A pharmaceutical composition for treating acute lymphoblastic leukemia and use thereof

Abstract

This invention relates to a pharmaceutical composition for treating acute lymphoblastic leukemia (ALL) and its application, belonging to the field of biomedical technology. Addressing the problem of poor efficacy of FHD-286 monotherapy due to adaptive survival of ALL cells, this invention provides a pharmaceutical composition comprising FHD-286 and the IRE1α inhibitor STF-083010. In this invention, STF-083010 is not simply added as a generic second anti-leukemia drug, but rather is used to block the residual IRE1α-dependent adaptive survival pathway after FHD-286 treatment. Synergistic analysis results show that the combination of the two drugs has synergistic anti-ALL activity, rather than a simple additive effect. In a PDX mouse model, the combination therapy significantly reduced the ALL burden compared to monotherapy, decreased liver and spleen weight and the proportion of human CD45-positive cells in peripheral blood, spleen, and bone marrow, reduced ALL cell infiltration, and prolonged survival.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a pharmaceutical composition for treating acute lymphoblastic leukemia and its application. Background Technology

[0002] Acute lymphoblastic leukemia (ALL) is a type of malignant clonal tumor originating from lymphocyte precursor cells, characterized by rapid clinical progression and poor prognosis. Although the complete remission rate of ALL has improved with the continuous development of new drugs and treatment regimens, efficacy and tolerability are limited in elderly patients or those with underlying diseases, and treatment-related toxicity, drug resistance, and relapse remain key bottlenecks. Therefore, the development of new targets and combination therapies is of significant clinical importance.

[0003] Gene expression in eukaryotic cells is precisely controlled by an epigenetic regulatory network. The SWI / SNF complex is an ATP-dependent chromatin remodeling complex that regulates the accessibility of transcription factors and enhancers / promoters by hydrolyzing ATP to move nucleosomes and remodeling chromatin structure. BRG1 is the core catalytic subunit of this complex and plays a crucial role in the survival and differentiation of leukemia stem cells / progenitor cells.

[0004] FHD-286 is a selective BRG1 / BRM ATPase inhibitor that induces leukemia cell differentiation and inhibits their proliferation by blocking the chromatin remodeling function of the SWI / SNF complex. Based on this epigenetic regulatory mechanism, the field has been exploring combination therapy strategies for FHD-286. Studies have shown that FHD-286, in combination with the BET inhibitor OTX015 or the demethylating drug decitabine, can produce a synergistic effect at the epigenetic level, demonstrating enhanced anti-leukemic activity in acute myeloid leukemia (AML) models with mixed lineage leukemia (MLL) gene rearrangements or nucleophosphorus protein 1 (NPM1) gene mutations; the combination regimen of FHD-286 with an FLT3 inhibitor has also shown superior efficacy compared to monotherapy in preclinical models.

[0005] However, the existing combination therapies still have key shortcomings. Although FHD-286 monotherapy has cytotoxic activity against leukemia stem cells / progenitor cells, preclinical and clinical studies have shown that some leukemia cells can survive under FHD-286 drug stress, becoming the root cause of subsequent relapse and treatment resistance. Existing combination strategies are still limited to epigenetic synergistic regulation and have not elucidated the mechanism of adaptive survival of leukemia cells after FHD-286 treatment, nor have they specifically blocked this survival pathway. Therefore, how to fundamentally overcome the adaptive survival of leukemia cells after FHD-286 treatment is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the issue of poor efficacy of FHD-286 monotherapy due to adaptive survival of ALL cells, this invention provides a pharmaceutical composition for treating acute lymphoblastic leukemia and its application.

[0007] The technical solution of the present invention:

[0008] A pharmaceutical composition for treating acute lymphoblastic leukemia, the pharmaceutical composition comprising FHD-286 and STF-083010,

[0009] The molecular formula of FHD-286 is C 24 H 30 N6O6S2, structural formula is

[0010] ;

[0011] The molecular formula of STF-083010 is C 15 H 11 NO3S2, structural formula is .

[0012] Furthermore, the molar ratio of FHD-286 to STF-083010 in the pharmaceutical composition is 1:35.

[0013] Furthermore, the pharmaceutical composition is a single compound preparation, or a combination of a first preparation containing FHD-286 and a second preparation containing STF-083010.

[0014] Furthermore, the first formulation and the second formulation are used for simultaneous, sequential, or alternating administration.

[0015] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0016] The use of a pharmaceutical composition provided by the present invention in the preparation of a treatment for acute lymphoblastic leukemia.

[0017] The beneficial effects of this invention are:

[0018] This invention reveals for the first time that BRG1 is a key factor regulating the activity of the ER / UPR pathway in ALL cells. While FHD-286 inhibits BRG1, it also downregulates the expression of key genes in the ER / UPR pathway, including IRE1α. However, the downregulation of IRE1 by FHD-286 is not equivalent to a complete blockade of IRE1 function; endoplasmic reticulum stress signals can still directly activate IRE1's endonuclease activity through other mechanisms. Therefore, although BRG1 inhibitors can downregulate IRE1 expression and function, they cannot completely block IRE1 protein activation and its downstream signaling. Residual IRE1α-dependent UPR signaling can still serve as a compensatory survival pathway for tumor cells, which may be an important mechanism for the continued survival and acquired resistance of ALL cells under FHD-286 treatment stress. Based on the above analysis, this invention proposes a dual-attack inhibition strategy that combines upstream and downstream vertical blockade: on the basis of using FHD-286 to inhibit BRG1, it is further combined with the IRE1α specific inhibitor STF-083010 to block residual IRE1α-dependent adaptive signaling and synergistically enhance the anti-leukemia effect.

[0019] FHD-286 inhibits BRG1 upstream, disrupting its oncogenic program and downregulating IRE1 expression and function; STF-083010 directly blocks IRE1 functional activity at the effector terminal, blocking the BRG1-independent IRE1 activation pathway. In this invention, the IRE1α-specific inhibitor STF-083010 is not simply added as a generic second anti-leukemia drug, but rather acts as a "functional enhancer" and "resistance eliminater" for FHD-286, blocking the residual IRE1α-dependent adaptive survival pathway after FHD-286 treatment. This combined design is not simply the superposition of two poor prognostic targets, but rather a synergistic attack along the upstream and downstream nodes of the same oncogenic axis. The two drugs are responsible for "weakening the source" and "blocking function," respectively, forming a complementary closed loop in mechanism. The combination of the two can achieve functional complementarity, realizing anti-tumor effects that cannot be achieved by single drugs or other combination regimens.

[0020] Experimental data confirmed that FHD-286 treatment resulted in changes in the expression levels of key genes in the ER / UPR pathway, such as IRE1, and an increase in the content of unfolded proteins, suggesting that BRG1 is a key factor regulating the activity of the ER / UPR pathway. Further verification showed that FHD-286 and STF-083010 treatment significantly promoted apoptosis of ALL cells in acute lymphoblastic leukemia (ALL) patient samples. Synergistic analysis results showed that the combination of the two drugs had synergistic anti-ALL activity in patient samples, confirming that the combination was not a simple additive effect. In the PDX mouse model, the combination therapy significantly reduced the ALL burden compared to single-drug therapy, decreased liver and spleen weight, and reduced the proportion of human CD45-positive cells in peripheral blood, spleen, and bone marrow. No obvious ALL cell infiltration was observed in peripheral blood smears, and the survival time of the model animals was significantly prolonged.

[0021] This invention proposes for the first time a treatment regimen combining STF-083010 and FHD-286, and provides the application of this pharmaceutical composition in the preparation of drugs for the treatment of acute lymphoblastic leukemia. The pharmaceutical composition of this invention can be prepared as a single compound preparation or a combination of two separate preparations, offering flexible administration methods and demonstrating good clinical translational potential and application prospects. Attached Figure Description

[0022] Figure 1 This is a comparison of the expression and function changes of key genes in the ER / UPR pathway after FHD-286 treatment of ALL cells in Example 1. A is the RT-qPCR result, B is the Western Blot result, and C is the TPE-MI fluorescence detection result.

[0023] Figure 2 This is a comparison of the apoptosis rate of primary leukemia cells from ALL patients after treatment with FHD-286, STF-083010, and siBRG1 in Example 2. A represents the FHD-286 group, B represents the STF-083010 group, and C represents the siBRG1 group.

[0024] Figure 3 This is a diagram based on the ZIP algorithm showing the synergistic effect of drugs after FHD-286 and STF-083010 were used to treat primary ALL leukemia cells in Example 3.

[0025] Figure 4 The images show the liver and spleen tissue morphology photographs and the liver and spleen weight / body weight ratio comparisons of the PDX mice in each group in Example 4. A is a liver and spleen tissue morphology photograph, B is the liver weight / body weight ratio, and C is the spleen weight / body weight ratio.

[0026] Figure 5 This is a comparison of the proportion of hCD45 positive cells in the spleen, bone marrow, and peripheral blood of PDX mice in each group in Example 4. A represents the spleen, B represents the bone marrow, and C represents the peripheral blood.

[0027] Figure 6 The images show the Wright-Giemsa staining comparison of peripheral blood smears from PDX mice in each group in Example 4. A is the control group, B is the STF-083010 single drug group, C is the FHD-286 single drug group, and D is the FHD-286 and STF-083010 dual drug combination group.

[0028] Figure 7 The image shows the Kaplan-Meier survival curves of each group of PDX mice in Example 4. Detailed Implementation

[0029] 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.

[0030] The experimental data from Examples 1-4 of this invention were analyzed using Prism 10. The homogeneity of variance between two groups was tested using a t-test, and the homogeneity of variance between three or more groups was tested using one-way ANOVA. All data are described using mean ± SEM. A p-value < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0031] Example 1

[0032] To verify the changes in mRNA and protein expression levels and functions of key genes in the ER / UPR pathway, such as IRE1α, after treatment of ALL cells with the BRG1 inhibitor FHD-286, this example conducted RT-qPCR, Western Blot, and TPE-MI fluorescence detection experiments.

[0033] I. Experimental Methods

[0034] The human ALL cell line KOPN-8 was divided into a DMSO control group and an FHD-286 treatment group (15 nM). Both groups were cultured at 37°C and 5% CO2 using RPM-1640 medium supplemented with 10% fetal bovine serum, and mycoplasma contamination was detected periodically.

[0035] (1) RT-qPCR

[0036] The relative mRNA expression levels of genes related to the endoplasmic reticulum stress pathway were detected using RT-qPCR. Target genes included IRE1, XBP1s, EIF2AK3, ATF4, and ATF6. β-actin was used as an internal reference gene for standardization. The specific procedures are as follows:

[0037] 1 μg of purified RNA was reverse transcribed using cDNA Synthesis SuperMix (YEASON, #11141ES60). RT-qPCR was performed on a BIOER real-time PCR system using the universal SYBR Green Master Mix (YEASON, #11184ES08). The relative expression levels of target gene mRNA were calculated using a method and standardized using β-actin Ct values.

[0038] (2) Western Blot

[0039] Western blotting was used to detect the expression levels of proteins related to the endoplasmic reticulum stress pathway. The proteins tested included phosphorylated PERK (p-PERK), total PERK, phosphorylated IRE1 (p-IRE1), total IRE1, phosphorylated EIF2A (p-EIF2A), total EIF2A, ATF6, ATF4, spliced ​​XBP1 (XBP1s), and unspliced ​​XBP1 (XBP1u). ACTIN was used as an internal control to correct the loading amount. The specific procedures are as follows:

[0040] Two groups of cells were collected, washed with PBS, and lysed on ice for 30 minutes with RIPA lysis buffer, vortexing every 10 minutes. The lysis buffer was centrifuged at 12,000 rpm for 15 minutes at 4°C to obtain the total protein supernatant. The protein concentration was determined using the BCA method, and a standard curve was plotted. The protein samples from each group were diluted to the same concentration. The protein samples were mixed with 5× loading buffer at a volume ratio of 4:1, boiled at 100°C for 5 minutes to denature the proteins, cooled, and briefly centrifuged. The mixture was vortexed before loading. 7.5% separating and stacking gels were prepared for SDS-PAGE electrophoresis. 20 μg of total protein was loaded into each lane. The stacking gel was electrophoresed at 80V for about 30 minutes, and the separating gel was electrophoresed at 120V for 60-90 minutes, stopping electrophoresis when the bromophenol blue front was close to the bottom of the gel. The PVDF membrane was activated with methanol for 1 minute and then rinsed with deionized water. The filter paper and sponge were pre-equilibrated in transfer buffer and assembled in the following order: black plate - sponge - filter paper - gel - membrane - filter paper - sponge - red plate. The transfer buffer (1L) was prepared by adding 3.03g Tris, 14.4g glycine, 200mL methanol, and deionized water to a final volume of 1L, adjusting the pH to 8.3. Transfer conditions were: wet transfer system at 300mA constant current, ice bath for 120 minutes. Membrane blocking was performed using 5% skim milk powder at room temperature for 2 hours. The membrane was washed three times with TBST for 5 minutes each time. The primary antibody was diluted according to the recommended dilution ratio and incubated overnight at 4°C with gentle agitation during incubation. Before secondary antibody incubation, the membrane was washed three times with TBST for 10 minutes each time. The HRP-conjugated secondary antibody was diluted and incubated at room temperature for 2 hours with continuous agitation. The membrane was then exposed and detected using an ECL luminescence imaging system.

[0041] (3) TPE-MI fluorescence detection

[0042] Intracellular unfolded protein content was detected using flow cytometry combined with TPE-MI fluorescence staining to reflect the activation level of endoplasmic reticulum stress and unfolded protein response (ER / UPR). The specific procedures are as follows:

[0043] Cells in the logarithmic growth phase were collected, washed twice with PBS, and then the cell density was adjusted to 1×10⁻⁶. 6Cells / mL, 100 μL of cell suspension was added to flow cytometry tubes; unstained blank group, DMSO control group, and FHD-286 drug-treated group were set up. TPE-MI staining solution to a final concentration of 2 mM was added to each group, and after incubation at 37°C for 45 minutes in the dark, the cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS. Then, 400 μL of PBS was added and mixed thoroughly. After 1 hour, the TPE-MI fluorescence signal intensity of each group was detected using flow cytometry. The unstained blank group was used to calibrate the flow cytometer voltage, set the fluorescence negative threshold, and subtract interference from cell autofluorescence. TPE-MI fluorescence staining was performed on cells in the DMSO control group and FHD-286 drug-treated group, and the percentage of TPE-MI positive cells with fluorescence signals above the negative threshold in each group was counted. This percentage is the relative content of unfolded protein (% of TPE-MI), used to quantify the proportion of cells with protein misfolding and evaluate the level of endoplasmic reticulum stress activation.

[0044] II. Experimental Results

[0045] RT-qPCR results are as follows Figure 1 As shown in Figure A, compared with the DMSO control group, the mRNA expression of key genes in the ER / UPR pathway, IRE1, XBP1s, EIF2AK3, ATF4, and ATF6, was significantly downregulated in the FHD-286-treated group (****, P<0.0001), indicating that FHD-286 can inhibit the expression of UPR-related genes at the transcriptional level. However, the mRNA expression of IRE1α was not completely eliminated, and a clear mRNA signal could still be detected, indicating that the transcription of IRE1α was not completely silenced.

[0046] Western Blot results are as follows: Figure 1 As shown in Figure B, after FHD-286 treatment, the expression of activated phosphorylated proteins and downstream effector proteins of the PERK pathway (p-PERK, p-EIF2A, ATF4), IRE1 pathway (p-IRE1, XBP1s, XBP1u), and ATF6 pathway was significantly reduced, but the protein bands did not disappear, and there was still detectable residual protein expression, suggesting that FHD-286 cannot completely eliminate ER / UPR pathway-related proteins in cells.

[0047] The TPE-MI fluorescence detection results are as follows: Figure 1 As shown in Figure C, only about 10% of the cells in the DMSO control group had a small amount of background unfolded protein; after FHD-286 treatment, the proportion of unfolded protein positive cells increased significantly to nearly 30% (****, P<0.0001), indicating that there are a large number of misfolded proteins in the cells that urgently need to be treated.

[0048] The above experimental results indicate that while FHD-286 can inhibit IRE1α expression, it does not completely eliminate it; IRE1α expression and function remain. Furthermore, after FHD-286 treatment, unfolded proteins not only do not decrease but accumulate in large quantities, constituting a persistent activation signal for IRE1α. In this state, the endoplasmic reticulum stress response triggered by residual IRE1α and other key ER / UPR factors becomes almost the only dependent pathway for cells to cope with unfolded protein stress, providing a molecular basis for the adaptive survival of residual ALL cells under drug stress. Based on this mechanism, this invention proposes, in addition to FHD-286's inhibition of BRG1, to further combine it with the IRE1α-specific inhibitor STF-083010 to completely block the residual activity of IRE1α at the functional level, thereby synergistically eliminating residual ALL cells and overcoming the adaptive survival problem caused by FHD-286 monotherapy.

[0049] Example 2

[0050] To verify whether FHD-286 targeting BRG1, STF-083010 targeting IRE1α, and direct knockdown of BRG1 can effectively induce apoptosis in primary cells of patients with acute lymphoblastic leukemia, this embodiment conducted an in vitro primary cell apoptosis experiment.

[0051] I. Experimental Methods

[0052] (1) Primary cell preparation and culture

[0053] In this embodiment, primary samples were selected from patients with acute lymphoblastic leukemia (ALL). All bone marrow samples were aseptically collected after obtaining informed consent from the patients.

[0054] To ensure the representativeness and reproducibility of the experimental results, all included primary ALL samples were clinically and pathologically confirmed and met the following screening criteria:

[0055] (1) Immunophenotype is B-ALL (cCD79a) + CD19 + CD10 + CD34 + 、TdT + , and sIg - ) or T-ALL (cCD3) + CD7 + CD5 + CD2 + And sCD3 - / + );

[0056] (2) Not carrying gene mutations known to be resistant to FHD-286 or STF-083010 (e.g., BCR-ABL1 fusion gene positive samples are excluded).

[0057] (3) The sample viability is >80% and the total number of cells meets the requirements for multiple parallel experiments.

[0058] Each donor sample was obtained from at least three independent patients, and the experiment was independently repeated at least three times to exclude the influence of individual differences on the experimental results.

[0059] Bone marrow mononuclear cells were isolated and purified using Ficoll density gradient centrifugation to remove contaminating cells such as erythrocytes and granulocytes, ensuring the purity of the experimental cells. The isolated primary cells were cultured in RPMI-1640 complete medium supplemented with 20% fetal bovine serum (FBS) at a constant temperature in a standard cell culture environment to maintain the viability and malignant biological characteristics of the primary leukemia cells for subsequent drug intervention experiments.

[0060] (2) Drug treatment methods

[0061] FHD-286 is a selective BRG1 / BRM ATPase inhibitor with the molecular formula C2. 24 H 30 N6O6S2, CAS No. 2671128-05-3, was purchased from Selleck Chemicals.

[0062] STF-083010 is an IRE1α-specific endonuclease inhibitor with the molecular formula C. 15 H 11 NO3S2, CAS number 307543-71-1, purchased from MCE.

[0063] Primary cells from patients with acute lymphoblastic leukemia were treated with FHD-286 and STF-083010, respectively.

[0064] FHD-286 final concentration 250nM, STF-083010 final concentration 750nM;

[0065] The same volume of DMSO was used as a blank control, and all samples were treated for 96 hours.

[0066] (3) siRNA transfection experiment

[0067] For ALL samples, BRG1 expression was knocked down using specific siBRG1, with the negative control siNC as a reference:

[0068] The justice chain sequence of siBRG1 is 5'-GGUCAAUGGUGUCCUCAAA-3';

[0069] The antisense strand sequence of siBRG1 is 5'-UUUGAGGACACCAUUGACC-3';

[0070] The justice chain sequence of siNC is 5'-UUCUCCGAACGUGUCACGUUU-3';

[0071] The antisense strand sequence of siNC is 5'-ACGUGACACGUUCGGAGAAUU-3';

[0072] To comply with WIPO ST.26 requirements, as shown in SEQ ID NO:1, the positive strand sequence of siBRG1 uses T to replace U at positions 3, 7, 10, 12, and 15 at the 5' end to represent uracil in RNA; as shown in SEQ ID NO:2, the negative strand sequence of siBRG1 uses T to replace U at positions 1, 2, 3, 14, and 15 at the 5' end to represent uracil in RNA; as shown in SEQ ID NO:3, the positive strand sequence of siNC uses T to replace U at positions 1, 2, 4, 12, 14, 19, 20, and 21 at the 5' end to represent uracil in RNA; as shown in SEQ ID NO:4, the negative strand sequence of siNC uses T to replace U at positions 4, 11, 12, 20, and 21 at the 5' end to represent uracil in RNA.

[0073] Both siBRG1 and siNC were prepared by GenePharma according to standard methods.

[0074] Lipofectamine RNAiMAX reagent was used according to the instructions, with a final transfection concentration of 10 nM. Cells were collected 72 h after transfection for subsequent apoptosis detection.

[0075] (4) Apoptosis detection methods

[0076] After treatment, cells were collected, washed with pre-cooled PBS, stained with Annexin V-FITC / PI apoptosis detection kit, and fluorescence signals were detected by flow cytometry. The apoptosis rate was analyzed using FlowJo software.

[0077] II. Experimental Results

[0078] The results are as follows Figure 2 As shown, compared with the DMSO control group, both FHD-286 and STF-083010 single-drug treatments significantly increased the apoptosis rate of primary ALL cells, and the differences between the groups were statistically significant; compared with the siNC control group, siBRG1 knockdown treatment also significantly increased the apoptosis rate of primary ALL cells.

[0079] The experimental results of this embodiment demonstrate that inhibiting BRG1 gene function through intervention with FHD-286 or siRNA knockdown, as well as targeting and inhibiting IRE1α pathway activity with STF-083010, can effectively induce apoptosis in primary ALL cells, fully validating the anti-ALL effects of both targets. However, the apoptosis rate induced by single drugs is still limited, suggesting that single-target inhibition is insufficient to completely eliminate ALL cells. This further supports the necessity of combined targeting of the BRG1 and IRE1α pathways, providing direct and reliable in vitro pharmacodynamic support for subsequent research on the combined use of FHD-286 and STF-083010 and the construction of novel synergistic treatment strategies.

[0080] Example 3

[0081] To verify whether the combined use of FHD-286 and STF-083010 has a synergistic anti-ALL effect, rather than a simple additive effect, this embodiment conducted a drug synergistic effect analysis based on primary cells from patients with acute lymphoblastic leukemia.

[0082] I. Experimental Methods

[0083] (1) Primary cell preparation and culture

[0084] The method for obtaining and culturing primary ALL cells is the same as in Example 2.

[0085] (2) Combined drug treatment method

[0086] This embodiment refers to the conventional gradient screening method for the synergistic effect of antitumor small molecule drugs, setting multiple sets of continuous concentration gradients for the two drugs to achieve cross-combination dosing treatment covering both high and low concentrations. Specifically, the gradient concentrations for FHD-286 were set at 250 nM, 500 nM, 1000 nM, and 2000 nM; and the gradient concentrations for STF-083010 were set at 2500 nM, 5000 nM, 10000 nM, and 20000 nM.

[0087] The two drugs were combined in pairs at various concentrations, with corresponding concentrations of FHD-286 and STF-083010 as monotherapy groups, and an equal volume DMSO treatment group as a blank control. All groups were simultaneously treated with primary ALL leukemia cells for 96 hours. Through comprehensive concentration gradient cross-screening, the simple additive effect of the drugs and the true synergistic effect were accurately distinguished, and the interaction mode of the two drugs was objectively determined.

[0088] (3) Cell viability detection and synergistic analysis

[0089] After treatment, the relative viability of cells in each group was measured using the CellTiter-Glo chemiluminescence immunoassay reagent and a microplate reader. The experimental data were imported into the Synergyfinder analysis platform, and the ZIP synergy score algorithm was used to calculate the synergistic effect of the two drugs. A higher synergy score indicates a stronger synergistic effect, and a score greater than 10 suggests a significant synergistic effect.

[0090] II. Experimental Results

[0091] The results are as follows Figure 3 As shown, in primary cells from ALL patients, the mean ZIP synergy score of the combination of FHD-286 and STF-083010 was 20.18, which was statistically significant (P=3.55e-07). This indicates that the combination of FHD-286 and STF-083010 exhibits a clear synergistic anti-ALL effect in primary ALL cells, rather than a simple additive effect.

[0092] The results of this embodiment show that STF-083010 can further inhibit the adaptive survival mechanism of ALL cells on the basis of FHD-286. The combination of the two has a significant synergistic effect, providing key evidence for combined drug use in subsequent in vivo animal experiments and clinical translation studies.

[0093] Example 4

[0094] To verify the synergistic anti-ALL effect of the combination of FHD-286 and STF-083010 in vivo, this embodiment uses a xenograft (PDX) mouse model derived from B-ALL patients to conduct pharmacodynamic verification from multiple dimensions, including tumor burden, organ invasion, and survival.

[0095] I. Experimental Methods

[0096] (1) PDX model construction and passaging amplification

[0097] Immunodeficient NKG mice of the same batch, age, sex, and similar weight were selected and purchased from Cyagen to construct a patient-derived xenotransplantation model.

[0098] Primary leukemia cells from B-ALL patients were passaged and expanded in vivo for three consecutive rounds to obtain PDX cells with stable growth and consistent tumor burden. The PDX cells from the third passage were then injected into mice via tail vein injection, with each mouse receiving 3 × 10⁶ cells. 6 Mice were monitored regularly after inoculation for vital signs and the proportion of hCD45-positive cells in peripheral blood to assess changes in leukemia burden.

[0099] (2) Grouping and administration methods

[0100] When the proportion of hCD45-positive cells in the peripheral blood of mice was >1%, leukemia engraftment was considered successful, and drug administration could begin. The experiment was set up with four groups:

[0101] Control group: Administered an equal volume of drug solution, with no drug intervention; all other feeding and treatment conditions were identical to the drug-treated group.

[0102] FHD-286 monotherapy group: FHD-286 solution was administered intraperitoneally at a dose of 1.5 mg / kg body weight;

[0103] STF-083010 monotherapy group: STF-083010 solution was administered intraperitoneally at a dose of 30 mg / kg body weight;

[0104] The combination therapy of FHD-286 and STF-083010: both drugs were administered intraperitoneally simultaneously, with FHD-286 at a dose of 1.5 mg / kg body weight and STF-083010 at a dose of 30 mg / kg body weight;

[0105] The administration frequency for each group was once a day until all mice in the control group died.

[0106] (3) Liver and spleen tumor burden detection

[0107] When the control group mice entered a near-death state, three mice from each group were randomly selected for euthanasia. The liver and spleen were dissected, photographed, and weighed. The ratio of liver and spleen weight to mouse body weight was calculated to assess the degree of ALL cell infiltration into the liver and spleen.

[0108] (4) Flow cytometry detection of the proportion of hCD45 positive cells

[0109] Peripheral blood, spleen, and femoral bone marrow samples were collected from euthanized mice. Single-cell suspensions were prepared and treated with schizoaflavins, followed by staining with human CD45 (hCD45) antibody. Fluorescence signals were detected by flow cytometry, and the proportion of hCD45-positive cells in each sample was analyzed using FlowJo software to assess the distribution and infiltration of ALL cells in mice.

[0110] (5) Wright-Gymsa staining of peripheral blood smears

[0111] Mouse peripheral blood smears were prepared and allowed to air dry before Wright-Giantsa staining. Staining procedure: Wright-Giantsa stain was added to cover the sample, and the sample was allowed to stand for 1 minute for initial staining. Then, 2-3 times the volume of the staining solution of PBS buffer was added along the edge of the slide, gently mixed, and staining continued for 5 minutes. After staining, the slide was rinsed with running water, air-dried at room temperature, and observed under a microscope to assess the degree of ALL cell infiltration in the peripheral blood.

[0112] (6) Monitoring and analysis of mouse survival

[0113] From the date of drug administration, the survival status and survival time of mice in each group were recorded daily. Kaplan-Meier survival curves were plotted using GraphPad Prism software, and the differences in survival time among groups were compared using the log-rank test.

[0114] II. Experimental Results

[0115] Liver and spleen tumor burden test results as follows Figure 4 As shown, compared with the control group, the liver and spleen weights of mice in the FHD-286 monotherapy group and the STF-083010 monotherapy group were significantly reduced, while the liver and spleen weights of the combined drug group decreased most significantly, and the degree of liver and spleen enlargement was significantly alleviated, suggesting that the combined drug can effectively inhibit the infiltration and proliferation of ALL cells in the liver and spleen.

[0116] Flow cytometry results as follows Figure 5 As shown, compared with the control group, the proportion of hCD45 positive cells in the spleen, bone marrow and peripheral blood of mice in each drug administration group was significantly reduced. The proportion of hCD45 positive cells in the combined drug administration group was the most significant, indicating that the combined drug administration can effectively clear ALL cells and reduce their infiltration in key organs.

[0117] Wright-Gymsa staining results as follows Figure 6 As shown, a large number of ALL cells infiltrated the peripheral blood of mice in the control group; the number of ALL cells in the peripheral blood of the single-drug treatment group was significantly reduced; while almost no ALL cells were found in the peripheral blood of the combined drug treatment group, suggesting that the combined drug treatment has a better effect on the in vivo clearance of ALL cells.

[0118] Mouse survival analysis results are as follows Figure 7 As shown, compared with the control group, the survival time of mice in the FHD-286 monotherapy group and the STF-083010 monotherapy group was significantly prolonged; while the survival time of mice in the combination therapy group was the longest, and the difference compared with the monotherapy group was statistically significant, indicating that the combination therapy can significantly prolong the survival time of mice in the leukemia PDX model.

[0119] The in vivo experimental results of this embodiment show that the combined use of FHD-286 and STF-083010 can effectively inhibit the in vivo proliferation and organ infiltration of ALL cells, and significantly prolong the survival of model mice. Its efficacy is significantly better than that of single-drug treatment, providing solid in vivo pharmacodynamic support for the clinical translation of this combination regimen.

Claims

1. A pharmaceutical composition for treating acute lymphoblastic leukemia, characterized in that, The pharmaceutical composition comprises FHD-286 and STF-083010, the molecular formula of the FHD-286 is C 24 H 30 N6O6S2, the structural formula is ； The molecular formula of STF-083010 is C 15 H 11 NO3S2, structural formula is .

2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of FHD-286 to STF-083010 in the pharmaceutical composition is 1:

35.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutical composition is a single compound preparation, or a combination of a first preparation containing FHD-286 and a second preparation containing STF-083010.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

5. Use of a pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating acute lymphoblastic leukemia.

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