New application of elaifolin

Abstract

The invention provides a novel application of elaifolin, and belongs to the technical field of acute myelogenous leukemia treatment. In particular to application of the elaifolin compound or pharmaceutically acceptable salt thereof in preparation of drugs for treating acute myelogenous leukemia, and when the elaifolin compound is used for treating an acute myelogenous leukemia cell line, the elaifolin compound has excellent effects of killing cells and remarkably inhibiting cell proliferation; in a PDX mouse model of AML, the elaifolin compound can significantly inhibit tumor growth and prolong the lifetime; when the compound is combined with a BCL-2 inhibitor Venetoclax, the compound shows a synergistic anti-AML effect, and the drug resistance risk can be reduced. Therefore, the elaifolin compound can be effectively used as a pharmaceutical composition for preventing or treating acute myelogenous leukemia.

Description

Technical Field

[0001] This invention relates to the field of acute myeloid leukemia treatment technology, and in particular to a novel use of olive leaf extract. Background Technology

[0002] Acute myeloid leukemia (AML) is a type of hematopoietic malignancy characterized by abnormal proliferation and differentiation arrest of myeloid blast cells in the bone marrow. The disease progresses rapidly and has a poor prognosis. Currently, first-line clinical treatment mainly involves chemotherapy and hematopoietic stem cell transplantation, but drug resistance and relapse are common problems. Natural products, due to their unique chemical skeletons and multi-target action modes, have become an important source of anti-tumor drugs. Elaiophylin is a drug derived from Streptomyces spp. Streptomyces The resulting C2 symmetrical macrolide antibiotic has the chemical formula C 54 H 88 O 18 It has CAS number 37318-06-2, a molecular weight of 1025.27 Da, and possesses various biological activities. However, there are currently few publicly available reports on its use in treating AML. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a new use for olive leaf extract in the treatment or prevention of acute myeloid leukemia.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention is to provide the use of oleuropein or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of acute myeloid leukemia, wherein oleuropein has the structure shown in Formula I: .

[0005] Furthermore, the treatment or prevention includes improvement of the subject's disease symptoms or health status.

[0006] Furthermore, the treatment or prevention includes inhibiting the proliferation of acute myeloid leukemia tumor cells, inhibiting the growth of acute myeloid leukemia tumor cells, or inducing the death of acute myeloid leukemia tumor cells.

[0007] Furthermore, the treatment or prevention includes delaying or overcoming the resistance of acute myeloid leukemia tumor cells to BCL-2 inhibitors; Furthermore, the treatment or prevention includes downregulating NAMPT protein expression, promoting NAMPT protein degradation, or inhibiting NAD. + Synthetic pathway.

[0008] Furthermore, the tumor cells of the acute myeloid leukemia include any one of Molm-13, THP-1, and MV4-11.

[0009] Furthermore, the oleuropein or a pharmaceutically acceptable salt thereof may be used concurrently with one or more drugs for the treatment or prevention of acute myeloid leukemia.

[0010] Furthermore, the drug is administered via intraperitoneal injection, intramuscular injection, intravenous injection, or oral administration.

[0011] Further, the dosage of the drug or oleuropein or its pharmaceutically acceptable derivative is 0.1 to 50 mg / kg per administration, calculated as oleuropein.

[0012] A second aspect of the invention is to provide the use of a pharmaceutical composition in the preparation of a medicament for treating acute myeloid leukemia, the pharmaceutical composition comprising the above-described oleuropein or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0013] Furthermore, the smallest unit of the pharmaceutical composition comprises or consists of a unit dose of 0.1 to 50 mg of olive leaf extract compound.

[0014] Furthermore, the pharmaceutical composition also comprises a BCL-2 inhibitor, which includes any one of venetoclax, risaxatolacria, and sotopracria.

[0015] definition Arsenosides or their salts can be chemically synthesized or manufactured through microbial fermentation. Extracts or refined products containing arsenosides or their salts, manufactured through microbial fermentation, can also be used. Arsenosides or their salts can also be isolated substances. The present invention preferably uses arsenosides or their salts manufactured through microbial fermentation.

[0016] The term "acute myeloid leukemia" is a malignant hematologic malignancy that originates from myeloid hematopoietic stem cells or progenitor cells in the bone marrow. It is characterized by the abnormal proliferation and impaired differentiation of immature myeloid cells (leukemia cells) in the bone marrow, which inhibits normal hematopoietic function and subsequently infiltrates other organs and tissues.

[0017] The term "treatment or prevention" refers to a method aimed at achieving a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relieving symptoms, reducing disease severity, stabilizing the condition (i.e., preventing deterioration), delaying or slowing disease progression, improving or alleviating the condition, and achieving detectable or undetectable partial or complete remission. Another meaning of "treatment" is extending a patient's survival compared to the expected survival rate without treatment. Therefore, "treatment" is an intervention aimed at altering the pathological process of a disease. Specifically, treatment can directly prevent, delay, or reduce pathological cellular degeneration or damage (e.g., organ cell damage in acute myeloid leukemia) or make cells more sensitive to other therapies or drugs.

[0018] The terms "therapeutic or preventative effective amount," "effective amount," or "sufficient amount" refer to an amount of an active agent sufficient to induce a specific biological state, effect, and / or response. Specifically, in this invention, it refers to an amount sufficient to achieve the desired outcome, such as an effective treatment of sepsis, when administered to a subject, including mammals, such as humans. The effective amount of the agents described herein can vary depending on factors such as the subject's disease state, age, sex, and weight. As will be understood by those skilled in the art, dosages or treatment regimens can be adjusted to provide an optimal therapeutic response. For example, in this invention, the dosage of oleuropein and / or its pharmaceutically acceptable derivatives is 0.1-50 mg / kg, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, calculated as oleuropein.

[0019] Furthermore, the effective dose of a treatment regimen for a subject may consist of a single administration or include a series of applications. The length of the treatment period depends on various factors, such as disease severity, the subject's age, reagent concentration, patient responsiveness to the reagent, or combinations thereof. It will also be understood that the effective dose of the reagent used for treatment may be increased or decreased during a specific treatment regimen. Dosage changes can be produced and become apparent through standard diagnostic analyses known in the art. In one aspect, the reagents of the present invention may be administered before, during, or after treatment with conventional therapies for the disease or condition in question, such as acute myeloid leukemia.

[0020] The term "subject" refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and agricultural animals, as well as zoo, sport, or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is human.

[0021] In this invention, pharmaceutically acceptable salts of oleuropein may include, but are not limited to, addition salts formed from inorganic acids, such as hydrochloride, sulfate, phosphate, hydrobromide, hydroiodide, nitrate, pyrosulfate, or metaphosphate; addition salts formed from organic acids, such as citrate, oxalate, benzoate, acetate, trifluoroacetate, propionate, succinate, fumarate, lactate, maleate, tartrate, glutarate, or sulfonate; or metal salts, such as lithium, sodium, potassium, magnesium, and calcium salts.

[0022] The pharmaceutical compositions of the present invention can be formulated into suitable forms with commonly used pharmaceutically acceptable carriers. "Pharmaceutically acceptable" means physiologically acceptable and generally does not cause allergic reactions or similar reactions, such as gastrointestinal symptoms and dizziness, when administered to humans. Furthermore, the pharmaceutical compositions of the present invention can be used after being formulated into oral and parenteral preparations according to conventional methods, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, etc., and such parenteral preparations as epidermal preparations, suppositories, or sterile injections.

[0023] Examples of carriers, excipients, and diluents that may be included in the composition include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. When formulated into dosage forms, diluents or excipients, such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants, may be used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms can be prepared by mixing the compounds of the present invention with at least one excipient, such as starch, microcrystalline cellulose, sucrose, lactose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups. Besides commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solutions or suspensions may contain propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable esters (e.g., ethyl oleate). As a base for suppositories, Tween 80, cocoa butter, lauryl butter, and glycerin gelatin can be used. To formulate a preparation for parenteral administration, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be mixed in water with sterile adjuvants and / or contain adjuvants such as preservatives, stabilizers, adjuvants (e.g., humidifying powders or emulsifying agents), salts and / or buffers for controlling osmotic pressure, and other therapeutically useful substances, to prepare a solution or suspension, which is then formulated into ampoules or vials for unit administration.

[0024] Pharmaceutical compositions including the oleuropein compound described in this invention as an active ingredient can be administered to mammals such as mice, livestock, and humans via various routes for the prevention or treatment of acute myeloid leukemia. All routes of administration are predictable, for example, via oral, rectal or intravenous, intramuscular, subcutaneous, endometrial, or cerebrovascular injection. Dosage varies depending on the age, sex, weight, specific disease or pathological condition being treated, severity of the disease or pathological condition, duration of administration, route of administration, rate of drug absorption, distribution, and excretion, type of other drugs used, and the prescribing authority's judgment. Dosage determination based on these factors is within the standard range for those skilled in the art. For the above dosage forms, the content of oleuropein or its pharmaceutically acceptable salt in a unit dosage form is 0.1~1000 mg, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 mg. 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 30 0, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg, calculated as oleuropein. Dosage does not limit the scope of the invention in any way.

[0025] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention provides the use of oleuropein compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment of acute myeloid leukemia. When oleuropein compounds are used to treat acute myeloid leukemia cell lines, they have excellent cell-killing and cell proliferation-inhibiting effects, and can therefore be effectively used as pharmaceutical compositions for the prevention or treatment of acute myeloid leukemia.

[0026] (2) In the PDX mouse model of AML, the olive leaf extract compound can significantly inhibit tumor growth and prolong survival.

[0027] (3) The combination of olive leaf extract compounds and BCL-2 inhibitor Venetoclax showed a synergistic anti-AML effect and could reduce the risk of drug resistance. Attached Figure Description

[0028] Figure 1 The figure shows the significant inhibitory effect of oleuropein on AML cell lines. Figure 2 Figure 1 shows the effect of oleuropein on the proliferation of Molm-13, MV-4-11, and THP-1 cell lines. A: EdU staining experiment: EdU (green) was used to label cell proliferation, and Hoechst 33342 (blue) was used to label cell nuclei. The morphology of THP-1, MV-4-11, and Molm-13 cell lines was observed under a bright-field microscope. After 24 hours of treatment with oleuropein (Ela, 0.3 μM), the number of EdU-positive cells (green) was significantly reduced, indicating that oleuropein effectively inhibited the proliferation of AML cell lines. BC: Flow cytometry analysis of EdU-positive cells: The EdU-positive cell population in each treatment group was analyzed by flow cytometry. Figure 3 Figure A shows the results of the apoptosis-inducing effect of oleuropein on Molm-13, MV-4-11, and THP-1 cell lines. Figure A: The apoptosis-inducing effect of oleuropein on Molm-13, MV-4-11, and THP-1 cells was detected by flow cytometry (Annexin V-FITC / PI double staining method), showing the changes in cell apoptosis under different concentrations (0.25 μM, 0.5 μM, 0.75 μM) of oleuropein treatment; Figure B: Quantitative analysis of cell apoptosis, based on the results in Figure A, the apoptosis rate of Molm-13, MV-4-11, and THP-1 cells after treatment with different concentrations of oleuropein was statistically analyzed. Figure 4 The figure shows the effect of oleuropein on the cell cycle in THP-1, MV-4-11, and Molm-13 cell lines. Figure A: The effect of oleuropein (0.25 μM and 0.50 μM) on the cell cycle of THP-1, MV-4-11, and Molm-13 cell lines was detected by flow cytometry (PI staining); Figure B: Quantitative analysis of each stage of the cell cycle, showing the cell cycle distribution of THP-1, MV-4-11, and Molm-13 cells under different concentrations of oleuropein treatment. Figure 5Figure A shows the therapeutic effects of oleuropein and the ferroptosis inhibitor (Fer1) on the MLL-AF9 mouse model. Figure A: Experimental design: MLL-AF9 gene-mutated hematopoietic stem cells were transplanted into the tail vein of C57BL / 6 mice and then randomly divided into four groups: AML group (untreated), Ela group (2.5 mg / kg), Fer1 group (10 mg / kg), and Ela + Fer1 group (Ela 2.5 mg / kg + Fer1 10 mg / kg); B: Bone marrow smears of successfully modeled mice (Wright staining, 1000X magnification); C: Survival time of MLL-AF9 mice in different treatment groups. Mice were randomly divided into four groups: AML group (untreated), Ela group (2.5 mg / kg), Fer1 group (10 mg / kg), and Ela + Fer1 group (Ela 2.5 mg / kg + Fer1 10 mg / kg); D: Spleen of mice: appearance and weight of spleen in mice in different treatment groups (AML, Ela, Fer1, Ela + Fer1); E: Liver of mice: appearance and weight of liver in mice in different treatment groups (AML, Ela, Fer1, Ela + Fer1). Figure 6 Figure 1 shows the results of the synergistic anti-AML activity of oleuropein and venetoclax; Figure A: Synergistic effect of oleuropein and venetoclax in THP-1 cells; Figure B: Synergistic effect of oleuropein and venetoclax in MV4-11 cells; Figure 7 Figure 1 shows the results of synergistic induction of apoptosis in AML cells by oleuropein and venetoclax; Figure A: synergistic induction of apoptosis by oleuropein and venetoclax in THP-1 and MV-4-11 cells, oleuropein at 0.5 μM and venetoclax at 10 nM; Figure BC: statistical graph of synergistic induction of apoptosis by oleuropein and venetoclax in MV4-11 cells. Figure 8 The effect of Ela leaf extract (Ela) treatment on NAMPT protein expression in acute myeloid leukemia cells; Figure 9 The effect of Ela leaf extract (Ela) treatment on the stability of NAMPT protein expression in acute myeloid leukemia cells; Figure 10 The figure shows the effect of NAMPT expression regulation on apoptosis in acute myeloid leukemia (AML) cells. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] In this invention, the Chinese name is: Olive Leaf Extract; English alias: Elaiophylin; Molecular formula: C 54 H 88 O 18 ; Molecular weight: 1025.27; CAS Registry Number: 37318-06-2.

[0031] This invention demonstrates the application of elaiophylin in the treatment and prevention of acute myeloid leukemia through in vitro and in vivo anti-AML experiments.

[0032] In vitro anti-AML studies.

[0033] Example 1 Elaiophylin has a significant inhibitory effect on a variety of AML cell lines.

[0034] After the cells have grown to the logarithmic growth phase, remove the cells and count them using a counting chamber. Adjust the concentration of the cell suspension, typically seeding 1 × 10⁶ cells per well. 4 Up to 5×10 4 Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well. After incubating the cells in an incubator for 24 hours, drug treatment was performed. Multiple concentration groups and a blank control group were set up. Each concentration group had at least three replicates. The antiproliferative effect of the drug was evaluated after 24 hours of cell culture. After the treatment time, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for another 2–4 hours. The WST-8 component in the CCK-8 reagent can be reduced by intracellular dehydrogenases to formazan, and the intensity of the color is directly proportional to the cell viability. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Based on the OD values ​​of each concentration group, the cell viability was calculated, and the half-maximal inhibitory concentration (IC50), which is the drug concentration at which cell viability decreases by 50%, was calculated by plotting a dose-response curve. Reference Figure 1The results showed that oleuropein had an IC50 value of 0.7391 μM (95% CI: 0.6831 - 0.7951) in MV-4-11 cells, 0.5732 μM (95% CI: 0.5516 - 0.5949) in Molm-13 cells, and 0.5146 μM (95% CI: 0.5013 - 0.5279) in THP-1 cells.

[0035] Example 2 Olive leaf extract has a significant inhibitory effect on the proliferation of various AML cell lines. EdU flow cytometry analysis method: THP-1, MV-4-11, and Molm-13 cells were seeded into 6-well or 96-well plates at a seeding density of 1 × 10⁶ cells per well. 4 Up to 5×10 4 Cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase. Different concentrations of oleuropein were added to the treatment groups, while the control group was treated with DMSO. After 24 hours of culture, cells were collected. Cell proliferation was marked with EdU (5-ethynyl-2'-deoxyuridine). EdU solution was added to the cell culture medium to a final concentration of 10 μM according to the manufacturer's instructions, and incubation continued for 2 hours to ensure EdU entry into the cells and incorporation into newly synthesized DNA. After treatment, cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 minutes to ensure no impact on cell morphology. Staining was performed using the EdU staining reagent according to the kit instructions. Cells were mixed with the EdU reaction reagent and incubated for 30 minutes to ensure sufficient staining. Flow cytometry analysis was then performed. The proportion of EdU-positive cells in different treatment groups was determined based on the EdU-positive (FITC) labeling, and the data were analyzed using FlowJo software.

[0036] Reference Figure 2 The results showed that after 24 hours of treatment with oleuropein (0.3 μM), the number of EdU-positive cells (green markers) was significantly reduced, indicating that oleuropein significantly inhibited the proliferation of these cell lines. Figure 2 BC used flow cytometry to quantitatively analyze changes in the EdU-positive cell population. In the DMSO control group, the proportion of EdU-positive cells was high, indicating active cell proliferation. However, in the oleuropein treatment group, the proportion of EdU-positive cells was significantly reduced, indicating that oleuropein effectively inhibited cellular DNA synthesis and proliferation.

[0037] Example 3 Experiments on apoptosis and cell cycle of AML cell lines using olive leaf extract.

[0038] After treating AML cells with different concentrations of oleuropein for 24 h, the cells were collected, and the culture medium was removed by centrifugation (1200 rpm, 5 min). The cells were washed twice and resuspended in PBS. The cell suspension was counted to ensure an appropriate cell concentration. Following the manufacturer's instructions, 100 μL of cell suspension (approximately 1 × 10⁻⁶ cells) was taken. 6 Cells were treated with 5 μL Annexin V-FITC (for early apoptotic cells) and 10 μL PI (for late apoptotic and necrotic cells). Then, 1×Annexin V binding buffer was added to a final volume of 500 μL, mixed thoroughly, and incubated in the dark for 15–20 minutes. The stained cells were then analyzed by flow cytometry (BD Accuri C6), collecting at least 10,000 cell events. Annexin V + / PI - Indicates early apoptotic cells, Annexin V + / PI + These are late-stage apoptotic cells, Annexin V - / PI + Indicates necrotic cells, Annexin V - / PI - For surviving cells, quantitative analysis of apoptosis was performed using CytExpert analysis software.

[0039] Reference Figure 3 The result, such as Figure 3 As shown in Figure A, Annexin V induces apoptosis in three AML cell lines (Molm-13, MV-4-11, and THP-1). Flow cytometry analysis using Annexin V-FITC / PI double staining showed that the proportion of apoptotic cells increased in a concentration-dependent manner under Annexin V treatment. In the 0.25 μM, 0.5 μM, and 0.75 μM Annexin V groups, with increasing Annexin V concentration, the proportions of early apoptosis (Annexin V+ / PI-) and late apoptosis (Annexin V+ / PI-) in AML cells increased. + / PI + The proportion of cells gradually increases. Figure 3 Figure B shows the quantitative analysis results of the effects of oleuropein at different concentrations on apoptosis in Molm-13, MV-4-11, and THP-1 cells. Data analysis showed that the apoptosis rate of the three cell lines gradually increased with increasing oleuropein concentration, reaching its maximum at a treatment concentration of 0.75 μM. This indicates that the apoptosis-inducing effect of oleuropein in AML cells is concentration-dependent, and the effect is more significant at higher concentrations.

[0040] Example 4 Experiment on the effect of oleuropein on the cell cycle of AML cell lines.

[0041] After treating AML cells with different concentrations of oleuropein for 24 h, the cells were collected and the culture medium was removed by centrifugation (1000 rpm, 5 min), followed by two washes. Cells were fixed with 70% ethanol at a volume 10 times the cell volume and stored at -20°C for at least 4 hours. After fixation, the cells were centrifuged to remove the ethanol and washed twice with PBS. Subsequently, 50 μg / mL of PI dye was added, and the cells were incubated in the dark for 30 min.

[0042] The stained cells were analyzed using flow cytometry, with at least 10,000 cells collected. The distribution of cells at different cell cycle stages (G0 / G1, S, and G2 / M phases) was analyzed by detecting PI staining intensity. Data were analyzed using FlowJo software.

[0043] Reference Figure 4 The result, such as Figure 4 As shown in Figure A, the cell cycle distribution under oleuropein treatment exhibited a concentration-dependent change. The DMSO group, serving as the solvent control, showed a normal cell cycle distribution with no significant arrest effect. However, with increasing oleuropein concentrations (0.25 μM and 0.50 μM), the proportion of cells in the G0 / G1 phase significantly increased, while the proportions in the S and G2 / M phases significantly decreased. Particularly in the 0.50 μM concentration group, the proportion of cells in the G0 / G1 phase reached its highest level, and the difference compared to the DMSO control group was statistically significant. ＊ P<0.05). These data indicate that oleuropein can significantly induce arrest in AML cells during the G0 / G1 phase and inhibit cells from entering the S and G2 / M phases. Figure 4 Figure B shows that at a concentration of 0.50 μM, oleuropein significantly increased the proportion of THP-1, MV-4-11, and Molm-13 cells in the G0 / G1 phase, while significantly decreasing the proportion of cells in the S and G2 / M phases, and the differences were statistically significant. ＊＊＊＊ P < 0.0001. Oleiferin significantly induced G0 / G1 phase arrest, especially at higher concentrations (0.50 μM), increasing the proportion of cells in the G0 / G1 phase in all cell lines, while decreasing the proportions in the S and G2 / M phases. These results indicate that oleiferin inhibits the proliferation of AML cell lines by suppressing cell cycle progression and arresting the G1 / S inflection point.

[0044] Example 5 In vivo anti-AML study of olive leaf extract.

[0045] Lin-bone marrow cells from C57BL / 6J mice were sorted and infected with MSCV-MLL-AF9-GFP retrovirus. The retrovirus-transfected cells were then injected via tail vein into C57BL / 6 recipient mice that had undergone sublethal radiation treatment. Radiation treatment was used to eliminate immune cells in the mice, ensuring the successful establishment of a leukemia model using exogenous cells. After injection, the proportion of GFP+ cells in the peripheral blood of the recipient mice was monitored regularly until acute myeloid leukemia developed in the mice. This model was established with the assistance of Professor Zhang Haojian's team at the Medical Research Institute of Wuhan University. [1,2] It has been widely used in research on the pathogenesis and treatment of AML.

[0046] To further investigate the antitumor effects of oleuropein, Fer1 was used as an inhibitor of ferroptosis. Fer1 effectively inhibits ferroptosis by suppressing excessive intracellular iron accumulation and slowing ROS production. In this experiment, Fer1 was used to investigate whether it could alter the anti-leukemic effect of oleuropein by blocking the ferroptosis mechanism. Twenty-four mice were randomly assigned to four groups of six each: AML group (untreated, as the control group), Ela group (oleuropein 2.5 mg / kg), Fer1 group (Fer1 10 mg / kg), and Ela + Fer1 group (oleuropein 2.5 mg / kg + Fer1 10 mg / kg). Each group of mice received different treatments to evaluate the efficacy of oleuropein alone in treating AML and the effect of Fer1 after inhibiting ferroptosis. To assess tumor burden in the bone marrow and spleen of mice, flow cytometry was used to detect GFP-positive cell populations in the bone marrow and spleen. Bone marrow samples were obtained from the femur and iliac crest, while spleen tissue was harvested from dissected mice. After collection, samples were washed with PBS buffer to remove blood and other impurities. Bone marrow and spleen samples underwent mechanical grinding and filtration to obtain single-cell suspensions. The cell suspensions were centrifuged (1000×g, 5 min), washed with PBS, and resuspended. Flow cytometry was used to detect the proportion of GFP-positive cells and to assess the effect of Fer1 inhibition on leukemia treatment efficacy.

[0047] Reference Figure 5In this mouse experiment, mice were randomly divided into four groups: the AML group (untreated, serving as the control group), the Ela group (oleiferin 2.5 mg / kg), the Fer1 group (Fer1 10 mg / kg), and the Ela + Fer1 group (oleiferin 2.5 mg / kg + Fer1 10 mg / kg). These groups were used to evaluate the therapeutic effect of oleiferin alone and the impact of Fer1's inhibition of ferroptosis on the efficacy of oleiferin. Mice in the AML group (untreated control group) had the shortest survival time, with all mice dying rapidly in the later stages of treatment. The Ela group (2.5 mg / kg) prolonged the survival time of mice, suggesting that oleiferin can effectively inhibit the growth of AML cells and promote tumor remission. Figure 5 As shown in the CD, when oleuropein was used alone (Ela group), the spleen of mice was significantly smaller than that of the AML group, with a significant reduction in spleen weight and volume, indicating that oleuropein can effectively slow the growth of tumor cells and inhibit tumor expansion. However, in the Ela + Fer1 group, the spleen reduction in the combined treatment group did not reach the effect of the Ela group, and its growth inhibition effect was significantly lower than that of the Ela-only group, indicating that the inhibitory effect of Fer1 weakened the efficacy of oleuropein.

[0048] Example 6 Experiments on the combined use of olive leaf extract and BCL-2 inhibitors.

[0049] Venetoclax is an oral small molecule inhibitor targeting BCL-2, playing an important role in the treatment of acute myeloid leukemia (AML). This study used a CCK8 assay to verify the cytotoxic effect of a combination of two drugs on AML cells, and calculated the synergy score between the two drugs using the SynergyFinder website. The dose-response matrix of the drug combination was used to evaluate whether there was a synergistic effect between the two drugs; a synergistic score greater than 10 indicated a synergistic effect. Figure 6 As shown in Figure A, the HSA synergistic fraction of oleuropein and venetoclax in THP-1 cells was 10.25; Figure 6 As shown in Figure B, the HSA synergistic score of oleuropein and venetoclax in MV4-11 cells was 12.8, indicating that the combination of the two drugs exerted a good synergistic anti-MV4-11 cell activity. These results demonstrate that oleuropein has a good chemosensitizing effect against venetoclax in vitro.

[0050] By examining the effects of oleuropein and venetoclax on apoptosis, it was found that oleuropein and venetoclax synergistically induced apoptosis in acute myeloid leukemia (AML) cells in THP-1 and MV-4-11 cell lines. In both THP-1 and MV-4-11 cells, oleuropein (0.5 μM) and venetoclax (10 nM) synergistically induced apoptosis. Figure 7 The apoptosis rate in the DMSO group (Figure A) was low. Treatment with Ela or Venetoclax alone significantly increased early and late apoptosis rates, while treatment with Ela in combination with Venetoclax further significantly increased the apoptosis rate, indicating that both have a synergistic effect in inducing apoptosis in AML cells. In THP-1 cells (such as...) Figure 7 In cells treated with Ela alone and Venetoclax, the apoptosis rates were moderate, while the apoptosis rate in the combined treatment group was significantly increased (P<0.0001). In MV-4-11 cells (such as...), the apoptosis rate was moderate in both the Ela single treatment group and the Venetoclax group, while the apoptosis rate in the combined treatment group was significantly increased (P<0.0001). Figure 7 A similar trend was observed in the combination therapy group (C), where the apoptosis rate was significantly higher than that of the single-drug treatment group.

[0051] Example 7 Research on the target of action of oleuropein.

[0052] This invention identifies NAMPT (nicotinamide phosphoribosyltransferase) as the main target of oleuropein through thermal proteomics and molecular docking simulation. Furthermore, it demonstrates through cell thermal migration experiments that oleuropein affects its thermal stability by directly interacting with NAMPT. It also verifies that NAMPT is the target of oleuropein through surface plasmon resonance experiments.

[0053] Further in vitro experiments were conducted to explore the mechanism of action between oleuropein and NAMPT.

[0054] (1) After AML cells were treated with different concentrations of oleuropein for 24 h, the expression level of NAMPT protein was detected, with β-actin as an internal control. The results are as follows: Figure 8 As shown, oleuropein can downregulate NAMPT protein expression in a concentration-dependent manner.

[0055] (2) AML cells were treated with CHX (50 μg / mL) alone or in combination with oleuropein, and NAMPT levels were detected at different time points. The results are as follows: Figure 9 As shown, oleuropein significantly accelerated the degradation of NAMPT under CHX-induced conditions, suggesting that oleuropein may reduce the stability of NAMPT protein by promoting its degradation.

[0056] (3) After NAMPT overexpression or knockdown treatment, AML cells were double-stained with Annexin V-FITC / PI and the apoptosis rate was detected by flow cytometry. The results are as follows: Figure 10 As shown, NAMPT knockdown significantly increased the proportion of early and late apoptotic cells, while NAMPT overexpression significantly reduced apoptosis levels. This demonstrates that NAMPT plays an important protective role in the survival of AML cells.

[0057] In summary, oleuropein (Ela) significantly downregulated NAMPT protein levels in acute myeloid leukemia (AML) cells in a concentration-dependent manner, suggesting that oleuropein can directly or indirectly inhibit NAMPT expression. In further CHX tracking experiments, when protein synthesis was inhibited by cycloheximide, the NAMPT level in the oleuropein-treated group decreased significantly faster than in the control group, indicating that oleuropein does not only inhibit NAMPT transcriptionally but also accelerates its protein degradation process.

[0058] Olive leaf extract may interfere with NAD by reducing the stability of NAMPT through promoting proteosomal degradation or ubiquitination modification pathways. + Synthetic pathways weaken the metabolic homeostasis and antioxidant capacity of AML cells.

[0059] Example 8 Tablet preparation.

[0060] 100 mg of olive leaf extract, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose and 2 mg of magnesium stearate were mixed, and then the mixture was compressed into tablets according to conventional tablet preparation methods.

[0061] Example 9 Preparation of powder.

[0062] Mix 1g of olive leaf extract and 1g of lactose and fill them into an airtight cloth to prepare a powder.

[0063] Example 10 Preparation of capsules.

[0064] 100 mg of olive leaf extract, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose and 2 mg of magnesium stearate were mixed, and then the above ingredients were mixed according to the conventional capsule preparation method and filled into gelatin capsules to prepare capsules.

[0065] Example 11 Preparation of injectable formulations.

[0066] Mix 10 mg of olive leaf extract, an appropriate amount of injectable sterile distilled water, and an appropriate amount of pH adjuster, and then prepare an injection using the above ingredients per ampoule (2 ml) according to conventional injection preparation methods.

[0067] References [1]Han G, Cui M, Lu P, et.al. Selective translation of nuclear mitochondrial respiratory proteins reprograms succinate metabolism in AML development and chemoresistance[J]. Cell Stem Cell, 2024, 31(12): 1777-1793.e9. [2]Wang J, Li Y, Wang P, et.al. Leukemogenic chromatin alterationspormote AML leukemia stem cells via a KDM4C-ALKBH5-AXL signaling axis[J]. Cell Stem Cell, 2020, 27(1): 81-97.e8. For any points not covered above, existing technologies shall apply.

[0068] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of oleuropein or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of acute myeloid leukemia, wherein, Olive leaf extract has the structure shown in Formula I: 。 2. The use according to claim 1, characterized in that, The treatment or prevention includes improvement in the subject's disease symptoms or health status.

3. The use according to claim 1, characterized in that, The treatment or prevention includes any of the following: A1) Inhibits the proliferation of acute myeloid leukemia tumor cells; A2) Inhibits the growth of acute myeloid leukemia tumor cells; A3) Induces cell death in acute myeloid leukemia tumors; A4) Delay or overcome the resistance of acute myeloid leukemia tumor cells to BCL-2 inhibitors; A5) Downregulates NAMPT protein expression; A6) Promotes the degradation of NAMPT protein; A7) Inhibits NAD + Synthetic pathway.

4. The use according to any one of claims 1-3, characterized in that, The tumor cells of the acute myeloid leukemia include any one of Molm-13, THP-1, and MV4-11.

5. The use according to claim 4, characterized in that, The oleuropein or its pharmaceutically acceptable salt is used concurrently with one or more drugs for the treatment or prevention of acute myeloid leukemia.

6. The use according to claim 5, characterized in that, The drug is administered via intraperitoneal injection, intramuscular injection, intravenous injection, or oral administration.

7. The use according to claim 6, characterized in that, The dosage of the drug or oleuropein or its pharmaceutically acceptable derivative is 0.1 to 50 mg / kg per administration, calculated as oleuropein.

8. Use of a pharmaceutical composition in the preparation of a medicament for the treatment or prevention of acute myeloid leukemia, the pharmaceutical composition comprising the oleuropein of claim 1 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The smallest unit of the pharmaceutical composition comprises or consists of a dose of 0.1 to 1000 mg of olive leaf extract.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further comprises a BCL-2 inhibitor, which includes any one of venetoclax, risatoclax, and sotopra.

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