Application of pharmaceutical composition containing brucea javanica bitter alcohol in preparation of medicine for treating leukemia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a pharmaceutical composition containing brucea javanica to preparation of a medicine for treating leukemia. The invention relates to application of a pharmaceutical composition containing brucea javanica in preparation of drugs for treating leukemia, in particular to application of a pharmaceutical composition containing brucea javanica and a pharmaceutical composition containing brucea javanica in preparation of drugs for treating acute myeloid leukemia or chronic myeloid leukemia. The components of the composition can be used at the same time or in any sequence.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an application of a pharmaceutical composition containing brucein in the preparation of a drug for treating leukemia. Background Art

[0002] Myeloid leukemia is a blood cancer caused by the abnormal proliferation of hematopoietic stem cells in the bone marrow, characterized by uncontrolled myeloid cell growth and impaired differentiation and apoptosis. Myeloid leukemia is divided into acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). AML is a highly heterogeneous, extremely lethal leukemia triggered by multiple oncogenes. The standard clinical treatment is cytarabine combined with daunorubicin or idarubicin. This regimen has been used for over 40 years, but only 25%-50% of patients survive long-term after treatment. AML can be categorized into two groups based on molecular biology and genetics: favorable and poor prognosis. Twenty-five percent of AML patients harbor FLT3-ITD mutations, placing them in the poor prognosis group. Currently, four FLT3-ITD inhibitors are approved for the treatment of AML, but these drugs only achieve short-lived remissions in patients and require combination with chemotherapy. The pathogenesis of CML is relatively simple. 90% of patients are induced by the t(9;22) BCR-ABL fusion gene, and the disease progresses slowly, making it a chronic disease. BCR-ABL is a mutated, self-activated tyrosine kinase. As early as 1990, the BCR-ABL kinase inhibitor imatinib (STI-571) was approved. Subsequently, second- and third-generation inhibitors that overcome imatinib resistance were developed, significantly improving the survival and quality of life of CML patients. However, patients require long-term medication and are not cured. Once drug resistance develops, the prognosis is extremely poor. Overcoming drug resistance and combining drugs are key to improving the treatment of myeloid leukemia.

[0003] Chemotherapy and targeted therapy exert their anti-leukemic effects by inducing apoptosis. Apoptotic escape is a major mechanism for the survival and drug resistance of myeloid leukemia. The mitochondrial pathway is a key apoptosis pathway regulated by the Bcl-2 family of proteins. The anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are three key proteins that protect cells from death. They are functionally complementary and jointly determine cell fate by controlling the activation of the apoptosis execution proteins Bak / Bax. AML cells express Bcl-2 and Mcl-1, while CML cells express Bcl-xL and Mcl-1. The Bcl-2 inhibitor venetoclax (ABT-199) has shown significant efficacy in CLL patients and is approved by the US FDA. However, in AML, its therapeutic effect is only transient in a minority of patients, possibly related to the inability of venetoclax to inhibit Mcl-1. High Mcl-1 expression reduces sensitivity to venetoclax and induces direct or indirect resistance. Mcl-1 is a key protein that maintains the survival of leukemia cells and plays a more important pro-survival role than Bcl-2 and Bcl-xL. Mcl-1 inhibitors are still under development. FLT3-ITD inhibitors reduce Mcl-1 protein-induced apoptosis. BCR-ABL inhibitors reduce Bcl-xL and Mcl-1-induced apoptosis and play a major role in the treatment of leukemia. Mcl-1 is elevated in drug-resistant cells, protecting against apoptosis. Drug-resistant cells are highly dependent on rapid protein synthesis, and targeting protein translation is one of the effective methods to overcome drug resistance. Brucea bruceiol, a quasinoid compound derived from the Brucea brucei plant, is a potential protein synthesis inhibitor that is worthy of further research and development. Summary of the Invention

[0004] In order to overcome the defects of the prior art, the present invention provides a pharmaceutical composition containing brucein for use in preparing a drug for treating leukemia.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The invention discloses an application of a pharmaceutical composition containing brucein in the preparation of a drug for treating leukemia.

[0007] The invention relates to an application of the brucea javanica-containing piroplasm alcohol and the pharmaceutical composition in preparing medicine for treating acute myeloid leukemia or chronic myeloid leukemia.

[0008] The invention relates to an application of the pharmaceutical composition containing bruceiol in the preparation of a drug for treating acute myeloid leukemia with FLT3-ITD mutation.

[0009] The invention relates to an application of the pharmaceutical composition containing bruceiol in the preparation of a drug for treating chronic myeloid leukemia with BCR-ABL mutation.

[0010] The pharmaceutical composition containing brucein is brucein and an inhibitor, wherein the inhibitor is one or more of a Bcl-2 inhibitor, a Bcl-2 / Bcl-xL inhibitor, a FLT3-ITD inhibitor, a BCR-ABL inhibitor or derivatives of the corresponding inhibitors.

[0011] The brucein can be brucein or its derivatives (derivatives are brucein derivatives, including brucein B, demethylbrucein and brucein B, etc.), and the brucein has a structure shown in Formula I as follows:

[0012]

[0013] The Bcl-2 inhibitor venetoclax is a structure shown in Formula II or an analog thereof:

[0014]

[0015] The Bcl-2 / Bcl-xL inhibitor navitoclax is a structure represented by Formula III or an analog thereof:

[0016]

[0017] The FLT3-ITD inhibitor sorafenib is a compound represented by formula IV or its analogues:

[0018]

[0019] The FLT3-ITD inhibitor gilteritinib is a compound represented by formula V or an analog thereof:

[0020]

[0021] The BCR-ABL inhibitor imatinib is a compound represented by formula VI or its analogues:

[0022]

[0023] The BCR-ABL inhibitor dasatinib is a compound represented by formula VII or an analog thereof:

[0024]

[0025] In the pharmaceutical composition of the present invention, the components are not limited to the above-mentioned compounds themselves, and drugs with the same mechanism of action include Bcl-2 inhibitor Sonrotoclax, Bcl-xL inhibitor A155463, FLT3-ITD inhibitors Midostaurin and Crenolanib, and BCR-ABL inhibitors Nilotinib, Ponatinib, Bosutinib, Asciminib, and Overembatinib.

[0026] The invention relates to the use of a composition of brucein and the Bcl-2 inhibitor venetoclax (ABT-199) or the Bcl-xL inhibitor navitoclax (ABT-263) in the preparation of a drug for treating acute myeloid leukemia and chronic myeloid leukemia; wherein the molar ratio of brucein to the Bcl-2 inhibitor venetoclax in the composition is in the range of 1:25-8:1; and the molar ratio of brucein to the Bcl-2 / Bcl-xL inhibitor navitoclax is in the range of 1:40-4:5.

[0027] The invention relates to the use of a composition of brucein and a FLT3-ITD inhibitor (sorafenib) or gilteritinib in the preparation of a drug for treating acute myeloid leukemia with FLT3-ITD mutation; wherein the molar ratio of brucein to the FLT3-ITD inhibitor in the composition is in the range of 1:160-10:1.

[0028] The invention relates to a method for preparing a composition of brucein and the BCR-ABL inhibitor imatinib or dasatinib for treating chronic myeloid leukemia with a BCR-ABL mutation; wherein the molar ratio of brucein to the BCR-ABL inhibitor in the composition is in the range of 1:8-4:1.

[0029] The brucea alcohol and the Bcl-2 inhibitor or Bcl-2 / Bcl-xL inhibitor or FLT3-ITD inhibitor or BCR-ABL inhibitor in the composition can be used simultaneously or in any order, for example, the brucea alcohol and the Bcl-2 inhibitor or Bcl-2 / Bcl-xL inhibitor or FLT3-ITD inhibitor or BCR-ABL inhibitor can be administered to the patient at the same time; the brucea alcohol can also be administered to the patient first, followed by the Bcl-2 inhibitor or Bcl-2 / Bcl-xL inhibitor or FLT3-ITD inhibitor or BCR-ABL inhibitor, or the Bcl-2 inhibitor or Bcl-2 / Bcl-xL inhibitor or FLT3-ITD inhibitor or BCR-ABL inhibitor can be administered first, followed by the brucea alcohol.

[0030] The above-mentioned various compositions can be prepared into pharmaceutical preparations suitable for gastrointestinal or parenteral administration by conventional methods in the art. In the present invention, the Bcl-2 inhibitor, Bcl-2 / Bcl-xL inhibitor, FLT3-ITD inhibitor, or BCR-ABL inhibitor is preferably prepared into a pharmaceutical preparation for gastrointestinal administration. The preparation can be in the form of conventional tablets or capsules, or controlled-release or sustained-release preparations.

[0031] In the present invention, there is no particular limitation on the method for preparing the pharmaceutical composition. Brucea bruceiol and the Bcl-2 inhibitor or Bcl-2 / Bcl-xL inhibitor can be directly mixed and then prepared into a preparation, or they can be mixed separately with / or with corresponding excipients to prepare preparations and then packaged together according to conventional methods in the art, or they can be mixed separately with corresponding excipients and then mixed to prepare a preparation.

[0032] The present invention evaluates the anti-tumor activity of brucein in drug-resistant AML cells and CML cells and its mechanism of action in reducing Mcl-1. The pharmaceutical combination of brucein and the Bcl-2 inhibitor venetoclax induces apoptosis of AML cell lines HL-60 and MOLM-13; the pharmaceutical combination of brucein and the Bcl-2 / Bcl-xL inhibitor navitoclax induces apoptosis of CML cell lines K562 and KCL22; the pharmaceutical combination of brucein and the FLT3-ITD inhibitors sorafenib and gilteritinib induces FLT3-ITD mutation. The results showed that the combination of bruceiol and the Bcl-2 inhibitor venetoclax or the Bcl-2 / Bcl-xL inhibitor navitoclax in the treatment of AML or CML, and the combination of bruceiol and the FLT3 inhibitor or BCR-ABL inhibitor in the treatment of FLT3-ITD AML or BCR-ABL CML had significant synergistic effects, which improved the efficacy of the drugs and reduced the dosage of individual drugs.

[0033] Advantages of the present invention: The brucea alcohol provided by the present invention inhibits the effect of Mcl-1 in overcoming drug resistance to treat leukemia. The brucea alcohol pharmaceutical composition one provided by the present invention contains brucea alcohol and the Bcl-2 inhibitor venetoclax (ABT-199). Brucea alcohol and venetoclax synergistically induce AML cell apoptosis. The brucea alcohol pharmaceutical composition two provided by the present invention contains brucea alcohol and the Bcl-2 / Bcl-xL inhibitor navitoclax (ABT-263). Brucea alcohol and navitoclax synergistically induce CML cell apoptosis. The brucea alcohol pharmaceutical composition three provided by the present invention contains brucea alcohol and the FLT3 inhibitor sorafenib or gilteritinib, and the composition synergistically induces cell apoptosis with sorafenib and gilteritinib. The present invention provides a fourth brucein pharmaceutical composition containing brucein and the BCR-ABL inhibitors imatinib and dasatinib. This composition synergistically induces cell apoptosis with imatinib and dasatinib. This composition can be developed into a combination drug regimen or formulation for the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram showing the protein expression effects of bruceiol in MOLM-13 and MOLM-13 / SOR cells at different concentrations provided in an embodiment of the present invention.

[0035] Figure 2 This is a diagram showing the protein expression effect of bruceiol in drug-resistant K562 / STI cells at different concentrations provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The essential contents of the present invention are further illustrated below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0037] The present invention's brucein induces apoptosis in drug-resistant cells by inhibiting Mcl-1, and the resistant cells are more sensitive than the parental cells. The present invention provides four pharmaceutical compositions containing brucein for use in preparing drugs for treating leukemia, specifically a pharmaceutical composition comprising brucein and the Bcl-2 inhibitor venetoclax (ABT-199) for treating AML. A pharmaceutical composition comprising brucein and the Bcl-2 / Bcl-xL inhibitor navitoclax (ABT-263) for treating CML. A pharmaceutical composition comprising brucein and the FLT3 inhibitors sorafenib and gilteritinib for use in preparing drugs for treating FLT3-ITD AML. A pharmaceutical composition comprising brucein and the BCR-ABL inhibitors imatinib and dasatinib for use in preparing drugs for treating BCR-ABL CML. The composition improves the current treatment status of AML and CML by synergistically inducing cell apoptosis.

[0038] Reagents and methods:

[0039] Cells: AML cells HL-60 were purchased from American Type Culture Collection (ATCC), AML cells MOLM-13 were purchased from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), and CML cells K562 and KCL22 were purchased from American Type Culture Collection (ATCC).

[0040] The drug-resistant cells of the above cells are MOLM-13 Sorafenib-resistant cell line MOLM-13 / SOR and K562 Imatinib-resistant cell line K562 / STI.

[0041] The above-mentioned MOLM-13 / SOR cell line was constructed using conventional methods. MOLM-13 cells were continuously stimulated with increasing doses of sorafenib, and cell growth was ultimately maintained at a dose of 500 nM sorafenib, thereby obtaining the sorafenib-resistant cell line MOLM-13 / SOR for future use. Before the experiment began, sorafenib was removed and cultured for one week.

[0042] K562 / STI cells were constructed using conventional methods. K562 cells were continuously stimulated with increasing doses of imatinib, and cell growth was ultimately maintained at a dose of 2 μM imatinib, thereby obtaining the imatinib-resistant cell line K562 / STI for future use. Before the experiment began, imatinib was removed and cultured for one week.

[0043] Drugs: The pharmaceutical compositions used in the following examples were prepared according to the following methods; dacholecalciferol was purchased from Chengdu Zhibiao Huachun Biotechnology Co., Ltd.; venetoclax, navitoclax, sorafenib, gilteritinib, imatinib and dasatinib were all purchased from Selleck.

[0044] Accurately weigh brucein, venetoclax, navitoclax, sorafenib, gilteritinib, imatinib, and dasatinib and dissolve them in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Store at -20°C and dilute to the appropriate concentration in culture medium before use. In all experiments, the final DMSO concentration was strictly controlled to ensure that it did not affect cell viability.

[0045] The cells were cultured in a medium containing 10% inactivated fetal bovine serum, 10 mmol / L L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C and 5% CO 2 in a saturated humidity environment.

[0046] In the following examples, the detection methods of cell apoptosis induction, growth inhibition and protein levels were carried out according to the following methods:

[0047] Method 1: Cells in logarithmic growth phase were divided into 10 5Cells were seeded at different densities in 24-well plates and treated with different drug concentrations. After treatment for different durations, the percentage of apoptotic cells was assessed using AOEB staining. After drug treatment, 1 ml of the mixed culture medium was centrifuged and washed once with PBS. After resuspending in PBS, the dye was added and 300 cells were counted under a fluorescence microscope to calculate the percentage of apoptotic cells. The synergy index (CI) of the drug combination was calculated using Compusyn software.

[0048] Method 2: Cells in logarithmic growth phase were divided into 10 5 Cells were seeded at varying density in 24-well plates and treated with various drug concentrations. After treatment for varying durations, cell growth inhibition was assessed using the trypan blue exclusion assay. After mixing, an equal volume of trypan blue dye was added and the cells were counted using a hemocytometer. Blue-stained cells were considered dead. The drug-induced cell growth inhibition rate and cell viability were calculated, and the GI50 (GI50) was calculated using GraphPad Prism 6 software.

[0049] Method 3: Cells in logarithmic growth phase were divided into 10 5 Cells were seeded at different densities in 6-well plates and different drug concentrations were added to the cells. The cells were collected and lysed by adding NP 40 cell lysis buffer. Protein concentration was determined using a BCA kit. Electrophoresis was performed using an 8%-12% denaturing polypropylene gel. After the electrophoresis, the proteins were transferred to a nitrocellulose membrane. The bands of the desired molecular weight were cut out and incubated with blocking solution at room temperature for 1 hour. The membranes were washed three times with TBST and incubated with the primary antibody at 4°C overnight. The next day, the bands were removed, washed three times with TBST, and the secondary antibody was added and incubated at room temperature for 1 hour. The membranes were washed three times with TBST and once with TBS. The membranes were then developed with ECL luminescent solution to detect protein expression levels.

[0050] Example 1

[0051] The growth inhibition effect of brusatol and sorafenib in parental MOLM-13 cells and sorafenib-resistant MOLM-13 / SOR cells and the function of reducing Mcl-1 were implemented, wherein the growth inhibition effect was determined according to the above method 2. 50 The Mcl-1-reducing function was determined according to the method described in Method 3 above. Figure 1 .

[0052] Table 1

[0053]

[0054] MOLM-13 contains FLT3-ITD mutations, representing FLT3-ITD AML, and is sensitive to the FLT3-ITD inhibitor sorafenib. As shown in Table 1 above, the resistance index of the resistant cell line MOLM-13 / SOR to sorafenib is more than 30 times higher than that of the parental cells. The growth inhibitory effect of Brusatol on the parental and resistant cells was investigated. The GI of Brusatol in the two cell lines was significantly different. 50 The values ​​were similar, suggesting that bruceiol could overcome the resistance to FLT3-ITD inhibitor Sorafenib.

[0055] like Figure 1 As shown, treatment of MOLM-13 and MOLM-13 / SOR cells with 10-40 nM bruceiol for 24 h could decrease Mcl-1 protein in a concentration-dependent manner.

[0056] Example 2

[0057] Brucea bruceiol and imatinib inhibit the growth of parental K562 cells and imatinib-resistant K562 / STI cells and reduce the function of Mcl-1, wherein the growth inhibition effect is determined according to the above method 2. 50 The Mcl-1-reducing function was determined according to the method described in Method 3 above. Figure 2 .

[0058] Table 2

[0059]

[0060] K562 cells are a CML cell line expressing the BCR-ABL gene and are sensitive to the BCR-ABL inhibitor Imatinib. As shown in Table 2 above, the resistance index of the resistant cell line K562 / STI cells to Imatinib is about 20 times higher than that of the parental cells. 50 The values ​​were 12.54±0.92nM and 13.94±0.99nM, respectively, indicating that there was no significant difference in the sensitivity of Brusatol to the two cells, suggesting that brusatol can overcome the resistance to the BCR-ABL inhibitor Imatinib.

[0061] like Figure 2 As shown, treatment of K562 and K562 / STI cells with 10-40 nM bruceiol for 24 h could decrease the Mcl-1 protein in a concentration-dependent manner.

[0062] Example 3

[0063] In the experiment of synergistically promoting apoptosis of HL-60 cells by the combination of brucein and venetoclax at different concentrations, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 3.

[0064] Table 3

[0065] BR (nM) Venetoclax (nM) Apoptosis rate (%) Combined Index 5 2.7 10 3.9 20 16.7 40 23.3 12.5 8.3 25 9.5 50 20.9 5 12.5 10 0.95090 5 25 16.7 0.79418 5 50 24.9 0.75808 10 12.5 18.9 0.58532 10 25 25 0.56682 10 50 30 0.66046 20 12.5 43.3 0.31714 20 25 50.3 0.28542 20 50 53.3 0.31929 40 12.5 50 0.46383 40 25 67 0.27376 40 50 68.3 0.28835

[0066] The AML cell line HL-60 was treated with brucein at 5-40 nM and venetoclax at 12.5-50 nM for 24 hours. Bruceinin and venetoclax alone induced <25% apoptosis. The combination of brucein and venetoclax exerted a synergistic apoptosis-inducing effect, with 40 nM brucein combined with 50 nM venetoclax inducing apoptosis in >60% of HL-60 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and venetoclax was <1, indicating synergistic activity between the two compounds.

[0067] Example 4

[0068] In the experiment of synergistically promoting apoptosis of MOLM-13 cells by the combination of brucein and venetoclax at different concentrations, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 4.

[0069] Table 4

[0070] BR (nM) Venetoclax (nM) Apoptosis rate (%) Combined Index 1.25 5 2.5 8.7 5 12 10 20 20 46.7 2.5 5.7 5 10.3 10 17 1.25 2.5 9.7 0.93643 1.25 5 16.7 0.73102 1.25 10 24.3 0.73700 2.5 2.5 14.7 0.80631 2.5 5 19.3 0.77744 2.5 10 40 0.38493 5 2.5 23 0.72378 5 5 26 0.74532 5 10 50 0.32389 10 2.5 60 0.23106 10 5 76.7 0.11113 10 10 78.7 0.11716

[0071] The AML cell line MOLM-13 was treated with 1.25-20 nM bruceanol and 2.5-10 nM venetoclax for 24 hours. Bruceanol alone induced apoptosis in a maximum of 46.7% of cells, while venetoclax alone induced apoptosis in a maximum of 17%. The addition of venetoclax significantly enhanced the apoptosis-inducing effect of bruceanol. The combination of 10 nM bruceanol and 5 nM venetoclax resulted in a 76.7% apoptosis rate in MOLM-13 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of bruceanol and venetoclax was <1, indicating synergistic effects between the two compounds.

[0072] Example 5

[0073] In the experiment of synergistically promoting apoptosis of KCL22 cells by the combination of brucein and navitoclax at different concentrations, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 5.

[0074] Table 5

[0075] BR (nM) Navitoclax (nM) Apoptosis rate (%) Combined Index 5 2.5 10 2.6 20 5.1 40 9.3 50 3.3 100 6 200 9.3 5 50 8.1 0.44383 5 100 10.9 0.49564 5 200 14.4 0.60556 10 50 14.9 0.23029 10 100 24 0.16767 10 200 30 0.19782 20 50 23.1 0.15806 20 100 45 0.05958 20 200 52.6 0.06552 40 50 29.4 0.15889 40 100 64.3 0.02814 40 200 71.1 0.02793

[0076] The CML cell line KCL22 was treated with brucein at concentrations of 5-40 nM and navitoclax at concentrations of 50-200 nM for 24 hours. Bruceinin and navitoclax alone had weak apoptosis-inducing abilities, with apoptosis rates of less than 10%. However, the combination of brucein and navitoclax exerted a synergistic apoptosis-inducing effect, with 40 nM brucein and 200 navitoclax inducing apoptosis in >70% of KCL22 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and navitoclax was <1, indicating synergistic activity between the two compounds.

[0077] Example 6

[0078] In the experiment of synergistically promoting apoptosis of K562 cells by the combination of brucein and navitoclax at different concentrations, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 6.

[0079] Table 6

[0080] BR (nM) Navitoclax (nM) Apoptosis rate (%) Combined Index 5 0.8 10 0.9 20 1.3 40 2.6 50 1.0 100 1.6 200 4 5 50 2.7 0.43462 5 100 3.3 0.62593 5 200 4.9 0.78349 10 50 3.6 0.36467 10 100 4.2 0.52160 10 200 5.5 0.71759 20 50 14.7 0.07249 20 100 19.9 0.08856 20 200 23.1 0.13978 40 50 17.9 0.06540 40 100 26.7 0.06352 40 200 50.2 0.04240

[0081] The CML cell line K562 was treated with brucein at concentrations of 5-40 nM and navitoclax at concentrations of 50-200 nM for 24 hours. Bruceinin and navitoclax alone had weak apoptosis-inducing abilities, with apoptosis rates of less than 10%. However, the combination of brucein and navitoclax exerted a synergistic apoptosis-inducing effect, with 40 nM brucein and 200 navitoclax inducing apoptosis in >50% of K562 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and navitoclax was <1, indicating synergistic activity between the two compounds.

[0082] Example 7

[0083] In the experiment of synergistically promoting apoptosis of MOLM-13 cells by the combination of brucein and sorafenib at different concentrations, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 7.

[0084] Table 7

[0085] BR (nM) Sorafenib (nM) Apoptosis rate (%) Combined Index 2.5 3.7 5 11 10 29.3 20 51.7 2 2.3 10 7.3 50 36.7 2.5 2 9.7 0.75552 2.5 10 26.3 0.54355 2.5 50 69 0.30506 5 2 14 0.98701 5 10 38 0.52861 5 50 76.7 0.27433 10 2 33.7 0.85955 10 10 55.3 0.54090 10 50 78.3 0.36800 20 2 59 0.84599 20 10 70. 0.65028 20 50 80 0.55299

[0086] MOLM-13 cells were treated with 2.5-20 nM bruceanol and 2-50 nM sorafenib for 24 hours. Bruceanol alone induced apoptosis in a maximum of 51.7% of cells, while sorafenib alone induced apoptosis in a maximum of 36.7%. The addition of sorafenib significantly enhanced the apoptosis-inducing effect of bruceanol. The combination of 20 nM bruceanol and 50 nM sorafenib resulted in an apoptotic rate of 80% in MOLM-13 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of bruceanol and sorafenib was <1, indicating synergistic activity between the two compounds.

[0087] Example 8

[0088] In the experiment of synergistically promoting apoptosis of MOLM-13 cells by the combination of different concentrations of brucein and gilteritinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 8.

[0089] Table 8

[0090] BR (nM) Gilteritinib (nM) Apoptosis rate (%) Combined Index 2.5 4.3 5 14.7 10 30 20 35.3 2 8.3 10 23.7 50 69 2.5 2 23.7 0.50814 2.5 10 63.7 0.28639 2.5 50 76.7 0.63363 5 2 36.7 0.44064 5 10 70 0.26680 5 50 80 0.55380 10 2 60 0.33365 10 10 76.7 0.26783 10 50 83.3 0.49883 20 2 70 0.42649 20 10 80. 0.35170 20 50 90 0.34838

[0091] MOLM-13 cells were treated with 2.5-20 nM bruceanol and 2-50 nM gilteritinib for 24 hours. Bruceanol alone induced apoptosis in a maximum of 35.5% of cells, while sorafenib alone induced apoptosis in a maximum of 69%. The addition of gilteritinib significantly enhanced the apoptosis-inducing effect of bruceanol. The combination of 20 nM bruceanol and 50 nM gilteritinib resulted in a 90% apoptosis rate in MOLM-13 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of bruceanol and sorafenib was <1, indicating synergistic effects between the two compounds.

[0092] Example 9

[0093] In the experiment of synergistically promoting apoptosis of MOLM-13 / SOR cells by the combination of different concentrations of brucein and sorafenib, the corresponding synergistic index (CI) was determined according to the above method, see Table 9.

[0094] Table 9

[0095] BR (nM) Sorafenib (nM) Apoptosis rate (%) Combined Index 2.5 2 5 6.7 10 20 20 30.3 100 1 200 1.7 400 11 2.5 100 3.3 0.99332 2.5 200 4.7 0.92584 2.5 400 21 0.25412 5 100 9.3 0.83219 5 200 14 0.61351 5 400 25 0.37484 10 100 23.3 0.72024 10 200 33.7 0.49897 10 400 42 0.39174 20 100 44.7 0.69663 20 200 46.7 0.65986 20 400 53.7 0.54141

[0096] MOLM-13 / SOR cells were treated with 2.5-20 nM bruceanol and 100-400 nM sorafenib for 24 hours. Bruceanol alone induced apoptosis in a maximum of 30.3% of cells, while sorafenib alone induced apoptosis in a maximum of 11%. The addition of sorafenib significantly enhanced the apoptosis-inducing effect of bruceanol. The combination of 20 nM bruceanol and 400 nM sorafenib resulted in a 53.7% apoptosis rate in MOLM-13 / SOR cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of bruceanol and sorafenib was <1, indicating synergistic activity between the two compounds.

[0097] Example 10

[0098] In the experiment of synergistically promoting apoptosis of MOLM-13 / SOR cells by the combination of different concentrations of brucein and gilteritinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 10.

[0099] Table 10

[0100] BR (nM) Gilteritinib (nM) Apoptosis rate (%) Combined Index 2.5 2.3 5 6 10 16.7 20 31.7 50 1 100 1.3 200 4.7 2.5 50 4 0.99565 2.5 100 9.7 0.58504 2.5 200 20.7 0.38915 5 100 14 0.69015 5 200 30 0.39090 10 50 24 0.69923 10 100 32.7 0.53396 10 200 40 0.46366 20 50 40 0.77330 20 100 50 0.58561 20 200 56.2 0.51002

[0101] MOLM-13 / SOR cells were treated with brucein at concentrations of 2.5-20 nM and gilteritinib at concentrations of 50-200 nM for 24 hours. Bruceinin alone induced apoptosis in a maximum of 31.7% of cells, while gilteritinib alone induced apoptosis in a maximum of 4.7%. Gilteritinib significantly enhanced the apoptosis-inducing effect of brucein. The combination of 20 nM brucein and 200 nM gilteritinib resulted in a 56.2% apoptosis rate in MOLM-13 / SOR cells. The combination index of the two drugs was calculated using Compusyn software. Both brucein and gilteritinib had a combination index of <1, indicating synergistic effects between the two compounds.

[0102] Example 11

[0103] In the experiment of synergistically promoting apoptosis of K562 cells by the combination of different concentrations of brucein and imatinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 11.

[0104] Table 11

[0105] BR (μM) Imatinib (μM) Apoptosis rate (%) Combined Index 0.125 1.67 0.25 5 0.5 6 1 8.67 0.25 3 0.5 4 1 7.33 0.125 0.25 6.33 0.51844 0.125 0.5 9.67 0.41998 0.125 1 11.67 0.52694 0.25 0.25 9 0.44445 0.25 0.5 12.67 0.35773 0.25 1 14 0.46316 0.5 0.25 10.67 0.56247 0.5 0.5 16.33 0.35282 0.5 1 18 0.40267 1 0.25 14 0.66519 1 0.5 20.67 0.39788 1 1 29.33 0.25406

[0106] K562 cells were treated with brucein 0.125-1 μM and imatinib 0.25-1 μM for 48 hours. Brucein and imatinib alone showed weak apoptosis-inducing activity, with apoptosis rates below 10%. The combination of imatinib and brucein exhibited a synergistic apoptosis-inducing effect, with a 1 μM dose of brucein combined with 1 μM imatinib resulting in a 29.3% apoptosis rate in K562 cells. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and imatinib was <1, indicating synergistic activity between the two compounds.

[0107] Example 12

[0108] In the experiment of synergistically promoting apoptosis of K562 cells by the combination of different concentrations of brucein and dasatinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 12.

[0109] Table 12

[0110] BR (μM) Dasatinib (μM) Apoptosis rate (%) Combined Index 0.125 1.67 0.25 3 0.5 4.67 1 7 0.25 15 0.5 18 1 20.67 0.125 0.25 19.67 0.34565 0.125 0.5 23 0.33512 0.125 1 26 0.37000 0.25 0.25 22.67 0.20787 0.25 0.5 26.33 0.19642 0.25 1 32.67 0.13169 0.5 0.25 24 0.19974 0.5 0.5 30.67 0.12178 0.5 1 50.67 0.02113 1 0.25 28.33 0.16048 1 0.5 35.67 0.09845 1 1 59 0.01893

[0111] K562 cells were treated with brucein 0.125-1μM and dasatinib 0.25-1μM for 48 hours. Brucein induced apoptosis in a maximum of 7% of cells when used alone, while dasatinib induced apoptosis in a maximum of 20.7% of cells when used alone. The addition of dasatinib significantly enhanced the apoptosis-inducing effect of brucein. The apoptosis rate in K562 cells reached 59% when brucein was combined with 1μM dasatinib. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and dasatinib was <1, indicating synergistic activity between the two compounds.

[0112] Example 13

[0113] In the experiment of synergistically promoting apoptosis of K562 / STI cells by the combination of different concentrations of brucein and imatinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 13.

[0114] Table 13

[0115] BR (μM) Imatinib (μM) Apoptosis rate (%) Combined Index 0.125 9 0.25 14.67 0.5 17 1 22.33 0.25 2 0.5 5.33 1 12.33 0.125 0.25 14 0.89224 0.125 0.5 19 0.92055 0.25 0.25 19.67 0.71606 0.25 0.5 26 0.71363 0.5 0.25 27 0.57433 0.5 0.5 33 0.59752 0.5 1 40 0.77415 1 0.25 32.67 0.57412 1 0.5 46.67 0.38718 1 1 57 0.46104

[0116] K562 / STI cells were treated with brucein 0.125-1μM and imatinib 0.25-1μM for 48 hours. Brucein induced apoptosis in a maximum of 22.3% of cells when used alone, while imatinib induced apoptosis in a maximum of 12.3% of cells when used alone. The addition of imatinib significantly enhanced the apoptosis-inducing effect of brucein. The apoptosis rate in K562 / STI cells reached 57% when brucein was combined with 1μM imatinib. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and imatinib was <1, indicating synergistic effects between the two compounds.

[0117] Example 14

[0118] In the experiment of synergistically promoting apoptosis of K562 / STI cells by the combination of different concentrations of brucein and dasatinib, the corresponding synergistic index (CI) was determined according to the above method, as shown in Table 14.

[0119] Table 14

[0120] BR (μM) Dasatinib (μM) Apoptosis rate (%) Combined Index 0.125 6 0.25 13 0.5 13.67 1 22 0.25 17.67 0.5 30 1 40 0.125 0.25 24 0.76163 0.125 0.5 40 0.55963 0.125 1 48 0.72706 0.25 0.25 28.33 0.64808 0.25 0.5 46.33 0.42863 0.25 1 60.67 0.39561 0.5 0.25 45.67 0.29203 0.5 0.5 55 0.30355 0.5 1 65.67 0.31644 1 0.25 49 0.32125 1 0.5 64 0.21801 1 1 83.67 0.09858

[0121] K562 / STI cells were treated with brucein 0.125-1μM and dasatinib 0.25-1μM for 48 hours. Brucein induced apoptosis in a maximum of 22% of cells when used alone, while dasatinib induced apoptosis in a maximum of 40% of cells when used alone. The addition of dasatinib significantly enhanced the apoptosis-inducing effect of brucein. The apoptosis rate in K562 / STI cells reached 83.7% when brucein was combined with 1μM dasatinib. The combination index of the two drugs was calculated using Compusyn software. The combination index of brucein and dasatinib was <1, indicating synergistic effects between the two compounds.

Claims

1. Use of a pharmaceutical composition containing brucein in the preparation of a drug for treating leukemia.

2. The use according to claim 1, characterized in that: The invention relates to an application of the brucea javanica-containing piroplasm alcohol and the pharmaceutical composition in preparing medicine for treating acute myeloid leukemia or chronic myeloid leukemia.

3. The use according to claim 2, characterized in that: The invention relates to an application of the pharmaceutical composition containing bruceiol in the preparation of a drug for treating acute myeloid leukemia with FLT3-ITD mutation.

4. The use according to claim 2, characterized in that: The invention relates to an application of the pharmaceutical composition containing bruceiol in the preparation of a drug for treating chronic myeloid leukemia with BCR-ABL mutation.

5. The use according to any one of claims 1 to 4, characterized in that: The pharmaceutical composition containing brucein is brucein and an inhibitor, wherein the inhibitor is one or more of a Bcl-2 inhibitor, a Bcl-2 / Bcl-xL inhibitor, a FLT3-ITD inhibitor, a BCR-ABL inhibitor or derivatives of the corresponding inhibitors.

6. The use according to claim 5, characterized in that: The invention relates to an application of a composition of brucein and the Bcl-2 inhibitor venetoclax (ABT-199) or the Bcl-xL inhibitor navitoclax (ABT-263) in the preparation of a drug for treating acute myeloid leukemia and chronic myeloid leukemia.

7. The use according to claim 5, characterized in that: The invention relates to an application of the composition of brucein and FLT3-ITD inhibitor sorafenib or gilteritinib in the preparation of a drug for treating acute myeloid leukemia with FLT3-ITD mutation.

8. The use according to claim 5, characterized in that: The invention relates to an application of a composition of brucein and a BCR-ABL inhibitor imatinib or dasatinib in the preparation of a drug for treating chronic myeloid leukemia with BCR-ABL mutation.

9. Use of bruceiol in the preparation of an inhibitor of Mcl-1-induced apoptosis of drug-resistant cells.

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