Use of cct018159 in combination with nelarabine in the preparation of drugs for acute leukemia

Abstract

The application of CCT018159 combined with nelarabine in the preparation of an acute leukemia drug belongs to the technical field of biological medicine. In view of the problems of high relapse rate and poor prognosis of acute leukemia, the application of CCT018159 combined with nelarabine in the preparation of an acute leukemia drug includes a DDX39B inhibitor CCT018159 and a nucleoside analogue nelarabine. The CCT018159 DDX39B inhibitor is combined with the nucleoside analogue nelarabine, wherein the ara-GTP metabolic product of nelarabine exerts a cytotoxic effect by selectively incorporating T cell DNA and inhibiting DNA polymerase activity; CCT018159 significantly improves the drug sensitivity of T-ALL cells to nelarabine, and achieves synergistic effect. The present application reduces the leukemia load through a double mechanism, prolongs the survival cycle, and can be used for developing T-ALL treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CCT018159 in combination with nerabine in the preparation of drugs for acute leukemia. Background Technology

[0002] Acute leukemia is a malignant clonal disease of hematopoietic stem cells, characterized by the proliferation of abnormal primitive and immature cells in the bone marrow, inhibiting normal hematopoiesis, and potentially infiltrating extramedullary organs such as the liver, spleen, and lymph nodes. Based on the affected cell types, acute leukemia is mainly divided into two categories: acute lymphoblastic leukemia and acute myeloid leukemia. With the application of targeted and immunotherapies, progress has been made in leukemia treatment; however, it still ranks sixth in cancer mortality in China. Developing novel targeted drugs and treatment regimens remains crucial for reducing relapse and mortality rates and prolonging patient survival.

[0003] DDX39B (DExD-Box Helicase 39B, also known as UAP56) is a conserved DEAD-box RNA helicase involved in multiple biological processes of RNA metabolism. DDX39B has been considered a potential prognostic biomarker for various solid tumors, including clear cell renal cell carcinoma, invasive breast cancer, and liver cancer. However, research on DDX39B and its ATPase inhibitor CCT018159 (CCT) in the field of leukemia has not yet been reported domestically or internationally. Acute leukemia is prone to relapse and has a high mortality rate, making the exploration of new targeted drugs and combination therapy regimens an urgent priority. Current reports on DDX39B and its ATPase inhibitor CCT mainly focus on their role in driving the progression of solid tumors. However, research on the application of DDX39B and its ATPase inhibitor CCT in the preparation of anti-leukemia drugs remains unclear both domestically and internationally. Summary of the Invention

[0004] The problem this invention aims to solve is the high relapse rate and poor prognosis of acute leukemia. This invention provides the application of CCT018159 in combination with nerabine in the preparation of drugs for acute leukemia.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The application of a combination of CCT and nerabine in the preparation of an acute leukemia drug, wherein the application of the combination of CCT and nerabine in the preparation of an acute leukemia drug includes the DDX39B inhibitor CCT018159 and the nucleoside analog nerabine.

[0007] Furthermore, the combined use of CCT and nerabine in the preparation of drugs for acute leukemia is either a single compound preparation or a combination of two separate preparations.

[0008] Furthermore, this includes one of the following methods: oral, injection, inhalation, spray, parenteral, and topical application.

[0009] Furthermore, CCT018159 is a DDX39B inhibitor with the chemical formula C 20 H 20 N2O4, structural formula is

[0010] .

[0011] Furthermore, nerabine is an antitumor drug with the chemical formula: The structural formula is

[0012] .

[0013] Furthermore, the acute leukemia mentioned is acute T-lymphoblastic leukemia.

[0014] The beneficial effects of this invention are:

[0015] This invention describes the application of the combination of CCT018159 and nerabine in the preparation of drugs for acute leukemia. The DDX39B inhibitor CCT018159 is combined with the nucleoside analog nerabine. The nerabine metabolite ara-GTP exerts its cytotoxic effect by selectively incorporating into T cell DNA and inhibiting DNA polymerase activity. CCT018159 significantly enhances the drug sensitivity of acute T-lymphoblastic leukemia (T-ALL) cells to nerabine, achieving a synergistic effect. This invention reduces leukemia burden and prolongs survival through a dual mechanism, and can be used to develop drugs for the treatment of acute leukemia. Attached Figure Description

[0016] Figure 1 This is a graph showing the results of analyzing the sensitivity of CCT in various tumors using the GDSC website in Example 1;

[0017] Figure 2 The image shows the CTG detection results in Example 2, where (a) is a CCT-treated leukemia cell line, and (b) is a CCT-treated healthy donor bone marrow sample, a T-ALL patient bone marrow sample, and PDX cells.

[0018] Figure 3 The image shows the cell counting results after treating Jurkat and CCRF-CEM with CCT018159 in Example 3, where (a) is Jurkat cells treated with CCT018159 and (b) is CCRF-CEM cells treated with CCT018159.

[0019] Figure 4This is a graph showing the apoptosis detection results of Jurkat cells treated with CCT018159 in Example 4;

[0020] Figure 5 This is a Giemsa staining result of Jurkat cells treated with CCT018159 in Example 5;

[0021] Figure 6 The table below shows the drug synergy analysis matrix and results graph after treating Jurkat cells with nerabine and CCT018159 in Example 6, where (a) is the experimental analysis matrix and (b) is the results graph.

[0022] Figure 7 The table shows the drug synergy analysis matrix and results graph after treating patient samples with the combined application of nerabine and CCT018159 in Example 7, where (a) is the analysis matrix and (b) is the results graph.

[0023] Figure 8 This is a diagram illustrating the drug treatment strategy for PDX mice in Example 8, using nerabine alone or in combination with CCT018159.

[0024] Figure 9 The images show a comparison of staining results after treating PDX mice with nerabine and CCT018159 in Example 8, where (a) is the Giemsa staining result of peripheral blood of mice, (b) is the HE staining result of liver, and (c) is the HE staining result of spleen.

[0025] Figure 10 The images and weight percentage analysis diagrams of PDX mice treated with nerabine and CCT018159 in Example 8 are shown, where (a) is a mouse liver image and (b) is a weight percentage analysis diagram.

[0026] Figure 11 The images and weight ratio analysis diagrams of the spleens of PDX mice treated with nerabine and CCT018159 in Example 8 are shown, where (a) is a picture of the liver of the mouse and (b) is a weight ratio analysis diagram.

[0027] Figure 12 This is a graph showing the survival analysis of PDX mice after treatment with nerabine and CCT018159 in Example 8, either as a single drug or in combination. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described specific embodiments are merely a part of the embodiments of the invention, and not all of them. The components of the specific embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations, and the invention may also have other embodiments.

[0029] Therefore, the following detailed description of specific embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected specific embodiments of the invention. All other specific embodiments obtained by those skilled in the art based on these specific embodiments without inventive effort are within the scope of protection of this invention.

[0030] To further understand the invention's content, features, and effects, the following specific embodiments are provided, along with accompanying drawings. Figure 1 - Appendix Figure 12 Detailed explanations are provided below. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the cell lines and experimental materials used in the following examples are commercially available products.

[0031] Example 1

[0032] Analysis of the sensitivity of CCT018159 in various tumors: CCT018159 (abbreviated as CCT, purchased from MCE) is a 3,4-diarylpyrazole resorcinol, an ATP-competitive inhibitor, with the molecular formula C... 20 H 20 N2O4.

[0033] The CAS number for CCT018159 is 171009-07-7;

[0034] The structural formula is CC1=C(C(=NN1)C2=CC(CC)=C(O)C=C2O)C=3C=C4C(=CC3)OCCO4; the predicted density is 1.322 g / cm³. 3 The specific steps for sensitivity analysis were as follows: Large-scale pharmacogenomic screening analysis of cancer cell lines was performed using the Genomics of Drug Sensitivity in Cancer (GDSC) website. The results are as follows: Figure 1 As shown, hematologic malignancies are most sensitive to CCT018159.

[0035] Example 2

[0036] Cell viability analysis of CCT018159: Acute leukemia cell lines, healthy donors, and bone marrow samples from T-ALL patients were treated with different concentrations of CCT018159 (DMSO, 2.5 μM, 5 μM, 7.5 μM, 10 μM). All cells were cultured in 96-well plates with 5000 cells per well. Cell viability was assessed using CellTiter-Glo (CTG) reagent after 48 hours, and the data were obtained and analyzed using a microplate reader. Results are as follows: Figure 2 As shown, CCT018159 exhibits cytotoxicity against all AL cells; CCT018159 shows minimal cytotoxicity against healthy donor samples, with an IC50 of 8.19 μM against T-ALL patient samples, and a cytotoxicity of 5.82 μM against leukemia PDX (human tissue xenograft) cells. The PDX cells are derived from mouse spleen cells obtained by transplanting bone marrow cells from T-ALL patients into immunodeficient mice (NKG) via the tail vein.

[0037] Example 3

[0038] Cell proliferation assay of CCT018159: Cells were adjusted to 1×10⁻⁶ cells / year. 5 Cells were seeded per well into 6-well plates, with 3 replicates per group. Acute leukemia cells were treated with different concentrations of CCT018159 (DMSO, 2.5 μM, 5 μM, 7.5 μM) and cultured in a cell culture incubator. Cells were counted after trypan blue staining every 2 days for a total of 10 days. Results are as follows: Figure 3 As shown, compared with the untreated DMSO group, cell proliferation in the treated group was inhibited, and the inhibition increased with increasing concentration.

[0039] Example 4

[0040] Apoptosis analysis of CCT018159 cells: Cells were adjusted to 1×10⁻⁶ cells / cells. 5 Cells were seeded per well in 6-well plates, with 3 replicates per group. Acute leukemia cells were treated with different concentrations of CCT018159 (DMSO, 2.5 μM, 5 μM, 7.5 μM) for 48 h. Cells were collected, washed once with pre-cooled PBS, and analyzed using an apoptosis detection kit. The fluorescence signals of Annexin V-FITC and PI-FE were measured by flow cytometry, and the data were analyzed using FlowJo software. Results are as follows: Figure 4 As shown, compared with the untreated DMSO group, the treatment group showed increased cell apoptosis, and the apoptosis increased with increasing concentration.

[0041] Example 5

[0042] Analysis of Giemsa staining in CCT018159: Cells were adjusted to 1×10⁻⁶ cells.5 Cells were seeded per well in 6-well plates, with 3 replicates per group. Acute leukemia cells were treated with different concentrations of CCT018159 (DMSO, 2.5 μM, 5 μM, 7.5 μM) for 48 h. Cells were collected, and the cell suspension was smeared onto glass slides, fixed with anhydrous methanol, dried, stained with working solution, gently rinsed with pH 7.0 buffer, and then air-dried for microscopic observation. Results are as follows: Figure 5 As shown, compared with the untreated group, the cells in the treated group showed significant differentiation and apoptosis.

[0043] Example 6

[0044] A drug synergy experiment was conducted on the application of CCT combined with nerabine in the preparation of drugs for acute leukemia. Jurkat cells were simultaneously treated for 48 hours with different concentrations of CCT018159 (0 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM) and different concentrations of nerabine (0 μM, 0.5 μM, 1 μM, 1.5 μM, 2.5 μM). Cells were collected, and cell viability was detected using CellTiter-Glo (CTG) reagent. Cell viability data were measured using an ELISA reader, and the drug synergy was analyzed using the Synergyfinder website. The results are as follows: Figure 6 As shown, the dose-response matrix heatmap is in red (red represents synergy, blue represents antagonism), with a mean of 32.46 and a synergy score (ZIP) of 13.26. P=2.57e-13 (strong synergy ≥10, no synergy <10), indicating that the above-mentioned dual-drug combined treatment has a synergistic effect on Jurkat cells, that is, after CCT018159 treatment, the sensitivity of leukemia cells to nerabine chemotherapy increases.

[0045] Example 7

[0046] A cellular drug synergistic experiment was conducted on the application of CCT combined with nerabine in the preparation of drugs for acute leukemia. Patient sample cells were simultaneously treated for 48 hours with different concentrations of CCT018159 (0 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM) and different concentrations of nerabine (0 μM, 0.5 μM, 1 μM, 1.5 μM, 2.5 μM). Cells were collected, cell viability was detected using CTG reagent, data were obtained using an ELISA reader, and the synergistic effect of the dual-drug treatment was analyzed using the Synergyfinder website. Results are as follows: Figure 7 The dose-response matrix heatmap shown is in red, with a mean of 24.1, a synergistic score (ZIP) of 15, and P=2.70e-04. This indicates that the above-mentioned dual-drug combined treatment has a synergistic effect on cancer cells in the patient sample, that is, after treatment with CCT018159, the sensitivity of the patient's leukemia cells to nerabine chemotherapy increased.

[0047] Example 8

[0048] A synergistic drug experiment in PDX mice on the application of CCT combined with nerabine in the preparation of drugs for acute leukemia: Specific steps for constructing a PDX model: Immunodeficient NKG mice of the same batch, age, sex, and weight were randomly selected and injected with PDX cells from T-ALL patients via the tail vein, 2.5 million cells / mouse.

[0049] Specific medication regimens, such as Figure 8 As shown, medication was started on day 10. CCT was administered once daily via intraperitoneal injection at a dose of 2 mg / mouse, for a total of 7 doses. Nerapheline was administered intraperitoneally from days 10 to 13 and from days 19 to 22, at a dose of 150 mg / kg. The mice were divided into four groups: DMSO group, CCT monotherapy group, nerapheline monotherapy group, and a combination therapy group. After treatment, disease burden and survival time were observed in the mice.

[0050] Giemsa staining: Peripheral blood smears from mice were collected at the same time after drug administration, mounted on glass slides, fixed with anhydrous methanol, dried, stained with working solution, gently rinsed with pH 7.0 buffer, and then air-dried for microscopic observation. Results are as follows: Figure 9 As shown in the upper section, compared with the untreated group, the DMSO monotherapy group showed reduced leukemia cell infiltration in the peripheral blood, while the dual-drug combination group showed no cancer cell infiltration in the peripheral blood.

[0051] HE staining: Three mice were randomly selected from each group and euthanized at the same time after drug administration. The liver and spleen were dissected and removed. After tissue fixation with tissue fixative, the liver and spleen were sealed with paraffin and sectioned. (1) Dewaxing and hydration: Xylene I → Xylene II (5-10 minutes each) → Anhydrous ethanol → 95% ethanol → 85% ethanol → 70% ethanol (2-5 minutes per stage) → Rinse with running water for 1 minute. (2) Hematoxylin staining of nuclei: Immerse in hematoxylin staining solution (such as Harris hematoxylin) for 5-8 minutes → Rinse with running water to remove excess stain. (3) Differentiation and blueing: Differentiation: 1% hydrochloric acid alcohol (1-3 seconds) → Rinse with running water immediately. Blueing: Rinse with running water for 10-15 minutes (or 0.5% ammonia water for blueing for 10 seconds) → Microscopic examination confirms that the nucleus is blue and the cytoplasm is colorless. (4) Eosin staining of cytoplasm: Immerse in eosin staining solution (0.5-1% aqueous solution or alcohol solution) for 1-3 minutes → Rinse briefly with running water. (5) Dehydration and clearing: 70% ethanol → 85% ethanol → 95% ethanol → anhydrous ethanol I → anhydrous ethanol II (1-2 minutes per stage) → xylene I → xylene II (2-5 minutes each). (6) Sealing with neutral resin, covering with a coverslip and scanning with a panoramic scanner. Results are as follows. Figure 9 As shown in the middle section, the livers of mice in the DMSO group showed extensive infiltration of leukemia cells, which was significantly reduced in the single-drug group and lowest in the dual-drug combination group. The results are as follows... Figure 9As shown in the lower part, the DMSO group showed extensive splenic leukemia cell infiltration, which was significantly reduced in the single-drug group and lowest in the dual-drug combination group.

[0052] Liver and spleen specific gravity assay: Three mice from each group were randomly selected and euthanized at the same time after drug administration. The liver and spleen were dissected and their weights were then measured. The results are as follows: Figure 10-11 As shown, compared with the control group, the liver and spleen of the treatment group were significantly reduced, and the combined treatment group had the best therapeutic effect.

[0053] Survival analysis: The survival time of the mice was recorded after drug administration and analyzed using GraphPad Prism. The results are as follows: Figure 12 As shown, the survival of the single-drug group was significantly longer than that of the DMSO group, while the survival period of the dual-drug combination group was the longest.

[0054] In this embodiment, analysis of the website predicted that CCT018159 is most sensitive to hematologic malignancies. When CCT018159 was used to treat leukemia cells, it successfully killed leukemia cells, increasing both apoptosis and differentiation. In this embodiment, the combination of CCT018159 and nerabine chemotherapy showed a higher growth inhibitory effect on leukemia cell lines and primary cells from patients than either treatment alone, demonstrating a significant sensitization effect. Compared with the nerabine chemotherapy alone group or the CCT018159 treatment alone group, the combined treatment group of CCT018159 and nerabine significantly reduced the leukemia burden and prolonged survival. This invention is the first to apply CCT018159 to the treatment of leukemia and the first to propose a treatment regimen of CCT018159 combined with nerabine, providing its application in the preparation of AL, especially T-ALL, therapeutic drugs.

[0055] Furthermore, when the combined use of CCT and nerabine in the preparation of acute leukemia drugs is a combination of two separate formulations, the administration method is simultaneous, alternating, or sequential.

[0056] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0057] Although this application has been described above with reference to specific embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of this application. In particular, as long as there is no structural conflict, the features in the specific embodiments disclosed in this application can be combined with each other in any way. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, this application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The application of a combination of CCT018159 and nerabine in the preparation of a drug for treating acute lymphoblastic leukemia; The acute lymphoblastic leukemia mentioned is acute T-lymphoblastic leukemia.

2. The application of the combination of CCT018159 and nerabine as described in claim 1 in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, The use of CCT018159 in combination with nerabine in the preparation of drugs for treating acute lymphoblastic leukemia is either a single compound preparation or a combination of two separate preparations.

3. The application of the combination of CCT018159 and nerabine as described in claim 2 in the preparation of a drug for treating acute lymphoblastic leukemia, characterized in that, This includes one of the following methods: oral administration, injection, or inhalation.

Images