Use of a co-active ingredient in the preparation of a medicament for the treatment of a tumor

Abstract

The application provides a use of vincristine and lithium carbonate as co-active ingredients in the preparation of a medicament for treating tumors. The vincristine and lithium carbonate as co-active ingredients of the application have a killing effect on various leukemia cells, including human acute lymphoblastic leukemia cell lines, chronic myeloid leukemia cells, acute monocytic leukemia cell lines; in particular, for human acute lymphoblastic leukemia cell lines, compared with single free drugs (lithium carbonate or vincristine), lithium carbonate and vincristine free drug combination, the leukemia cell proliferation can be more effectively inhibited; in addition, the vincristine and lithium carbonate as co-active ingredients of the application can obviously stimulate the generation of granulocyte colony-stimulating factor, and alleviate the neutropenia side effect caused by chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of vincristine and lithium carbonate as common active ingredients in the preparation of drugs for treating tumors. Background Technology

[0002] Leukemia has a complex pathogenesis, and monotherapy rarely achieves ideal therapeutic effects. Therefore, in both clinical and basic research, multi-drug combination therapy can achieve better synergistic effects. Multiple drugs exert synergistic effects through different molecular mechanisms, blocking disease progression through multiple signaling pathways, reducing the toxic side effects and drug resistance caused by monotherapy, inducing remission, and improving treatment outcomes. Drugs used to treat leukemia mainly include cytarabine, vincristine, prednisone, doxorubicin, daunorubicin, and asparaginase. Generally, after achieving complete remission with combination chemotherapy, the decision to perform hematopoietic stem cell transplantation (HSCT) is made based on the patient's condition. Data analysis of relapsed leukemia patients has found that patients who have not undergone high-dose chemotherapy have almost no chance of cure after autologous or allogeneic HSCT transplantation. However, chemotherapy drugs have significant toxic side effects; therefore, there is an urgent need to develop more effective treatment strategies to improve the prognosis of leukemia patients.

[0003] Vincristine (VCR) is a bisindole alkaloid extracted from the periwinkle (Catharanthus roseus), a plant in the Apocynaceae family. It belongs to the cytotoxic class of antitumor drugs and has been used clinically since the 1960s. It remains a first-line treatment for T-ALL (tuberculous leukemia). Vincristine is a cell cycle-dependent compound; its antitumor mechanism of action involves inhibiting tubulin polymerization, preventing spindle microtubule formation, arresting cancer cell mitosis at metaphase, and inducing tumor cell apoptosis. However, vincristine suffers from drawbacks such as a short half-life, rapid elimination, and severe neurotoxicity and gastrointestinal toxicity, limiting its clinical application. Developing new formulations of vincristine to prolong its duration of action, reduce dosage and side effects, and improve efficacy has become a research hotspot. Furthermore, neutropenia is the most common hematological toxicity reaction to chemotherapy drugs. Severe neutropenia increases the risk of invasive infections and often leads to dose reduction or delayed treatment due to fever and infection, ultimately affecting antitumor efficacy. Literature reports that the degree and duration of granulocytopenia are closely related to the patient's risk of infection and even death.

[0004] Lithium carbonate is a medication used for mental illnesses, primarily for treating mania and bipolar disorder. However, recent studies have shown that lithium carbonate also plays an important role in the treatment of leukemia. Firstly, lithium carbonate treatment can reduce the side effects of chemotherapy in leukemia patients. It can also reduce bacterial infections caused by neutropenia and neutropenic fever, thus improving quality of life. A 73-year-old patient with hairy cell leukemia who received lithium carbonate treatment before and after splenectomy showed that lithium's positive effect on granulocyte count was related to stimulating granulocyte production, not just redistribution. In patients with persistent neutropenia, over 85% of patients could effectively correct their neutrophil count with short-term use of lithium carbonate. Due to its high tolerability, lithium carbonate treatment can also be used prophylactically to prevent neutropenia. The mechanism by which lithium carbonate enhances neutrophil production has been extensively studied; the drug has been shown to enhance colony-stimulating factor activity levels, with lithium stimulating the secretion and production of colony-stimulating factors. In addition, some believe that lithium regulates granulocyte production by stimulating the proliferation of pluripotent stem cells. Lithium treatment can increase the number of CD34+ cells in the peripheral blood of patients and can affect the proliferation, apoptosis and cell cycle of different cell lines.

[0005] Besides preventing bacterial infections caused by neutropenia from chemotherapy, the combination of lithium carbonate with other anti-leukemia drugs may be an effective approach for future leukemia treatment. Synergistic effects can enhance the cell-inhibiting and cytotoxic response rates of chemotherapy drugs. Studies have found that the combination of lithium carbonate and retinoic acid is well-tolerated; lithium carbonate, as a GSK3 inhibitor, can enhance the differentiation effect of retinoic acid by inhibiting GSK3, thus inducing differentiation of acute myeloid leukemia cells.

[0006] In recent years, various nano-drug delivery systems have been developed, which can improve drug solubility, alter the pharmacokinetics and tissue distribution of molecular drugs, reduce toxic side effects, and enhance anti-tumor effects or achieve integrated diagnosis and treatment by utilizing the synergistic effects between different molecules.

[0007] Currently, there are no reports on the combined use of vincristine and lithium carbonate for tumor treatment or the preparation of nanomedicines. Summary of the Invention

[0008] Therefore, the purpose of this invention is to overcome the deficiencies in the prior art and to provide the application of vincristine and lithium carbonate as common active ingredients in the preparation of drugs for treating tumors. By utilizing the synergistic effect of lithium carbonate and vincristine, and the fact that lithium carbonate can reduce neutropenia caused by chemotherapy, the efficacy is improved and the toxic side effects of chemical drugs are reduced.

[0009] Before describing the content of this invention, the following terms are defined as follows:

[0010] The term "VCR" refers to vincristine, with the chemical formula C0. 46 H 56 N4O 10 VCR, medically known as vegetative-cause compound, is an alkaloid extracted from the periwinkle, a plant belonging to the genus Vinca in the family Apocynaceae.

[0011] The term "LM" refers to: free lithium carbonate micelles.

[0012] The term "VM" refers to: free vincristine micelles.

[0013] The term "VLM" refers to: dual-drug nanomicelle assembly.

[0014] The term "CCRF-CEM cells" refers to a human acute lymphoblastic leukemia cell line.

[0015] The term "Jurkat cell" refers to an immortalized human T lymphocyte line of suspension cells used to study acute T-cell leukemia, T-cell signaling, and the expression of various chemokine receptors, particularly HIV, for the entry of susceptible viruses.

[0016] The term "U937 cell" refers to the human acute monocytic leukemia cell line.

[0017] The term "K562 cells" refers to human chronic myeloid leukemia cells.

[0018] The term "PBS solution" refers to phosphate buffer solution.

[0019] To achieve the above objectives, a first aspect of the present invention provides the use of vincristine and lithium carbonate as common active ingredients in the preparation of a medicament for treating tumors.

[0020] According to the first aspect of the invention, in the common active ingredient, the mass ratio of vincristine to lithium carbonate is 2-10:2-200, preferably 2-8:2-150, and more preferably 2-6:2-50.

[0021] According to the first aspect of the present invention, the drug is a nanomedicine;

[0022] Preferably, the nanomedicine is selected from one or more of the following: nanoliposomes, solid lipid nanoparticles, nanocapsules and nanospheres, polymer micelles, nano-sized drug suspensions, protein and cell-derived nanoparticles, more preferably selected from one or more of the following: nanoliposomes, solid lipid nanoparticles, nanocapsules and nanospheres, polymer micelles, and most preferably polymer micelles.

[0023] According to the application of the first aspect of the present invention, the dosage form of the drug is selected from one or more of the following: solution, sol, emulsion, suspension, gas dispersion, solid dispersion, and particulate dispersion; wherein,

[0024] The solution type is selected from one or more of the following: aqueous solution, solvent, and injection;

[0025] The sol type is selected from one or more of the following: adhesive, coating agent, gel;

[0026] The emulsion type includes emulsions, preferably selected from one or more of the following: ordinary emulsions, microemulsions, and nanoemulsions;

[0027] The suspension type is selected from one or more of the following: suspension agent, mixture, lotion;

[0028] The gas dispersion is an aerosol;

[0029] The solid dispersion is selected from one or more of the following: powders, granules, tablets, capsules; and / or

[0030] The particulate dispersion is a microcapsule or nanocapsule.

[0031] According to the application of the first aspect of the present invention, the method of administration of the drug is selected from one or more of the following: oral administration, injection administration, local administration, inhalation administration, rectal administration, and preferably selected from one or more of the following: oral administration, injection administration, local administration, and inhalation administration;

[0032] Preferably, the injection administration is selected from one or more of the following: intramuscular injection, subcutaneous injection, and intravenous injection.

[0033] According to the first aspect of the invention, the carrier in the polymer micelles is a block copolymer, preferably a linear block copolymer or a nonlinear block copolymer; wherein,

[0034] The linear block copolymer is more preferably selected from one or more of the following: AB block, ABA block, ABC triblock, ABABA pentablock linear copolymer, and even more preferably selected from one or more of the following: AB block, ABA block, ABC triblock; wherein A, B, and C are different polymers from each other, A is further preferably selected from one or more of the following: polystyrene, polycaprolactone, polyethylene glycol, B is further preferably selected from one or more of the following: polybutadiene, polypropylene, polyoxypropylene, polyoxyethylene, polystyrene, polyvinylcaprolactam, and C is further preferably selected from one or more of the following: polymethyl methacrylate, polyacrylic acid, polymethyl methacrylate, polylactic acid, polyvinyl acetate; and / or

[0035] The nonlinear block copolymer is more preferably selected from one or more of the following: star-shaped block copolymer, comb-shaped block copolymer, dendritic block copolymer, cyclic block copolymer, H-shaped block copolymer, cross-linked network block copolymer, and even more preferably selected from one or more of the following: star-shaped block copolymer, comb-shaped block copolymer, dendritic block copolymer, cross-linked network block copolymer.

[0036] According to the first aspect of the invention, the block copolymer is formed from two or more polymers selected from the following: polyvinyl caprolactam, polyvinyl acetate, polyethylene glycol, polystyrene, polybutadiene, polyoxypropylene, polyoxyethylene, polyacrylic acid, and polylactic acid; preferably formed from two or more polymers selected from the following: polyvinyl caprolactam, polyvinyl acetate, polyethylene glycol, polystyrene, and polybutadiene; more preferably formed from polyvinyl caprolactam, polyvinyl acetate, and polyethylene glycol.

[0037] According to the first aspect of the invention, wherein the block copolymer contains:

[0038] The molecular weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 40–80:20–50:5–20, preferably 50–70:25–40:10–15, and most preferably 57:30:13; and / or

[0039] The weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 1–20:1–50:4–200; preferably 1–10:3–30:10–200; more preferably 1–6:3–16:20–200.

[0040] According to the first aspect of the invention, when the nanomedicine is a polymer micelle, the mass ratio of the vincristine, the lithium carbonate and the block copolymer is 2-10:2-200:20-200, preferably 2-8:2-150:20-200, and more preferably 2-6:2-50:20-200.

[0041] According to the first aspect of the present invention, the tumor is selected from one or more of the following: leukemia, breast cancer, gastrointestinal cancer, multiple myeloma, malignant lymphoma, germ cell tumor, preferably selected from one or more of the following: leukemia, malignant lymphoma, multiple myeloma, and most preferably leukemia;

[0042] Preferably, the leukemia is selected from one or more of the following: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia.

[0043] According to a specific embodiment of the present invention, a first aspect of the present invention provides a dual-drug nanomicelle for treating leukemia, which is prepared from a block copolymer, lithium carbonate and vincristine.

[0044] The weight ratio of the block copolymer is (1-20):(1-50):(4-200); preferably (1-6):(3-16):(40-200).

[0045] The mass ratio of vinblastine:lithium carbonate:block copolymer is (2-6):(2-20):(24-200).

[0046] The molecular weight ratio of polyvinylcaprolactam, polyvinyl acetate, and polyethylene glycol in the block copolymer is 57:30:13.

[0047] The concentration of the block copolymer solution is 1-50 mg / mL.

[0048] The particle size of the dual-drug nanomicelles is 65nm to 85nm, preferably 68nm to 83nm, and more preferably 70nm to 80nm.

[0049] The second aspect provides a method for preparing the aforementioned dual-drug nanomicelles, comprising the following steps: dissolving block copolymer, lithium carbonate, and vincristine in phosphate buffer, heating and stirring thoroughly, allowing the suspension to stand at room temperature for about half an hour until it becomes clear and transparent, and then filtering it through a 0.22 μm filter membrane to obtain lithium carbonate and vincristine dual-drug nanomicelles.

[0050] The heating temperature is 25-80℃; the stirring speed is 100-1000 r / min; and the stirring time is 30-60 min.

[0051] The third aspect provides the application of the aforementioned dual-drug nanomicelles in the preparation of drugs for treating leukemia, wherein the leukemia is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia.

[0052] According to another specific embodiment of the present invention, the present invention provides a lithium carbonate and vincristine dual-drug nanomicelle, which utilizes the synergistic effect of lithium carbonate and vincristine, and the ability of lithium carbonate to reduce neutropenia caused by chemotherapy, thereby improving efficacy and reducing the toxic side effects of chemotherapy drugs.

[0053] Secondly, this invention provides a method for preparing the above-mentioned dual-drug nanomicelles. Lithium vincristine dual-drug nanomicelles are synthesized using a self-assembly method. The specific steps are as follows:

[0054] (1) Dissolve 24.0-200.0 mg of block copolymer, 2.0-10.0 mg of lithium carbonate and 2.0-6.0 mg of vincristine in 4 mL of phosphate buffer;

[0055] (2) Heat the mixture at 25-80℃ and stir at 300-900r / min for 30-60min;

[0056] (3) Let the suspension stand at room temperature for about half an hour until it becomes clear and transparent, then filter it through a 0.22 μm filter membrane to obtain lithium vincristine dual-drug nanomicelles, as shown in the picture. Figure 1 As shown.

[0057] Thirdly, the present invention provides the application of the above-mentioned dual-drug nanomicelles in the preparation of anti-leukemia drugs.

[0058] According to another specific embodiment of the present invention, the present invention provides a method for preparing dual-drug nanomicelles, the method comprising the following steps:

[0059] (1) Dissolve the block copolymer, lithium carbonate and alkaloid in a buffer solution to obtain a mixture;

[0060] (2) Heat and stir the mixture prepared in step (1) to obtain a suspension;

[0061] (3) After the suspension prepared in step (2) is allowed to stand and filtered, the dual-drug nanomicelles are obtained.

[0062] In step (1): the buffer solution is a phosphate buffer solution.

[0063] In step (2): the heating temperature is 25-80℃, preferably 25-70℃, more preferably 25-60℃; the stirring speed is 300-900r / min, preferably 300-800r / min, more preferably 500-800r / min; and / or the stirring time is 30-90min, preferably 30-70min, more preferably 30-60min.

[0064] In step (3): the settling time is 20-80 min, preferably 30-60 min, more preferably 30-50 min; and / or the pore size of the filter membrane used for filtration is 0.15-0.3 μm, most preferably 0.22 μm.

[0065] The dual-drug nanomicelles of the present invention, with vincristine and lithium carbonate as common active ingredients, may have, but are not limited to, the following beneficial effects:

[0066] The dual-drug nanomicelles of the present invention, with vincristine and lithium carbonate as common active ingredients, exhibit cytotoxic effects on various leukemia cell lines, including human acute lymphoblastic leukemia cell lines, chronic myeloid leukemia cell lines, and acute monocytic leukemia cell lines. In particular, for human acute lymphoblastic leukemia cell lines, compared with single free drugs (lithium carbonate or vincristine) or combinations of lithium carbonate and vincristine free drugs, it can more effectively inhibit the proliferation of leukemia cells. In addition, the dual-drug nanomicelles of the present invention can significantly stimulate the production of granulocyte colony-stimulating factor, alleviating the neutropenia side effect caused by chemotherapy. Attached Figure Description

[0067] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0068] Figure 1 A photograph of the dual-drug nanomicelles prepared in Example 1, in which vincristine and lithium carbonate are used as common active ingredients, is shown.

[0069] Figure 2 The particle size distribution curve of the dual-drug nanomicelles of the present invention in PBS is shown in Example 2.

[0070] Figure 3 The bar chart shown illustrates the effect of dual-drug nanomicelles (lithium carbonate and vincristine in a weight ratio of 1:1) on the viability of CCRF-CEM cells in Example 3 of this invention.

[0071] Figure 4 The bar chart shown illustrates the effect of dual-drug nanomicelles (lithium carbonate and vincristine in a weight ratio of 1:1) on Jurkat cell viability in Example 3 of this invention.

[0072] Figure 5 The bar chart shown illustrates the effect of dual-drug nanomicelles (lithium carbonate and vincristine in a weight ratio of 1:1) on the viability of U937 cells in Example 3 of this invention.

[0073] Figure 6 The bar chart shown illustrates the effect of dual-drug nanomicelles (lithium carbonate and vincristine in a weight ratio of 1:1) on the viability of K562 cells in Example 3 of this invention.

[0074] Figure 7 This invention illustrates the effect of dual-drug nanomicelles (lithium carbonate and vincristine in a 1:1 weight ratio) on granulocyte colony-stimulating factor levels in human peripheral blood mononuclear cells (hPBMCs) in Example 4 of this invention. Detailed Implementation

[0075] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0076] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0077] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0078] Unless otherwise specified, the human acute lymphoblastic leukemia cell lines CCRF-CEM and Jurkat, and the human acute monocytic leukemia cell line U937 used in the following examples were all purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0079] Unless otherwise specified, the PBS solutions used in the following examples are all 1×PBS solutions, and the aqueous solutions used are all double-distilled water.

[0080] The reagents and instruments used in the following examples are as follows:

[0081] Reagents:

[0082] RPMI 1640 medium was purchased from Hyclone.

[0083] The CCK-8 reagent kit was purchased from Dojin Chemical Research Institute, Japan.

[0084] The granulocyte colony-stimulating factor ELISA kit was purchased from Xinbosheng Biotechnology Co., Ltd.

[0085] instrument:

[0086] Nanoparticle size and potential analyzer, purchased from Malvern, UK, model NS-90Z;

[0087] The microplate reader was purchased from Thermo Scientific, USA, model Varioskan LUX.

[0088] Example 1: Construction of Lithium Carbonate Vincristine Bi-drug Nanomicelles

[0089] Lithium vincristine dual-drug nanomicelles were synthesized using a self-assembly method, and the specific steps are as follows:

[0090] (1) Dissolve 24.0 mg of block copolymer, 3 mg of lithium carbonate and 3 mg of vincristine in 4 mL of phosphate buffer;

[0091] (2) Heat the mixture at 55°C and stir at 800 r / min for 60 min;

[0092] (3) Let the suspension stand at room temperature for about half an hour until it becomes clear and transparent, then filter it through a 0.22 μm filter membrane to obtain lithium vincristine dual-drug nanomicelles, as shown in the picture. Figure 1 As shown.

[0093] Example 2: DLS analysis of the particle size distribution of lithium vincristine dual-drug nanomicelles

[0094] 1 mL of lithium vincristine dual-drug nanomicelle solution was placed in a cuvette, and the particle size and potential were measured using a Nano ZS90 nanoparticle size analyzer. The results are as follows: Figure 2 As shown, the average hydrated particle size of the empty micelles was 60.08 nm, and the polydispersity index (PDI) was 0.038; the particle size of the micelles loaded with drugs increased, with the average hydrated particle size of the dual-drug nanomicelles being 79.55 nm and the PDI being 0.107.

[0095] Example 3: Single free drug, combination of two free drugs, and lithium carbonate vincristine dual-drug nanomicelles The proliferation of various leukemia cells and normal cells is affected

[0096] The three cell lines CCRF-CEM, Jurkat, and U937 were all cultured in RPMI 1640 medium. The medium contained 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin. Cells were cultured at 37°C in a 5% CO2 incubator. When the cells reached the logarithmic growth phase, they were passaged at a 1:3 ratio. The specific implementation method is as follows:

[0097] Using 96-well U-shaped cell culture plates, the cell seeding density was 4 × 10⁶ cells / well. 4 Cells per well. Groups included: (1) control group; (2) free lithium carbonate group; (3) free vincristine group; (4) lithium carbonate micelle group; (5) vincristine micelle group; (6) free drug combination group; (7) dual-drug nanomicelle group (dual-drug nanomicelles prepared in Example 1). Cells in the culture plate were incubated in a 37°C, 5% CO2 incubator for 24 h, then washed with PBS, and 110 μL of CCK-8 dilution solution was added to each well for 2 h. After incubation, the cells were centrifuged at 1500 rpm for 5 minutes, and 90 μL of supernatant was aspirated into a 96-well microplate. The absorbance values ​​at 450 nm and 630 nm were measured, with CCK-8 working solution as a blank control.

[0098] The formula for calculating cell viability is:

[0099] Cell viability (%) = [(OD) 450 -OD 630 Experimental group - (OD) 450 -OD 630 (blank group)] / [(OD) 450 -OD 630 Control group - (OD) 450 -OD 630 (Blank group) × 100%

[0100] Figure 3-6 The results showed that free lithium carbonate itself had no cytotoxicity to various leukemia cell lines within the experimental concentration range, while free vincristine exhibited significant killing effects to varying degrees. In the combination of lithium carbonate and vincristine, lithium carbonate enhanced the cytotoxic effect of vincristine. Compared to the combination of lithium carbonate and vincristine, the dual-drug nanomicelles more effectively inhibited the proliferation of leukemia cells. They showed killing effects on various leukemia cell lines, including human acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and acute monocytic leukemia (AML), particularly against ALL. Therefore, at the same drug concentration, the dual-drug nanomicelles of this invention are more effective in inhibiting the proliferation of leukemia cells than single free drugs or combinations of free drugs.

[0101] For CCRF-CEM cells, when the concentrations of vincristine and lithium carbonate in the dual-drug nanomicelles were both 10 ng / mL, the cell viability rates of free lithium carbonate, free vincristine, lithium carbonate micelles, vincristine micelles, free drug combination, and dual-drug nanomicelles prepared in Example 1 of this invention were 98.5%, 71.0%, 98.0%, 60.1%, 52.4%, and 35.5%, respectively.

[0102] For Jurkat cells, when the concentrations of vincristine and lithium carbonate in the dual-drug nanomicelles were both 20 ng / mL, the cell viability rates of free lithium carbonate, free vincristine, lithium carbonate micelles, vincristine micelles, free drug combination, and dual-drug nanomicelles prepared in Example 1 of this invention were 97.6%, 76.4%, 98.3%, 69.2%, 61.8%, and 48.6%, respectively.

[0103] For U937 cells, when the concentrations of vincristine and lithium carbonate in the dual-drug nanomicelles were both 50 ng / mL, the cell viability rates of free lithium carbonate, free vincristine, lithium carbonate micelles, vincristine micelles, free drug combination, and dual-drug nanomicelles prepared in Example 1 of this invention were 94.8%, 83.2%, 96.8%, 60.3%, 79.3%, and 65.8%, respectively.

[0104] For K562 cells, when the concentrations of vincristine and lithium carbonate in the dual-drug nanomicelles were both 60 ng / mL, the cell viability rates of free lithium carbonate, free vincristine, lithium carbonate micelles, vincristine micelles, free drug combination, and dual-drug nanomicelles prepared in Example 1 of this invention were 96.4%, 80.1%, 98.0%, 74.2%, 72.4%, and 68.5%, respectively.

[0105] Example 4: Isolation of human peripheral blood mononuclear cells and detection of granulocyte colony-stimulating factor levels

[0106] Transfer 10 mL of whole blood to a 50 mL centrifuge tube, dilute with 10 mL of PBS solution, and mix gently. Take two 15 mL centrifuge tubes and add 5 mL of Ficoll solution to each. Then, add 10 mL of diluted blood from each centrifuge tube to the Ficoll layer in both tubes and centrifuge at 2000 rpm for 20 min. After centrifugation, the cell layer containing hPBMCs will be white. Use a pipette to transfer the white cell layer to a new 15 mL centrifuge tube. Add 10 mL of PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cells in culture medium, count them, and seed them into 6-well plates at 5 × 10⁶ cells per well. 5 Cells were divided into groups: (1) control group; (2) lithium carbonate micelle group (LM); (3) vincristine micelle group (VM); and (4) dual-drug nanomicelle group (VLM, dual-drug nanomicelles prepared in Example 1), with a weight ratio of lithium carbonate to vincristine of 1:1. After incubation in an incubator for 24 hours, cells were collected, washed three times with cold PBS, and lysed with fresh lysis buffer. The cells were then centrifuged at 4°C and 1500×g for 10 minutes, and the precipitate was discarded. The supernatant was collected strictly according to the ELISA kit instructions.

[0107] Depend on Figure 7 It is known that although vincristine can significantly reduce the level of granulocyte colony-stimulating factor in human peripheral blood mononuclear cells, the dual-drug nanomicelles prepared in Example 1 of this invention can significantly stimulate the generation of granulocyte colony-stimulating factor.

[0108] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements.

Claims

1. The application of vincristine and lithium carbonate as common active ingredients in the preparation of drugs for treating tumors and inhibiting the proliferation of leukemia cells; among which, The leukemia cells are acute T-lymphoblastic leukemia cells and / or chronic myeloid leukemia cells.

2. The application according to claim 1, characterized in that, In the common active ingredient, the mass ratio of vincristine to lithium carbonate is 2~10:2~200.

3. The application according to claim 2, characterized in that, In the common active ingredient, the mass ratio of vincristine to lithium carbonate is 2~8:2~150.

4. The application according to claim 3, characterized in that, In the common active ingredient, the mass ratio of vincristine to lithium carbonate is 2~6:2~50.

5. The application according to any one of claims 1 to 4, characterized in that, The drug is a nanomedicine.

6. The application according to claim 5, characterized in that, The nanomedicine is selected from one or more of the following: nanoliposomes, solid lipid nanoparticles, nanocapsules and nanospheres, polymer micelles, nano-sized drug suspensions, protein and cell-derived nanoparticles.

7. The application according to claim 6, characterized in that, The nanomedicine is selected from one or more of the following: nanoliposomes, solid lipid nanoparticles, nanocapsules and nanospheres, and polymer micelles.

8. The application according to claim 7, characterized in that, The nanomedicine is a polymer micelle.

9. The application according to any one of claims 1 to 4, characterized in that, The dosage form of the drug is selected from one or more of the following: solution, sol, emulsion, suspension, gas dispersion, solid dispersion, and particulate dispersion; wherein, The sol type is selected from one or more of the following: adhesive, coating agent, gel; The emulsion type includes emulsions; The gas dispersion is an aerosol; The solid dispersion is selected from one or more of the following: powders, granules, tablets, capsules; and / or The particulate dispersion is a microcapsule or nanocapsule.

10. The application according to claim 9, characterized in that, The solution is an aqueous solution.

11. The application according to claim 9, characterized in that, The emulsion is selected from one or more of the following: ordinary emulsion, microemulsion, and nanoemulsion.

12. The application according to any one of claims 1 to 4, characterized in that, The drug is in the form of a lotion.

13. The application according to any one of claims 1 to 4, characterized in that, The drug is in the form of an injection.

14. The application according to any one of claims 1 to 4, characterized in that, The administration method of the drug is selected from one or more of the following: oral administration, injection administration, local administration, inhalation administration, and rectal administration.

15. The application according to claim 14, characterized in that, The administration method of the drug is selected from one or more of the following: oral administration, injection administration, local administration, and inhalation administration.

16. The application according to claim 15, characterized in that, The injection administration method is selected from one or more of the following: intramuscular injection, subcutaneous injection, and intravenous injection.

17. The application according to claim 8, characterized in that, The carrier in the polymer micelles is a block copolymer.

18. The application according to claim 17, characterized in that, The carrier in the polymer micelles is a linear block copolymer or a nonlinear block copolymer.

19. The application according to claim 18, characterized in that, The linear block copolymer is selected from one or more of the following: AB block, ABA block, ABC triblock, ABABA pentablock linear copolymer; wherein A, B, and C are different polymers; and / or The nonlinear block copolymer is selected from one or more of the following: star-shaped block copolymer, comb-shaped block copolymer, dendritic block copolymer, cyclic block copolymer, H-shaped block copolymer, and cross-linked network block copolymer.

20. The application according to claim 19, characterized in that, The linear block copolymer is selected from one or more of the following: AB block, ABA block, ABC triblock; wherein, A is selected from one or more of the following: polystyrene, polycaprolactone, polyethylene glycol; B is selected from one or more of the following: polybutadiene, polypropylene, polyoxypropylene, polyoxyethylene, polystyrene, polyvinylcaprolactam; C is selected from one or more of the following: polymethyl methacrylate, polyacrylic acid, polymethyl methacrylate, polylactic acid, polyvinyl acetate; and / or The nonlinear block copolymer is selected from one or more of the following: star-shaped block copolymer, comb-shaped block copolymer, dendritic block copolymer, and cross-linked network block copolymer.

21. The application according to claim 20, characterized in that, The block copolymer is formed from two or more polymers selected from the following: polyvinyl caprolactam, polyvinyl acetate, polyethylene glycol, polystyrene, polybutadiene, polyoxypropylene, polyoxyethylene, polyacrylic acid, and polylactic acid.

22. The application according to claim 21, characterized in that, The block copolymer is formed from two or more polymers selected from the following: polyvinyl caprolactam, polyvinyl acetate, polyethylene glycol, polystyrene, and polybutadiene.

23. The application according to claim 22, characterized in that, The block copolymer is formed from polyvinylcaprolactam, polyvinyl acetate and polyethylene glycol.

24. The application according to claim 23, characterized in that, In the block copolymer: The molecular weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 40~80:20~50:5~20; and / or The weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 1~20:1~50:4~200.

25. The application according to claim 24, characterized in that, In the block copolymer: The molecular weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 50~70:25~40:10~15; and / or The weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 1~10:3~30:10~200.

26. The application according to claim 25, characterized in that, In the block copolymer: The molecular weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 57:30:13; and / or The weight ratio of the polyvinyl caprolactam, the polyvinyl acetate, and the polyethylene glycol is 1~6:3~16:20~200.

27. The application according to claim 17, characterized in that, When the nanomedicine is a polymer micelle, the mass ratio of vincristine, lithium carbonate and the block copolymer is 2~10:2~200:20~200.

28. The application according to claim 27, characterized in that, When the nanomedicine is a polymer micelle, the mass ratio of vincristine, lithium carbonate and the block copolymer is 2~8:2~150:20~200.

29. The application according to claim 28, characterized in that, When the nanomedicine is a polymer micelle, the mass ratio of vincristine, lithium carbonate and the block copolymer is 2~6:2~50:20~200.

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