A combined drug for treating acute t-lymphocyte leukemia and central nervous system leukemia and application thereof

Abstract

The application provides a combined drug for treating acute T lymphocyte leukemia and central nervous system leukemia and application thereof, and the combined drug for treating T-ALL comprises dasatinib and dabrafinib. The application creatively combines the drug dasatinib and the drug dabrafinib as a drug for treating T-ALL, and the combination has a significant synergistic effect, has a significant effect of inhibiting T-ALL cell proliferation than a single drug, has an effect of prolonging the survival time of a T-ALL mouse model, and relieves the progress of T-ALL combined with central nervous system leukemia. The application provides a new strategy and idea for treating acute T lymphocyte leukemia and acute T lymphocyte leukemia combined with central nervous system leukemia, and has a very significant meaning.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a combination drug for treating acute T-lymphoblastic leukemia and its application. Background Technology

[0002] Acute lymphoblastic leukemia (ALL) is a lymphocytic hematologic malignancy. Acute T-cell lymphoblastic leukemia (T-ALL) is a subtype of ALL originating from the T-cell lineage, accounting for 10-15% of pediatric ALL patients and 20-25% of adult ALL patients. T-ALL often presents with more aggressive clinical manifestations than other types of ALL and is prone to complications such as central nervous system leukemia (CNSL). Combined chemotherapy is the first-line treatment for T-ALL, but its efficacy is not ideal. Approximately 20% of children and 50% of adults with T-ALL fail to achieve durable complete remission and die due to disease progression. To improve efficacy and prognosis, more second-line treatment options, including hematopoietic stem cell transplantation (HSCT) and CAR-T (Chimeric antigen receptor T cells), have been applied in clinical trials and practice. However, these cell therapy methods are costly, complex to manage throughout the process, and lack medical accessibility in some developing regions. Therefore, the high invasiveness of T-ALL, limited clinical treatment options, and poor prognosis remain clinical pain points, and there is still a need to research effective and relatively economical T-ALL treatments, especially in relapsed or refractory T-ALL.

[0003] Dasatinib is an FDA-approved second-generation BCR-ABL tyrosine kinase inhibitor (TKI), originally developed for the treatment of chronic myeloid leukemia (CML) and currently also used to treat Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Considering dasatinib's targeted inhibition of activated ABL1 protein, it has also been used to treat T-ALL patients with ABL1 gene amplification and carrying the NUP214-ABL1 fusion gene. However, BCR / ABL1-positive TALL is rare, unlike CML, which always presents with BCR / ABL1 positivity. Subsequently, researchers began exploring the use of dasatinib to treat T-ALL patients without ABL1 gene abnormalities. Studies have shown that dasatinib and protein degradation-targeting chimeras (PROTACs) using dasatinib as a ligand exhibit targeted killing effects against T-ALL in computer simulations (in silicon), in vitro experiments, and in vivo experiments. After in vitro cell experiments verified that dasatinib effectively kills patient-derived T-ALL cells, some patients with refractory or relapsed T-ALL have used dasatinib as salvage therapy and achieved remission. This unexpected effect is mainly due to dasatinib's ability to inhibit LCK, a key regulator of the T cell receptor (TCR) signaling pathway and T cell proliferation, and one of the most vulnerable targets in T-ALL.

[0004] Although some studies have shown that dasatinib is effective in some TALL patients, nearly half of the TALL cells derived from these patients exhibit resistance to dasatinib in in vitro experiments. Studies have shown that in cell line experiments, although LCK gene expression was significantly inhibited by dasatinib, the activity and proliferation of TALL cells were not affected. Furthermore, the correlation between the sensitivity of samples from TALL patients to dasatinib and LCK expression has been inconsistent across different studies. All these previous studies suggest that, in addition to TCR-LCK signaling, there may be other important kinases or pathways regulating the response of tumor cells to dasatinib.

[0005] Compared to CML, BCR-ABL fusion gene positivity is rarer in T-ALL, and the incidence of central nervous system leukemia is higher. Therefore, the pathogenesis and treatment focus of these two diseases differ. Dasatinib, as one of the standard treatments for CML, has not yet been elucidated in its efficacy and resistance mechanisms in T-ALL. Therefore, further research is needed on the pharmacological targets of dasatinib and the molecular mechanisms influencing its action to identify potential combination drug targets, improve treatment response, and enhance patient prognosis. Summary of the Invention

[0006] The present invention aims to overcome the defects and shortcomings of existing dasatinib in the treatment of acute T-cell lymphoblastic leukemia, and provides a new treatment option for acute T-cell lymphoblastic leukemia.

[0007] The first objective of this invention is to provide the use of dabrafenib in the preparation of dasatinib as an adjuvant for the prevention, improvement or treatment of acute T-lymphoblastic leukemia.

[0008] A second objective of this invention is to provide the use of dasatinib and dabrafenib in combination in the preparation of a medicament for the prevention, improvement or treatment of acute T-lymphoblastic leukemia.

[0009] Another object of the present invention is to provide a combination drug for treating acute T-lymphoblastic leukemia, characterized in that it comprises dasatinib and dabrafenib, and pharmaceutically acceptable excipients.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention creatively combines dasatinib and dabrafenib as a treatment for acute T-lymphoblastic leukemia (T-ALL). Dasatinib induces apoptosis in T-ALL cells via the LCK pathway, but other mechanisms limit its efficacy, resulting in limited treatment of T-ALL. Mechanistic studies reveal that dasatinib may transactivate the MAPK pathway; inhibiting the MAPK pathway enhances dasatinib's inhibitory effect on T-ALL cell proliferation. Dabrafenib, by inhibiting the MAPK pathway, exhibits a synergistic killing effect on T-ALL cells with dasatinib, and both dasatinib and dabrafenib can cross the blood-brain barrier. This invention demonstrates that the combination of dasatinib and dabrafenib significantly inhibits T-ALL cell proliferation more than either dasatinib alone, significantly prolongs the survival time of T-ALL mouse models more than either dasatinib alone, and alleviates the progression of T-ALL complicated by central nervous system leukemia.

[0012] In a first aspect, the present invention provides the use of dabrafenib in the preparation of an adjuvant to dasatinib for the prevention, improvement or treatment of acute T-lymphoblastic leukemia.

[0013] Secondly, the present invention provides the use of dasatinib and dabrafenib in combination in the preparation of a medicament for the prevention, improvement or treatment of acute T-lymphoblastic leukemia.

[0014] Thirdly, the present invention provides the use of dasatinib and dabrafenib in combination in the preparation of an inhibitor of acute T-lymphocytic leukemia cell proliferation or an inducer of apoptosis.

[0015] Fourthly, the present invention provides the use of dasatinib and dabrafenib in combination in the preparation of an inhibitor of clonogenic formation in acute T-lymphocytic leukemia cells.

[0016] Fifthly, the present invention provides the use of dasatinib and dabrafenib in combination in the preparation of an inhibitor for acute T-lymphoblastic leukemia complicated with central nervous system leukemia.

[0017] Specifically, in the above-mentioned combined use of dasatinib and dabrafenib, the mass ratio of dasatinib to dabrafenib is 1:(1-100).

[0018] Preferably, in the application of dasatinib and dabrafenib in combination as described above, the mass ratio of dasatinib to dabrafenib is 1:(3-60).

[0019] More preferably, the mass ratio of dasatinib to dabrafenib is 1:(3-15).

[0020] More preferably, the mass ratio of dasatinib to dabrafenib is 1:(3-11).

[0021] In a sixth aspect, the present invention provides a combination drug for treating acute T-lymphoblastic leukemia, characterized in that it comprises dasatinib and dabrafenib, and pharmaceutically acceptable excipients.

[0022] The combined medication may be a single compound preparation or a combination of two separate preparations. When the combined medication is a combination of two separate preparations, the administration method may be simultaneous administration of the two separate preparations, alternating administration, or sequential administration. The preparation may be any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0023] The combined drug can be administered alone or in combination with pharmaceutical excipients to form an appropriate dosage form for administration. The pharmaceutical excipients include any one or a combination of at least two of the following: carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers. The carrier includes liposomes, micelles, dendritic macromolecules, etc.

[0024] Preferably, the pharmaceutical carrier includes liposomes, micelles, dendritic macromolecules, microspheres, or microcapsules.

[0025] The present invention has the following beneficial effects:

[0026] This invention creatively combines dasatinib and dabrafenib as a treatment for T-ALL. Dasatinib induces apoptosis in T-ALL cells through the LCK pathway, but its efficacy in treating T-ALL is limited due to other mechanisms. Mechanistic studies in this invention have revealed that dasatinib may transactivate the MAPK pathway; inhibiting the MAPK pathway enhances dasatinib's inhibitory effect on T-ALL cell proliferation. This invention demonstrates that the combination of dasatinib and dabrafenib significantly inhibits T-ALL cell proliferation and significantly prolongs the survival time of T-ALL mouse models compared to either dasatinib alone. Furthermore, both dasatinib and dabrafenib can cross the blood-brain barrier, mitigating the progression of T-ALL complicated with central nervous system leukemia. This invention provides a new strategy and approach for the treatment of acute T-lymphoblastic leukemia and acute T-lymphoblastic leukemia complicated with central nervous system leukemia, and is of significant importance. Attached Figure Description

[0027] Figure 1 The results show the inhibitory effects of different drug combinations (dasatinib and different MAPK pathway inhibitor combinations) on the proliferation of HAP1 cell lines in Example 1.

[0028] Figure 2 The figure shows the results of the inhibitory effect of dasatinib and different concentrations of dabrafenib on the proliferation of HAP1 cell line in Example 1.

[0029] Figure 3 The figure shows the results of the inhibitory effect of different concentrations of drug combinations on the proliferation of T-ALL cell line in Example 2.

[0030] Figure 4 The figure shows the results of the inhibitory effect of different concentrations of drug combinations on the proliferation of primary T-ALL cells in Example 3.

[0031] Figure 5 This is a graph showing the weight statistics of each group of NSG mice in Example 4.

[0032] Figure 6 The survival curves of each group of NSG mice in Example 4 are shown.

[0033] Figure 7 This is an image showing the HE staining results of pathological sections of the spinal cord and subarachnoid space in the control group of Example 4.

[0034] Figure 8 This is an image showing the HE staining results of pathological sections of the spinal cord and subarachnoid space in the drug combination group in Example 4. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] The drugs dasatinib and dabrafenib mentioned in the following examples were provided by Shanghai Lanmu Chemical Co., Ltd.

[0038] The experimental animals, SPF-grade NSG mice, were provided by the Animal Center of the First Affiliated Hospital of Sun Yat-sen University. The mice were 6-8 weeks old and female. All operations on the mice were performed in a sterile laminar flow room.

[0039] Clinical case specimens (bone marrow specimens from 2 T-ALL patients) were obtained from the Department of Pediatrics II, The First Affiliated Hospital of Sun Yat-sen University.

[0040] The T-ALL cell lines (including Jurkat and CCRF-CEM) were kindly donated by Professor Chen Yueqin of the School of Life Sciences, Sun Yat-sen University.

[0041] Example 1: Drug Screening Combination Drugs

[0042] The experimental procedure was as follows: HAP1 cells that had been passaged to the 4th or 5th generation and were in good condition were injected at a dose of 1.5 × 10⁻⁶. 4 HAP1 cells were seeded into 96-well plates at 1 μM each and placed in a cell culture incubator. After 24 hours, the culture medium was discarded, and 100 nM dasatinib and 1 μM of different MAPK pathway inhibitors (lapatinib, sorafenib, regorafenib, lonafarnib, sottorazib (AMG-510), dabrafenib, vemurafenib, selumetinib, cobimetinib, trametinib, and Asiatic acid) were added to the 96-well plates. A DMSO control group and a control group without dasatinib (i.e., DMSO replacing dasatinib) were also included. After 48 hours, the culture medium was discarded, and the proliferation of HAP1 cells in different groups was detected using a CCK8 assay kit. The results are shown below. Figure 1 As shown.

[0043] In addition, HAP1 cells that have been passaged to the 4th or 5th generation and are in good condition are treated at a dose of 1.5 × 10⁻⁶. 4 HAP1 cells were seeded into 96-well plates and incubated in a cell culture incubator. After 24 hours, the culture medium was discarded, and 100 nM dasatinib and different concentrations of dabrafenib (concentration gradients of 0 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM) were added to the 96-well plates. A control group without dasatinib was also included. After 48 hours, the culture medium was discarded, and the proliferation of HAP1 cells in different groups was detected using a CCK8 assay kit. Results are as follows: Figure 2 As shown.

[0044] Depend on Figure 1 It was found that the combination of dasatinib and dabrafenib significantly inhibited the proliferation of HAP1 cell lines, showing a more significant inhibitory effect than either dasatinib or dabrafenib alone. This experiment demonstrates that not all MAPK pathway inhibitors can synergistically enhance dasatinib's effects; inhibitors of RTK, RAS, p38, and MEK1 / 2 proteins did not significantly inhibit HAP1 cell line proliferation when combined with dasatinib. Even vemurafenib, an inhibitor targeting the same RAF protein, did not synergize with dabrafenib, suggesting a specific mechanism underlying the synergistic effect of the combination of dasatinib and dabrafenib. Figure 2 The results showed that dabrafenib alone had a weak inhibitory effect on the HAP1 cell line. However, when used in combination with 100 nM dasatinib, the inhibitory effect on cell proliferation increased with increasing dabrafenib concentration. At concentrations of 0.5 μM and 1 μM, dabrafenib achieved an inhibitory effect of more than 50% on the proliferation of HAP1 cells. This indicates that the combination of dasatinib and dabrafenib has a significant concentration-dependent inhibitory effect on the proliferation of HAP1 cells.

[0045] Example 2: Inhibitory effect of combined drug combination on the proliferation of T-ALL cell line

[0046] The experimental procedure was as follows: T-ALL cell lines (including Jurkat and CCRF-CEM) passaged to the 4th-5th generation and in good condition were divided into cells at a concentration of 1.5 × 10⁻⁶ cells / mL. 4 T-ALL cells were seeded into 96-well plates and incubated in a cell culture incubator. After 24 hours, the culture medium was discarded, and 100 nM dasatinib and different concentrations of dabrafenib were added to the 96-well plates (concentration gradients of 0 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM). A control group without dasatinib was also included. After 48 hours, the culture medium was discarded, and the proliferation of T-ALL cells in different groups was detected using a CCK8 assay kit.

[0047] The results of the inhibitory effects of different drug concentration combinations on the proliferation of T-ALL cell lines are as follows: Figure 3 As shown, Figure 3 Figure A shows the results of treatment with the Jurkat cell line for 48 hours. Figure 3 Figure B shows the results of CCRF-CEM cell line treatment for 48 hours. Figure 3 It is known that dabrafenib alone can inhibit the proliferation of CCRF-CEM cell line to a certain extent, but has almost no inhibitory effect on Jurkat cell line. However, the combination of dasatinib and dabrafenib significantly inhibits the proliferation of T-ALL cell line, and the inhibitory effect is more significant than that of dasatinib or dabrafenib alone. Furthermore, the inhibitory effect of the combination of dasatinib and dabrafenib on the proliferation of T-ALL cell line is significantly concentration-dependent, with dabrafenib at 1 μM combined with 100 nM dasatinib showing better efficacy.

[0048] Example 3: Evaluation of the effects of the combination drug regimen on T-ALL cells at the primary cell level.

[0049] The experimental procedure was as follows: Bone marrow samples were collected from two high-risk T-ALL patients, and mononuclear cells were extracted using lymphocyte separation medium. The cells were then analyzed at a concentration of 1.5 × 10⁻⁶ cells / mL. 4 Seed each cell in a 96-well plate and incubate them in a cell culture incubator. After 24 hours, discard the culture medium and add 100 nM dasatinib and different concentrations of dabrafenib (concentration gradients of 0 μM, 1 μM, and 5 μM) to the 96-well plates. A control group without dasatinib was also included. After 48 hours, discard the culture medium and use a CCK8 assay kit to detect the proliferation of primary T-ALL cells in different groups.

[0050] The results of the inhibitory effects of different drug concentration combinations on the proliferation of primary T-ALL cells are as follows: Figure 4 As shown. By Figure 4 It is known that 100 nM dasatinib alone may not necessarily inhibit the proliferation of primary T-ALL cell lines. However, the combination of dasatinib and dabrafenib significantly inhibits the proliferation of primary T-ALL cell lines, with a more significant inhibitory effect than either dasatinib or dabrafenib alone. Furthermore, the inhibitory effect of the dasatinib-dabrafenib combination on the proliferation of primary T-ALL cell lines is significantly concentration-dependent; the inhibitory effect on cell line proliferation increases with increasing dabrafenib concentration.

[0051] Example 4: Evaluation of the effects of the combination drug on T-ALL cells at the animal level.

[0052] The experimental procedure is as follows:

[0053] (1) An NSG mouse tumor-bearing model was constructed using the T-ALL cell line Jurkat.

[0054] Jurkat cells were resuspended in 100 μL of PBS (containing 10 μg / mL of PBS). 6 (a cell) was injected into NSG mice via the tail vein, and in vivo drug administration experiments began 4 weeks later.

[0055] (2) A blank control group (using an equal volume of solvent instead of the drug), a dasatinib group (10 mg / kg / d), a dabrafenib group (30 mg / kg / d), and a dasatinib and dabrafenib combination group (10 mg / kg / d and 30 mg / kg / d) were set up, with 8 mice in each group. The drugs were administered daily (orally) until the human endpoint. The weight of the mice was monitored daily, and survival curves were plotted.

[0056] from Figure 5 The statistical results of weight changes in each group show that the weight loss in the combination drug group was slower than that in the control group and the single drug treatment group, indicating that the combination of dasatinib and dabrafenib has a smaller impact on animal weight and lower toxicity to the body.

[0057] from Figure 6 The survival curves showed that, at 40 days, the mice treated with the combination of dasatinib and dabrafenib had a survival rate of approximately 80%, compared to approximately 40% in the dabrafenib group and only about 30% in the dasatinib group. Regarding the time to death for all mice, the mice in the dasatinib and dabrafenib combination group died at 45 days, a significantly longer survival time than the control group and the single-drug group. In conclusion, the median survival time of the combination therapy group was significantly longer than that of the control group and the single-drug treatment group.

[0058] from Figure 7 control group and Figure 8 HE staining results of spinal cord and subarachnoid space pathological sections in the combined drug group showed that the combined drug treatment significantly reduced leukemia cell infiltration in the subarachnoid space and alleviated T-ALL combined with central nervous system leukemia.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of dasatinib in the preparation of dasatinib as an adjuvant for the prevention, improvement or treatment of acute T-lymphoblastic leukemia, wherein the mass ratio of dasatinib to dasatinib is 1:(3~11).

2. The use of dasatinib and dabrafenib in combination in the preparation of drugs for the prevention, improvement or treatment of acute T-lymphoblastic leukemia, wherein the mass ratio of dasatinib to dabrafenib is 1:(3~11).

3. The application of dasatinib and dabrafenib in the preparation of an inhibitor of cell proliferation or an inducer of apoptosis in acute T-lymphocytic leukemia, wherein the mass ratio of dasatinib to dabrafenib is 1:(3~11).

4. The application of dasatinib and dabrafenib in the preparation of an inhibitor of clonogenicity in acute T-lymphocytic leukemia, wherein the mass ratio of dasatinib to dabrafenib is 1:(3~11).

5. The use of dasatinib and dabrafenib in combination in the preparation of drugs for the prevention, improvement or treatment of acute T-lymphoblastic leukemia complicated with central nervous system leukemia, wherein the mass ratio of dasatinib to dabrafenib is 1:(3~11).

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