An antitumor pharmaceutical composition and application thereof in preparation of ETP-ALL treatment drugs

Abstract

This invention relates to an antitumor pharmaceutical composition and its application in the preparation of therapeutic drugs for ETP-ALL, belonging to the field of biomedical technology. To address the problems of natural resistance to nerabine in ETP-ALL and the limited clinical treatment options, this invention provides an antitumor pharmaceutical composition and its application in the preparation of therapeutic drugs for acute early-stage prodrug T-lymphoblastic leukemia. The antitumor pharmaceutical composition of this invention combines the BRG1 / BRM ATPase inhibitor FHD-286 with the nucleoside analog nerabine, producing a significant synergistic effect and effectively overcoming the natural resistance of ETP-ALL to nerabine. Experiments have confirmed that the combined drug treatment significantly enhances the inhibitory effect on the proliferation and apoptosis-inducing effect on ETP-ALL cells, providing sufficient experimental evidence for the application of this pharmaceutical composition in the preparation of therapeutic drugs for ETP-ALL.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an antitumor drug composition and its application in the preparation of ETP-ALL therapeutic drugs. Background Technology

[0002] Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an ultra-high-risk subtype of acute T-cell acute lymphoblastic leukemia (T-ALL) characterized by differentiation arrest and poor differentiation, accounting for approximately 10-15% of all T-ALL cases. Unlike ordinary T-ALL, ETP-ALL exhibits stronger chemotherapy resistance, poor early chemotherapy response, and extremely poor long-term prognosis.

[0003] Nelarabine is a targeted drug used to treat relapsed / refractory T-ALL. However, clinical studies have shown that nelarabine has limited efficacy in ETP-ALL. Adding nelarabine to the Hyper-CVAD regimen significantly improves the 5-year overall survival in non-ETP-ALL patients, but no similar benefit has been observed in ETP-ALL patients. Related mechanism studies reveal that nelarabine activation depends on deoxycytidine kinase (dCK), and the dCK promoter region in ETP-ALL cells undergoes epigenetic silencing due to H3 / H4 histone deacetylation, leading to decreased dCK expression and inhibited nelarabine activation. This ultimately manifests as natural resistance to nelarabine in ETP-ALL cells, which is the core reason for the poor response to nelarabine treatment in clinical practice. This resistance mechanism is unique to ETP-ALL and is fundamentally different from the resistance mechanisms in non-ETP-ALL and AML.

[0004] FHD-286 is a BRG1 / BRM ATPase inhibitor that has been shown to exert anti-tumor effects by inducing myeloid differentiation of acute myeloid leukemia (AML) cells through remodeling chromatin accessibility and inhibiting key myeloid transcription factors such as c-Myc and PU.1. However, ETP-ALL and AML belong to the T lymphoid and myeloid lineages respectively, and there are significant differences between them in gene expression regulatory networks, epigenetic landscapes, and drug sensitivity. This makes it difficult to directly extrapolate the applicability of drugs effective in AML to ETP-ALL.

[0005] At present, there is still a severe lack of effective treatment strategies to intervene in the progression of ETP-ALL, especially to overcome its nerabine resistance. This is a key technical challenge that urgently needs to be overcome in the field of hematologic malignancies. Summary of the Invention

[0006] To address the issues of natural resistance to nerabine and limited clinical treatment options in ETP-ALL, this invention provides an antitumor drug composition and its application in the preparation of ETP-ALL therapeutic drugs.

[0007] The technical solution of the present invention: An antitumor drug composition comprising a first formulation containing FHD-286 and a second formulation containing nerabine, wherein the molecular formula of FHD-286 is C 24 H 30 N6O6S2, structural formula is .

[0008] Furthermore, the molar ratio of FHD-286 to nerabine is 1:1 to 5.

[0009] Furthermore, the molar ratio of FHD-286 to nerabine is 3:10.

[0010] Furthermore, the first formulation containing FHD-286 is an oral formulation, and the second formulation containing nerabine is an intravenous injection formulation.

[0011] Furthermore, the first formulation and the second formulation are administered simultaneously or sequentially.

[0012] Furthermore, both the first and second formulations contain pharmaceutically acceptable excipients.

[0013] Application of an antitumor pharmaceutical composition provided by the present invention in the preparation of a therapeutic drug for acute early prodrug T-lymphocytic leukemia Furthermore, the acute early precursor T-lymphoblastic leukemia is an acute early precursor T-lymphoblastic leukemia resistant to nerabine.

[0014] The beneficial effects of this invention are: This invention provides a combination drug composition for the treatment of ETP-ALL, the core of which lies in the combination of the BRG1 / BRM ATPase inhibitor FHD-286 and the nucleoside analog nelabine. This invention is the first to discover that FHD-286 itself has a significant killing effect on ETP-ALL cells, and that its combination with nelabine produces a significant synergistic effect, effectively overcoming the natural resistance of ETP-ALL to nelabine. Simultaneously, this combination drug composition, while efficiently killing tumor cells, exhibits no significant cytotoxicity to normal bone marrow cells, demonstrating good safety. Through a series of experiments including cell viability detection, proliferation inhibition experiments, cell morphology observation, flow cytometry apoptosis analysis, and Western blot, this invention confirms that the combination drug significantly enhances the effects of proliferation inhibition and apoptosis induction on ETP-ALL cells, opening a new path to overcome the treatment bottleneck of ETP-ALL and providing sufficient experimental evidence for the application of this drug composition in the preparation of ETP-ALL therapeutic drugs. Attached Figure Description

[0015] Figure 1 This is a ranking chart of the sensitivity of different T-ALL cell lines to nerabine based on the GDSC database analysis in Example 1; Figure 2 This is a comparison of the inhibitory activity of nelarabine monotherapy on the proliferation of T-ALL cell lines in Example 2; Figure 3 This is a comparison of the inhibitory activity of FHD-286 monotherapy on the proliferation of T-ALL cell lines in Example 2; Figure 4 This is a dose-response matrix diagram of Loucy cells treated with FHD-286 and nerabine in Example 3; Figure 5 This is a ZIP synergistic fraction analysis diagram of Loucy cells treated with FHD-286 and nerabine in Example 3; Figure 6 This is a statistical chart showing the relative cell viability of bone marrow cells from healthy donors treated with FHD-286 and nerabine in Example 4. Figure 7 This is a comparison of the long-term growth curves of Loucy cells under different treatment conditions in Example 5; Figure 8 Giemsa staining images of Loucy cells under different treatment conditions in Example 6; Figure 9 This is a comparison of the apoptosis rate of Loucy cells under different treatment conditions in Example 7; Figure 10 This is a comparison of the expression levels of apoptosis-related proteins in Loucy cells under different treatment conditions in Example 8. Detailed Implementation

[0016] 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.

[0017] Embodiment 3 of the present invention utilizes SynergyFinder + The ZIP model used in Example 4 (https: / / synergyfinder.org / ) was used to analyze the effects of drug combination therapy. Data from Example 4 were analyzed using Prism10. Two-way ANOVA was performed. Data from Example 6 were analyzed using Prism10. Unpaired two-tailed Student's t-test was used. All data are described as mean ± standard deviation. A p-value < 0.05 was considered statistically significant. .

[0018] Example 1 To investigate the sensitivity of ETP-ALL cell lines to nerabine, this embodiment systematically analyzes nerabine drug sensitivity data of various publicly available T-ALL cell lines based on the Genomics of Drug Sensitivity in Cancer (GDSC) database.

[0019] The half-maximal inhibitory concentration (IC50) of nerabine against various T-ALL cell lines was determined using the GDSC database (version GDSC2, https: / / www.cancerrxgene.org / ). 50 (Unit: μM) data, this indicator is defined as the drug concentration required to inhibit 50% of tumor cell survival. IC 50 The lower the value, the more sensitive the cells are to the drug; conversely, a higher value indicates decreased sensitivity or the presence of drug resistance.

[0020] A total of 22 cell lines derived from T-ALL were screened and retained for subsequent drug sensitivity analysis. The nerabine IC50 values ​​of each cell line were then compared. 50The values ​​were sorted from low to high, and sensitivity distribution curves were plotted to compare the differences in drug sensitivity among different cell lines. The upper limit of clinically accessible drug concentration was used as a reference to evaluate the clinical treatment response potential of each cell line to nerabine.

[0021] The results are as follows Figure 1 As shown, typical cell lines of different differentiation subtypes were selected and labeled, including the common T-ALL cell lines CCRF-CEM, SUP-B15, MOLT-4, and MOLT-16, and the ETP-ALL cell line Loucy. The dashed line in the figure represents the upper limit of clinically accessible nerabine concentration. The sensitivity characteristics of the cell lines are defined as follows: Highly sensitive conventional T-ALL cell lines: IC50 of CCRF-CEM and SUP-B15 50 The value was low, significantly below the upper limit of clinically accessible drug concentration, suggesting that conventional clinical doses of nerabine can effectively inhibit the proliferation of this type of cell. Intermediately sensitive common T-ALL cell line: IC50 of MOLT-16 50 The value was significantly elevated, approaching the upper limit of clinically accessible drug concentration, indicating that its sensitivity to nerabine had significantly decreased, and conventional doses of nerabine were unlikely to achieve the desired tumor-suppressing effect. Low-sensitivity / drug-resistant cell lines: IC50 of Loucy and MOLT-4 cell lines 50 Both values ​​were significantly higher than the upper limit of clinically achievable drug concentrations, indicating strong resistance to nerabine. Specifically, the IC50 values ​​for Loucy cells were significantly higher. 50 The value was at a relatively high level among the 22 cell lines, but the sensitivity was significantly low.

[0022] The results of this embodiment indicate that, compared with the ordinary T-ALL cell line, the ETP-ALL cell line Loucy exhibits significant natural resistance to nerabine, with its IC50 value being significantly lower. 50 The concentration exceeded the range achievable with conventional treatment, consistent with the poor efficacy of nerabine monotherapy in ETP-ALL in clinical practice. This result provides a direct experimental background and theoretical basis for subsequent studies employing combination therapy strategies to overcome nerabine resistance in ETP-ALL and improve treatment outcomes.

[0023] Example 2 To verify the natural resistance of nerabine to ETP-ALL cells and to evaluate the single-drug killing effect of FHD-286 on ETP-ALL cells, this embodiment uses cell viability assays to detect the inhibitory effects of different concentrations of nerabine or FHD-286 on the proliferation of various T-ALL cell lines.

[0024] (1) Experimental materials and methods Cell lines: This experiment used T-ALL cell lines, including the ETP-ALL cell line Loucy, and the common T-ALL cell lines CCRF-CEM, Jurkat, and MOLT-3.

[0025] Drug treatment: Neraphine treatment: Cells were seeded in 96-well plates and neraphine was prepared at different logarithmic gradient concentrations of 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM. The plates were then incubated at 37 °C in a 5% CO2 incubator for 48 h.

[0026] FHD-286 treatment: Loucy cells were seeded in 96-well plates and different gradient concentrations of FHD-286 were set at 0 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM and 5 μM, and cultured under the same conditions for 48 h.

[0027] Cell viability assay: After culture, cell viability was assessed using the CellTiter-Glo chemiluminescence assay. An equal volume of CellTiter-Glo reagent was added to each well, and after incubation at room temperature in the dark, the chemiluminescence signal value of each well was measured using a microplate reader. The signal intensity was positively correlated with the number of viable cells. Using the cell viability of the untreated group as 100%, the relative cell viability at each drug concentration was calculated, and the half-maximal inhibitory concentration (IC50) of each drug for different cell lines was calculated by fitting dose-response curves. 50 ).

[0028] (2) Experimental results The results are as follows Figure 2 and Figure 3 As shown, there is a significant difference in the inhibitory activity of nerabine and FHD-286 on the proliferation of T-ALL cell lines: Neraphine exhibits strong inhibitory effects on the proliferation of common T-ALL cell lines (CCRF-CEM, Jurkat, MOLT-3). Cell viability decreases significantly in a dose-dependent manner with increasing drug concentration, and can drop to near zero at higher concentrations. However, the killing effect of neraphine on the ETP-ALL cell line Loucy is significantly weakened. Even at high concentrations, Loucy cell viability remains at a high level without a significant downward trend, confirming that Loucy cells have significant natural resistance to neraphine.

[0029] FHD-286 exhibited a significant dose-dependent inhibitory effect on the proliferation of Loucy cells; cell viability gradually decreased with increasing drug concentration, and its IC50 effect on Loucy cells... 50 The value was 3.855 μM, indicating that FHD-286 can effectively kill ETP-ALL cells and has the potential to overcome their drug resistance background.

[0030] The results of this embodiment are consistent with the analysis conclusions of the GDSC database, further verifying the natural resistance of the ETP-ALL cell line Loucy to nerabine. Simultaneously, this experiment confirmed that FHD-286 has clear single-drug killing activity against Loucy cells, providing direct experimental evidence for subsequent research on the combined use of FHD-286 and nerabine, and for exploring new strategies to overcome nerabine resistance in ETP-ALL.

[0031] Example 3 To investigate the inhibitory effect and synergistic effect of the combined use of FHD-286 and nerabine on the proliferation of ETP-ALL cells, Loucy cells were treated with different gradient concentrations of nerabine and FHD-286 in this embodiment. Cell viability was detected by CellTiter-Glo luminescence assay, and synergistic scores were calculated using the SynergyFinder platform to evaluate the combined action mode of the two drugs.

[0032] (1) Experimental materials and methods Cell line: ETP-ALL cell line Loucy.

[0033] Drug treatment: Loucy cells were seeded in 96-well plates and nerabine and FHD-286 were administered at different gradient concentrations to construct a two-dimensional dose-response matrix for combined treatment.

[0034] The nerabine concentration gradients were set to: 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, and 25 μM; The FHD-286 concentration gradient was set to: 0 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, and 4 μM; All treatment groups were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0035] Cell viability assay: After culture, the cell viability of each well was detected by CellTiter-Glo luminescence assay. The chemiluminescence signal value was measured by microplate reader. The cell viability of the untreated group was taken as 100%, and the cell inhibition rate at each drug combination concentration was calculated.

[0036] Synergistic effect analysis: The dose-response matrix data were uploaded to the SynergyFinder platform, and a synergistic score was calculated using the Zero Interaction Potential Model (ZIP model) to assess the combined action mode of FHD-286 and nerabine. A synergistic score greater than 10 indicates a synergistic effect between the drugs, and the higher the score, the stronger the synergistic effect.

[0037] (2) Experimental results like Figure 4The dose-response matrix showed that the inhibition rate of Loucy cells increased significantly in a dose-dependent manner with increasing concentrations of FHD-286 and nerabine. When the concentration of FHD-286 was 4 μM and the concentration of nerabine was 25 μM, the cell inhibition rate reached 75.42%, significantly higher than the single-drug treatment groups. The inhibition rate of nerabine monotherapy at 25 μM was 48.68%, and the inhibition rate of FHD-286 monotherapy at 4 μM was 51.84%.

[0038] like Figure 5 As shown, the mean synergistic score calculated based on the ZIP model was 14.908, which is greater than 10, indicating that FHD-286 and nerabine have a significant synergistic inhibitory effect on Loucy cells. The three-dimensional synergistic score heatmap shows that the synergistic score is greater than 10 at multiple drug concentration combinations, with the most significant synergistic effect observed in the medium concentration combination region. This suggests that FHD-286 can effectively enhance the sensitivity of Loucy cells to nerabine and reverse their natural resistance to nerabine.

[0039] The results of this embodiment demonstrate that the combined use of FHD-286 and nerabine has a significant synergistic inhibitory effect on the proliferation of the ETP-ALL cell line Loucy, effectively enhancing the killing effect of nerabine on drug-resistant cells. This result provides crucial experimental evidence for the subsequent use of this drug combination in the treatment of ETP-ALL and also verifies the feasibility of the proposed combination therapy strategy in overcoming nerabine resistance in ETP-ALL.

[0040] Example 4 To investigate the safety of the combination of FHD-286 and nerabine on healthy donor bone marrow cells, this embodiment uses cell viability assays to detect the inhibitory effect of the combined drug combination on the proliferation of healthy donor cells.

[0041] (1) Experimental materials and methods Cells: Bone marrow cells from a healthy donor.

[0042] Cell seeding and grouping: Healthy donor bone marrow mononuclear cells were seeded at a rate of 2 × 10⁻⁶ cells / year. 4 The wells were seeded at a density of 1 well per well, with 3 replicates per group. The experiment was divided into 2 groups: Control group: Add 1‰ DMSO; Combination therapy group: nerabine final concentration 10 μM, FHD-286 final concentration 3 μM; All treatment groups were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0043] Cell viability assay: After culture, the cell viability of each well was detected by CellTiter-Glo luminescence assay. The chemiluminescence signal value was measured by microplate reader. The cell viability of the untreated group was taken as 100%, and the relative cell viability of the combined drug treatment group was calculated.

[0044] (2) Experimental results like Figure 6 As shown, compared with the cell viability of the DMSO blank control group at the same time, the relative cell viability of the combined drug group did not decrease significantly. Therefore, the combined drug therapy of FHD-286 and nerabine has no significant killing effect on bone marrow cells from healthy donors, which confirms that the combined drug therapy strategy is safe.

[0045] The results of this embodiment show that treatment with 10 μM nerabine combined with 3 μM FHD-286 for 48 h did not produce significant proliferation inhibition or cell killing effects on healthy donor bone marrow cells. This drug combination showed good safety for normal hematopoietic cells under the experimental conditions, suggesting that the combined drug strategy of FHD-286 and nerabine will not cause significant cytotoxicity to normal bone marrow cells. This provides experimental support from a safety perspective for the subsequent application of this combination regimen to the efficacy study of ETP-ALL cells.

[0046] Example 5 To investigate the inhibitory effect of the combination of FHD-286 and nerabine on the long-term proliferation of ETP-ALL cells, this example uses trypan blue staining and counting to continuously monitor the growth curves of Loucy cells under different treatment conditions and evaluate the long-term inhibitory effects of single and combined drug therapy.

[0047] (1) Experimental materials and methods Cell line: ETP-ALL cell line Loucy.

[0048] Cell seeding and grouping: Loucy cells were adjusted to 1×10⁶ cells / year. 5 An initial concentration of [number] cells / mL was inoculated into 6-well plates, with 3 replicates per group. The experiment was divided into 4 groups: Control group: Add 1‰ DMSO; Neraphine monotherapy group: final concentration 10 μM; FHD-286 monotherapy group: final concentration 3μM; Combination therapy group: nerabine final concentration 10 μM, FHD-286 final concentration 3 μM; All groups were incubated in a 37°C, 5% CO2 incubator, with the culture medium containing the corresponding drug replaced every 2 days.

[0049] Cell counting: On days 0, 1, 2, 3 and 4 after treatment, viable cells in each group were counted using trypan blue staining, growth curves were plotted, and statistical methods were used to analyze differences between groups.

[0050] (2) Experimental results The results are as follows Figure 7 As shown, the growth curves of Loucy cells in each group showed significant differences: the DMSO control group cells, without drug intervention, exhibited typical exponential proliferation; nerabine and FHD-286 single-drug treatments only partially inhibited Loucy cell proliferation, and cells still showed slow regeneration. Therefore, the cell viability of the control group only decreased slowly, without a significant decrease in cell number; the combined drug treatment could almost completely block cell proliferation and induce a large number of cell deaths, with the relative cell viability approaching 0 on the 4th day of culture.

[0051] The above results indicate that the combined use of FHD-286 and nerabine can significantly enhance the inhibitory effect on the long-term proliferation of Loucy cells, and its effect is far superior to the two single-drug treatments, further verifying the synergistic anti-tumor effect of the two.

[0052] The results of the long-term cell proliferation experiment in this embodiment are consistent with the conclusions of the short-term cell viability experiment in Example 3, further confirming that the combined use of FHD-286 and nerabine can effectively overcome the natural resistance of ETP-ALL cells to nerabine, significantly enhance the inhibitory effect on tumor cell proliferation, and provide more sufficient experimental evidence for the clinical application of this drug composition.

[0053] Example 6 To observe the effects of FHD-286 and nerabine monotherapy and combination therapy on the morphology of ETP-ALL cells, this example uses Giemsa staining to observe the morphological changes of Loucy cells under different treatment conditions under a microscope, and to evaluate the effects of the drugs on cell apoptosis and differentiation status.

[0054] (1) Experimental materials and methods Cell line: ETP-ALL cell line Loucy.

[0055] Drug treatment: Loucy cells were divided into 4 groups, and the treatment conditions were the same as in Example 5: Control group: 1‰ DMSO; Neraphine monotherapy group: final concentration 10 μM; FHD-286 monotherapy group: final concentration 3μM; Combination therapy group: nerabine final concentration 10 μM, FHD-286 final concentration 3 μM; Each group of cells was treated in a 37℃, 5% CO2 incubator for 48 h.

[0056] Giemsa staining and observation: Collect cells from each group, prepare cell smears, fix with methanol, air dry, add Giemsa staining working solution for staining, gently rinse with phosphate buffer at pH 7.0, air dry again, and observe and photograph under an optical microscope to record cell morphological characteristics.

[0057] (2) Experimental results The results are as follows Figure 8 As shown, Loucy cells in the DMSO control group had intact morphology, with round and centrally located nuclei, uniform and dense chromatin, and a moderate cytoplasm ratio, without obvious vacuoles or cell membrane protrusions. Cells in both the nerabine monotherapy group and the FHD-286 monotherapy group showed abnormal morphological changes such as nuclear displacement, cytoplasmic vacuolation, and cell membrane protrusions, which were indicative of pre-apoptotic or abnormal differentiation. The combination therapy group showed the most significant morphological changes, with obvious nuclear condensation, highly condensed chromatin, severe cytoplasmic vacuolation, and significantly aggravated cell membrane vacuolization and fragmentation, suggesting that the process of apoptosis or programmed cell death was significantly enhanced.

[0058] The Giemsa staining results in this embodiment visually confirm from a cell morphology perspective that FHD-286 can significantly enhance the killing effect of nerabine on ETP-ALL cells, which corroborates the conclusions of the aforementioned cell viability and proliferation experiments, providing multi-dimensional experimental evidence for the synergistic effect of this drug composition.

[0059] Example 7 To investigate the apoptosis-inducing effects of FHD-286 and nerabine monotherapy and combination therapy on ETP-ALL cells, this study employed Annexin V-FITC / PI double staining flow cytometry to quantitatively detect the apoptosis rate of Loucy cells under different treatment conditions and to evaluate the pro-apoptotic effects of the drugs.

[0060] (1) Experimental materials and methods Cell line: ETP-ALL cell line Loucy.

[0061] Drug treatment: Loucy cells were divided into 4 groups, and the treatment conditions were the same as in Example 5: Control group: 1‰ DMSO; Neraphine monotherapy group: final concentration 10 μM; FHD-286 monotherapy group: final concentration 3μM; Combination therapy group: nerabine final concentration 10 μM, FHD-286 final concentration 3 μM; Each group of cells was treated in a 37℃, 5% CO2 incubator for 48 h.

[0062] Flow cytometry detection of apoptosis: After treatment, cells from each group were collected, washed once with pre-cooled PBS, and resuspended in binding buffer. Annexin V-FITC and PI dye were added, and the cells were incubated at room temperature in the dark. Fluorescence signals were detected by flow cytometry, and data were analyzed using FlowJo software to calculate the total apoptosis rate (percentage of early apoptotic cells + percentage of late apoptotic cells) for each group.

[0063] (2) Experimental results The results are as follows Figure 9 As shown, the baseline apoptosis rate of Loucy cells in the DMSO control group was low and within the normal range; the apoptosis rates of both the neprabine monotherapy group and the FHD-286 monotherapy group were significantly higher than those of the control group, indicating that both drugs can induce apoptosis in Loucy cells; the apoptosis rate of the combined drug group was further significantly increased and was significantly higher than that of the two single drug treatment groups, and the difference between the groups was highly statistically significant.

[0064] The above results indicate that the combined use of FHD-286 and nerabine can significantly enhance the apoptosis-inducing effect on Loucy cells, and its effect is significantly better than that of single treatment, further verifying the synergistic anti-tumor effect of the two at the molecular level.

[0065] The flow cytometry apoptosis experiment results in this embodiment corroborate the conclusions of the aforementioned cell viability, proliferation, and morphology experiments, clarifying that one of the mechanisms of action of the combined drug is to significantly induce apoptosis in ETP-ALL cells, overcoming their natural resistance to nerabine, and providing key mechanistic evidence for the clinical application of this drug composition.

[0066] Example 8 To further investigate the molecular mechanism by which FHD-286 and nerabine induce apoptosis in ETP-ALL cells, this study used Western blot technology to detect the expression levels of apoptosis-related proteins in Loucy cells under different treatment conditions, thereby elucidating the drug's target and signaling pathway.

[0067] (1) Experimental materials and methods Cell line: ETP-ALL cell line Loucy.

[0068] Drug treatment: Loucy cells were divided into 4 groups, and the treatment conditions were the same as in Example 5: Control group: 1‰ DMSO; Neraphine monotherapy group: final concentration 10 μM; FHD-286 monotherapy group: final concentration 3μM; Combination therapy group: nerabine final concentration 10 μM, FHD-286 final concentration 3 μM; Each group of cells was treated in a 37℃, 5% CO2 incubator for 48 h.

[0069] Western blot experiment: After treatment, cells from each group were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined and quantified using the BCA method. Equal amounts of protein samples were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk at room temperature, and then incubated overnight at 4°C with anti-BCL-2, BAX, Caspase-3, Cleaved caspase-3, and β-Tubulin primary antibodies, respectively. The next day, HRP-labeled secondary antibody was added and incubated at room temperature. The samples were then exposed in an imaging system using ECL chemiluminescence developing solution to detect the signal of each protein band. β-Tubulin was used as an internal control to analyze the relative expression levels of proteins in each group.

[0070] (2) Experimental results The results are as follows Figure 10 As shown, compared with the DMSO control group, the expression level of the anti-apoptotic protein BCL-2 was only slightly decreased in the nelabine monotherapy group and the FHD-286 monotherapy group, while the expression levels of the pro-apoptotic proteins BAX and Cleaved caspase-3 were slightly upregulated. In the combination therapy group, the expression level of BCL-2 was significantly reduced, while the expression level of BAX was significantly increased, and the BAX / BCL-2 ratio was significantly upregulated. At the same time, the expression levels of total Caspase-3 and its activated form Cleaved Caspase-3 both showed a significant upregulated trend, suggesting that the endogenous mitochondrial apoptosis pathway was strongly activated.

[0071] The above results indicate that the combined use of FHD-286 and nerabine can significantly induce apoptosis in Loucy cells by regulating the BCL-2 / BAX apoptosis pathway and activating the Caspase cascade reaction, with a pro-apoptotic effect far superior to that of single-drug treatment.

[0072] The Western blot results of this embodiment reveal the key pathway of combined drug therapy inducing apoptosis in ETP-ALL cells at the molecular mechanism level, which is completely consistent with the conclusions of the aforementioned flow cytometry and morphological experiments, providing direct molecular evidence for the synergistic mechanism of this drug composition in overcoming nerabine resistance in ETP-ALL.

Claims

1. An antitumor drug composition, characterized in that, This includes a first formulation containing FHD-286 and a second formulation containing nerabine, wherein the molecular formula of FHD-286 is C 24 H 30 N6O6S2, structural formula is 。 2. The antitumor pharmaceutical composition according to claim 1, characterized in that, The molar ratio of FHD-286 to Nairabin is 1:1~5.

3. The antitumor drug composition according to claim 2, characterized in that, The molar ratio of FHD-286 to Nairabin is 3:

10.

4. The antitumor drug composition according to any one of claims 1-3, characterized in that, The first formulation containing FHD-286 is an oral formulation, and the second formulation containing nerabine is an intravenous injection formulation.

5. The antitumor drug composition according to claim 4, characterized in that, The first and second formulations also contain pharmaceutically acceptable excipients.

6. The use of an antitumor pharmaceutical composition as described in any one of claims 1-5 in the preparation of a therapeutic agent for acute early precursor T-lymphocytic leukemia.

7. The application according to claim 6, characterized in that, The therapeutic drug targets acute early precursor T-lymphoblastic leukemia cells that are resistant to nerabine.

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