. For young AML patients, the treatment of the young AML patients is carried out by a '7+3' (daunorubicin+cytarabine) induction scheme which is 40 years ago, the remission rate of the treatment scheme is between 30 and 80 percent, but the long-term survival rate and the cure rate are lower due to recurrence, and the treatment effect needs to be improved.
Apoptosis retardation is a common characteristic of AML, abnormal expression of anti-apoptosis proteins Bcl-2 and Mcl-1 increases the apoptosis threshold of AML cells, is one of main reasons for AML pathogenesis and drug tolerance, venetoclax is a Bcl-2 family anti-apoptosis protein small molecule inhibitor only approved for CLL and AML treatment at present, but the effect of single application in AML is limited, and in clinical research, venetoclax is found to improve the therapeutic effect of azacitidine, decitabine or low dose of cytarabine on AML.
Anti-apoptotic protein Mcl-1 is believed to be the primary cause of mediating Venetoclax tolerance, venetoclax induces apoptosis in cells by inhibiting Bcl-2 release of Bim, but the release of Bim is sequestered by Mcl-1 resulting in Venetoclax tolerance. Mcl-1 can also be transferred into the nucleus to bind to chromatin to reduce DNA damage and induce p-Chk1 to initiate DNA damage repair, mcl-1 also being involved in the tolerizing process of cytarabine. Targeted inhibition of Mcl-1 is expected to improve the therapeutic effect of the venetoclax and cytarabine combination regimen on AML. Intracellular Mcl-1 protein levels are regulated by Noxa and Bim, and Noxa can displace Bim in Mcl-1 and induce Mcl-1 to degrade, so that drugs inducing Noxa can inhibit Mcl-1 by reducing protein levels of Mcl-1 in a targeted manner, and the therapeutic effect of venetoclax combined with cytarabine is enhanced.
At present, researchers in China have summarized the combined scheme of artesunate and cytarabine, and meanwhile, the combined scheme of venetoclax and cytarabine is clinically approved, but the two schemes generate drug resistance due to the feedback upregulation of other anti-apoptosis proteins, and have limited therapeutic effects on AML.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a pharmaceutical composition containing artesunate, vennoclax and cytarabine and application thereof in preparing medicines for treating acute myelogenous leukemia. Further, artesunate in the composition can be used for inducing apoptosis and combining and synergistically inducing apoptosis with venetoclax by inducing Noxa, reducing Mcl-1 protein, overcoming Mcl-1 mediated cytarabine tolerance and enhancing cytarabine-induced DNA damage effect, and the three pharmaceutical compositions can be used for inducing apoptosis and DNA damage of acute myeloid leukemia cells, so that the current treatment situation of the combined scheme of venetoclax and cytarabine on AML is improved.
In order to achieve the above purpose, the invention adopts the technical scheme that:
a composition containing artemisinin derivatives is prepared by mixing an A component, a B component and a C component according to a ratio of 8-40:1-2:1-80; wherein, the liquid crystal display device comprises a liquid crystal display device,
the component A is one or more of artesunate and analogues thereof; the analogues can be artemisinin, dihydroartemisinin, artemether, arteether, etc.;
the component B is one or more of venetoclax and analogues thereof; the analog may be navitocrax, etc.;
the component C is one or more of cytarabine and analogues thereof; the analog may be azacitidine, decitabine, gemcitabine, and the like;
preferably, the A component is artesunate; the component B is vennetoclax; and the component C is cytarabine.
The artesunate has a structural formula shown in the specification.
Bcl-2 inhibitor venetoclax is a compound of formula II:
cytarabine is a compound represented by formula iii:
the application of a composition containing artemisinin derivatives in preparing medicines for treating hematological tumors including leukemia and lymphoma.
The composition is used for inducing leukemia cell apoptosis and DNA damage in a coordinated manner to prepare the medicine for treating acute myelogenous leukemia.
The acute myelogenous leukemia is THP-1 or MOLM-13 cells.
In the preparation of the THP-1 medicine, the A component, the B component and the C component of the composition are mixed according to the proportion of 8:1: mixing in proportion of 5;
in the preparation of the medicine for treating MOLM-13, the components A, B and C of the composition are prepared according to the weight ratio of 40:2: mixing in proportion of 1.
A preparation of a composition containing artemisinin derivatives, which is prepared by mixing active ingredients and a pharmaceutically acceptable carrier, wherein the active ingredients are the composition as claimed in claim 1, and the active ingredients account for 0.01-99% of the mass of the preparation.
The preparation is in the form of tablet, capsule or granule.
The preparation is prepared by mixing the components in the composition and then mixing the components with a pharmaceutically acceptable carrier, or preparing the preparation by mixing the components in the composition and the pharmaceutically acceptable carrier respectively and then mixing the components.
Further, in the case of preparing the composition of the present invention into a pharmaceutical preparation for simultaneous administration, artesunate, bcl-2 inhibitor venetoclax and cytarabine may be contained in the same pharmaceutical preparation such as a tablet or capsule, which is administered to a patient according to instructions, or three components of the above composition are prepared into a controlled release preparation, which releases the components of the composition sequentially, and which is administered to a patient only; in the preparation scheme of preparing the composition into the cross-administration, artesunate, bcl-2 inhibitor venetoclax and cytarabine can be respectively prepared into different preparations, and the artesunate, bcl-2 inhibitor venetoclax and cytarabine are packaged or combined together in a conventional manner in the field, and then the patient takes the composition according to the cross sequence indicated by the medicine instruction, or the pharmaceutical composition is prepared into a controlled release preparation for cross release of the artesunate, bcl-2 inhibitor venetoclax and cytarabine.
The invention has the advantages that:
the invention provides an artesunate, bcl-2 inhibitor venetoclax and cytarabine composition, and the mechanism clarifies that artesunate can induce apoptosis by inducing Noxa and cooperating with venetoclax, overcomes tolerance of venetoclax, simultaneously down-regulates Mcl-1 protein, overcomes feedback activation and DNA damage repair process of cytarabine induced p-Chk1, and enhances killing effect of cytarabine on leukemia cells. The invention overcomes the drug resistance generated by the feedback upregulation of other anti-apoptosis proteins in the combined scheme of artesunate and cytarabine and the combined scheme of venetoclax and cytarabine in the prior art, and obviously improves the treatment effect of the combined scheme of venetoclax and cytarabine on AML.
Drawings
FIG. 1 is a graph of the growth inhibitory effect of the composition of the invention on AML cells in THP-1 and MOLM-13 cells, as provided in the examples of the invention;
FIG. 2 is a graph showing the effect of the composition of the present invention on apoptosis induction of AML cells in THP-1 and MOLM-13 cells;
FIG. 3 is a graph of the effect of the composition of the invention on killing AML cells in THP-1 and MOLM-13 cells;
FIG. 4 is a graph showing the effect of the composition of the present invention on the inhibition of colony formation in THP-1 and MOLM-13 cells.
FIG. 5 is a graph showing the effect of inhibiting tumor growth in NOD-SCID mice examined by using the composition of the present invention, wherein A is a tumor picture of the mice, B is a tumor weight of the mice, and C is a body weight of the mice.
FIG. 6 is a graph of the survival of a prolonged xenograft mouse examined in vivo in NOD-SCID mice using a composition of the present invention, as provided in the examples of the present invention.
FIG. 7 is a graph showing the effect of the composition of the invention on inducing pro-apoptotic Noxa, down-regulating anti-apoptotic Mcl-1, inhibiting replication stress checkpoint kinase p-Chk1, and inducing DNA damage protein ≡ (-H2A.X) in THP-1 and MOLM-13 cells. .
Detailed Description
The following examples are provided to further illustrate the substance of the present invention, but are not intended to limit the scope of the present invention.
The cells and drugs used in the examples of the present application are as follows:
and (3) cells: human acute myelogenous leukemia cell THP-1 was purchased from American Type Culture Collection (ATCC). Human acute myelogenous leukemia cells Molm-13 were purchased from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig Germany). Medicine: the pharmaceutical compositions used in the following examples were prepared as described below; artesunate was purchased from Sigma and stored at a concentration of 10mM; venetoclax was purchased from Selleck and stored at a concentration of 10mM; cytarabine was purchased from MedChemExpress and stored at a concentration of 20mM.
Example 1
Reagents and methods:
artesunate and ABT-199 were accurately weighed, dissolved in dimethyl sulfoxide, and prepared into 10mM stock solutions, and stored at-20deg.C, and diluted with ethanol to appropriate concentration when used. Accurately weighing cytarabine, dissolving in PBS filtered by a 0.22 mu m filter membrane; artesunate, venetoclax and cytarabine were then diluted to the appropriate concentrations with fresh medium (1640 medium) when used. In all experiments, the final concentration of dimethyl sulfoxide and ethanol is strictly controlled, the final concentration of dimethyl sulfoxide is not more than 1 per mill, the final concentration of ethanol is not more than 1 percent, and the cell activity is not influenced.
THP-1 cells and Molm-13 cells were cultured in 1640 medium containing 10% inactivated fetal bovine serum, 10mmol/L L-glutamine,100U/mL penicillin, and 100. Mu.g/mL streptomycin, 37℃and 5% CO 2 Culturing under saturated humidity.
The logarithmic phase of cells was then treated at 10 5 Density inoculation in 24-well plates, treatment with different drug concentrations was added to the cells (see tables 1-6). After 72h, dead cells were observed by trypan blue staining. The homogenized cells were mixed with trypan blue dye at 1:1 (v/v), and after staining, normal cells and blue-stained dead cells were counted with an optical microscope and a blood cell counting plate (see fig. 1). The cell growth inhibition ratio (GI) was calculated according to the following formula, and the half growth inhibition concentration (GI 50 : drug concentration to achieve 50% inhibition of cell growth).
Gi= (control cell number-dosing cell number)/control cell number x 100%
The synergy index (CI) of the pharmaceutical composition was calculated using Compusyn software.
TABLE 1 synergistic inhibition of THP-1 cell growth experiments with combinations of artesunate and venetoclax at different concentrations
| Artesunate (mu M) | Venetoclax(μM) | Growth inhibition ratio (%) | Combination index CI |
| 0.2 | -- | 10.29 | -- |
| 0.4 | -- | 17.65 | -- |
| 0.8 | -- | 31.80 | -- |
| -- | 0.1 | 15.51 | -- |
| -- | 0.5 | 22.96 | -- |
| -- | 1 | 40.82 | -- |
| 0.2 | 0.1 | 35.52 | 0.47 |
| 0.2 | 0.5 | 49.58 | 0.58 |
| 0.2 | 1 | 78.82 | 0.49 |
| 0.4 | 0.1 | 42.02 | 0.34 |
| 0.4 | 0.5 | 55.83 | 0.39 |
| 0.4 | 1 | 85.41 | 0.42 |
| 0.8 | 0.1 | 59.13 | 0.12 |
| 0.8 | 0.5 | 64.83 | 0.10 |
| 0.8 | 1 | 88.14 | 0.11 |
As can be seen from Table 1, the acute myelogenous leukemia cell line THP-1 cells were treated with artesunate 0.2-0.8. Mu.M in combination with venetoclax 0.1-1. Mu.M for 72h. When artesunate and venetoclax are taken alone, the inhibition ratio of the growth of cells is less than 50 percent. The addition of artesunate obviously enhances the effect of venetoclax in inhibiting cell growth, and the combination index of the two medicines is calculated by using Compusyn software, wherein the combination index is more than 1, the two compounds are antagonism, and the combination index is less than 1, and the synergism is achieved. The combined index of artesunate and venetoclax is less than 0.6, and the synergistic effect of the two compounds is shown. The combined application of artesunate with the concentration of 0.8 mu M and venetoclax with the concentration of 0.1 mu M can inhibit the growth proportion of THP-1 cells to 59%, and CI=0.12, thus showing strong synergistic inhibition of cell growth.
TABLE 2 synergistic inhibition of MOLM-13 cell growth experiments with combinations of artesunate and venetoclax at different concentrations
| Artesunate (mu M) | Venetoclax(μM) | Growth inhibition ratio (%) | Combination index CI |
| 0.1 | -- | 7.89 | -- |
| 0.2 | -- | 13.61 | -- |
| 0.4 | -- | 27.83 | -- |
| -- | 0.0025 | 29.57 | -- |
| -- | 0.005 | 36.02 | -- |
| -- | 0.01 | 45.02 | -- |
| 0.1 | 0.0025 | 60.6 | 0.11 |
| 0.1 | 0.005 | 62.06 | 0.11 |
| 0.1 | 0.01 | 66.79 | 0.08 |
| 0.2 | 0.0025 | 66.49 | 0.15 |
| 0.2 | 0.005 | 69.9 | 0.13 |
| 0.2 | 0.01 | 74.24 | 0.08 |
| 0.4 | 0.0025 | 73.99 | 0.18 |
| 0.4 | 0.005 | 78.11 | 0.15 |
| 0.4 | 0.01 | 84.03 | 0.07 |
As can be seen from Table 2, the sensitivity of the acute myelogenous leukemia cell line MOLM-13 cells to artesunate and venetoclax was greater than that of THP-1 cells, and treatment with artesunate 0.1-0.4. Mu.M in combination with venetoclax 0.0025-0.01. Mu.M for 72h. When artesunate and venetoclax are taken alone, the inhibition ratio of the growth of cells is less than 50 percent. The addition of artesunate obviously enhances the effect of venetoclax in inhibiting cell growth, and the combination index of the artesunate and the venetoclax is less than 0.2 by calculating the combination index of the two medicines by using Compusyn software, so that the two compounds have very strong synergistic effect. When artesunate (0.2 mu M) and venetoclax (0.01 mu M) are combined, the growth inhibition ratio of MOLM-13 cells reaches 74%, CI=0.08, and the synergistic inhibition effect is strong.
According to experiments, artesunate and venetoclax perform the effect of synergistically killing AML cells by synergistically inducing apoptosis of the cells, but have limited effect on animal experiment level in vivo, so that a new combination scheme needs to be searched.
TABLE 3 Combined inhibition of THP-1 cell growth experiments with artesunate and cytarabine at different concentrations
As can be seen from Table 3, THP-1 cells were treated with artesunate at 0.2-0.8. Mu.M in combination with cytarabine at 0.25-1. Mu.M for 72h. When artesunate and cytarabine are taken alone, the cell growth inhibition ratio is less than 50%. Artesunate cannot obviously enhance the effect of cytarabine in inhibiting cell growth, and the combination index of the two medicines is calculated by using Compusyn software, wherein the combination index is more than 1, so that the two compounds have antagonism. The combination of artesunate at 0.8. Mu.M with venetoclax at 0.5. Mu.M inhibited THP-1 cell growth at 49%, CI=1.23, showed antagonism.
TABLE 4 Combined inhibition of MOLM-13 cell growth experiments with artesunate and cytarabine at different concentrations
| Artesunate (mu M) | Cytarabine (mu M) | Growth inhibition ratio (%) | Combination index CI |
| 0.1 | -- | 21.73 | -- |
| 0.2 | -- | 35.35 | -- |
| 0.4 | -- | 46.44 | -- |
| -- | 0.00125 | 3.74 | -- |
| -- | 0.0025 | 7.36 | -- |
| -- | 0.005 | 13.94 | -- |
| 0.1 | 0.00125 | 17.24 | 1.75 |
| 0.1 | 0.0025 | 26.97 | 0.95 |
| 0.1 | 0.005 | 24.81 | 1.31 |
| 0.2 | 0.00125 | 37.64 | 0.84 |
| 0.2 | 0.0025 | 40.47 | 0.82 |
| 0.2 | 0.005 | 44.68 | 0.73 |
| 0.4 | 0.00125 | 48.17 | 0.98 |
| 0.4 | 0.0025 | 50.94 | 0.88 |
As can be seen from Table 4, MOLM-13 cells were treated with artesunate at 0.1-0.4. Mu.M in combination with cytarabine at 0.00125-0.005. Mu.M for 72h. When artesunate and cytarabine are taken alone, the cell growth inhibition ratio is less than 50%. Artesunate cannot obviously enhance the effect of cytarabine in inhibiting cell growth, and the combination index of the two medicines is calculated by using Compusyn software, wherein the combination index is more than 0.7, and the synergy index is more than 1 at partial dosage, so that no obvious synergy effect exists between the two compounds. The combined application of artesunate with the concentration of cytarabine with the concentration of 0.005 mu M, wherein the growth inhibition ratio of THP-1 cells is 45%, CI=0.73, shows weak synergic inhibition effect on cell growth.
The experimental data show that artesunate and cytarabine cannot play a significant synergistic effect at the level of AML cells.
TABLE 5 synergistic inhibition of THP-1 cell growth experiments with combinations of venetoclax and cytarabine at different concentrations
| Venetoclax(μM) | Cytarabine (mu M) | Growth inhibition ratio (%) | Combination index CI |
| 0.1 | -- | 8.52 | -- |
| 0.5 | -- | 20.81 | -- |
| 1 | -- | 29.66 | -- |
| -- | 0.25 | 19.84 | -- |
| -- | 0.5 | 29.99 | -- |
| -- | 1 | 43.5 | -- |
| 0.1 | 0.25 | 37.61 | 0.51 |
| 0.1 | 0.5 | 49.82 | 0.52 |
| 0.1 | 1 | 61.22 | 0.49 |
| 0.5 | 0.25 | 43.18 | 0.57 |
| 0.5 | 0.5 | 53.25 | 0.53 |
| 0.5 | 1 | 67.52 | 0.41 |
| 1 | 0.25 | 52.43 | 0.63 |
| 1 | 0.5 | 59.68 | 0.56 |
| 1 | 1 | 72.68 | 0.41 |
As can be seen from Table 5, THP-1 cells were treated with venetoclax 0.1-1. Mu.M in combination with cytarabine 0.25-1. Mu.M for 72h. When Venetoclax and cytarabine are taken alone, the inhibition ratio of the growth of the cells is less than 50 percent. The addition of Venetoclax can obviously enhance the effect of cytarabine in inhibiting cell growth, and the combination index of the two medicines is calculated by using Compusyn software, wherein the combination index is less than 0.6, so that the two compounds have the effect of synergistically inhibiting cell growth. When vennetoclax of 0.1 μm was used in combination with cytarabine of 0.5 μm, the THP-1 cell growth inhibition ratio was 50%, ci=0.52, showing synergistic inhibition of cell growth.
TABLE 6 synergistic inhibition of MOLM-13 cell growth experiments with combinations of venetoclax and cytarabine at different concentrations
| Venetoclax(μM) | Cytarabine (mu M) | Growth inhibition ratio (%) | Combination index CI |
| 0.0025 | -- | 20.27 | -- |
| 0.005 | -- | 26.35 | -- |
| 0.01 | -- | 35.98 | -- |
| -- | 0.00125 | 9.2 | -- |
| -- | 0.0025 | 11.28 | -- |
| -- | 0.005 | 22.3 | -- |
| 0.0025 | 0.00125 | 43.63 | 0.2 |
| 0.0025 | 0.0025 | 56.27 | 0.13 |
| 0.0025 | 0.005 | 60.96 | 0.15 |
| 0.005 | 0.00125 | 48.8 | 0.24 |
| 0.005 | 0.0025 | 61.88 | 0.13 |
| 0.005 | 0.005 | 67.83 | 0.12 |
| 0.01 | 0.00125 | 62.64 | 0.17 |
| 0.01 | 0.0025 | 71.64 | 0.1 |
| 0.01 | 0.005 | 79.11 | 0.07 |
As can be seen from Table 6, MOLM-13 cells were treated with venetoclax 0.0025-0.01. Mu.M in combination with cytarabine 0.00125-0.005. Mu.M for 72h. When Venetoclax and cytarabine are taken alone, the inhibition ratio of the growth of the cells is less than 50 percent. The addition of Venetoclax can obviously enhance the effect of cytarabine in inhibiting cell growth, and the combination index of the two medicines is calculated by using Compusyn software, wherein the combination index is less than 0.3, so that the two compounds have stronger synergistic effect in inhibiting cell growth. When vennetoclax of 0.01 μm was used in combination with cytarabine of 0.005 μm, the THP-1 cell growth inhibition ratio was 79%, ci=0.07, showing a strong synergistic inhibition of cell growth.
Although cytarabine in combination with venetoclax has been approved for the treatment of elderly AML patients, clinical treatment has limited efficacy and there is a need to find new combination regimens.
In both AML cell lines, artesunate in combination with venetoclax and venetoclax in combination with cytarabine synergistically inhibited AML cell growth, and artesunate in combination with cytarabine did not exert a significant synergistic effect. According to the above experiments, the killing effect on AML cells was further investigated using artesunate in combination with three drug concentrations of venetoclax and cytarabine.
The experimental groups were then set up based on the effects of the individual substances and combinations of the two substances described above and the growth inhibition of AML cells was examined in the manner described above, see fig. 1.
Each experimental group for THP-1 cells is as follows: (1) a blank; (2) artesunate group (0.8 μm); (3) Venetoclax group (0.1. Mu.M); (4) cytarabine group (0.5 μm); (5) artesunate (0.8. Mu.M) +vennetoclax (0.1. Mu.M); (6) Artesunate (0.8 μm) +cytarabine (0.5 μm); (7) Venetoclax (0.1. Mu.M) +Cytarabine (0.5. Mu.M) group; (8) Artesunate (0.8. Mu.M) +venetoclax (0.1. Mu.M) +cytarabine (0.5. Mu.M).
Each experimental group for MOLM-13 cells is as follows: (1) a blank; (2) artesunate group (0.2 μm); (3) Venetoclax group (0.01. Mu.M); (4) cytarabine group (0.005 μm); (5) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M); (6) Artesunate (0.2 μm) +cytarabine (0.005 μm) group; (7) Venetoclax (0.01. Mu.M) +cytarabine (0.005. Mu.M) group; (8) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M) +cytarabine (0.005. Mu.M).
As can be seen from FIG. 1, the growth inhibition effect of different experimental groups on AML cells in THP-1 cells can be achieved, the artesunate+venetoclax group and the venetoclax+cytarabine group can inhibit 70% and 59% of cell growth, the activity is better than that of any single administration group (25% of artesunate, 31% of venetoclax and 38% of cytarabine), the artesunate and cytarabine are combined to inhibit 44% of cell growth, the growth inhibition effect of cytarabine or artesunate is not obviously enhanced, and the artesunate+venetoclax+cytarabine group can inhibit 85% of cell growth, and the activity is better than that of any two-medicine combined group.
Because MOLM-13 cells are more sensitive to artesunate, venetoclax and cytarabine than THP-1 cells, the effects of each group are that the artesunate+venetoclax group and the venetoclax+cytarabine group can inhibit 71% and 70% of cell growth, the activity is better than that of any single administration group (17% of artesunate, 38% of venetoclax and 26% of cytarabine), the artesunate and cytarabine are combined to inhibit 35% of cell growth, the growth inhibition effect of the cytarabine or the artesunate is not obviously enhanced, and the artesunate+venetoclax+cytarabine group can inhibit 92% of cell growth, and the activity is better than that of any two-medicine combined group. The artesunate is suggested to be capable of obviously enhancing the growth inhibition effect of the combination scheme of venetoclax and cytarabine on AML cells.
Example 2
Taking THP-1 cells and Molm-13 cells in logarithmic growth phase according to 10 respectively 5 Density inoculation is carried out in 6-well plates, and different medicines are added to different THP-1 and MOLM-13 cells for 24 hours or 12 hours respectively or in combination. THP-1 cells were grouped into (1) blank control groups as follows; (2) artesunate group (0.8 μm); (3) Venetoclax group (0.1. Mu.M); (4) cytarabine group (8 μm); (5) artesunate (0.8. Mu.M) +vennetoclax (0.1. Mu.M); (6) artesunate (0.8. Mu.M) +cytarabine (8. Mu.M) group; (7) Venetoclax (0.1. Mu.M) +Cytarabine (8. Mu.M) group; (8) Artesunate (0.8. Mu.M) +venetoclax (0.1. Mu.M) +cytarabine (8. Mu.M).
The MOLM-13 cell experiments were grouped as follows (1) blank; (2) artesunate group (0.2 μm); (3) Venetoclax group (0.01. Mu.M); (4) cytarabine group (0.08 μm); (5) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M); (6) Artesunate (0.2 μm) +cytarabine (0.08 μm) group; (7) Venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M) group; (8) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M). Detecting apoptosis using AV-PI kit (see figure 2);
as shown in the graph, in THP-1 cells, the artesunate+venetoclax group can induce 28% of cells to undergo apoptosis, the activity is better than that of any single administration group (4% of artesunate and 5% of venetoclax), the artesunate+cytarabine (8%) and the venetoclax+cytarabine group (9%) can not obviously induce the cells to undergo apoptosis, and the artesunate+venetoclax+cytarabine group can induce 38% of cells to undergo apoptosis, so that the activity is better than that of any two-drug combination group. In MOLM-13 cells, venetoclax alone can induce apoptosis of 25%, the apoptosis rate is increased to 43% or 45% in combination with artesunate or cytarabine, the activity is better than that of any single administration group (11% of artesunate, 25% of venetoclax and 14% of cytarabine), 15% of apoptosis can be induced in combination with cytarabine, the apoptosis induction effect of cytarabine or artesunate is not obviously enhanced, and 59% of apoptosis can be induced in the artesunate+venetoclax+cytarabine group, and the activity is better than that of any two-medicine combination group. The artesunate is suggested to be capable of obviously enhancing the apoptosis induction effect of the combination scheme of venetoclax and cytarabine on AML cells.
Example 3
Taking THP-1 cells and Molm-13 cells in logarithmic growth phase according to 10 respectively 5 Density inoculation in 6-well plates, THP-1 and MOLM-13 cells were treated with different drugs either alone or in combination for 24h or 12h. THP-1 cells were grouped into (1) blank control groups as follows; (2) artesunate group (0.8 μm); (3) Venetoclax group (0.1. Mu.M); (4) cytarabine group (8 μm); (5) artesunate (0.8. Mu.M) +vennetoclax (0.1. Mu.M); (6) artesunate (0.8. Mu.M) +cytarabine (8. Mu.M) group; (7) Venetoclax (0.1. Mu.M) +Cytarabine (8. Mu.M) group; (8) Artesunate (0.8. Mu.M) +venetoclax (0.1. Mu.M) +cytarabine (8. Mu.M).
The MOLM-13 cell experiments were grouped as follows (1) blank; (2) artesunate group (0.2 μm); (3) Venetoclax group (0.01. Mu.M); (4) cytarabine group (0.08 μm); (5) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M); (6) Artesunate (0.2 μm) +cytarabine (0.08 μm) group; (7) Venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M) group; (8) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M). Cell cycle changes were detected using PI single staining (see figure 3).
As shown in the graph, in THP-1 cells, the artesunate+venetoclax group and the venetoclax+cytarabine group can induce 26% and 19% of the increase of the cells in the Sub-G1 phase, the activity is better than that of any single administration group (4% of artesunate, 4% of venetoclax and 8% of cytarabine), the artesunate+cytarabine group cannot obviously induce the increase of the proportion of the cells in the Sub-G1 phase, and the artesunate+venetoclax+cytarabine group can induce 32% of the increase of the cells in the Sub-G1 phase, so that the activity is better than that of any two-drug combination group. In MOLM-13 cells, the venetoclax alone can induce 33% of the increase of the cell proportion of the Sub-G1 phase, the cell proportion of the Sub-G1 phase is increased to 45% and 40% by combining with artesunate and cytarabine, the activity is better than that of any single administration group (9% of artesunate, 33% of venetoclax and 11% of cytarabine), the artesunate and cytarabine can induce 14% of the increase of the cell proportion of the Sub-G1 phase by combining without obviously enhancing the effect of the cytarabine or the artesunate, and the artesunate+venetoclax+cytarabine group can induce 51% of the increase of the cell proportion of the Sub-G1 phase, and the activity is better than that of any two-medicine combination group. The artesunate is suggested to be capable of obviously enhancing the effect of combining venetoclax and cytarabine to induce AML cells to generate DNA fracture. The above results demonstrate that artesunate can significantly enhance the killing effect of venetoclax in combination with cytarabine on AML cells.
Example 4
The effect on single cell proliferation potential was examined using a soft agar colony formation assay in THP-1 and MOLM-13 cells.
20. Mu.L of the test drug was mixed into the bottom layer of soft agar, 5000 THP-1 cells and Molm-13 cells in the logarithmic phase were mixed into the upper layer of soft agar, respectively, and colonies with diameters of more than 75 μm (more than 50 cells) were counted under a dig microscope after 14 days (see FIG. 4).
THP-1 cells were grouped into (1) blank control groups as follows; (2) artesunate group (0.8 μm); (3) Venetoclax group (0.1. Mu.M); (4) cytarabine group (0.5 μm); (5) artesunate (0.8. Mu.M) +vennetoclax (0.1. Mu.M); (6) Artesunate (0.8 μm) +cytarabine (0.5 μm); (7) Venetoclax (0.1. Mu.M) +Cytarabine (0.5. Mu.M) group; (8) Artesunate (0.8. Mu.M) +venetoclax (0.1. Mu.M) +cytarabine (0.5. Mu.M).
The MOLM-13 cell experiments were grouped as follows (1) blank; (2) artesunate group (0.2 μm); (3) Venetoclax group (0.01. Mu.M); (4) cytarabine group (0.005 μm); (5) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M); (6) Artesunate (0.2 μm) +cytarabine (0.005 μm) group; (7) Venetoclax (0.01. Mu.M) +cytarabine (0.005. Mu.M) group; (8) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M) +cytarabine (0.005. Mu.M). As shown in the experimental results, artesunate alone cannot inhibit colony formation; venetoclax and cytarabine alone inhibit colony formation by no more than 30%, and have limited inhibition effect; the artesunate+vennetoclax group and the vennetoclax+cytarabine group inhibit 70% of colony formation, and the effect is better than that of any single administration group; the artesunate and cytarabine are combined to inhibit 50% of colony formation, and the inhibition effect of cytarabine on colony formation is not obviously enhanced; the artesunate+venetoclax+cytarabine group inhibits the formation of colonies by about 90 percent, and has better effect than any two-medicine combined group.
Example 5
To investigate the in vivo anti-AML effect of artesunate in combination with venetoclax and cytarabine:
right axillary subcutaneous inoculation of MOLM-13 cells 5X 10 in 40 female NOD-SCID mice 6 And/or just. When the tumor volume reaches 100mm 2 Randomly dividing the cells into 8 groups, (1) blank control group; (2) artesunate group (100 mg/kg); (3) Venetoclax group (100 mg/kg); (4) cytarabine group (50 mg/kg); (5) Artesunate (100 mg/kg) +venetoclax (100 mg/kg); (6) Artesunate (100 mg/kg) +cytarabine (50 mg/kg); (7) Venetoclax (100 mg/kg) +cytarabine (50 mg/kg); (8) Artesunate (100 mg/kg) +venetoclax (100 mg/kg) +cytarabine (50 mg/kg). Artesunate is dissolved in 10% (5% NaHCO) 3 ) +90% physiological saline, administered by intraperitoneal injection, 1 time per day, for 10 days; venetoclax was dissolved in 10% ethanol +30%PEG 400+60%Phosal50PG and administered orally, 1 time per day, for 10 consecutive days; cytarabine is dissolved in physiological saline and is administrated by intraperitoneal injection, 1 time a day, and 10 days continuously.
As can be seen from FIG. 5, the tumor inhibition effect of the artesunate group is relatively weak, and the tumor inhibition rate is 22.9%. Venetoclax and cytarabine can effectively inhibit tumor growth, and the tumor inhibition rates are 64.2% and 44.8% respectively. The tumor inhibition rate of the artesunate and cytarabine group is 44.8%, which is the same as that of the cytarabine group, and the artesunate cannot enhance the tumor inhibition effect of cytarabine. The tumor inhibition rates of the artesunate+venetoclax group and the venetoclax+cytarabine group are 68.2% and 75% respectively, and the tumor inhibition effect of the venetoclax is not obviously enhanced. The tumor inhibition rate of the artesunate, venetoclax and cytarabine group is 92%, and the tumor inhibition effect is superior to that of any two-medicine combined group. The body weight of mice in the artesunate+venetoclax+cytarabine group was not significantly changed during the course of the test.
Example 6
To simulate the true AML model, 35 female NOD-SCID mice were vaccinated with MOLM-13 cells 2X 10 via the tail vein 6 And/or just. After three days, the mice were randomly divided into 5 groups according to body weight, which were (1) blank control groups, respectively; (2) Venetoclax (100 mg/kg) group; (3) Artesunate (100 mg/kg) +venetoclax (100 mg/kg); (4) Venetoclax (100 mg/kg) +cytarabine (50 mg/kg); (5) Artesunate (100 mg/kg) +venetoclax (100 mg/kg) +cytarabine (50 mg/kg). Artesunate is dissolved in 10% (5% NaHCO) 3 ) +90% physiological saline, and is administered by intraperitoneal injection; venetoclax was dissolved in 10% ethanol +30%PEG 400+60%Phosal50PG and administered orally; cytarabine is dissolved in physiological saline and is administrated by intraperitoneal injection. Artesunate and venetoclax were administered 5 days per week for 4 weeks continuously; cytarabine was administered for 7 consecutive days. The body weight and survival time of the mice were recorded and survival curves of the mice were plotted using the Kaplan-Meier method. The THP-1 cell xenograft model and dosing regimen were identical to the MOLM-13 cell regimen (see FIG. 6).
As can be seen from fig. 6, the veneoclax group, the artesunate+veneoclax group, the venenoclax+cytarabine group and the artesunate+veneoclax+cytarabine group significantly prolonged the survival time of xenograft mice compared with the control group, and the artesunate+venenoclax+cytarabine group further prolonged the survival time of the veneoclax group, the artesunate+venenoclax group and the venenoclax+cytarabine group compared with the control group, and the ILS% reached 118.8%. In the tail vein xenograft THP-1 cell model, the survival time of xenograft mice is obviously prolonged in the artesunate+venetoclax group, the venetoclax+cytarabine group and the artesunate+venetoclax+cytarabine group compared with the control group, and the survival time of the artesunate+venetoclax+cytarabine group is further prolonged in the artesunate+venetoclax+cytarabine group compared with the venetoclax group, the artesunate+venetoclax group and the venetoclax+cytarabine group, and the ILS% reaches 45.1%. The above results demonstrate that artesunate is effective in enhancing the anti-AML effect of the venetoclax and cytarabine combination regimen in vivo.
Example 7
Taking THP-1 cells and Molm in logarithmic growth phase13 cells, respectively according to 10 5 Density inoculation is carried out in a 6-hole plate, different medicines are added into THP-1 and MOLM-13 cells to treat for 24 hours or 12 hours singly or in combination, and THP-1 cell experiments are grouped into a blank control group as follows (1); (2) artesunate group (0.8 μm); (3) Venetoclax group (0.1. Mu.M); (4) cytarabine group (8 μm); (5) artesunate (0.8. Mu.M) +vennetoclax (0.1. Mu.M); (6) artesunate (0.8. Mu.M) +cytarabine (8. Mu.M) group; (7) Venetoclax (0.1. Mu.M) +Cytarabine (8. Mu.M) group; (8) Artesunate (0.8. Mu.M) +venetoclax (0.1. Mu.M) +cytarabine (8. Mu.M). The MOLM-13 cell experiments were grouped as follows (1) blank; (2) artesunate group (0.2 μm); (3) Venetoclax group (0.01. Mu.M); (4) cytarabine group (0.08 μm); (5) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M); (6) Artesunate (0.2 μm) +cytarabine (0.08 μm) group; (7) Venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M) group; (8) Artesunate (0.2. Mu.M) +venetoclax (0.01. Mu.M) +cytarabine (0.08. Mu.M). The mechanism study of the combination of artesunate, venetoclax and cytarabine was performed by using the western-blot technique, and apoptosis-related proteins, DNA damaging proteins and replication stress checkpoint kinases were examined in THP-1 and MOLM-13 cells (see FIG. 7).
As shown in the experimental results, the artesunate-based combination scheme induces up-regulation of Noxa, which can exert an indirect inhibitory effect on Mcl-1 through binding to Mcl-1, whereas up-regulation of Noxa alone is insufficient to induce apoptosis and DNA damage in cells, requiring simultaneous inhibition of Bcl-2, venetoclax in combination with artesunate to synergistically induce cleavage of PARP and caspase-3 and up-regulation of gamma-H2A.X, further increases in the artesunate+venetoclax+cytarabine group, and concomitant down-regulation of Mcl-1. Cytarabine is the basic stone for AML treatment, cytarabine can be doped into DNA, DNA synthesis is inhibited, cell cycle retardation is caused, DNA damage is induced to play an anti-tumor role, but cytarabine treatment can simultaneously induce phosphorylation activation of replication stress checkpoint kinase Chk, a DNA damage repair process is started, further, a combination scheme based on cytarabine induces up-regulation of p-Chk1, and combination of artesunate and venetoclax induces down-regulation of caspase with dependent Mcl-1, so that up-regulation of p-Chk1 induced by cytarabine is overcome, and p-Chk1 and Mcl-1 levels of the artesunate+venetoclax+cytarabine group are lower compared with cytarabine groups, the artesunate+venetoclax+cytarabine groups. The results prove that artesunate enhances the apoptosis induction effect of venetoclax by inducing Noxa up-regulation, and simultaneously reduces the protein level of Mcl-1, thereby overcoming the activation of p-Chk1 induced by cytarabine and enhancing the DNA damage effect of cytarabine.
According to the invention, the expression levels of basic anti-apoptosis proteins of THP-1 and MOLM-13 cells are different, and the MOLM-13 cells express Bcl-2 in a high mode and simultaneously express Mcl-1 and Bim in a low mode, so that the MOLM-13 cells are sensitive to venetoclax and cytarabine; THP-1 cells highly express Bcl-2, mcl-1 and Bim and are relatively tolerant to venetoclax and cytarabine, so that artesunate, venetoclax and cytarabine with different concentrations are selected for treatment of two cells, and the combination of three drugs is found to produce stronger effect of killing AML cells than any combination of two drugs in the two cells.
In conclusion, compared with single artesunate, venetoclax, cytarabine or any two drug combinations, the invention combines the three drugs, namely, the artesunate induces apoptosis by inducing Noxa and synergizing venetoclax, and the protein level of Mcl-1 is down-regulated to overcome the p-Chk1 tolerance mechanism of cytarabine, so that the three drugs combined have stronger synergic effect of killing AML cells than any two drugs combined.
Claims (8)
1. A composition containing artemisinin derivatives is characterized in that the composition is prepared by mixing an A component, a B component and a C component according to the proportion of 8-40:1-2:1-80; wherein, the liquid crystal display device comprises a liquid crystal display device,
the component A is one or more of artesunate and analogues thereof;
the component B is one or more of venetoclax and analogues thereof;
the component C is one or more of cytarabine and analogues thereof.
2. The composition containing artemisinin derivatives according to claim 1, which is characterized in that: the component A is artesunate; the component B is vennetoclax; and the component C is cytarabine.
3. Use of the composition comprising artemisinin derivatives according to claim 1, characterized in that: the application of the composition in preparing medicines for treating blood tumor.
4. Use of a composition comprising an artemisinin derivative according to claim 3 characterized in that: the composition is applied to preparing medicines for treating acute myelogenous leukemia by inducing Noxa to reduce Mcl-1 and cooperatively inducing leukemia cells to apoptosis and enhancing DNA damage.
5. Use of the composition comprising artemisinin derivatives according to claim 4, characterized in that: the acute myelogenous leukemia is THP-1 or MOLM-13 cells.
6. A formulation of the artemisinin derivative-containing composition according to claim 1, characterized in that: the active ingredients of the preparation and a pharmaceutically acceptable carrier are mixed, wherein the active ingredients are the composition of claim 1, and the active ingredients account for 0.01-99% of the mass of the preparation.
7. The preparation of the composition containing artemisinin derivatives according to claim 6, which is characterized in that: the preparation is in the form of tablet, capsule or granule.
8. The preparation of the composition containing the artemisinin derivatives according to claim 7, which is prepared by mixing the components in the composition and then mixing the components with a pharmaceutically acceptable carrier, or by preparing the components in the composition and then mixing the components with pharmaceutically acceptable carriers.
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