Application of brucein D and Vinetoram in preparation of medicine for treating acute myelogenous leukemia

Abstract

The invention discloses an application of brucein D and Vinetoram in preparation of a medicine for treating acute or drug-resistant myelogenous leukemia. Through a large number of experimental screening, the brucein D and the Venotocork are combined for medication, and the test result shows that the brucein D and the Venotocork act on different targets and different signal channels of AML cells to generate a remarkable synergistic anti-tumor effect, and after the brucein D and the Venotocork are combined, the proliferation inhibition and apoptosis induction effects on the AML cells can be directly enhanced, and the anti-tumor effect of the Venotocork D and the AML cells can be improved. The compound can also reverse the drug resistance possibly occurring when the Vinetoram is singly used, and also has a remarkable killing effect on chemotherapy drug-resistant AML cells at the same time. The composition provided by the invention can reduce the dosage of antitumor drugs, reduce drug resistance, reduce adverse reactions and improve the life quality of patients, and has important application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of the combined use of crotonin D and venetoclax in the preparation of drugs for treating acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy originating from hematopoietic stem cells. The standard treatment for AML is intensive chemotherapy ("7+3" therapy) primarily using cytarabine (Ara-C) and daunorubicin, but disease heterogeneity and the presence of leukemia stem cells lead to a high relapse rate. Adult AML patients have a poor prognosis (5-year survival rate of 30%), with a survival rate of less than 10% for patients over 75 years of age. Venetoclax (VEN), a molecularly targeted inhibitor of the anti-apoptotic protein Bcl-2, has been approved for use in combination with DNA hypomethylating agents or low-dose Ara-C to treat AML patients over 75 years of age or those unsuitable for intensive chemotherapy, and has shown unprecedented remission rates in AML clinical trials. However, strong drug resistance and high relapse rates remain major clinical challenges. Therefore, the search for new drugs and treatment regimens targeting chemotherapy resistance and improving survival rates in elderly patients is of significant clinical and social value.

[0003] Combination therapy for cancer is currently a hot research topic. However, the combination of different drugs is a complex process. Different drugs have different mechanisms of action and may have different effects, such as antagonism or synergy. Summary of the Invention

[0004] Purpose of the Invention: To address the aforementioned problems, this invention, through extensive experimental screening, provides a novel combination therapy for newly diagnosed or relapsed / refractory acute myeloid leukemia (AML), particularly for patients resistant to conventional chemotherapy (cytarabine) or existing targeted therapies (such as the BCL-2 inhibitor venetoclax). This invention combines bruceline D with venetoclax, which synergistically overcomes resistance to venetoclax monotherapy and cytarabine treatment, enhancing the ability to induce apoptosis in AML cells. This provides a potentially significant combination therapy regimen for clinical treatment, especially for chemotherapy-resistant or high-risk AML patients.

[0005] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows: Application of crotonin D and venetoclax as the sole active ingredients in the preparation of drugs for the treatment of acute myeloid leukemia.

[0006] As an alternative, crotonin D and venetoclax are used as the sole active ingredients in the preparation of drugs for treating chemotherapy-resistant acute myeloid leukemia.

[0007] As a further preferred option, the use of crotonin D and venetoclax as the sole active ingredients in the preparation of drugs for treating acute myeloid leukemia that has developed resistance to cytarabine and venetoclax.

[0008] For the applications described above, the preferred molar ratio of crotonin D to venetoclax is 1:1 to 4:1.

[0009] As a further preferred option, the molar ratio of crotonin D to venetoclax is in the range of 1:1 to 2:1.

[0010] This invention relates to the preparation of pharmaceutical formulations from crotonin D, venetoclax, and a pharmaceutically acceptable carrier. These pharmaceutical formulations include tablets, pills, capsules, granules, oral liquids, and injections.

[0011] Beneficial effects: (I) This invention combines bruceine D with venetoclax, which act on different targets and signaling pathways in AML cells, producing a significant synergistic anti-tumor effect. Bruceine D works by inhibiting AML cell proliferation signaling pathways, inducing cell cycle arrest, and promoting apoptosis, while venetoclax primarily inhibits the anti-apoptotic function of BCL-2 protein, thus relieving the inhibition of AML cell apoptosis. The combination not only directly enhances the inhibitory effect on AML cell proliferation and the induction of apoptosis, but also reverses the drug resistance that occurs when venetoclax is used alone, and also has a significant killing effect on chemotherapy-resistant AML cells. In vitro experiments have confirmed that the combined drug combination increases the proliferation inhibition rate of newly diagnosed AML cell lines by 30%–83% or more compared to the single-drug group, and increases the proliferation inhibition rate of chemotherapy-resistant AML cell lines by 60%–87% or more compared to the single-drug group. In vivo animal experiments show that the combined drug group has significantly reduced human CD45 content and significantly prolongs the survival time of tumor-bearing mice.

[0012] (ii) Expand the scope of treatment and address clinical pain points

[0013] The combined drug combination of this invention is not only applicable to patients with newly diagnosed acute myeloid leukemia, providing them with a more effective treatment option, but also shows significant efficacy in patients with chemotherapy-resistant AML. Chemotherapy resistance is a major challenge in AML treatment, and currently there is a lack of effective treatment methods. The combined drug combination of this invention can overcome the limitations of chemotherapy resistance, effectively kill drug-resistant AML cells, alleviate the patient's condition, prolong the patient's survival time, fill a gap in clinical treatment, and has important clinical application value.

[0014] (iii) Reduce drug dosage and improve safety

[0015] Due to their significant synergistic effect, the dosages of Bruceine D and Venetoclax used in this invention are lower than those used in single-drug therapy, while achieving the same or better therapeutic effects. This reduces the potential toxic side effects of high-dose single-drug therapy. In vitro toxicity experiments and in vivo animal experiments have confirmed that the combined drug group has significantly lower toxicity to normal blood cells and vital organs (such as the heart, liver, kidneys, and lungs) than the high-dose single-drug group, exhibiting better tolerability and higher drug safety, which is beneficial for long-term clinical use and multi-cycle treatment.

[0016] (iv) The drug sources are wide-ranging and the formulation process is simple.

[0017] Bruceine D can be extracted and purified from the natural plant Brucea javanica, or it can be prepared by chemical synthesis. It is widely available and relatively inexpensive. Venetoclax is a commonly used clinical drug and is readily available. The combined drug formulation of this invention has a simple process and can be prepared using conventional pharmaceutical processes. It requires no special equipment or technology, is easy to industrialize, and can meet the needs of large-scale clinical application.

[0018] (v) Broad application prospects

[0019] The combined drug combination of the present invention can not only be used directly for the treatment of acute myeloid leukemia, but can also be used in combination with other treatment methods (such as chemotherapy, radiotherapy, hematopoietic stem cell transplantation, etc.) to further improve the treatment effect. In addition, based on its synergistic anti-AML mechanism, it can also provide new ideas and references for the treatment of other hematological malignancies (such as chronic myeloid leukemia, lymphoma, etc.), and its application prospects are very broad. Attached Figure Description

[0020] Figure 1 Bruceine D from croton seeds kills acute myeloid leukemia cells, cytarabine-resistant cells, and case cells.

[0021] Figure 2 Bruceine D overcomes Venetoclax resistance in vitro.

[0022] Figure 3 The combination of Bruceine D and Venetoclax has a synergistic effect, exhibits low cytotoxicity to normal cells, and partially kills AML cells through endogenous apoptosis.

[0023] Figure 4 Bruceine D combined with Venetoclax in vivo xenograft model synergistically kills drug-resistant AML. Detailed Implementation

[0024] The present invention will be further explained below with reference to embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0025] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all those that can be purchased through legitimate channels.

[0026] Example 1: Bruceine D from croton seeds kills acute myeloid leukemia cells, cytarabine-resistant cells, and case cells.

[0027] 1.1 Materials and Methods

[0028] 1.1.1 Experimental Materials

[0029] Drugs: Cytarabine (Ara-C) was purchased from AbMole (CAS No.: 147-94-4); Bruceine D was purchased from Chenguang Biotech (CAS No.: 21499-66-1), and prepared as a 10 mM stock solution with DMSO.

[0030] Materials: RPMI-1640 medium was purchased from Gibico; fetal bovine serum was purchased from ABW; CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) was purchased from ABBKine; apoptosis kit was purchased from Bestbio; lymphocyte separation medium was purchased from Solarbio; ITS was purchased from Beyotime.

[0031] 1.1.2 Cell lines and cell culture

[0032] Human acute myeloid cells MV4-11, MOLM-13, and THP-1 were donated to the Cancer Translational Medicine Laboratory of the College of Life Sciences, Jilin University, and cultured in RPMI-1640 complete medium containing 10% FBS.

[0033] Cytarabine Ara-C resistant cells: Cells were cultured for six months with the concentration of Ara-C gradually increased in the culture medium to obtain AML cell lines resistant to Ara-C, named MOLM-13 / AraC-R and MV4-11 / AraC-R, respectively. They were stably grown in RPMI-1640 complete medium containing 10% FBS with 1000 nM and 1100 nM Ara-C, respectively.

[0034] AML case cells: cultured in RPMI-1640 complete medium containing 20% ​​FBS, 20% 5637 cell supernatant (providing a source of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-1β, macrophage colony-stimulating factor and stem cell factor) and 1*ITS.

[0035] All complete culture media contained 100 U / mL penicillin and streptomycin. All cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0036] 1.1.3 Density gradient centrifugation of AML clinical samples

[0037] Dilute AML blood samples with an equal volume of sterile PBS. Add an appropriate amount of separation buffer to a centrifuge tube (add 3 mL of separation buffer if the diluted blood volume is <3 mL; add an equal volume of separation buffer if ≥3 mL. However, the total volume of both should not exceed two-thirds of the centrifuge tube, otherwise it will affect the centrifugation effect). Use a disposable sterile plastic dropper to aspirate the diluted blood sample and spread it evenly above the separation buffer surface, ensuring a clear interface between the volumes. Centrifuge at room temperature, horizontal rotor, 500-1000 g for 20-30 min. After centrifugation, the cells will separate into four layers: the top layer is diluted plasma, the middle layer is clear separation buffer, the white membrane layer between the plasma and separation buffer is the lymphocyte layer, and the bottom of the centrifuge tube contains erythrocytes and granulocytes. Carefully aspirate the white lymphocyte or mononuclear cell layer with a dropper. Add this to another centrifuge tube containing 7-8 mL of PBS, mix well, centrifuge at 250 g for 10 min, discard the supernatant, resuspend the pellet in complete culture medium, and count the cells.

[0038] 1.1.4 Effect of drugs on the proliferation of acute myeloid leukemia cells detected by CCK8 assay

[0039] The drug stock solution was 10 mM. The dosage was calculated based on the required concentration and added to each well of a 96-well plate at a rate of 10 μL, for a final solution volume of 100 μL. Serial dilution was used, with serum-free 1640 as the diluent and 10 μL of diluent added to the blank control. Logarithmic growth phase AML cells were collected, and the cell suspension concentration was adjusted. 90 μL of the cell suspension was seeded into each well of a 96-well plate to achieve a cell density of 3000-6000 cells / well. The edge wells were filled with sterile PBS. After culturing for 24 h at 37℃, 5% CO2, and saturated humidity, 10 μL of CCK8 was added to each well, and the cells were cultured for another 1-4 h. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability and inhibition rate were calculated. Cell viability (%) = (OD drug - OD blank) / (OD control - OD blank) × 100%.

[0040] 1.1.5 Annexin V-FITC / PI double staining flow cytometry detection of cell apoptosis

[0041] After drug treatment, cells were collected by centrifugation at 2000 rpm for 5 min, washed once with 1 mL PBS, and the supernatant was discarded. 50 µL of binding buffer was added to the cell pellet, followed by 3 µL of Annexin V staining solution. The mixture was incubated at room temperature in the dark for 15 min. 6 µL of PI staining solution was then added, and the mixture was incubated at room temperature in the dark for 5 min. 450 µL of binding buffer was added to each tube, and the mixture was filtered through a single layer of 300-mesh nylon mesh into a flow cytometry tube for analysis.

[0042] 1.1.6 Effect of shRNA silencing of Bax / Bak on Bruceine D-induced apoptosis.

[0043] Bax, Bak, and non-targeting control (NTC) shRNA lentiviral vectors were purchased from Sigma-Aldrich. pMD-VSV-G, delta8.2, and the lentiviral shRNA constructs were transfected into TLA-HEK293T cells using Lipofectamine and Plus transfection reagent (Thermo Fisher Scientific). Cell stability assays were performed after transfection.

[0044] 1.1.7 Statistical Analysis

[0045] Experimental data are expressed as mean ± standard error (X̄ ± SEM). Graphpad 9.5.1 software was used for data processing. Error bars in the graph represent ± standard error of mean (SEM). Significant differences were determined using an unpair-wise two-sample t-test; p < 0.05 indicated a statistically significant difference.

[0046] 1.2 Experimental Results

[0047] like Figure 1 As shown in AD, the inhibition rate IC50 was calculated by performing cell proliferation experiments with different doses of Bruceine D. 50 Bruceine D showed good activity in different AML maternal cells, cytarabine-resistant cells, and case cells, with an IC50 value of [missing information]. 50 Between 100 nM and 3 µM. Figure 1 E&F assays were performed to detect the cell-killing ability of bruceine D in maternal AML and drug-resistant AML. The results showed that low concentrations of bruceine D still exhibited good activity in drug-resistant cell lines. To investigate the effect of bruceine D on endogenous apoptosis, the key endogenous apoptosis proteins Bax and Bak were silenced. Figure 1 G&H results showed that even after silencing Bax and Bak, Bruceine D could still partially kill cells, thus Bruceine D kills AML cells through partial endogenous apoptosis.

[0048] Example 2: Bruceine D overcomes Venetoclax resistance in vitro

[0049] 2.1 Materials and Methods

[0050] 2.1.1 Experimental Materials

[0051] Venetoclax was purchased from AbMole (CAS No.: 1257044-40-8), otherwise as per 1.1.1.

[0052] 2.1.2 Cell lines and cell culture

[0053] Venetoclax-resistant cells: Cells were cultured for six months with progressively increasing Venetoclax concentrations to obtain AML cell lines resistant to Ara-C, named MOLM-13 / VEN-R and MV4-11 / VEN-R, respectively. These cells were stably grown in RPMI-1640 complete medium containing 1000 nM Venetoclax, 10% fetal bovine serum, and 100 U / mL penicillin / streptomycin. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0054] 2.1.3 Effect of CCK8 assay on the proliferation of venetoclax-resistant cells

[0055] The drug stock solution was 10 mM. The dosage was calculated based on the required concentration and added to each well of a 96-well plate at a rate of 10 μL, for a final solution volume of 100 μL. Serial dilution was used, with serum-free 1640 as the diluent and 10 μL of diluent added to the blank control. Logarithmic growth phase AML cells were collected, and the cell suspension concentration was adjusted. 90 μL of the cell suspension was seeded into each well of a 96-well plate to achieve a cell density of 3000-6000 cells / well. The edge wells were filled with sterile PBS. After culturing for 24 h at 37℃, 5% CO2, and saturated humidity, 10 μL of CCK8 was added to each well, and the cells were cultured for another 1-4 h. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability and inhibition rate were calculated. Cell viability (%) = (OD drug - OD blank) / (OD control - OD blank) × 100%.

[0056] 2.1.4 Annexin V-FITC / PI double staining flow cytometry detection of cell apoptosis

[0057] After drug treatment, cells were collected by centrifugation at 2000 rpm for 5 min, washed once with 1 mL PBS, and the supernatant was discarded. 50 µL of binding buffer was added to the cell pellet, followed by 3 µL of Annexin V staining solution. The mixture was incubated at room temperature in the dark for 15 min. 6 µL of PI staining solution was then added, and the mixture was incubated at room temperature in the dark for 5 min. 450 µL of binding buffer was added to each tube, and the mixture was filtered through a single layer of 300-mesh nylon mesh into a flow cytometry tube for analysis.

[0058] 2.1.5 Statistical Analysis

[0059] Experimental data are expressed as mean ± standard error (X̄ ± SEM). Graphpad 9.5.1 software was used for data processing. Error bars in the graph represent ± standard error of mean (SEM). Significant differences were determined using an unpair-wise two-sample t-test; p < 0.05 indicated a statistically significant difference.

[0060] 2.2 Experimental Results

[0061] like Figure 2 As shown in AC, the inhibition rate IC was calculated by performing cell proliferation experiments with different doses of Bruceine D. 50 Venetoclax-resistant cells showed more than 30 times the resistance of the parent cell line. Different concentrations of Bruceine D were added to the Venetoclax-resistant cells... Figure 2 D&E results showed that the IC50 of Bruceine D was... 50 Decreasing or increasing venetoclax concentrations can enhance the sensitivity of bruceline D. Furthermore, flow cytometry experiments (…) Figure 2 F&G confirmed that the combination of Bruceine D and Venetoclax can kill more Venetoclax-resistant cells.

[0062] Example 3: The combination of Bruceine D and Venetoclax has a synergistic effect, with low cytotoxicity to normal cells and partial killing of AML cells through endogenous apoptosis.

[0063] 3.1 Materials and Methods

[0064] 3.1.1 Experimental Materials

[0065] Drugs: Cytarabine (Ara-C) was purchased from AbMole (CAS No.: 147-94-4); Venetoclax was purchased from AbMole (CAS No.: 1257044-40-8); Bruceine D was purchased from Chenguang Biotech (CAS No.: 21499-66-1).

[0066] 3.1.2 Cell lines and cell culture

[0067] Same as 1.1.1 and 2.1.2

[0068] 3.1.3 Annexin V-FITC / PI double staining flow cytometry detection of cell apoptosis

[0069] After drug treatment, cells were collected by centrifugation at 2000 rpm for 5 min, washed once with 1 mL PBS, and the supernatant was discarded. 50 µL of binding buffer was added to the cell pellet, followed by 3 µL of Annexin V staining solution. The mixture was incubated at room temperature in the dark for 15 min. 6 µL of PI staining solution was then added, and the mixture was incubated at room temperature in the dark for 5 min. 450 µL of binding buffer was added to each tube, and the mixture was filtered through a single layer of 300-mesh nylon mesh into a flow cytometry tube for analysis.

[0070] Table 1. Combined concentrations of BruceineD and Venetoclax for AML cells over 24 h.

[0071] 3.1.4 Calculation of Combination Drug Index (CI) to evaluate the anti-pancreatic cancer activity of combination drugs

[0072] The inhibition rate of cells under different drug doses was obtained using the CCK8 assay. The inhibition rate and corresponding drug dose were input into CompuSyn software to calculate the CI value. A CI value <1 indicates synergy, a CI value = 1 indicates additive, and a CI value >1 indicates antagonism.

[0073] 3.1.5 Application of Data Models

[0074] SynergyFinder 3.0 software was used to estimate the expected drug combination response based on the ZIP reference model. This software allows for interactive analysis and visualization of multi-drug combination data. Positive and negative synergy scores can be observed in the dose region of the synergy map, representing synergy (red) and antagonism (green), respectively.

[0075] 3.1.6 Statistical Analysis

[0076] Experimental data are expressed as mean ± standard error (X̄ ± SEM). Graphpad 9.5.1 software was used for data processing. Error bars in the graph represent ± standard error of mean (SEM). Significant differences were determined using an unpair-wise two-sample t-test; p < 0.05 indicated a statistically significant difference.

[0077] 3.2 Experimental Results

[0078] In AML resistant to cytarabine and maternal AML, Bruceine D was combined with Venetoclax. Figure 3The AC results showed that the synergy coefficients (CI) were all less than 0.2, and the ZIP model score calculated using SynergyFinder was greater than 10 (Mean: 46.21, p<0.05). Figure 3 (D) The results indicate that the combined effect of the two drugs is excellent. Flow cytometry was used to detect the toxicity of the combined drugs in normal human blood cells, and the results showed that the combined drugs could not induce apoptosis in normal cells, indicating that the combined drugs have low toxicity (Figure EG). Furthermore, after silencing Bax and Bak, the combined drugs partially killed AML cells through endogenous apoptosis ( Figure 3 HK).

[0079] Example 4: Bruceine D combined with Venetoclax in an in vivo xenograft model synergistically kills drug-resistant AML.

[0080] 4.1 Materials and Methods

[0081] 4.1.1 Experimental Materials

[0082] Drugs: Cytarabine (Ara-C) was purchased from AbMole (CAS No.: 147-94-4); Venetoclax was purchased from AbMole (CAS No.: 1257044-40-8); Bruceine D was purchased from Chenguang Biotech (CAS No.: 21499-66-1).

[0083] Animals: NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Animal Qualification Certificate: A202509120457), female, 6 weeks old, and housed at the Experimental Animal Center of Nanjing University of Chinese Medicine.

[0084] 4.2 Animal Experiments

[0085] 4.2.1 Establishment of AML hematologic malignancy model

[0086] NCG mice were housed in a sterile laboratory with purified air on a laminar flow rack under controlled humidity and temperature. After one week of adaptive growth, experiments were conducted to establish a tail vein injection xenograft model. Animal experiments strictly adhered to the regulations for the management and protection of experimental animals of Nanjing University of Chinese Medicine. MOLM-13 / Ara-C resistant cells in the logarithmic growth phase were collected and resuspended in an appropriate amount of PBS (1x10⁻⁶ cells / mL). 5 1 cell / mouse; 0.2 mL / injection solution; day 0).

[0087] 4.2.2 Animal grouping and drug dosage

[0088] (1) Drug-resistant control group (5 animals; IP; physiological saline; QD)

[0089] (2) Ara-C monotherapy group (5 animals; IP; 30 mg / kg Bruceine D; 0.1 mL / 10g; QD, administered for 14 days);

[0090] (3) Bruceine D monotherapy group (5 animals; IP; 1.5 mg / kg Bruceine D; 0.1 mL / 10g; QOD, administered for 14 days);

[0091] (4) Venetoclax monotherapy (5 animals; PO; 50 mg / kg; 0.1 mL / 10g; QOD, administered for 14 days);

[0092] (5) The Bruceine D + Venetoclax combination group had the same dosage as the single drug group and was administered for 14 days;

[0093] (Drug administration was discontinued when one mouse in the drug-resistant control group developed leukemia symptoms). During the study, mice were assessed at least twice daily, and their weight was recorded daily.

[0094] 4.2.3 Observation and detection indicators

[0095] (1) Observation of animal behavior or special indicators

[0096] Weight measurement: The weight of all mice was recorded daily using an electronic scale and statistically analyzed.

[0097] (2) Observation of behavior and signs as experimental endpoint indicators: All animals exhibited AML disease model characteristics such as hind limb paralysis, arched back, ruffled fur, anemia, and weight loss. If disease characteristics were not observed, the experiment was terminated on day 100 of modeling. (Day 0 was the day when AML cells were injected to establish the AML model).

[0098] (3) Euthanasia is used as a merciful endpoint when animals cannot tolerate the experiment and euthanasia is necessary. For example:

[0099] a. Observe the survival status of mice daily.

[0100] b. The mice suffered from hind limb paralysis and were unable to eat or drink.

[0101] c. Weight loss (>20% of initial body weight)

[0102] d. Loss of appetite for 24 hours or poor appetite (less than 50% of normal) for three days.

[0103] e. Weakness (inability to eat or drink): When unable to eat or drink for up to 24 hours without anesthesia or sedation, or only able to stand with difficulty.

[0104] 4.2.4 Organization of collection, testing indicators and methods.

[0105] (1) Euthanasia is performed by dislocating the cervical spine.

[0106] (2) Pathological examination of the spleen and liver of mice

[0107] Spleen and liver were harvested, washed with physiological saline, and the size of the spleen was observed and photographed on calipers. After blotting with filter paper, the spleen was weighed using a precision balance and the weight recorded. The spleen and liver were then preserved in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The structure and lesions of the spleen and liver were observed under a microscope, and photographs were taken and recorded. Positive findings: Destruction of the red and white pulp structures in the spleen, with diffuse infiltration of leukemia cells. Leukemia cell aggregation was observed around the portal vein or in the sinusoidal space of the liver.

[0108] (3) Flow cytometry detection of hCD45+ expression in mouse bone marrow

[0109] a. Preparation of bone marrow single-cell suspension: Femur and tibia were taken, both ends were cut off, and bone marrow cells were flushed out with pre-cooled physiological saline using a 1 mL syringe. The residue was filtered through a 70 μm cell sieve, and 300 g of the collected cell suspension was centrifuged at 4°C for 5 min, and the supernatant was discarded.

[0110] b. Red blood cell lysis: Wash bone marrow cells with 1 ml of pre-cooled PBS, centrifuge at 300 g, 4°C for 5 min, and discard the supernatant; add 1 ml of red blood cell lysis buffer, resuspend and mix well, incubate at room temperature for 2-3 minutes, stop immediately after the liquid becomes clear, centrifuge at 300 g, 4°C for 5 min, and discard the supernatant; wash bone marrow cells with 1 ml of pre-cooled PBS (containing 2% FBS) 1-2 times, centrifuge at 300 g, 4°C for 5 min, and discard the supernatant.

[0111] c. Antibody staining: Resuspend and wash cells in 100 μL FACS buffer, centrifuge at 300g, 4℃ for 5 min, and discard the supernatant; add antibodies according to the instructions. For the experimental group, add human CD45-APC. Add 2 mL FACS buffer, centrifuge at 300g for 5 min, repeat twice. Resuspend in 300 μL FACS buffer, pass through a 300-mesh cell sieve, and load into flow cytometry tubes for instrumentation.

[0112] d. Flow cytometry detection and analysis: Gating strategy: FSC-A / SSC-A to select lymphocyte populations. Quantification of AML cell proportion using human CD45-APC⁺ gating. Data analysis using FlowJo software.

[0113] 4.2.5 Statistical Analysis

[0114] Experimental data are expressed as mean ± standard error (X̄ ± SEM). Graphpad 9.5.1 software was used for data processing. Error bars in the graph represent ± standard error of mean (SEM). Significant differences were determined using an unpair-wise two-sample t-test; p < 0.05 indicated a statistically significant difference.

[0115] 4.3 Experimental Results

[0116] Dosing regimen such as Figure 4 As shown in Figure A, the results indicated that compared with the control group, the combined drug administration group significantly prolonged the survival time of mice. Figure 4 B), and can reduce spleen weight in mice (B), and can reduce spleen weight in mice. Figure 4 D). The proportion of human CD45 was detected by flow cytometry. Figure 4 E&F results showed that the combined drug administration group significantly reduced the proportion of human leukemia cells, and the combined drug group mice did not show significant changes in body weight, indicating low toxicity. Figure 4 C).

Claims

1. Application of crotonin D and venetoclax as the sole active ingredients in the preparation of drugs for the treatment of acute myeloid leukemia.

2. Application of Brucea javanica extract D and venetoclax as the sole effective components in the preparation of drugs for treating chemotherapy-resistant acute myeloid leukemia.

3. The application according to claim 2, characterized in that, Application of crotonin D and venetoclax as the sole active ingredients in the preparation of drugs for treating acute myeloid leukemia that has developed resistance to cytarabine and venetoclax.

4. The application according to claim 1, characterized in that, The molar ratio of crotonin D to venetoclax ranges from 1:2 to 2:

1.

5. The application according to claim 3, characterized in that, The molar ratio of crotonin D to venetoclax ranges from 4:1 to 1:

20.

6. The application according to any one of claims 1 to 5, characterized in that, The crotonin D and venetoclax were prepared into a pharmaceutical formulation using a pharmaceutically acceptable carrier.

7. The application according to claim 6, characterized in that, The pharmaceutical preparations mentioned include tablets, pills, capsules, granules, oral liquids, and injections.

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