Application of glycogen synthase kinase GSK3 inhibitor composition in prevention and treatment of leukemia

Abstract

The invention discloses application of a glycogen synthase kinase GSK3 inhibitor composition in prevention and treatment of leukemia, and belongs to the technical field of medicines. The glycogen synthase kinase GSK3 inhibitor composition is prepared from CHIR99021, SB216763 and LY2090314, and can be used for preparing the glycogen synthase kinase GSK3 inhibitor composition. The invention discloses that the glycogen synthase kinase GSK3 inhibitors CHIR99021, SB216763 and LY2090314 have a synergistic effect in the aspect of treating the leukemia, a foundation is laid for researching the action mechanism of the glycogen synthase kinase GSK3 inhibitors in treating the leukemia, and a technical support is provided for further developing leukemia treatment medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of glycogen synthase kinase GSK3 inhibitor compositions in the prevention and treatment of leukemia. Background Technology

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Its complex pathogenesis is associated with multiple factors, including biological, physical, chemical, genetic, and other hematological disorders. Uncontrolled proliferation of leukemia cells in the bone marrow and other hematopoietic tissues suppresses normal hematopoiesis and leads to leakage into other organs. Clinically, the typical symptoms of leukemia are often less pronounced in the early stages compared to common diseases. Acute leukemia may present with high fever or severe bleeding, while chronic leukemia progresses more slowly and may present with anemia and bleeding symptoms, enlarged lymph nodes and spleen, and abnormal proliferation of lymphocytes. Treatment options for leukemia include molecularly targeted therapy, chemotherapy, radiotherapy, blood or bone marrow transplantation, and adjuvant therapies targeting various symptoms. However, these methods have limited effectiveness for many patients, especially high-risk and relapsed / refractory acute myeloid leukemia (AML), for which new treatment strategies are urgently needed to overcome the challenges.

[0003] Glycogen synthase kinase 3 (GSK3) is a conserved cytoplasmic serine / threonine protein kinase that can negatively regulate leukemia-related signaling pathways such as classical Wnt and Akt, and positively regulate nuclear factor-κB, non-classical Wnt, and lysosomal apoptosis signaling pathways, thereby modulating the proliferation, differentiation, and apoptosis of leukemia cells. Currently, various GSK3β inhibitors and activators have been reported in leukemia research, playing important roles in reducing chemotherapy side effects, reversing drug resistance, and enhancing the killing effect on leukemia cells. Therefore, GSK3 holds promise as a target for molecular targeted therapy of leukemia, enabling the development of novel drugs with highly effective therapeutic effects. Summary of the Invention

[0004] The purpose of this invention is to provide the application of glycogen synthase kinase 3 (GSK3) inhibitor compositions in the prevention and treatment of leukemia, thereby addressing the problems existing in the prior art. This invention reveals that the GSK3 inhibitors CHIR99021, SB216763, and LY2090314 have a synergistic effect in the treatment of leukemia, laying the foundation for studying the mechanism of action of GSK3 inhibitors in treating leukemia and providing technical support for further development of leukemia treatment drugs.

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

[0006] This invention provides the use of a glycogen synthase kinase GSK3 inhibitor composition in the preparation of a drug for the prevention and treatment of leukemia, wherein the glycogen synthase kinase GSK3 inhibitor composition includes CHIR99021, SB216763 and LY2090314.

[0007] Optionally, the mass ratio of CHIR99021, SB216763 and LY2090314 is 1-4:1-4:1-4.

[0008] Optionally, the mass ratio of CHIR99021, SB216763, and LY2090314 is 1:1:3.

[0009] Optionally, the glycogen synthase kinase GSK3 inhibitor composition may further include pharmaceutically acceptable excipients.

[0010] The present invention also provides a composition having the effect of preventing and treating leukemia, the composition comprising CHIR99021, SB216763 and LY2090314.

[0011] Optionally, the mass ratio of CHIR99021, SB216763 and LY2090314 is 1-4:1-4:1-4.

[0012] Optionally, the mass ratio of CHIR99021, SB216763, and LY2090314 is 1:1:3.

[0013] Optionally, the composition may also include pharmaceutically acceptable excipients.

[0014] The present invention discloses the following technical effects:

[0015] This invention has discovered that the glycogen synthase kinase 3 inhibitors CHIR99021, SB216763, and LY2090314 have inhibitory effects on the in vitro proliferation of leukemia cells, but their inhibitory effects are relatively low. This invention combines CHIR99021, SB216763, and LY2090314 in a scientifically reasonable ratio, significantly improving the inhibition rate of KG-1a and K562 cell proliferation in vitro, reaching 80%, thus greatly enhancing the therapeutic effect on leukemia. Animal experiments further confirm that the combined use of these three inhibitors significantly improves the survival rate of leukemia mice compared to using them individually. This invention reveals that the synergistic effect of glycogen synthase kinase 3 inhibitors CHIR99021, SB216763, and LY2090314 in the treatment of leukemia lays the foundation for studying the mechanism of action of glycogen synthase kinase 3 inhibitors in the treatment of leukemia and provides technical support for the further development of leukemia treatment drugs. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] In this invention, the glycogen synthase kinase GSK3 inhibitor composition includes CHIR99021, SB216763 and LY2090314.

[0022] In this invention, CHIR99021 (Cas: 1797989-42-4) is a potent inhibitor of GSK-3, inhibiting GSK3α and GSK3β by competing for their ATP-binding sites. CHIR-99021 acts on CHO-IR cells expressing the insulin receptor, inducing glycogen synthase (GS) activation. It has been reported that CHIR99021 can also maintain the pluripotent state of stem cells, and removal of CHIR-99021 alone causes hCiPS induction failure.

[0023] In this invention, SB216763 (Cas: 280744-09-4) is a selective and potent inhibitor of GSK-3. SB216763 competitively inhibits the activity of GSK-3α and GSK-3β with ATP. SB-216763 can protect neurons in the central and peripheral nervous systems from PI3K pathway-mediated cell death. It has been reported that SB-216763 can maintain the pluripotency of mouse embryonic stem cells (mESCs) in mouse embryonic fibroblasts (MEF) cultured without exogenous leukemia inhibitory factor (LIF). SB-216763 has also been widely used in various animal models to study the role of GSK-3. In a bleomycin-induced mouse pulmonary fibrosis model, SB-216763 protected mice from lung inflammation and subsequent fibrosis by reducing the release of macrophage inflammatory cytokines. In addition, in a rat model of perfusion injury, SB-216763 reduced the infarct area and prevented myocardial ischemia.

[0024] In this invention, LY2090314 (Cas: 603288-22-8) is a potent inhibitor of GSK-3, inhibiting both GSK-3α and GSK-3β. LY2090314 has been reported to exhibit high selectivity for GSK3, demonstrated by its fold selectivity relative to a wide range of kinases. LY2090314 effectively induced apoptosis in a group of melanoma cell lines, independent of BRAF mutation status. LY2090314-induced cell death was β-catenin-dependent, and GSK3β gene silencing increased cellular sensitivity to LY2090314. LY2090314 remained active in PLX4032-resistant cell lines and exhibited an independent mechanism of action.

[0025] In some embodiments of the present invention, the glycogen synthase kinase GSK3 inhibitors CHIR99021, SB216763 and LY2090314 have inhibitory effects on the in vitro proliferation of leukemia cells; when the three inhibitors are used in combination, the inhibitory effect on the in vitro proliferation of leukemia cells is significantly increased.

[0026] In some embodiments of the present invention, the mass ratio of the glycogen synthase kinase 3 inhibitors CHIR99021, SB216763, and LY2090314 used in combination is 1-4:1-4:1-4; in some specific embodiments, the mass ratio of the three inhibitors used in combination can be 1:1:1, 3:1:1, 1:3:1, or 1:1:3; in some specific embodiments, the inhibitory effect on the in vitro proliferation of leukemia cells is strongest when the mass ratio of the three inhibitors used in combination is 1:1:3.

[0027] In some embodiments of the present invention, the glycogen synthase kinase GSK3 inhibitors CHIR99021, SB216763 and LY2090314, when applied to leukemia mice, can improve the survival rate of leukemia mice; when the three inhibitors are used in combination, the survival rate of leukemia mice is significantly improved compared with the use of the drugs alone.

[0028] In some embodiments of the present invention, the glycogen synthase kinase 3 inhibitor composition is administered orally; in some embodiments of the present invention, the composition may further contain pharmaceutically acceptable excipients; in some embodiments of the present invention, pharmaceutically acceptable excipients include fillers, binders, preservatives, flavoring agents, etc.; fillers may be selected from lactose, starch, sucrose, microcrystalline cellulose, etc.; binders may be selected from starch paste, dextrin, sodium carboxymethyl cellulose (CMC-Na), etc.; preservatives may be selected from parabens, sodium benzoate, potassium sorbate, etc.; flavoring agents may be selected from sucrose, simple syrups, aromatic syrups (such as orange peel syrup, citron syrup, cherry syrup, licorice syrup, etc.), stevioside, etc.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are conventional laboratory instruments and equipment; unless otherwise specified, the reagents and materials used in the following examples are commercially available reagents and materials.

[0030] Example 1

[0031] CHIR99021 (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), SB216763 (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), and LY2090314 (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were used as solvent to prepare the following drugs:

[0032] Drug A: CHIR99021 solution at a concentration of 0.5 μg / mL;

[0033] Drug B: CHIR99021 solution at a concentration of 1.5 μg / mL;

[0034] Drug C: 0.5 μg / mL SB216763 solution;

[0035] Drug D: 1.5 μg / mL SB216763 solution;

[0036] Drug E: LY2090314 solution at a concentration of 0.5 μg / mL;

[0037] Drug F: LY2090314 solution at 1.5 μg / mL;

[0038] Drug G: A mixed solution containing CHIR99021 at a final concentration of 0.5 μg / mL, SB216763 at a final concentration of 0.5 μg / mL, and LY2090314 at a final concentration of 0.5 μg / mL;

[0039] Drug H: A mixed solution containing CHIR99021 at a final concentration of 0.9 μg / mL, SB216763 at a final concentration of 0.3 μg / mL, and LY2090314 at a final concentration of 0.3 μg / mL;

[0040] Drug I: A mixed solution containing CHIR99021 at a final concentration of 0.3 μg / mL, SB216763 at a final concentration of 0.9 μg / mL, and LY2090314 at a final concentration of 0.3 μg / mL;

[0041] Drug J: A mixed solution containing CHIR99021 with a final concentration of 0.3 μg / mL, SB216763 with a final concentration of 0.3 μg / mL, and LY2090314 with a final concentration of 0.9 μg / mL.

[0042] The effect of drug AJ on the in vitro proliferation of leukemia cells was verified using the acute myeloid leukemia cell line KG-1a (purchased from Shanghai Kanglang Biotechnology Co., Ltd.) and the chronic myeloid leukemia cell line K562 (purchased from Nanjing Kebai Biotechnology Co., Ltd.), respectively. The specific procedure is as follows:

[0043] KG-1a cells and K562 cells in the exponential growth phase were respectively fed with 5.0 × 10⁻⁶ cells. 5Cells were seeded at a density of 10 cells / well in 96-well culture plates (RPMI 1640 medium containing 5% fetal bovine serum) and incubated in a cell culture incubator at 37°C with 5% CO2. After 1 hour of culture, 10 μL of drug AJ was added to each well, with a blank control group (10 μL DMSO) included. Each drug was used in triplicate. After 48 hours of drug treatment, the inhibition rate of each drug on KG-1a and K562 cells was detected using the CCK8 assay. Inhibition rate (%) = 1 - [OD value of drug group / OD value of blank control group] × 100%. The results are shown in Tables 1 and 2.

[0044] Table 1. Inhibition rate of each drug on KG-1a cells

[0045] drug Inhibition rate Blank control group / Drug A 25.6％ Drug B 38.4％ Drug C 22.8％ Drug D 42.5％ Drug E 31.2％ Drug F 40.7％ Drug G 72.1％ Drug H 65.9％ Drug I 60.3％ Drug J 64.3％

[0046] Table 2. Inhibition rate of each drug on K562 cells

[0047]

[0048]

[0049] As shown in Tables 1 and 2, CHIR99021 and SB216763 exhibited certain inhibitory effects on the in vitro proliferation of KG-1a and K562 cells. The inhibitory effect increased with increasing drug concentration, but the overall inhibitory effect remained low. LY2090314 also showed some inhibitory effect on the in vitro proliferation of KG-1a and K562 cells, but this effect was also relatively low. However, the combined use of all three drugs significantly improved the inhibitory effect on KG-1a and K562 cells compared to their individual use, although the inhibitory effect was related to the ratio of the three drugs. The best inhibitory effect was observed when CHIR99021, SB216763, and LY2090314 were mixed in a mass ratio of 1:1:3.

[0050] Example 2

[0051] A mouse model of leukemia was constructed to verify the therapeutic effects of CHIR99021, SB216763, LY2090314, and their combinations on leukemia. The specific process is as follows:

[0052] Construction of a mouse model of leukemia: HL-60 cells were expanded to the logarithmic growth phase using RPMI-1640 medium containing 10% fetal bovine serum, and the cell concentration was adjusted to 1×10⁻⁶. 6Cells / mL. Eight-week-old NSG mice were selected and subjected to 2 Gy whole-body irradiation to enhance the success rate of implantation. 200 μL of HL-60 cell suspension was injected into each mouse via the tail vein at a rate controlled below 50 μL / s to avoid embolism. Mice were housed separately, and blood was collected weekly via the tail vein. The proportion of human CD45+ cells was analyzed by flow cytometry; a proportion >10% was considered a successful model establishment.

[0053] Leukemia mice were randomly divided into 5 groups of 10 mice each: a model control group (DMSO), a treatment group 1 (CHIR99021), a treatment group 2 (SB216763), a treatment group 3 (LY2090314), and a treatment group 4 (CHIR99021+SB216763+LY2090314, with a mass ratio of 1:1:3). Healthy mice served as a blank control group. All mice were administered the drug via gavage at a dose of 10 mg / kg body weight, once daily, for 60 days. During the treatment period, the survival status of the mice was observed, the number of deaths was recorded, and the survival rate was calculated. The results are shown in Table 3.

[0054] Table 3. Mortality and survival rate of mice in each group

[0055] Group Death count Survival rate Blank control group 0 100％ Model control group 9 10％ One group of drugs 6 40％ Group 2 of drugs 6 40％ Three groups of drugs 4 60％ Four groups of drugs were administered 0 100％

[0056] Table 3 shows that CHIR99021, SB216763, and LY2090314 all had significant protective effects against leukemia in mice, but the effects were not significant. However, the combined use of the three drugs significantly improved the protective effect against leukemia in mice compared to their individual use. This indicates that there is a significant synergistic effect among the three drugs, which can improve the shortcomings of using glycogen synthase kinase 3 inhibitors as monotherapy.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a glycogen synthase kinase GSK3 inhibitor composition in the preparation of drugs for the prevention and treatment of leukemia, characterized in that, The glycogen synthase kinase GSK3 inhibitor composition includes CHIR99021, SB216763, and LY2090314.

2. The application according to claim 1, characterized in that, The mass ratio of CHIR99021, SB216763 and LY2090314 is 1-4:1-4:1-4.

3. The application according to claim 2, characterized in that, The mass ratio of CHIR99021, SB216763 and LY2090314 is 1:1:

3.

4. The application according to claim 1, characterized in that, The glycogen synthase kinase GSK3 inhibitor composition also includes pharmaceutically acceptable excipients.

5. A composition having an effect in preventing and treating leukemia, characterized in that, The composition includes CHIR99021, SB216763 and LY2090314.

6. The composition according to claim 5, characterized in that, The mass ratio of CHIR99021, SB216763 and LY2090314 is 1-4:1-4:1-4.

7. The composition according to claim 6, characterized in that, The mass ratio of CHIR99021, SB216763 and LY2090314 is 1:1:

3.

8. The composition according to claim 5, characterized in that, The composition also includes pharmaceutically acceptable excipients.

Images