Use of tamoxifen in the preparation of a medicament for the treatment of leukemia

Abstract

The present invention discloses the use of tamoxifen or its pharmaceutically acceptable salt, solvate or hydrate in the preparation of a medicament for treating leukemia.

Description

[0001] Related applications This application claims priority to Chinese Patent Application No. 202610680199X, filed on May 18, 2026 with the China National Intellectual Property Administration, entitled “Use of Tamoxifen in the Preparation of a Drug for Treating Leukemia”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of the selective estrogen receptor modulator tamoxifen or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of medicaments for the treatment of leukemia. Background Technology

[0003] Tamoxifen (chemical name: (Z)-2-[4-(1,2-diphenyl-1-butene)phenoxy]-N,N-dimethylethylamine; English name: Tamoxifen) is a selective estrogen receptor modulator (SERM). It exerts its anti-tumor effect by binding to estrogen receptors and regulating estrogen signaling pathways. Currently, tamoxifen is mainly used for the treatment of estrogen receptor-positive breast cancer and is a widely used endocrine therapy drug in clinical practice.

[0004] Tamoxifen structural formula Recent studies have shown that the estrogen signaling pathway plays an important role in hematologic malignancies, particularly in acute myeloid leukemia, where estrogen receptor-related signaling is involved in the regulation of cell proliferation, differentiation, and apoptosis. However, the clinical application of this pathway remains relatively limited.

[0005] Acute monocytic leukemia (AMoL) is an important subtype of acute myeloid leukemia (AML), originating from the M5 type in the French-American-British (FAB) classification system. According to the FAB classification criteria, AMoL is a type of leukemia characterized primarily by the abnormal proliferation of monocytic cells, with a significantly elevated proportion of monocytic cells in the bone marrow or peripheral blood, including primitive monocytes, immature monocytes, and mature monocytes. Based on the degree of cell differentiation, AMoL can be further divided into M5a (undifferentiated acute monocytic leukemia) and M5b (partially differentiated acute monocytic leukemia). M5a is predominantly composed of primitive monocytes with a low degree of cell differentiation, while M5b is predominantly composed of more mature monocytes, exhibiting a certain degree of differentiation. While there are some differences in their biological behavior and clinical manifestations, both are generally considered highly aggressive types of leukemia.

[0006] The main characteristics of AMoL are abnormal proliferation of mononuclear cell lines accompanied by differentiation arrest. It usually has a rapid onset and progression, and is prone to extramedullary infiltration, such as involvement of the skin, gums, and central nervous system. In addition, AMoL patients often have high white blood cell counts and obvious inflammatory manifestations. Some patients may also develop complications such as coagulation disorders, which further increases the complexity of the disease and the difficulty of treatment.

[0007] In terms of molecular mechanisms, abnormalities in multiple proto-oncogenes and signaling pathways are involved in the development and progression of AMoL. For example, KMT2A ( MLL The gene is located on chromosome 11q23, and its translocation is relatively common in AMoL, which can lead to the formation of abnormal fusion proteins, thereby regulating… HOX Abnormal expression of gene clusters promotes the self-renewal and proliferation of leukemia cells. FLT3 Gene mutations, especially FLT3-ITD Mutations, which also occur more frequently in AMoL patients, can continuously activate downstream signaling pathways, thereby promoting cell proliferation and inhibiting apoptosis. Furthermore, NPM1 Gene mutations are also present in some AMoL patients, participating in the development and progression of leukemia cells by affecting nucleocytoplasmic transport and gene transcription regulation. These gene abnormalities often work synergistically, leading to uncontrolled cell proliferation, impaired differentiation, and inhibited apoptosis, thereby promoting the occurrence and progression of AMoL.

[0008] Patients with acute myeloid leukemia (AMoL) often present with high white blood cell counts and significant organ infiltration. Some patients respond poorly to conventional chemotherapy regimens, such as induction therapy based on cytarabine combined with anthracyclines, and have a high relapse rate and poor prognosis. Although targeted therapies have shown some efficacy in some patients in recent years, AMoL still lacks a safe, effective, and universally applicable treatment. Currently, there is no consensus on the optimal treatment for this type of leukemia. International research on this type of leukemia is currently at a bottleneck. Discovering new related pathways and drug targets to ultimately provide safe, effective, and inexpensive drugs for the whole society is a problem of great significance to both basic and applied research in my country's biomedicine. Given the increasing investment in new drug development for corresponding targets internationally, coupled with the growing risk of failure, exploring new indications for existing drugs (drug repurposing or repositioning) can effectively mitigate research risks, reduce costs, and accelerate drug launches to quickly meet clinical needs. Therefore, this has become a strategy valued and adopted by many international pharmaceutical companies.

[0009] Comparative functional genomics breaks down the analytical barriers hindering the combined application of multi-omics data, enabling the organic integration of disease, gene, and drug. Applying this method and clinical bioinformatics strategies, potential therapeutic compounds for AMoL can be discovered from a large pool of marketed compounds, significantly reducing the number of compounds required for experimental screening, saving financial and human resources, and potentially leading to better treatment outcomes. Currently, there are no reports on the use of tamoxifen in the treatment of acute monocytic leukemia. Summary of the Invention

[0010] According to one aspect of the invention, an object of the invention is to provide the use of tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for treating acute monocytic leukemia.

[0011] Preferably, the leukemia is acute monocytic leukemia.

[0012] According to another aspect of the invention, another object of the invention is to provide the use of tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, in combination with additional anticancer therapeutic agents in the preparation of a medicament for the treatment of leukemia.

[0013] Preferably, the leukemia is acute monocytic leukemia.

[0014] Preferably, the additional anticancer therapeutic agent is selected from one or more of the following: mitosis inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, antiangiogenic agents, topoisomerase I and II inhibitors, plant alkaloids, hormones and antagonists, growth factor inhibitors, radiation, signal transduction inhibitors such as protein tyrosine kinase and / or serine / threonine kinase inhibitors, cell cycle inhibitors, biological response modifiers, enzyme inhibitors, antisense oligonucleotides or oligonucleotide derivatives, cytotoxins, and immunotumor agents.

[0015] According to another aspect of the invention, another object of the invention is to provide the use of a pharmaceutical composition in the preparation of a medicament for treating leukemia, said pharmaceutical composition comprising the active ingredient tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable excipient or carrier. Preferably, the leukemia is acute monocytic leukemia.

[0016] Preferably, the pharmaceutical composition comprises 1-99 wt% of tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, and 1-99 wt% of a pharmaceutically acceptable excipient or carrier.

[0017] Preferably, the pharmaceutical composition can be prepared into tablets, capsules, pills, powders, immediate-release dosage forms, sustained-release dosage forms, solutions, suspensions, emulsions, ointments, creams, or suppositories, etc. Tablets, capsules, pills, powders, immediate-release dosage forms, sustained-release dosage forms, solutions, and suspensions are preferred.

[0018] Preferably, the dosage of the pharmaceutical composition is determined according to the patient's age, condition, etc., and the unit dose of the formulation contains 0.05-200 mg of the tamoxifen or its pharmaceutically acceptable salt, solvate or hydrate. Preferably, the unit dose of the formulation contains 1 mg-100 mg of the tamoxifen or its pharmaceutically acceptable salt, solvate or hydrate.

[0019] Preferably, the dosage of the pharmaceutical composition is typically 0.001-100 mg / kg body weight per day, and more preferably 0.1-20 mg / kg body weight per day, taken once or in divided doses.

[0020] According to another aspect of the invention, another object of the invention is to provide a method for treating leukemia, the method comprising administering to a patient a therapeutically effective amount of tamoxifen or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or the pharmaceutical composition thereof. Preferably, the leukemia is acute monocytic leukemia. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 To analyze the differential expression of transcriptome sequencing data from patients with GSE9476 acute monocytic leukemia, a volcano plot was used to screen for 1476 differentially expressed genes, including 656 upregulated genes and 820 downregulated genes.

[0023] Figure 2 The results of flow cytometry analysis showed that tamoxifen induced apoptosis in leukemia cells. Detailed Implementation

[0024] The present invention will now be described in detail. Before proceeding with the description, it should be understood that the terminology used in this specification and the appended claims should not be construed as limited to its general or dictionary meaning, but rather should be interpreted according to the meaning and concept corresponding to the technical aspects of the invention, based on the principle that the inventors are allowed to appropriately define the terms for the best interpretation. Therefore, the description presented herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention. It should be understood that other equivalents or modifications can be obtained from it without departing from the spirit and scope of the invention.

[0025] In this document, the terms “comprising,” “including,” “having,” “containing,” or any other similar terms are open-ended conjunctions intended to cover non-exclusive inclusions. For example, a composition or article containing a plurality of elements is not limited to those listed herein, but may also include other elements not explicitly listed but typically inherent to the composition or article. Furthermore, unless explicitly stated to the contrary, the term “or” is inclusive, not exclusive. For example, the condition “A or B” is satisfied in any of the following cases: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); A and B are both true (or exist). Moreover, in this document, the terms “comprising,” “including,” “having,” and “containing” should be interpreted as specifically disclosed and simultaneously cover closed or semi-closed conjunctions such as “composed of” and “substantially composed of.”

[0026] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual values ​​within those ranges, particularly integer values. For example, a range description of "1 to 8" should be considered as specifically disclosing all secondary ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., particularly secondary ranges defined by all integer values, and should be considered as specifically disclosing individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Unless otherwise specified, the foregoing interpretation applies to all content throughout this invention, regardless of its scope.

[0027] If a quantity or other numerical value or parameter is expressed as a range, a preferred range, or a series of upper and lower limits, it should be understood that this document has specifically disclosed all ranges consisting of any upper or preferred value of that range and the lower or preferred value of that range, regardless of whether such ranges are separately disclosed. Furthermore, when a range of numerical values ​​is mentioned herein, unless otherwise stated, the range shall include its endpoints and all integers and fractions within the range.

[0028] In this document, numerical values ​​are to be understood as having a precision with significant digits, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49.

[0029] definition The term "pharmaceutically acceptable salt" refers to those salts that, within reasonable medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. The pharmaceutically acceptable salts of tamoxifen described herein include those derived from suitable inorganic acids and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods known in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepanoate, glycerophosphate, glucuronate, hemisulfate, heptahydrate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate, lysine, dodecyl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, trimethylacetate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4- salts. Representative base or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate ions.

[0030] The term "solvent" refers to a compound form that is typically bound to a solvent via a solvent decomposition reaction. This physical binding can include hydrogen bonds. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates, and also include stoichiometric and non-stoichiometric solvates. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. "Solvent" includes both solution phases and separable solvates. Representative solvates include hydrates, ethoxides, and methoxides.

[0031] The term "hydrate" refers to a compound that is bound to water. Typically, the number of water molecules in a hydrate is proportional to the number of compound molecules in the hydrate. Therefore, a hydrate of a compound can, for example, be formed by the general formula R. x H2O represents a compound, where R is a compound and x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1), such as hemihydrates (R0H2O ... 0.5 H2O) and polyhydrates (x is a number greater than 1, such as dihydrates (R) 2 H2O) and hexahydrate (R 6 H2O).

[0032] As used herein, the term “treatment” means the elimination, reduction, or improvement of a disease or condition and / or its associated symptoms. While not excluded, treating a disease or condition does not require the complete elimination of its associated symptoms. As used herein, the term “treatment” and similar terms can include “preventive treatment,” which refers to reducing the likelihood of the recurrence of a disease or condition or the relapse of a previously controlled disease or condition in subjects who are not at risk or are at risk of developing or being at risk of developing or experiencing a disease or condition or its recurrence. The term “treatment” and its synonyms are considered in relation to the administration of a therapeutically effective amount of the compound described herein to a subject who requires such treatment.

[0033] For pharmaceuticals or pharmacologically active agents, the term "effective amount" or "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. For example, in the oral dosage form of this invention, the "effective amount" of one active substance in the composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. A suitable effective amount in any given case can be determined by a person skilled in the art through routine testing.

[0034] The term "pharmaceutically acceptable excipient or carrier" refers to any formulation or carrier medium capable of delivering an effective amount of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, transdermal penetration enhancers, etc. Pharmaceutically acceptable excipients that can be used to manufacture the pharmaceutical compositions described herein include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and fragrances may also be present in the composition.

[0035] Pharmaceutical compositions can be formulated for any route of administration, such as oral administration. Typically, pharmaceutical compositions are in solid dosage form. However, in some embodiments, other dosage forms, such as liquids, suspensions, or semi-solid dosage forms, may also be used.

[0036] Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is combined with at least one inert, pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and / or (a) a filler or extender (e.g., starch, lactose, sucrose, glucose, mannitol, and silica), (b) a binder (e.g., carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic), (c) a humectant (e.g., glycerin), (d) a disintegrant (e.g., agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate), (e) a retarder (e.g., paraffin wax), (f) an absorption enhancer (e.g., quaternary ammonium compounds), (g) a wetting agent (e.g., cetyl alcohol and glyceryl monostearate), (h) an absorbent (e.g., kaolin and bentonite), and (i) a lubricant (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. For capsules, tablets, and pills, the dosage form may contain a buffer.

[0037] The terms "subject," "patient," or "object" refer to an animal, preferably a mammal, and most preferably a human, that has become the subject of treatment, observation, or experimentation. In any of the embodiments described herein, the subject may be a human.

[0038] Various dosage forms of the pharmaceutical compositions disclosed herein can be prepared according to conventional pharmaceutical methods. Each unit dose of the formulation contains 0.05-200 mg of the compound, preferably 1-100 mg of the compound.

[0039] The compounds and pharmaceutical compositions disclosed herein are for clinical use in mammals, including humans and animals, and can be administered via oral, nasal, skin, lung, or gastrointestinal routes. The optimal daily dose is 0.1-20 mg / kg body weight, administered once or in divided doses. Regardless of the method of administration, the optimal dose for a patient should be determined based on the specific treatment. Clinical trials typically begin with a low dose and gradually increase it until the most suitable dose is found.

[0040] In a common embodiment of the treatment method provided in this disclosure, the abnormal cell growth is cancer. The compounds of the present invention can be administered as a single agent or in combination with other anticancer therapeutic agents, particularly with standard of care agents applicable to specific cancers.

[0041] As used in this disclosure, the term "additional anticancer therapeutic agent" refers to any one or more therapeutic agents other than the compounds of the present invention, which are used or can be used to treat leukemia. In some embodiments, such additional anticancer therapeutic agents include compounds derived from the following classes: mitosis inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, antiangiogenic agents, topoisomerase I and II inhibitors, plant alkaloids, hormones and antagonists, growth factor inhibitors, radiation, signal transduction inhibitors such as inhibitors of protein tyrosine kinases and / or serine / threonine kinases, cell cycle inhibitors, biological response modifiers, enzyme inhibitors, antisense oligonucleotides or oligonucleotide derivatives, cytotoxins, immunotumor agents, etc.

[0042] In some embodiments, administration of the compounds of the present invention is provided by any method capable of delivering the compound to the site of action. These methods include oral administration, duodenal administration, parenteral administration (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), local administration, and rectal administration.

[0043] The pharmaceutical composition may be in, for example, a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained-release formulations, solutions, or suspensions; a form suitable for parenteral injection, such as a sterile solution, suspension, or emulsion; a form suitable for topical application, such as an ointment or cream; or a form suitable for rectal administration, such as a suppository. Preferably, it is in the form of tablets, capsules, pills, powders, immediate-release dosage forms, sustained-release dosage forms, solutions, or suspensions.

[0044] The following embodiments are merely examples illustrating implementations of the present invention and do not constitute any limitation on the present invention. Those skilled in the art will understand that modifications made without departing from the spirit and concept of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0045] Example 1. Bioinformatics screening: 1.1 Data Sources and Screening of Differentially Expressed Genes The dataset GSE9476 was selected from the Gene Expression Omnibus (GEO) database. The experimental group consisted of peripheral blood mononuclear cell (PBMC) samples from 5 patients with acute monocytic leukemia (AMoL), while the control group consisted of PBMC samples from 10 healthy donors. This dataset was used to analyze gene expression differences between AMoL patients and healthy individuals. Preferably, both the experimental and control samples were derived from the same research dataset to ensure data comparability and reduce the impact of batch effects on the analysis results. This dataset has been widely used in leukemia research in publicly available databases and is highly representative.

[0046] The obtained data and matrices were processed using R 1.4.1717 software. After grouping, the dataset was screened for differentially expressed genes using the limma 3.49.4 package, with a screening threshold of P < 0.05 and |log2FoldChange| > 1. The differentially expressed genes (DEGs) were plotted using the ggplot2 package.

[0047] Disease association analysis 1.2 Drug Screening Based on the theories of "systems biology" and "comparative functional genomics," an "integrated multi-omics analysis" algorithm was designed to integrate and analyze the transcriptome of diseases or patients with that of drugs, establishing a clinical bioinformatics epigenomic precision medicine prediction platform (EpiMed). This platform was used to perform correlation analysis between the aforementioned DEGs (disease, disease, and genetic information) and drug transcriptome data to identify drugs that can effectively treat leukemia among FDA-approved clinical drugs and traditional Chinese medicines. The screening criteria were a correlation coefficient >|0.1| and P < 0.05; drugs with a negative correlation were considered to have therapeutic effects.

[0048] 2. Experimental verification: 2.1 Experimental Materials: 2.1.1 Main reagents: PRIM 1640 culture medium (Gibco, USA), fetal bovine serum (Gibco, USA), penicillin-streptomycin-amphotericidal (100X triple antibody) (Gibco, USA), CCK-8 assay kit (Dojindo, Japan), Annexin V / PI apoptosis detection kit (Novozymes, China), tamoxifen (Shanghai Myrui Biochemical Technology Co., Ltd.) 2.1.2 Main Instruments: Micropipettes (2.5ul, 10ul, 20ul, 100ul, 200ul, 1000ul, 5000ul, Eppendorf, Germany), Clean Bench (Thermo Fisher Scientific, USA), Cell Culture Incubator (Thermo Fisher Scientific, USA), Microplate Reader (Thermo Fisher Scientific, USA), Flow Cytometer (BD Scientific, USA) 2.1.3 Cell lines: MV4-11 (human myeloid monocytic leukemia cells), THP-1 (human myeloid monocytic leukemia cells), MOLM-13 (human acute myeloid leukemia cells, derived from acute monocytic leukemia patients). 2.2 Experimental Methods: 2.2.1 Preparation of complete cell culture medium: Take 6 ml of penicillin-streptomycin-amphotericidal (100X triple antibody) and 56 ml of fetal bovine serum, add them to 500 ml of PRIM 1640 culture medium to prepare complete culture medium, and store it at 4 degrees Celsius for later use.

[0049] 2.2.2 Cell Culture and Passaging: MV4-11, THP-1 and MOLM-13 cell lines were cultured in complete culture medium and placed in a 37°C, 5% incubator.

[0050] 2.2.3 Drug Preparation: Weigh a certain dose of the drug using an electronic balance, dissolve the drug in DMSO to prepare a 10 mmol / L solution, and store at -30°C. Before use, dilute the drug in culture medium to prepare concentrations of 40 μmol / L, 30 μmol / L, 20 μmol / L, 10 μmol / L, and 5 μmol / L.

[0051] 2.2.4 CCK-8 Assay: Cells were passaged at approximately 80% confluence at a ratio of 1:3, about 3 times. After passage, cells were cultured in serum-free medium for 24 hours. Cells were then selected for the CCK-8 assay. After centrifugation, the cells were resuspended in 10 ml of culture medium. 20 μL of cells were added to 80 μL of culture medium, and 10 μL was added to a cell counting chamber to calculate the total cell count. An appropriate number of cells were selected for the experiment. 100 μL of cell suspension was seeded into each well, resulting in approximately 5 × 10⁶ cells per well. 4Each group of 96-well plates had three replicates. 100 μL of different drug concentrations were added to each well, resulting in final concentrations of 20 μmol / L, 15 μmol / L, 10 μmol / L, 5 μmol / L, and 2.5 μmol / L. 100 μL of PBS was added to the outermost layer of each well. At three time points (24h, 48h, and 72h), 20 μL of CCK-8 solution (10% concentration per well) was added to each well, and the plates were incubated for 2 hours. The OD values ​​of each well were then read at 450 nm using a microplate reader and recorded.

[0052] 2.2.5 The drug killing inhibition rate is calculated as follows: Drug killing inhibition rate (%) = 1 - (mean OD of experimental group - blank OD) / (mean OD of control group - blank OD) × 100%.

[0053] 2.2.6 Annexin V / PI double staining method for detecting cell apoptosis: When the MV4-11 cell line reached 80-90% confluence during the logarithmic growth phase, it was collected in 15ml centrifuge tubes, centrifuged at 1000rpm for 3 minutes, and resuspended in 10ml of RPMI 1640 complete medium. The cell density of the cell suspension was adjusted to 2×10⁻⁶ cells / mL. 5 / ml, 5ml of cell suspension per bottle. Add 10umol / L tamoxifen for treatment, and set up a negative control group without drug treatment. Collect cells after incubation at 37℃, 5% CO2 for 24 h or 48 h.

[0054] Cells from each group were collected by centrifugation, washed twice with pre-chilled cell staining buffer, centrifuged at 1000 rpm / min for 3 minutes, and the supernatant was discarded. Cells were then resuspended in 500 μL of Annexin V binding buffer. Next, 5 μL of Annexin V-FITC solution was added, and the cells were gently vortexed and incubated for 15 minutes at room temperature in the dark. Before flow cytometry, 10 μL of PI solution was added to the mixture. Finally, 400 μL of Annexin V binding buffer was added to each tube, and the cells were immediately analyzed by flow cytometry.

[0055] Apoptosis was analyzed in each group using flow cytometry. Annexin V-positive / PI-negative (Q3) cells were defined as early apoptotic cells, and Annexin V-positive / PI-positive cells (Q2) cells were defined as late apoptotic cells. The sum of the two was used as the total apoptosis rate. The difference in total apoptosis rate between the tamoxifen-treated group and the control group at different treatment times was compared to evaluate the induction of apoptosis in leukemia cells by tamoxifen.

[0056] 2.2.7 Flow cytometry analysis: The flowjo software was used to process the images from the experiment and calculate the proportion of cells in each quadrant.

[0057] 2.2.8 Statistical methods: Three independent experiments were conducted, and the average value of the results was taken.

[0058] 3. Bioinformatics screening results 3.1 Results of differentially expressed gene screening By analyzing the transcriptome sequencing data of GSE9476 acute monocytic leukemia patients, 1476 differentially expressed genes were screened. Figure 1 (Volcano plot of differential expression analysis), which showed that 656 genes were upregulated and 820 genes were downregulated.

[0059] 3.2 Results of Differential Gene Drug Prediction The differentially expressed genes were introduced into EpiMed and integrated with the transcriptomes of the disease or patient and the drug transcriptomes for analysis. Drugs that can effectively treat this type of leukemia were searched among FDA-approved clinical drugs and traditional Chinese medicines. The screening criteria were a correlation coefficient >|0.1| and P<0.05. Drugs with negative correlation were considered to have therapeutic effects. Tamoxifen was selected as a potential treatment drug.

[0060] 4. Experimental verification of the drug's killing effect on leukemia cells. 4.1 Results of Differential Gene Drug Prediction 4.1.1 The results of the MV4-11 cell line are shown in Table 1 below. Table 1

[0061] The results showed that tamoxifen significantly inhibited the proliferation of human myeloid monocytic leukemia cells MV4-11, and this inhibitory effect was concentration- and time-dependent. The cell inhibition rate increased with increasing drug concentration and duration of action, indicating that tamoxifen can effectively inhibit the growth of leukemia cells and is a preferred drug for acute monocytic leukemia.

[0062] 4.1.2 The results of the THP-1 cell line are shown in Table 2 below. Table 2

[0063] Furthermore, CCK-8 assays were performed on human myeloid monocytic leukemia cells (THP-1) using the same method. The results showed that tamoxifen also had a significant inhibitory effect on the proliferation of human myeloid monocytic leukemia cells (THP-1), and the inhibitory effect generally increased with increasing drug concentration and duration of action.

[0064] 4.1.3 The results of the MOLM-13 cell line are shown in Table 3 below. Table 3

[0065] Furthermore, CCK-8 assays were performed on MOLM-13 cells derived from patients with acute monocytic leukemia using the same method. The results showed that tamoxifen also had a significant inhibitory effect on the proliferation of MOLM-13 cells derived from patients with acute monocytic leukemia, and the inhibitory effect generally increased with increasing drug concentration and duration of action.

[0066] The above results indicate that tamoxifen has a good inhibitory effect on different leukemia cell lines, suggesting that its killing effect on leukemia cells is not limited to a single cell line.

[0067] 4.2 Flow cytometry results of tamoxifen-induced apoptosis in leukemia cells are as follows: Figure 2 .

[0068] The effect of tamoxifen on apoptosis of leukemia cells was detected by Annexin V-FITC / PI double staining combined with flow cytometry. The results showed that, compared with the control group, the early apoptosis rate, late apoptosis rate and total apoptosis rate of MV4-11 cells treated with tamoxifen were significantly increased, and the total apoptosis rate showed an overall upward trend with the increase of treatment time.

[0069] The above results indicate that tamoxifen can not only inhibit the proliferation of leukemia cells, but also induce apoptosis in leukemia cells, thus further proving that tamoxifen has a clear anti-tumor activity against leukemia cells and is preferably used for the treatment of acute monocytic leukemia.

[0070] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of tamoxifen or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of medicaments for the treatment of leukemia.

2. The use according to claim 1, characterized in that, The leukemia mentioned is acute monocytic leukemia.

3. Use of tamoxifen or its pharmaceutically acceptable salts, solvates or hydrates in combination with additional anticancer agents in the preparation of a medicament for the treatment of leukemia.

4. The use according to claim 3, characterized in that, The leukemia mentioned is acute monocytic leukemia.

5. The use according to claim 3, characterized in that, The additional anticancer therapeutic agent is selected from one or more of the following: mitosis inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, antiangiogenic agents, topoisomerase I and II inhibitors, plant alkaloids, hormones and antagonists, growth factor inhibitors, radiation, signal transduction inhibitors such as protein tyrosine kinase and / or serine / threonine kinase inhibitors, cell cycle inhibitors, biological response modifiers, enzyme inhibitors, antisense oligonucleotides or oligonucleotide derivatives, cytotoxins, immunotumor agents, etc.

6. Use of a pharmaceutical composition in the preparation of a medicament for treating leukemia, said pharmaceutical composition comprising the active ingredient tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable excipient or carrier. Preferably, the leukemia is acute monocytic leukemia.

7. The use according to claim 6, characterized in that, The pharmaceutical composition comprises 1-99 wt% of tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, and 1-99 wt% of a pharmaceutically acceptable excipient or carrier.

8. The use according to claim 6, characterized in that, The pharmaceutical composition can be prepared into tablets, capsules, pills, powders, immediate-release dosage forms, sustained-release dosage forms, solutions, suspensions, emulsions, ointments, creams, or suppositories, etc.; preferably tablets, capsules, pills, powders, immediate-release dosage forms, sustained-release dosage forms, solutions, or suspensions.

9. A method of treating leukemia, the method comprising administering to a patient a therapeutically effective amount of tamoxifen or a pharmaceutically acceptable salt, solvate or hydrate thereof, or the pharmaceutical composition thereof.

10. The method according to claim 9, characterized in that, The leukemia mentioned is acute monocytic leukemia.

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