The composition of arsenic trioxide and veneclade and its application in the preparation of drugs for treating leukemia.

Abstract

This invention relates to a composition of arsenic trioxide and veneclade and its application in the preparation of drugs for treating leukemia, belonging to the field of biomedical technology. To address the technical problem in existing technologies where arsenic trioxide, although capable of repairing mutant p53 function, cannot effectively induce apoptosis in TP53-mutant AML, this invention provides a composition of arsenic trioxide and veneclade, with a molar ratio of arsenic trioxide to veneclade of 1:10-25. This invention demonstrates the synergistic antitumor effect of the combined treatment of arsenic trioxide and veneclade in TP53-mutant AML. Experiments show that the combination index remains stable at 0.3-0.8. In animal experiments, the tumor inhibition rate in the combined treatment group reached 67%, the median survival was extended to 46.5 days, and no serious toxic reactions occurred. This invention provides a novel combined treatment regimen for patients with TP53-mutant AML and has significant clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to the composition of arsenic trioxide and Veneclair and its application in the preparation of drugs for the treatment of leukemia. Background Technology

[0002] TP53-mutant acute myeloid leukemia (AML) accounts for 15-20% of newly diagnosed AML cases and is a recognized independent poor prognostic factor in AML. This disease originates from hematopoietic stem cells in the bone marrow. Due to the failure of the p53 pathway caused by TP53 gene mutations, patients generally exhibit chemotherapy resistance, high relapse rates, and short overall survival, making treatment extremely difficult. Traditional chemotherapy regimens are ineffective, and hematopoietic stem cell transplantation is limited by donor matching and the patient's physical condition. Therefore, there is an urgent need to develop new and effective treatment strategies.

[0003] Arsenic trioxide (ATO) is a small molecule drug proven to repair the function of mutated p53 in the human body. Theoretically, functional p53 repaired by ATO could exert anti-cancer effects by inhibiting tumor cell proliferation, regulating the cell cycle, and initiating endogenous apoptosis. However, current studies have shown that ATO's monotherapy efficacy in non-APL-type acute myeloid leukemia is extremely limited, with no significant inhibition of tumor burden or prolongation of patient survival. This indicates that even if ATO successfully repairs mutated p53, a single drug is still insufficient to overcome multiple apoptosis resistance barriers in tumor cells and cannot effectively drive apoptosis in leukemia cells.

[0004] Existing research clearly shows that TP53 mutation-mediated apoptosis resistance is driven by multiple pathways, including abnormal upregulation of MCL-1, inhibition of the caspase pathway, high expression of Survivin, and the acquisition of new functions by mutated p53 and activation of compensatory survival pathways. This is also the core reason why arsenic trioxide repair of p53 still fails to induce apoptosis. How to overcome the above-mentioned multiple apoptosis resistance and amplify the pro-apoptotic effect after p53 activation is a technical challenge that urgently needs to be solved in this field.

[0005] Venetoclax (VEN), a highly selective BCL-2 inhibitor, exerts its anti-AML effect by blocking the anti-apoptotic function of BCL-2 and relieving mitochondrial apoptosis inhibition, and has become a key drug in AML treatment. However, TP53-mutant AML patients are prone to primary or acquired resistance to veneclax, involving multiple factors such as p53 pathway failure, MCL-1 / BCL-XL upregulation, and activation of compensatory survival pathways. This means that the strategy of inhibiting BCL-2 has limited effectiveness in TP53-mutant AML and is difficult to meet clinical needs. Under this technical guidance, researchers have never attempted to combine arsenic trioxide with veneclax for the treatment of TP53-mutant acute myeloid leukemia. Summary of the Invention

[0006] To address the technical problem that while arsenic trioxide can repair the function of mutant p53 cells, it cannot effectively induce apoptosis in TP53-mutant AML, this invention provides a composition of arsenic trioxide and Veneclare and its application in the preparation of leukemia treatment drugs.

[0007] The technical solution of this invention:

[0008] A composition of arsenic trioxide and veneclade, wherein the molar ratio of arsenic trioxide to veneclade is 1:10~25, and the molecular formula of veneclade is C2. 43 H 44 ClN7O5S, with the following structural formula:

[0009] .

[0010] Furthermore, the molar ratio of arsenic trioxide to Venecra is 1:22.7.

[0011] Furthermore, the composition is a combination of a first formulation containing arsenic trioxide and a second formulation containing Veneclare.

[0012] Furthermore, the first formulation is an injectable formulation, and the second formulation is an oral formulation.

[0013] Furthermore, the first formulation and the second formulation are used for simultaneous, sequential, or alternating administration.

[0014] Furthermore, the composition also contains pharmaceutically acceptable excipients.

[0015] The use of a composition of arsenic trioxide and veneclade in the preparation of a medicament for the treatment of TP53-mutant acute myeloid leukemia.

[0016] Furthermore, the TP53-mutant acute myeloid leukemia is a TP53 missense mutation or a TP53 truncated mutation.

[0017] The beneficial effects of this invention are:

[0018] This invention demonstrates the synergistic antitumor effect of arsenic trioxide combined with veneclade in the treatment of TP53-mutant acute myeloid leukemia (AML). In vitro experiments showed that arsenic trioxide and veneclade exhibited a clear synergistic inhibitory effect on proliferation in TP53-mutant AML cell lines, with a combination index (CI) consistently between 0.3 and 0.8. Animal experiments showed that the tumor growth inhibition rate in the arsenic trioxide and veneclade combination treatment group reached 67%, higher than the 32% in the veneclade monotherapy group. Histopathological analysis of the spleen showed that the combination treatment effectively repaired the red and white pulp structure of the spleen in AML mice and reduced hCD45-positive cell infiltration. Survival analysis showed that the median survival in the combination treatment group was prolonged to 46.5 days, longer than the 21 days in the control group and the 30 days in the veneclade monotherapy group. No lethal or severe toxic reactions were observed in the combination group mice, and no abnormalities were found in body weight or behavioral indicators; the benefits of the combination regimen outweighed the risks. Furthermore, in this invention, arsenic trioxide and veneclade can produce a synergistic tumor-suppressing effect under low-dose conditions, providing a good safety basis for clinical translation.

[0019] This invention discovers that although arsenic trioxide can repair the function of mutated p53, the continued high expression of BCL-2 constitutes an obstacle to apoptosis. By combining it with Veneclazide, this obstacle is removed. This not only overcomes the limitations of arsenic trioxide in the treatment of TP53-mutant acute myeloid leukemia, but also enhances the anti-tumor effect of Veneclazide, achieving unexpected technical results. This provides a novel combination therapy for TP53-mutant AML patients and has significant clinical application prospects. Attached Figure Description

[0020] Figure 1 This is a comparison of cell viability of TP53 mutant human acute myeloid leukemia cells in different treatment groups in Example 1. A represents THP-1 cells, and B represents Kasumi-1 cells.

[0021] Figure 2 This is a scatter plot of the combined arsenic trioxide and Veneclare index calculated based on the Chou-Talalay method in Example 1. A represents THP-1 cells, and B represents Kasumi-1 cells.

[0022] Figure 3 The image shows the Western Blot results of p53 pathway and apoptosis-related protein expression in THP-1 cells from different treatment groups in Example 2. A represents p21 protein, B represents BAX protein, and C represents BCL-2 protein.

[0023] Figure 4This is a flow cytometry scatter plot of Annexin V / PI double staining for apoptosis in THP-1 cells from different treatment groups in Example 3.

[0024] Figure 5 This is a bubble diagram showing the enrichment of the GSEA, a hallmark pathway, in THP-1 cells after treatment with arsenic trioxide and Veneclair in Example 4.

[0025] Figure 6 This is a graph showing the GSEA enrichment curve of the p53 signaling pathway in THP-1 cells after treatment with arsenic trioxide and Veneclair in Example 4.

[0026] Figure 7 The images show a comparison of in vivo imaging and quantitative statistics of tumor load in each group of tumor-bearing mice in Example 5. A is an in vivo fluorescence imaging image of each group of mice, and B is a quantitative comparison of total fluorescence flux in the whole body of mice.

[0027] Figure 8 Comparison of the gross morphology of the spleens of mice in each group in Example 5;

[0028] Figure 9 The images show CD45 immunohistochemical staining of spleen tissue from each group of mice in Example 5.

[0029] Figure 10 Kaplan-Meier survival curves of TP53-mutant AML tumor-bearing mice in each group in Example 5. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0031] All data from Examples 1-5 were statistically analyzed using GraphPad Prism 9.5.1 and R4.3.1 software. Quantitative data are expressed as mean ± standard error (mean ± SEM). Independent samples t-tests were used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and Tukey's method was used for pairwise comparisons. The Kaplan-Meier method was used for survival analysis, and the Log-rank test was used to analyze differences between groups. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicated no statistically significant difference.

[0032] Example 1

[0033] To verify whether the combined use of arsenic trioxide and veneclade has a synergistic antiproliferative effect in TP53 mutant AML cells, this embodiment used the CCK-8 assay to detect the effects of different concentrations of veneclade alone and in combination with arsenic trioxide on the activity of THP-1 and Kasumi-1 cells, and used the Chou-Talalay method to calculate the combination index (CI) to evaluate the synergistic effect of the two.

[0034] I. Experimental Materials

[0035] 1. Cell lines:

[0036] TP53-mutant human acute myeloid leukemia cell lines THP-1 (mutation sites: p.Arg174fs*3, R174LfsTer3) and Kasumi-1 (mutation sites: p.Arg248Gln, R248Q) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The cell lines were identified as free of cross-contamination by STR testing and were negative for mycoplasma.

[0037] 2. Main reagents and drugs

[0038] Arsenic trioxide (ATO): National standard material (No. GBW08666), purity ≥99.9%, purchased from the National Institute of Metrology, China. It was dissolved in sterile PBS to prepare a 200 μg / mL stock solution, aliquoted and stored at -20℃.

[0039] Venecra (VEN): Purity ≥99%, purchased from Selleck Chemicals, dissolved in sterile PBS to prepare a 200 μg / mL stock solution, stored at -20°C protected from light.

[0040] Cell culture reagents: RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco; penicillin-streptomycin solution was purchased from HyClone.

[0041] Cell viability assay reagent: Cell Counting Kit-8 (CCK-8) was purchased from Seven Biotech.

[0042] II. Experimental Methods and Results

[0043] (a) Cell viability assay (CCK-8 assay)

[0044] THP-1 and Kasumi-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and routinely cultured in a 37°C, 5% CO2 saturated humidity incubator. Cell passage ratios were 1:3–1:5, and cells in logarithmic growth phase were used at 5 × 10⁻⁶ cells / year. 3 The cells were seeded at a density of 100 μL per well in a 96-well plate and cultured adherently for 24 h before drug treatment.

[0045] Drug treatment grouping:

[0046] ① Control group: Only an equal volume of culture medium was added;

[0047] ②ATO monotherapy group: Arsenic trioxide was added to achieve a final concentration of 2 μM;

[0048] ③VEN monotherapy group: Venecra was added to achieve final concentrations of 0, 100, 200, 500, 1000, and 2000 nM;

[0049] ④ Combined drug group: Arsenic trioxide was added to a final concentration of 2 μM, and vernacle was added to a final concentration of 0, 100, 200, 500, 1000, and 2000 nM, respectively.

[0050] All cells were treated for 24 hours. After treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader.

[0051] The cell viability calculation formula is: Cell viability (%) = (OD value of drug treatment group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%. Each concentration was set up with 3 replicates, and the experiment was independently repeated 3 times.

[0052] The results are as follows Figure 1As shown, arsenic trioxide monotherapy (2 μM) had a weak inhibitory effect on the proliferation of THP-1 and Kasumi-1 cells, indicating that the efficacy of arsenic trioxide monotherapy in non-APL acute myeloid leukemia is extremely limited. Venecella monotherapy inhibited the proliferation of both cell types in a concentration-dependent manner. Compared with the monotherapy groups, the activity of both cell types decreased after treatment with arsenic trioxide combined with venecella: in THP-1 cells, the cell activity in the 100 nM venecella monotherapy group remained at around 80%, while the cell activity dropped below 60% after the combination with 2 μM arsenic trioxide (P<0.05); in Kasumi-1 cells, the cell activity in the 500 nM venecella combined with arsenic trioxide group decreased by about 30% compared with the monotherapy group (P<0.01). This indicates that arsenic trioxide can effectively enhance the sensitivity of TP53 mutant AML cells to venecella, and the combined effect of the two on inhibiting cell proliferation is superior to monotherapy.

[0053] (II) Synergistic Effect Analysis

[0054] The combination index (CI) of arsenic trioxide and veneclade was calculated using CompuSyn software based on the Chou-Talalay method to evaluate the synergistic effect of arsenic trioxide and veneclade. Based on cell activity data, a synergistic effect curve was plotted with the effect size (FA) on the x-axis and the combination index on the y-axis. The judgment criteria were: CI < 1.0 for synergistic effect, CI = 1.0 for additive effect, and CI > 1.0 for antagonistic effect.

[0055] The results are as follows Figure 2 As shown, when arsenic trioxide and veneclade were used in combination to treat THP-1 and Kasumi-1 cells, the CI values ​​corresponding to most effect values ​​(FA) were less than 1.0. Within the effective concentration range of FA = 30%-80%, the CI values ​​remained stable between 0.3 and 0.8, suggesting that the two drugs exhibit a clear synergistic inhibitory effect on proliferation in TP53 mutant AML cells, rather than a simple additive effect. This result quantitatively confirms that the combined use of arsenic trioxide and veneclade can enhance antitumor activity through a synergistic mechanism.

[0056] This embodiment confirms that arsenic trioxide and veneclade have a clear synergistic inhibitory effect on proliferation in TP53 mutant AML cell lines, and the combined antiproliferative effect of the two is better than that of the single drug.

[0057] Example 2

[0058] To elucidate the molecular mechanism of the combined use of arsenic trioxide and veneclade, this embodiment used Western blotting to detect the expression levels of p21, a downstream target protein of p53, BAX, a pro-apoptotic protein, and BCL-2, in THP-1 cells after treatment with the control group, the arsenic trioxide monotherapy group, the veneclade monotherapy group, and the combined treatment group, in order to clarify the role of the two in the apoptosis pathway.

[0059] I. Experimental Materials

[0060] TP53 mutant human acute myeloid leukemia cell line THP-1 (same as Example 1).

[0061] Protein detection reagents: p21, BAX, BCL-2 and β-actin primary antibody were purchased from Cell Signaling Technology.

[0062] HRP-labeled secondary antibody was purchased from Jackson Immuno Research.

[0063] The ECL chemiluminescence reagent kit was purchased from Seven Biotech.

[0064] II. Western Blot Analysis and Results

[0065] Logarithmic growth phase THP-1 cells were seeded at 2 × 10⁵ cells / well in 6-well plates and cultured for 24 h before being divided into groups for treatment:

[0066] ① Control group: Only an equal volume of culture medium was added;

[0067] ②ATO monotherapy group: Arsenic trioxide was added to achieve a final concentration of 2 μM;

[0068] ③VEN monotherapy group: Veneclare was added to achieve a final concentration of 500 nM;

[0069] ④ Combined drug group: Arsenic trioxide was added to achieve a final concentration of 2 μM, and Veneclare was added to achieve a final concentration of 500 nM.

[0070] THP-1 cells from each group were collected, lysed on ice for 30 min with RIPA lysis buffer (containing protease and phosphatase inhibitors), centrifuged at 12000 rpm for 15 min (4℃), and the supernatant was collected as total protein. Protein concentration was determined using the BCA method. After adjusting the protein concentration, loading buffer was added, and the mixture was denatured at 95℃ for 5 min. 30 μg of protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 2 h, and incubated overnight at 4℃ with primary antibody (p21, BAX, BCL-2, β-actin, diluted 1:1000). After washing three times with TBST, HRP-labeled secondary antibody (diluted 1:5000) was added and incubated at room temperature for 1 h. After washing three times with TBST, the cells were developed using an ECL chemiluminescence assay kit, and the band gray values ​​were quantitatively analyzed using ImageJ software. The relative expression level of the target protein was calculated using β-actin as an internal control. Each experiment was independently repeated three times.

[0071] The results are as follows Figure 3 As shown, although arsenic trioxide monotherapy can effectively upregulate the expression of p53 downstream target protein p21 and pro-apoptotic protein BAX, the expression of anti-apoptotic protein BCL-2 is not affected; after combination with veneclade, BCL-2 is effectively inhibited at the same time as BAX upregulation by arsenic trioxide.

[0072] This embodiment confirms that persistently high expression of BCL-2 is a key barrier to inhibiting apoptosis induced by arsenic trioxide. After removing this barrier by combining with Veneclare, the upregulated BAX from arsenic trioxide can exert a pro-apoptotic effect and effectively promote apoptosis in TP53 mutant AML cells.

[0073] Example 3

[0074] To verify whether the combined use of arsenic trioxide and veneclade can effectively induce apoptosis in TP53 mutant AML cells, this embodiment used Annexin V-FITC / PI double staining combined with flow cytometry to detect the apoptosis rate of THP-1 cells after treatment in the control group, arsenic trioxide monotherapy group, veneclade monotherapy group, and combined treatment group, in order to evaluate the functional outcome of the combined treatment.

[0075] I. Experimental Materials

[0076] TP53 mutant human acute myeloid leukemia cell line THP-1 (same as Example 1).

[0077] Apoptosis detection reagent: Annexin V-FITC / PI apoptosis detection kit was purchased from BD Biosciences. Other main reagents are the same as in Example 1.

[0078] II. Apoptosis Detection (Annexin V-FITC / PI Double Staining Method and Results)

[0079] After treatment with the grouped THP-1 cells in Example 2, the cells were washed twice with PBS, resuspended in 100 μL of binding buffer, and then incubated with 5 μL of Annexin V-FITC and 5 μL of PI staining solution at room temperature in the dark for 15 min. Immediately after adding 400 μL of binding buffer, the cells were analyzed by flow cytometry. Apoptotic cells were divided into early apoptotic cells (Annexin V...) and early apoptotic cells (...). + / PI - ) and late apoptosis (Annexin V) + / PI + Total apoptosis rate = early apoptosis rate + late apoptosis rate. The experiment was independently repeated 3 times.

[0080] The results are as follows Figure 4 As shown, the total apoptosis rate of THP-1 cells in the arsenic trioxide monotherapy group was not significantly different from that in the control group (approximately 5%), and had almost no effect on the apoptosis level of THP-1 cells; the total apoptosis rate in the veneclade monotherapy group was approximately 15%, mainly early apoptosis; while the total apoptosis rate in the arsenic trioxide combined with veneclade group was effectively increased to over 30%, with both early and late apoptosis significantly increased (P<0.01).

[0081] This embodiment confirms that arsenic trioxide alone can hardly induce apoptosis in TP53 mutant AML cells, but the apoptosis rate increases after being combined with veneclade, indicating that the combination of veneclade can effectively overcome the obstacle of arsenic trioxide restoring p53 but not inducing apoptosis.

[0082] Example 4

[0083] To verify the activation effect of arsenic trioxide combined with veneclade on the p53 pathway at the global transcriptional level, this embodiment performed transcriptome sequencing on THP-1 cells treated with the control group, arsenic trioxide monotherapy group, veneclade monotherapy group, and combination therapy group. The characteristic pathways of differentially expressed genes were analyzed by GSEA to corroborate the key signaling pathways activated by the combination therapy.

[0084] I. Experimental Materials

[0085] TP53 mutant human acute myeloid leukemia cell line THP-1 (same as Example 1).

[0086] Trizol reagent was purchased from Invitrogen.

[0087] The transcriptome sequencing was commissioned to Beijing Novogene Bioinformatics Technology Co., Ltd.

[0088] II. Experimental Methods and Results

[0089] (I) Drug grouping and processing method for TP53 mutant AML cells before sequencing

[0090] THP-1 cells were collected and treated for 24 hours according to the following groups:

[0091] ① Control group: Only an equal volume of culture medium was added;

[0092] ②ATO monotherapy group: Arsenic trioxide was added to achieve a final concentration of 2 μM;

[0093] ③VEN monotherapy group: Veneclare was added to achieve a final concentration of 500 nM;

[0094] ④ Combined drug group: Arsenic trioxide was added to achieve a final concentration of 2 μM, and Veneclare was added to achieve a final concentration of 500 nM.

[0095] (II) Transcriptome sequencing and bioinformatics analysis

[0096] Cells from each group were collected, and total RNA was extracted using the Trizol method. RNA purity and integrity were assessed (RNA integrity coefficient ≥ 7.0). Transcriptome sequencing was performed on the Illumina NovaSeq 6000 platform by Beijing Novogene Bioinformatics Technology Co., Ltd. After obtaining the raw sequencing data, quality control, reference genome alignment, and quantitative gene expression analysis were conducted sequentially. The pathway enrichment of differentially expressed genes was analyzed based on marker gene sets using Gene Set Enrichment Analysis (GSEA) software.

[0097] The results are as follows Figure 5 and Figure 6 As shown, compared with the control group, the p53 signaling pathway was enriched in the arsenic trioxide combined with Veneclair group, with a standardized enrichment score (NES) of -1.66 and a corrected P value of 0.000253, demonstrating that the combined drug treatment can effectively activate the p53 signaling pathway. At the same time, the apoptosis pathway was also enriched, while cancer-promoting pathways such as oxidative phosphorylation and inflammatory response were significantly inhibited.

[0098] This embodiment demonstrates at the transcriptomic level that combined treatment with arsenic trioxide and Veneclare can effectively activate the p53 signaling pathway and simultaneously initiate the apoptosis program, further supporting the fact that combined Veneclare can overcome the obstacle of arsenic trioxide restoring p53 but not inducing apoptosis.

[0099] Example 5

[0100] To verify the antitumor efficacy and safety of the combined use of arsenic trioxide and Veneclare in vivo, this embodiment established a TP53 mutant AML mouse model. The tumor burden was assessed by in vivo imaging, the organ damage repair was assessed by spleen histopathological analysis, the survival benefit was assessed by survival curve analysis, and the toxicity of the combined drug was observed.

[0101] I. Experimental Materials

[0102] Six-week-old female NCG mice (athymic and severely immunodeficient) were purchased from the Nanjing Model Animal Resource Bank, weighing 18-22g. They were housed in a specific pathogen-free (SPF) grade animal facility under the following environmental conditions: temperature 22±1℃, relative humidity 55±5%, 12-hour light-dark cycle, and free access to food and water. All animal experimental procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Ethics Approval No.: HMU-IACUC-2024-003) and strictly adhered to the 3R principle.

[0103] TP53 mutant human acute myeloid leukemia cell line THP-1 (same as Example 1).

[0104] Immunohistochemical reagents: hCD45 antibody was purchased from Abcam; immunohistochemical detection kit (SP method) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0105] Main instruments: In vivo imaging system (PerkinElmer IVIS Spectrum), pathological microtome (Leica RM2245), fluorescence microscope (Olympus BX53).

[0106] II. Experimental Methods and Results

[0107] (I) Animal model establishment and drug administration methods

[0108] Take THP-1 cells in the logarithmic growth phase, wash twice with sterile PBS, and adjust the concentration to 1×10⁻⁶. 7 1 × 10⁶ cells / mL was injected into NCG mice via the tail vein. 6 1 cell / mouse. 14 days after injection, in vivo fluorescence signals in mice were detected using an in vivo imaging system to confirm AML cell infiltration and successful establishment of the model.

[0109] Mice that successfully developed the model were randomly divided into 4 groups (n=14 / group):

[0110] ①AML control group (PBS group): Intraperitoneal injection of an equal volume of sterile PBS;

[0111] ②ATO monotherapy group: intraperitoneal injection of arsenic trioxide, dose 1 mg / kg, once daily;

[0112] ③VEN monotherapy group: Veneclare was administered orally at a dose of 100 mg / kg once daily;

[0113] ④ Combination therapy group: intraperitoneal injection of arsenic trioxide (1 mg / kg) + oral administration of veneclade (100 mg / kg), once daily, with a molar ratio of arsenic trioxide to veneclade of 1:22.7. All groups were administered the medication continuously for 7 days.

[0114] (ii) Tumor burden detection

[0115] After the administration was completed, 4 mice per group were randomly selected, and the fluorescence signal value in vivo was detected by in vivo imaging system. The tumor burden was quantitatively analyzed, and the tumor growth inhibition rate (TGI) was calculated: TGI (%) = (average fluorescence signal value of control group - average fluorescence signal value of administration group) / average fluorescence signal value of control group × 100%.

[0116] The results are as follows Figure 7 As shown, the fluorescence signal value in the AML control group mice was as high as 4×10⁻⁶. 11 The results indicated an extremely high tumor burden; the fluorescence signal value of the arsenic trioxide monotherapy group was not significantly different from that of the control group (P>0.05), indicating that arsenic trioxide monotherapy had no significant inhibitory effect on the tumor burden of the established TP53-mutant AML model; the fluorescence signal value of the veneclade monotherapy group decreased to 2.5×10⁻⁶. 11 Around 1000 mg / L, the tumor growth inhibition rate was 32% (P<0.05); while the fluorescence signal value in the arsenic trioxide combined with Veneclare group decreased to 1×10⁻⁶. 11 The tumor growth inhibition rate reached 67% (P<0.005), which was lower than that of the veneclade monotherapy group (P<0.05).

[0117] This result confirms that the combined use of arsenic trioxide and Veneclair can synergistically inhibit the proliferation and infiltration of TP53 mutant AML cells in vivo, effectively reducing tumor burden.

[0118] (III) Pathological and Histological Analysis of the Spleen

[0119] Spleen tissue was harvested from euthanized mice, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm) for hCD45 immunohistochemical staining. The specific steps were: dewaxing to water, incubation with 3% H2O2 at room temperature for 10 min to inactivate endogenous peroxidase, antigen retrieval followed by blocking, incubation with hCD45 primary antibody (1:200) at 4℃ overnight, secondary antibody incubation followed by DAB staining, hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin. The structure of the red and white pulp of the spleen and the infiltration of hCD45-positive cells were observed under a fluorescence microscope.

[0120] Actual photograph of spleen tissue, as shown Figure 8As shown, the spleen of mice in the AML control group was significantly enlarged, while there was no significant difference between the arsenic trioxide monotherapy group and the control group. The degree of spleen enlargement was reduced in the veneclade monotherapy group, and the spleen volume of the arsenic trioxide combined with veneclade group was significantly reduced to close to the normal physiological size. Gross observation showed no obvious necrosis or atrophy.

[0121] Immunohistochemical results as follows Figure 9 As shown, the spleen red and white pulp structure of mice in the AML control group was destroyed, and hCD45 positive cells were widely infiltrated; there was no significant difference between the arsenic trioxide monotherapy group and the control group; hCD45 positive cell infiltration was reduced in the veneclade monotherapy group, but obvious pathological damage was still visible; while the spleen red and white pulp structure of mice in the combined treatment group was basically restored, and hCD45 positive cell infiltration was significantly reduced.

[0122] This result suggests that combined medication can effectively inhibit the proliferation and infiltration of AML cells in the spleen and repair pathological damage to hematopoietic organs.

[0123] (iv) Survival analysis

[0124] The remaining 10 mice / groups were observed for 50 days, and the survival time of each group was recorded to plot survival curves.

[0125] The results are as follows Figure 10 As shown, the median survival of mice in the AML control group was 21 days; the median survival of the arsenic trioxide monotherapy group was 22 days, which was not significantly different from the control group (P>0.05); the median survival of the veneclade monotherapy group was extended to 30 days (P<0.05); and the median survival of the arsenic trioxide combined with veneclade group was effectively extended to 46.5 days (P<0.001), and some mice were still alive at the end of the observation period (50 days).

[0126] This result indicates that the synergistic effect of arsenic trioxide and veneclade can effectively improve the prognosis and prolong the survival time of TP53 mutant AML mice, providing important evidence of survival benefits for the treatment of this subtype of AML.

[0127] This embodiment demonstrates through in vivo experiments that the combined treatment of arsenic trioxide and veneclade can effectively reduce tumor burden, repair pathological damage to the spleen, and prolong survival under low-dose conditions, without obvious toxic reactions, and has good safety and synergistic anti-tumor effects.

Claims

1. A composition of arsenic trioxide and vernacle, characterized in that, The molar ratio of arsenic trioxide to veneclade is 1:10~25, and the molecular formula of veneclade is C2. 43 H 44 ClN7O5S, with the following structural formula: 。 2. The composition of arsenic trioxide and Veneclair according to claim 1, characterized in that, The molar ratio of arsenic trioxide to Venecra is 1:22.

7.

3. The composition of arsenic trioxide and Veneclair according to claim 1 or 2, characterized in that, The composition is a combination of a first formulation containing arsenic trioxide and a second formulation containing Veneclare.

4. The composition of arsenic trioxide and Veneclair according to claim 3, characterized in that, The first formulation is an injectable formulation, and the second formulation is an oral formulation.

5. The composition of arsenic trioxide and Veneclair according to claim 4, characterized in that, The first and second formulations are used for simultaneous, sequential, or alternating administration.

6. The composition of arsenic trioxide and Veneclair according to claim 5, characterized in that, The composition also contains pharmaceutically acceptable excipients.

7. The use of a composition of arsenic trioxide and veneclade as described in any one of claims 1-6 in the preparation of a medicament for treating TP53-mutant acute myeloid leukemia.

8. The application according to claim 7, characterized in that, The TP53-mutant acute myeloid leukemia is either a TP53 missense mutation or a TP53 truncated mutation.

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