Application of fangchinoline in the preparation of therapeutic drugs for STAG2 mutation acute myeloid leukemia

Abstract

The present invention provides the use of fangchinoline in preparing a drug for treating STAG2 mutation acute myeloid leukemia, and relates to the field of leukemia therapeutics. The use of fangchinoline in preparing a drug for treating STAG2 mutation acute myeloid leukemia according to the present invention addresses the problems of high incidence, poor prognosis and lack of effective drugs in STAG2 mutation acute myeloid leukemia. Fangchinoline can inhibit the proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1 and U-937, induce apoptosis, cell cycle arrest and cell differentiation in these cells, and thus exert an anti-STAG2 mutation acute myeloid leukemia effect.

Description

Technical Field

[0001] The present invention relates to the field of leukemia therapeutic drugs, and in particular to the use of fangchinoline in the preparation of STAG2 mutation therapeutic drugs for acute myeloid leukemia. Background Art

[0002] Leukemia is a malignant clonal disease of hematopoietic stem cells, characterized by abnormal bone marrow proliferation and impaired normal hematopoietic function. Clinical manifestations include anemia, bleeding, infection, and organ infiltration symptoms (such as hepatosplenomegaly). Leukemia is primarily categorized into acute and chronic leukemias based on disease course, and myeloid and lymphocytic leukemias based on cell origin. These include chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, and acute myeloid leukemia.

[0003] Chronic myeloid leukemia (CML) is a malignant tumor formed by the clonal proliferation of hematopoietic stem cells in the bone marrow. The presence of the Ph chromosome and / or the BCR-ABL fusion gene is a major contributing factor to the disease. Tyrosine kinase inhibitors such as imatinib are commonly used as first-line treatment options.

[0004] Acute lymphoblastic leukemia (ALL) is caused by the abnormal proliferation of lymphocyte progenitor cells in the bone marrow and is associated with gene mutations or chromosomal aberrations. Treatment regimens typically include vincristine or vindesine, anthracyclines / anthraquinones (such as daunorubicin, idarubicin, doxorubicin, and mitoxantrone), and glucocorticoids (such as prednisone and dexamethasone). The mechanism of action of vincristine is to inhibit microtubule polymerization, arresting the cell cycle in the M phase.

[0005] Acute myeloid leukemia (AML) is a hematologic malignancy caused by the abnormal proliferation of hematopoietic stem cells. It is the most common type of adult leukemia, accounting for approximately 70% of cases and associated with high morbidity and mortality. Specifically, AML is a malignant tumor that forms through the clonal proliferation of hematopoietic stem cells in the bone marrow. Existing studies have identified gene mutations as a key factor in the development of AML. Mutations or chromosomal aberrations in multiple genes, such as FLT3, NPM1, mLL, and STAG2, alone or in combination, contribute to the disease. Among them, the detection rate of STAG2 (stromal antigen 2) mutation in acute myeloid leukemia and myelodysplastic syndrome is about 5-15%, and is closely associated with poor prognosis of patients [Bachelot A, Bouvier A, Riou J, Thepot S, Giltat A, Nunes Gomes C, Paillassa J, Jouanneau-Courville R, Renard M, Beucher A, Cottin L, Wiber M, Ribourtout B, Geneviève F, Luque Paz D, Tanguy-Schmidt A, Ugo V, Hunault-Berger M, Blanchet O, Orvain C. Relationship between comorbidities, mutational profile, and outcome after intensive chemotherapy in patients older than 60 years with acute myeloid leukemia: Assessment of different risk scores. Am J Hematol. 2023 Jun;98(6):922-931. Tariq H, Barnea Slonim L,Coty Fattal Z,Alikhan MB,SegalJ,Gurbuxani S,Helenowski IB,Zhang H,Sukhanova M,Lu X,Altman JK,Chen QC,BehdadA.Therapy-related myeloid neoplasms with normal karyotype showdistinctgenomic and clinical characteristics compared to their counterpartswith abnormal karyotype. Br J Haematol.2022 Jun;197(6):736-744. Anwar N,MemonFA,Shahid S,Shakeel M,Irfan M,Arshad A,Naz A,Ujjan ID,Shamsi T. The Dawnof next generation DNA sequencing in myelodysplastic syndromes- experiencefromPakistan. BMC Genomics.2021 Dec 16;22(1):903. Eckardt JN,Stasik S,RölligC,Sauer T,Scholl S,Hochhaus A,Crysandt M,BrümmendorfTH,Naumann R,Steffen B etal:Alterations of cohesin complex genes in acute myeloid leukemia:differential co-mutations,clinical presentation and impact onoutcome.BloodCancer J 2023,13(1):18.]。.

[0006] STAG2 is a key subunit of the cohesin complex, located on the X chromosome. It is responsible for maintaining the tight adhesion of sister chromatids during cell division and ensuring the correct separation of chromosomes in mitosis. Its mutation destroys the differentiation ability of hematopoietic stem cells (HSCs) and enhances the self-renewal potential of HSCs, leading to the occurrence of leukemia [Ochi Y, Kon A, Yoshida K, Kataoka K, Nakagawa MM, Makishima H, Nakayama M, Koseki H, Takaori-Kondo A, Ogawa S: Stag2 regulates Hematopoietic Differentiation and Self-Renewal. Blood 2017, 130(Supplement 1):790-790.].

[0007] Currently, there is no effective and unified treatment for STAG2-mutant acute myeloid leukemia. Studies have shown that STAG2 mutations lead to reduced cohesin-DNA binding ability, thereby enhancing sensitivity to PARP inhibitors [Tothova Z, Valton AL, Gorelov RA, Vallurupalli M, Krill-Burger JM, Holmes A, Landers CC, Haydu JE, Malolepsza E, Hartigan C, Donahue M, Popova KD, Koochaki S, Venev SV, Rivera J, Chen E, Lage K, Schenone M, D'Andrea AD, Carr SA, Morgan EA, Dekker J, Ebert BL. Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS / AML. JCI Insight. 2021 Feb 8;6(3):e142149.]. Given the high incidence, poor prognosis and lack of effective drugs in clinical practice of STAG2 mutation AML, it is urgent to find effective drugs for the treatment of STAG2 mutation acute myeloid leukemia.

[0008] Fangchinoline is a bisbenzylisoquinoline alkaloid with the molecular formula Figure 1As shown, it is derived from the widely existing Stephania tetrandra in nature [Jiang, F., Ren, S., Chen, Y., Zhang, A., Zhu, Y., Zhang, Z., Li, Z., & Piao, D. (2021). Fangchinoline exerts antitumour activity by suppressing the EGFR-PI3K / AKT signalling pathway in colonadenocarcinoma. Oncology reports, 45(1), 139–150.]. Fangchinoline has multiple biological and pharmacological activities, including antioxidant, anti-osteoporotic, anti-inflammatory, immune-enhancing and histamine release inhibition [Winardi, D., Chu, PY, Chen, GY, Wang, K., Hsu, WY, Hsieh, CL, Chen, YH, Wu, YC, & Yang, JC (2022). Novel Aurora A Kinase Inhibitor Fangchinoline Enhances Cisplatin-DNA Adducts and Cisplatin Therapeutic Efficacyin OVCAR-3 Ovarian Cancer Cells-Derived Xenograft Model. International journal of molecular sciences, 23(3), 1868.].Studies have shown that fangchinoline can inhibit the proliferation of tumor cell lines such as gastric cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, bladder cancer, breast cancer, multiple myeloma, chronic myeloid leukemia, and acute lymphocytic leukemia, induce their apoptosis, and inhibit tumor cell metastasis. In addition, existing in vivo experiments have also confirmed that fangchinoline can inhibit tumor growth [Chen B, Song Y, Zhan Y, Zhou S, Ke J, Ao W, Zhang Y, Liang Q, He M, Li S, Xie F, Huang H, Chan WN, Cheung AHK, Ma BBY, Kang W, To KF, Xiao J. Fangchinoline inhibits non-small cell lung cancer metastasis by reversing epithelial-mesenchymal transition and suppressing the cytosolic ROS-related Akt-mTOR signaling pathway. Cancer Lett. 2022 Sep 1;543:215783. Jung YY, Um JY, Sethi G, Ahn KS. Fangchinolineabrogates growth and survival of hepatocellular carcinoma by negative regulation of c-met / HGF and its associated downstream signaling pathways. Phytother Res. 2022 Dec;36(12):4542-4557.].

[0009] The prior art has disclosed technical means for using stephaniatetrandra to treat chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia. The specific treatment mechanism is as follows: in the acute lymphoblastic leukemia cell line Jurkat, stephaniatetrandra inhibits the AKT signaling pathway, promotes the production of reactive oxygen species, oxidative stress induces DNA damage, and then promotes cell apoptosis [Shao Y, Li C, Miao G, Xu Y. Fangchinoline, an Extract of the Stephaniatetrandra S. Moore Root, Promoted Oxidative Stress-induced DNA Damage and Apoptosis and Inhibited Akt Signaling in Jurkat T Cells. Curr Mol Pharmacol. 2024; 17(1): e100223213590.]. In the chronic myeloid leukemia cell line KBM5 and the multiple myeloma cell line U266, fangchinoline can inhibit NF-κB and AP-1 activation, including attenuating the phosphorylation of IκB kinase (IKK) and p65, and can also significantly enhance TNFα-driven cell apoptosis [Jung YY, Shanmugam MK, Chinnathambi A, Alharbi SA, Shair OHM, Um JY, Sethi G, Ahn KS. Fangchinoline,aBisbenzylisoquinoline Alkaloid can Modulate Cytokine-Impelled Apoptosis viathe Dual Regulation of NF-κB and AP-1 Pathways. Molecules. 2019 Aug 28;24(17):3127.]. In addition, Fangchinoline can regulate the expression of CDKN1A and CCND2 and induce cell arrest in the G0 / G1 cycle in the chronic myelogenous leukemia cell line K562 [Wang Y, Chen J, Wang L, Huang Y, Leng Y, Wang G. Fangchinoline induces G0 / G1 arrest by modulating the expression of CDKN1Aand CCND2 in K562 human chronic myelogenous leukemia cells. Exp Ther Med. 2013Apr;5(4):1105-1112.]

[0010] Compared with the aforementioned chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia, STAG2 mutation acute myeloid leukemia is caused by impaired function of the cohesin complex [Fischer A, Hernández-Rodríguez B, Mulet-Lazaro R, Nuetzel M, Hölzl F, van Herk S, Kavelaars FG, Stanewsky H, AckermannU, Niang AH, Diaz N, Reuschel E, Strieder N, Hernández-López I, Valk PJM, Vaquerizas JM, Rehli M, Delwel R, Gebhard C. STAG2 mutations reshape the cohesin-structured spatialchromatin architecture to drive gene regulation in acutemyeloid leukemia. Cell Rep. 2024 Aug 27;43(8):114498.] and dysregulated gene expression [Fischer A,Hernández-Rodríguez B,Mulet-Lazaro R,Nuetzel M,Hölzl F,van Herk S,KavelaarsFG,Stanewsky H,AckermannU,Niang AH,Diaz N,Reuschel E,Strieder N,Hernández-López I,Valk PJM,Vaquerizas JM,Rehli M,Delwel R,Gebhard C.STAG2 mutations reshape the cohesin-structuredspatial chromatin architecture to drive generegulation in acute myeloid leukemia. Cell Rep. 2024 Aug 27;43(8):114498.], hematopoietic stem cell differentiation blockade [Mullenders J, Aranda-Orgilles B, Lhoumaud P, Keller M, Pae J, Wang K, Kayembe C, Rocha PP, RaviramR, Gong Y, Premsrirut PK, Tsirigos A, Bonneau R, Skok JA, Cimmino L, Hoehn D, Aifantis I. Cohesin loss alters adult hematopoieticstem cell homeostasis, leading to myeloproliferative neoplasms. J Exp Med. 2015Oct 19;212(11):1833-50.] and synergistic effects with other gene mutations [Chin CV, Antony J, Ketharnathan S, Labudina A, Gimenez G, Parsons KM, HeJ, George AJ, Pallotta MM, Musio A, Braithwaite A, Guilford P, Hannan RD, Horsfield JA. Cohesin mutations are synthetic lethal with stimulation of WNT signaling. Elife. 2020 Dec 7;9:e61405.], leading to the occurrence of leukemia.

[0011] Chronic myeloid leukemia is primarily driven by the BCR-ABL fusion gene and is caused by overactivation of tyrosine kinases, with minimal epigenetic changes. Therefore, tyrosine kinase inhibitors have a good therapeutic effect on chronic myeloid leukemia, while demethylation inhibitors alone are ineffective.

[0012] Multiple myeloma is a hematologic malignancy that originates in plasma cells in the bone marrow. Mutations or chromosomal aberrations in various genes, such as KRAS, NRAS, DIS3, and BRAF, contribute to the disease. Currently, the most commonly used treatment for this disease is a proteasome inhibitor (such as bortezomib) combined with an immunomodulator (such as lenalidomide) and dexamethasone. Proteasome inhibitors primarily inhibit the function of the 26S proteasome in myeloma cells, leading to the accumulation of abnormal proteins and inducing apoptosis.

[0013] The molecular mechanisms that differentiate acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) primarily lie in specific chromosomal translocations, fusion genes, and mutations. BCR-ABL1, ETV6-RUNX1, and TCF3-PBX1 fusion genes, as well as NOTCH1, FBXW7, and KRAS / NRAS mutations, are common in ALL. RUNX1-RUNX1T1, CBFB-MYH11, and PmL-RARA fusion genes, as well as FLT3, NPM1, DNMT3A, and IDH1 / IDH2 mutations, are common in AML. Both involve genomic instability, signaling pathway aberrations, and altered epigenetic regulation, but the genes mutated are fundamentally different. Hypomethylating agents such as azacitidine and decitabine, when used alone, are effective in treating AML with mutations in DNMT3A, IDH1, IDH2, and TET2, but are generally less effective in treating chronic myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma.

[0014] In short, the molecular mechanisms of STAG2 mutation acute myeloid leukemia are different from those of chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia, which leads to completely different treatment strategies.

[0015] At the same time, existing studies have shown that the STAG2 mutant cell line SKM-1 is insensitive to commonly used chemotherapy drugs for acute myeloid leukemia, such as cytarabine [Liang SM, Zhou XJ, Cai D, Zhou Q, Wang L. SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP / mLKL. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct; 30(5): 1508-1514. Kimura S, Kuramoto K, Homan J, Naruoka H, Ego T, Nogawa M, Sugahara S, Naito H. Antiproliferative and antitumor effects of azacitidine against the humanmyelodysplastic syndrome cell line SKM-1. Anticancer Res. 2012 Mar; 32(3): 795-8.]. This directly leads to the fact that there is currently no effective treatment for STAG2 mutation acute myeloid leukemia except hematopoietic stem cell transplantation.

[0016] It can be seen that in view of the high incidence, poor prognosis and lack of effective drugs in clinical practice of STAG2 mutation acute myeloid leukemia, providing effective drugs for the targeted treatment of STAG2 mutation acute myeloid leukemia has important technical significance and research value. Summary of the Invention

[0017] In response to the problems of high incidence, poor prognosis and lack of effective drugs in the clinical setting of STAG2 mutation acute myeloid leukemia, the present invention provides the use of fangchinoline in the preparation of a drug for treating STAG2 mutation acute myeloid leukemia.

[0018] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0019] Application of fangchinoline in the preparation of therapeutic drugs for STAG2 mutation acute myeloid leukemia.

[0020] Furthermore, the drug for treating STAG2 mutation acute myeloid leukemia contains fangchinoline or its salt as an active ingredient or main active ingredient.

[0021] A pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia uses fangchinoline or its salt as an active ingredient or a main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0022] Preferably, the pharmaceutical composition comprises: fangchinoline liposomes.

[0023] Preferably, the fangchinoline liposomes comprise the following raw materials in parts by weight: 0.6-0.8 parts of fangchinoline, 1-1.3 parts of cholesterol, and 6-8 parts of phospholipids.

[0024] Furthermore, the fangchinoline liposomes are prepared by the following method:

[0025] S1. adding phospholipids, fangchinoline, and cholesterol to an ethanol solution and dispersing them evenly to obtain a mixed organic phase;

[0026] S2. The mixed organic phase was injected into pure water, heated to remove alcohol, and then added with pure water to the volume before removing alcohol to obtain the initial liposome solution;

[0027] S3. The initial liposome solution was extruded and filtered using filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the fanghemoline liposomes.

[0028] Preferably, in step S1, the weight ratio of fangchinoline, cholesterol and phospholipid is 0.6-0.8:1-1.3:6-8.

[0029] Preferably, in step S1, the volume concentration of the ethanol solution is 70-80%.

[0030] Preferably, in step S2, the mixed organic phase is injected into 4-7 times the volume of pure water.

[0031] Preferably, in step S3, the average particle size of the obtained fangchinoline liposomes is less than 300 nm.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The test of the present invention showed that the use of the fangqinoline can significantly induce apoptosis of STAG2 mutant acute myeloid leukemia cell lines SKM-1, Kasumi-6, THP-1 and U-937, with a 24-hour IC 50 The concentrations of STAG2 in the serum of AML cells were 6.147±0.261μmol / L, 7.070±0.275μmol / L, 13.66±0.584μmol / L and 18.53±0.342μmol / L, respectively. It can be seen that STAG2 mutant acute myeloid leukemia cells are more sensitive to fangchinoline than wild-type AML cells.

[0034] (2) According to the test of the present invention, the use of the described fangqinoline can significantly induce STAG2 mutation acute myeloid leukemia cell cycle arrest; after drug treatment, SKM-1 cells arrested in the G1 phase, and Kasumi-6 cells arrested in the S phase; at the same time, fangqinoline can also induce STAG2 mutation acute myeloid leukemia cell differentiation and inhibit the formation of AML cell colonies.

[0035] (3) The present invention has shown that the use of the described fanghemoline can significantly increase the levels of lipid peroxides and ROS in SKM-1 and Kasumi-6 cells, regulate SLC7A11 expression through NRF2, and ultimately trigger ferroptosis in STAG2 mutant acute myeloid leukemia cell lines.

[0036] (4) The use of the fangchinoline of the present invention in the preparation of a therapeutic drug for STAG2 mutation acute myeloid leukemia. The fangchinoline or its salt can be used as an effective ingredient or a main effective ingredient in the preparation of the therapeutic drug, and can inhibit the proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1 and U-937, induce these cells' apoptosis, cell cycle arrest and cell differentiation, and thus exert an anti-STAG2 mutation acute myeloid leukemia effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the chemical formula of fangchinoline.

[0038] Figure 2These are transmission electron micrographs of fangchinoline liposomes. In the figure, Figure A is a transmission electron micrograph of fangchinoline liposomes (×2000); Figure B is a transmission electron micrograph of fangchinoline liposomes (×20000).

[0039] Figure 3 Figure 2 shows the expression of STAG2 in AML cell lines SKM-1, Kasumi-6, THP-1, and U-937. Panel A shows the STAG2 mRNA expression levels in STAG2 mutant cell lines SKM-1 and Kasumi-6 and STAG2 wild-type cell lines THP-1 and U-937; Panel B shows the STAG2 protein expression levels in STAG2 mutant cell lines SKM-1 and Kasumi-6 and STAG2 wild-type cell lines THP-1 and U-937 (* in each figure). P <0.05;** P <0.01).

[0040] Figure 4 Graphs showing the proliferation results of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 and wild-type AML cell lines THP-1 and U-937 after treatment with different concentrations of fangchinoline for 24 and 48 hours in the examples of the present invention.

[0041] Figures 5 to 8 The apoptosis results and statistical results of STAG2 mutant and wild-type AML cell lines SKM-1, Kasumi-6, THP-1 and U-937 treated with fangchinoline for 24h and 48h in the embodiment of the present invention are shown in the figure (* in each figure) P <0.05;** P <0.01).

[0042] Figure 9 Figure 1 shows the cell cycle and statistical results of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 after 24 hours of treatment with fangchinoline in the present invention. Figure A shows the effect of fangchinoline on the cell cycle of SKM-1; Figure B shows the effect of fangchinoline on the cell cycle of Kasumi-6 (* in each figure indicates the effect of fangchinoline on the cell cycle of SKM-1). P <0.05;** P <0.01).

[0043] Figure 10 Figure 1 shows the effects of fangchinoline on the differentiation of STAG2-mutant AML cell lines SKM-1 and Kasumi-6, as well as statistical results, according to an embodiment of the present invention. Figure A shows the effect of fangchinoline on SKM-1 cell morphology; Figure B shows the effect of fangchinoline on Kasumi-6 cell morphology.

[0044] Figure 11The expression of CD15 in SKM-1 and Kasumi-6 cells was changed after fangchinoline was applied to SKM-1 and Kasumi-6 cells in the embodiment of the present invention (* in the figure) P <0.05;** P <0.01).

[0045] Figure 12 Figure 1 shows the colony formation and statistical results of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 after 15 days of treatment with fangchinoline in an embodiment of the present invention. Figure A shows the colony formation results of SKM-1 and Kasumi-6 cells; Figure B shows the number of colonies formed by SKM-1 and Kasumi-6 cells (* in each figure). P <0.05;** P <0.01).

[0046] Figures 13 to 15 This is a diagram showing the mechanism of action of fangchinoline in the examples of the present invention in inhibiting STAG2 mutant AML cell lines. Figure 13 Figure A is a volcano plot of differentially expressed genes; Figure B is a heat map of differentially expressed genes; Figure C is the KEGG pathway analysis of differentially expressed genes; Figure 14 Figure D in the middle shows the morphological changes of mitochondria in SKM-1 cells observed by transmission electron microscopy; Figure 15 Figure E in the middle shows the changes in mitochondrial morphology of Kasumi-6 cells observed by transmission electron microscopy.

[0047] Figures 16 and 17 Graph showing the effects of fangqinoline on lipid peroxidation and ROS accumulation in STAG2 mutant AML cell lines (* in each figure) P <0.05;** P <0.01).

[0048] Figure 18 This is a graph showing the results of Fangqinoline down-regulating the expression of NRF2 and SLC7A11 in STAG2 mutant AML cells in the embodiment of the present invention (* in each figure P <0.05;** P <0.01).

[0049] Figure 19 Graph showing the mouse survival curve in an embodiment of the present invention. DETAILED DESCRIPTION

[0050] In order to provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described. It should be noted that the following detailed description is illustrative and is intended to further illustrate the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] It should be noted that the terms used herein are intended only to describe specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, "first," "second," and the like are used to distinguish similar objects and are not used to describe a specific order or precedence. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0052] The embodiments of the present invention provide the use of fangchinoline in preparing a drug for treating STAG2 mutation acute myeloid leukemia.

[0053] Furthermore, the drug for treating STAG2 mutant acute myeloid leukemia contains fenvalerate or its salt as the active ingredient or main active ingredient.

[0054] An embodiment of the present invention also provides a pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia, which uses fangchinoline or a salt thereof as an active ingredient or a main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0055] Wherein, the aforementioned compound salt refers to a derivative of the fanghemoline; the parent compound is modified by making an acid salt or an alkaline salt of the parent compound. Examples of compound salts include, but are not limited to: inorganic acid salts or organic acid salts of basic residues (such as amine groups); alkaline salts or organic salts of acidic residues (such as carboxylic acids, etc.). The compound salts of the present invention can be synthesized from a parent compound containing a basic part or an acidic part by conventional chemical methods. In addition, the aforementioned compound salts can be prepared in situ during the final separation and purification of the compound.

[0056] The aforementioned excipients refer to any excipient, solvent, dispersion medium, absorption retardant, diluent, or adjuvant that does not cause secondary reactions such as allergic reactions in humans or animals; such as preservatives or antioxidants, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial agents, isotonic agents, and absorption delaying agents. Typical examples of excipients include, but are not limited to, mannitol, lactose, magnesium stearate, sodium saccharide, talc, cellulose, cross-linked sodium carboxymethyl cellulose, glucose, gelatin, starch, lactose, dicalcium phosphate, sucrose, kaolin, magnesium carbonate, wetting agents, emulsifiers, solubilizers, sterile water, physiological saline, pH buffers, nonionic surfactants, lubricants, stabilizers, binders, and edible oils (such as peanut oil and sesame oil). The use of the aforementioned excipients in the pharmaceutical composition or feed additive is contemplated.

[0057] Furthermore, the pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia is a fangchinoline liposome; the fangchinoline liposome comprises the following raw materials in parts by weight: 0.6-0.8 parts of fangchinoline, 1-1.3 parts of cholesterol, and 6-8 parts of phospholipids.

[0058] Furthermore, the preparation method of the fangchinoline liposome comprises the following steps:

[0059] S1. Dissolve phospholipids in an ethanol solution with a volume concentration of 70-80% to prepare a phospholipid ethanol solution with a mass concentration of 3-5%. Continue to add fanghemoline and cholesterol and disperse them evenly by ultrasonication to obtain a mixed organic phase.

[0060] Among them, the weight ratio of fanghenoline, cholesterol and phospholipid is 0.6-0.8:1-1.3:6-8.

[0061] S2. Inject the mixed organic phase into 4-7 times the volume of pure water through a syringe pump, raise the temperature to 55-60°C, stir while keeping warm to remove ethanol, and add pure water to the original volume (i.e., the volume of the material before removing alcohol) to obtain the initial liposome solution.

[0062] S3. The above-mentioned liposome initial solution is extruded and filtered using filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain fanghemoline liposomes with an average particle size of less than 300 nm.

[0063] The present invention will be further described below with reference to some specific embodiments.

[0064] Example 1

[0065] This example provides the preparation of a pharmaceutical composition (fangchinoline liposomes) for treating STAG2 mutation acute myeloid leukemia, as follows:

[0066] Materials and reagents: Tetrandrine (dissolved in DMSO to 20 mM, purchased from MedChemExpress, Shanghai, China), cholesterol (purchased from MedChemExpress, Shanghai, China), phospholipids, ethanol solution (75% by volume), purified water, 0.45 nm and 0.22 nm filters, 1.5 mL centrifuge tubes, and 10 μL sterile pipette tips.

[0067] 2. Procedure: Dissolve 25 mg of phospholipids in 0.8 mL of ethanol (75% by volume). Stir to dissolve. Add 2.5 mg of effective weight of fangchinoline and 4.1 mg of cholesterol weighed on an electronic balance. Ultrasonic dispersion is performed for 10 minutes to thoroughly mix the mixture to obtain a mixed organic phase. Add 5 mL of pure water to a small beaker. While magnetically stirring, inject the mixed organic phase into the pure water using a syringe pump. Heat to 55°C and maintain. Stir at 20 rpm for 1 hour to remove the ethanol. Add pure water to the original volume (i.e., the volume before alcohol removal) to obtain a liposome initial solution. Filter the initial liposome solution three times using 0.45 μm and 0.22 μm pore size filters, respectively, to obtain a fangchinoline liposome dispersion.

[0068] 3. Preparation results: After testing, the particle size of the fangchinoline liposomes prepared in this example was 244.0 nm, the particle size dispersion coefficient was 0.262, and the encapsulation efficiency was 93.3%; the transmission electron microscopy image of the fangchinoline liposomes is as follows: Figure 2 shown.

[0069] Example 2

[0070] This example provides real-time fluorescence quantitative PCR and immunoblotting assays, specifically:

[0071] 1. Materials and reagents: TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, RIPA, 4× loading buffer, reverse transcription and real-time fluorescence quantitative PCR kit (purchased from Acryl Biotechnology Co., Ltd., Changsha, China), GAPDH, HRP-conjugated goat anti-rabbit antibody, HRP-conjugated goat anti-mouse antibody (purchased from ABclonal, Wuhan, China), STAG2 antibody (purchased from Abways, Shanghai, China), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0072] 2. Procedure: Total RNA was extracted from untreated SKM-1, Kasumi-6, THP-1, and U-937 cells using TRIzol. 1 μg of total RNA was reverse transcribed to generate cDNA. STAG2 was quantified by qPCR using the Applied Biosystems 7,500 Fast Real-Time PCR System based on the SYBR Green method. —△△Ct Method. The primer sequences used are as follows:

[0073] The forward primer sequence is shown in the sequence listing as SEQ ID NO.1;

[0074] The reverse primer sequence is shown in SEQ ID NO.2 in the sequence listing.

[0075] Equal amounts of cell lysate were loaded onto SDS-PAGE gels and electrophoresed. Proteins were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST and incubated overnight with antibodies against GAPDH and STAG2. Subsequently, the membranes were incubated in a solution of HRP-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse antibodies for 1 hour. Finally, protein bands were visualized using enhanced chemiluminescence reagents and an Amersham Imager 600.

[0076] 3. Experimental results: Figure 3 As shown, it can be seen that both qPCR and immunoblotting results showed that STAG2 expression in SKM-1 cells and Kasumi-6 cells was significantly reduced, confirming the presence of truncation mutations in the two cell lines.

[0077] Example 3

[0078] This example uses fangchinoline to conduct a cell proliferation assay (CCK-8 method), specifically:

[0079] Materials and reagents: Tetrandrine (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), CCK-8 (Solebol), penicillin-streptomycin, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 96-well cell culture plates.

[0080] 2. Procedure: SKM-1, Kasumi-6, THP-1, and U-937 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted, and seeded into 96-well plates at a cell count of 1 × 10 cells per well. 4. After 6 hours, different concentrations of fangchinoline (0, 5, 10, 20, 40, 60, 80, 100 μM) were added, and the DMSO group was used as the control. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24, 48, and 72 hours. 4 hours before the end of incubation, 10 μL of CCK-8 was added to each well and incubated in a cell culture incubator for 4 hours. The total volume in each well was 100 μL. Finally, the absorbance at 450 nm was detected using an enzyme reader. The experiment was repeated 3 times, with 3 replicate wells for each experiment.

[0081] 3. Experimental results: Figure 4 As shown in Table 1, it can be seen that fangqinoline significantly inhibits the proliferation of STAG2 mutant AML cells. 50 The results showed that STAG2 mutant AML cell lines were more sensitive to the inhibitory effect of fangchinoline than wild-type AML cell lines. 50 About the latter IC 50 In addition, with the increase of the incubation time and concentration of fangchinoline, the IC 50 This indicated that the inhibitory effect of fanghenoline on AML cell lines was affected by time and dose.

[0082] Table 1 IC values of fangchinoline against various cell lines 50 Value (μM)

[0083]

[0084] Note: SKM-1 and Kasumi-6 were 1.5×10 6 / mL were inoculated in 96-well plates, and THP-1 and U-937 were seeded at a density of 1×10 6 The density of the cells was inoculated into 96-well plates, with 100 μL in each well. The results were expressed as IC 50 ±SD.

[0085] Example 4

[0086] This example uses fanghemoline to conduct cell apoptosis and cell cycle experiments, specifically:

[0087] 1. Materials and reagents: Fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, Annexin-V-FITC / PI apoptosis kit (BD Bioscience), PI (containing RNase, purchased from BD Bioscience), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0088] 2. Procedure: SKM-1, Kasumi-6, THP-1, and U-937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were harvested and counted when they entered the logarithmic growth phase and incubated with different concentrations of fangchinoline (0, 3, 6, and 9 μM). The DMSO-treated group served as a control. After 24 and 48 hours of incubation at 37°C and 5% CO2, cells were harvested, washed twice with binding buffer, and incubated with Annexin V-FITC and PI for 30 minutes at room temperature in the dark. Cell apoptosis was detected and analyzed by flow cytometry.

[0089] SKM-1 and Kasumi-6 cells in the logarithmic growth phase were seeded into 6-well plates at a cell density of 2 × 10 cells per well. 5 , 2mL cell suspension per well. 50 Appropriate concentrations of fangchinoline were selected for cell treatment: 3 μM, 6 μM, and 9 μM for SKM-1 cells; 2 μM, 4 μM, and 6 μM for Kasumi-6 cells. Cells were incubated with different concentrations of fangchinoline for 24 hours and then harvested. Cells were fixed with 70% pre-cooled ethanol. Subsequently, 200 μL of PI / RNase staining solution was added and incubated at room temperature in the dark for 30 minutes. Cell cycle analysis was performed using flow cytometry.

[0090] 3. Experimental results: Figure 5 As shown in the figure, it can be seen that at 24h and 48h of incubation, SKM-1 showed late apoptosis and increased with the increase of the concentration of fangqinoline. Figure 6 As shown in the figure, it can be seen that when treated with 9μM fangchinoline for 24h and 48h, the apoptosis rate of SKM-1 was 28.56% and 42.19%, respectively. The results of Kasumi-6 incubated for 24h and 48h showed that the late and early apoptosis increased with the increase of fangchinoline concentration. Figure 7-8As shown in the figure, it can be seen that when treated with 6μM fangchinoline for 24h and 48h, the apoptosis rates of Kasumi-6 cells were 38.85% and 58.50%, respectively. However, when THP-1 and U-937 cells were treated with 9μM fangchinoline for 24h and 48h, the apoptosis rates of cells were less than 20%. Figure 9 As shown, it can be seen that in SKM-1 cells, the proportion of cells in the G1 phase (58.50% vs 62.43% vs 66.90%) increased with the increase of the concentration of fangchinoline; in Kasumi-6 cells, the proportion of cells in the S phase (33.60% vs 40.37% vs 42.67%) increased with the increase of the concentration of fangchinoline.

[0091] These data confirm that fangqinoline is more effective in inducing apoptosis in STAG2 mutant AML cell lines. The effect on the cell cycle of STAG2 mutant AML cell lines is cell type-dependent, with the drug inhibiting cell proliferation by inducing cell cycle arrest in the G1 or S phase.

[0092] Example 5

[0093] This example uses fanghemoline to perform cell morphology and surface antigen analysis, specifically:

[0094] Materials and reagents: Tetrandrine (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), Wright-Giemsa stain, APC mouse anti-human CD15 antibody (Cat #551376, purchased from BD Biosciences), PBS, glass slides, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0095] 2. Procedure: SKM-1 and Kasumi-6 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. When cells entered the logarithmic growth phase, they were harvested and counted. They were then incubated with varying concentrations of fangchinoline (SKM-1: 0, 3, 6, and 9 μM; Kasumi-6: 0, 2, 4, and 6 μM). The DMSO-treated group served as a control. After 24 and 48 hours of incubation at 37°C and 5% CO2, cells were harvested, slides prepared, and stained with Wright-Giemsa. Finally, cell morphology was observed under light microscopy. After 24 hours of incubation with fangchinoline at the above concentrations, SKM-1 and Kasumi-6 cells were harvested, washed with PBS, and incubated with APC mouse anti-human CD15 antibody. Changes in cell surface antigens were assessed by flow cytometry.

[0096] 3. Experimental results: Figure 10-11 As shown in the figure, it can be seen that after treatment with fangchinoline, the nuclear / cytoplasmic ratio of SKM-1 and Kasumi-6 cells decreased with the increase of drug concentration, showing a phenotypic change consistent with cell differentiation. Figure 11 As shown in the figure, fangchinoline significantly upregulated the differentiation gene CD15 (a marker of granulocyte differentiation) in both SKM-1 and Kasumi-6 cells. These results suggest that fangchinoline induces granulocyte differentiation in STAG2 mutant AML cell lines. Therefore, fangchinoline may induce cell cycle arrest by promoting differentiation in STAG2 mutant AML cell lines.

[0097] Example 6

[0098] In this example, fanghemoline was used to count the number of cell colonies using a fluorescence inverted phase contrast microscope, specifically:

[0099] Materials and reagents: Tetrandrine (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 24-well cell culture plates.

[0100] 2. Procedure: SKM-1 and Kasumi-6 cells were mixed with different concentrations of fangchinoline (SKM-1: 0, 6, 9, 12 μM; Kasumi-6: 0, 3, 6, 9 μM) in 500 μL MethoCult TM H4435, which contains 10% fetal bovine serum, 10% cell suspension (cell number 5×10 3 ) and 1% penicillin / streptomycin, then seeded into 24-well plates. The cells were incubated in a 37°C, 5% CO2 cell culture incubator for 14 days, with the DMSO-added group serving as a control. The number of cell colonies in the different groups was counted using an inverted fluorescence phase-contrast microscope.

[0101] 3. Experimental results: Figure 12 As shown in Figures AB, both SKM-1 and Kasumi-6 showed a significant decrease in colony number with increasing concentrations of fangchinoline. Furthermore, neither SKM-1 nor Kasumi-6 was able to form colonies at sufficiently high concentrations of fangchinoline (SKM-1: 12 μM; Kasumi-6: 9 μM). This suggests that STAG2-mutant AML cell lines that have undergone granulocyte differentiation have lost their ability to form colonies. In summary, fangchinoline effectively inhibits colony formation in STAG2-mutant AML cell lines.

[0102] Example 7

[0103] In this example, fangchinoline was used for transcriptome sequencing and transmission electron microscopy observation, specifically:

[0104] 1. Materials and reagents: Fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, glutaraldehyde, PBS, osmium phosphate, double-distilled water, ethanol, resin, 15-mL sterile centrifuge tubes, 1.5-mL centrifuge tubes, 10-μL sterile pipette tips, 200-μL sterile pipette tips, 1-mL sterile pipette tips, and 6-well cell culture plates.

[0105] 2. Operation steps: RNA was extracted using TRIzol, and the quality of the extracted RNA samples was assessed. The library was sequenced using the Illumina Novasek 6000 sequencing platform. Fastp software was used for data processing. Then, the obtained clean reads were used for subsequent data analysis. HISAT2 software was used for reference genome alignment and FPKM calculation, and the read count of each gene was obtained by HTSeq-count. KEGG enrichment, GO enrichment, and PPI analysis were performed on differentially expressed genes. SKM-1 and Kasumi-6 cells were cultured with the same concentration of fangchinoline for 12 hours, 24 hours, and 36 hours. The cells were collected and fixed with glutaraldehyde for 2 hours. Then, the cells were washed 4 times with 0.1M PBS, fixed with 1% osmium phosphate for 2 hours, washed once with 0.1M PBS, and washed 3 times with double-distilled water. Subsequently, the cells were dehydrated in an ethanol gradient and embedded in the resin. Finally, the samples were embedded. The intracellular structure was observed using a transmission electron microscope.

[0106] 3. Experimental results: Figure 13-15 As shown, 52 genes were upregulated and 217 genes were downregulated in SKM-1 cells. KEGG analysis revealed that the differentially expressed genes were enriched in the ferroptosis pathway. Transmission electron microscopy confirmed this finding, revealing that drug-treated SKM-1 and Kasumi-6 cells exhibited morphological changes consistent with ferroptosis (mitochondrial shrinkage and deformation, increased mitochondrial membrane density, decreased or absent mitochondrial cristae, and the presence of flocculent amorphous structures within the mitochondrial lumen). These results suggest that fangchinoline can induce cell death in STAG2-mutant AML cells through the ferroptosis pathway.

[0107] Example 8

[0108] This example uses fangchinoline to detect lipid peroxides and ROS, specifically:

[0109] 1. Materials and reagents: Tetrandrine (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), HBSS, Liperfluo solution, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0110] 2. Operation steps: SKM-1 and Kasumi-6 cells in the logarithmic growth phase were seeded into 6-well plates; fangchinoline was added, and solvent controls and replicates were set up. After culturing at 37°C and 5% CO2 for 12 hours, the cells were collected, centrifuged, the supernatant was discarded, and the cells were washed twice with 200 μL HBSS and HBSS was removed; 200 μL of 1 μmo1 / L Liperfluo solution diluted with HBSS was added and cultured in a 37°C, 5% CO2 incubator for 30 minutes; the solution was removed, and the cells were washed twice with 200 μL HBSS. The intracellular lipid peroxide content and ROS level were determined by flow cytometry.

[0111] 3. Experimental results: Figure 16-17 As shown in the figure, fangchinoline significantly increased the levels of lipid peroxides and ROS in SKM-1 and Kasumi-6 cells. Furthermore, lipid peroxide levels increased with increasing fangchinoline concentrations, and ROS levels increased with prolonged fangchinoline treatment. These results provide strong evidence that fangchinoline induces ferroptosis in STAG2-mutant AML cell lines.

[0112] Example 9

[0113] This example uses fangchinoline to conduct a test to confirm the mechanism of ferroptosis, specifically:

[0114] 1. Materials and reagents: Fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, RIPA, 4× loading buffer, SLC7A11 / xCT (abcam, USA), NRF2 (Proteintech, Wuhan, China), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0115] 2. Procedure: Equal amounts of cell lysate were loaded onto SDS-PAGE gels and electrophoresed. Proteins were then transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST and incubated overnight with GAPDH, Nrf2, and SLC7A11 antibodies. The membranes were then incubated in HRP-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse antibody solutions for 1 hour. Protein bands were visualized using enhanced chemiluminescence reagents and an Amersham Imager 600.

[0116] 3. Experimental results: Figure 18 As shown in the figure, compared with the control group, fangchinoline significantly reduced the protein level of SLC7A11 in SKM-1 and Kasumi-6 cells. NRF2 protein levels were also reduced in STAG2 mutant AML cell lines treated with fangchinoline. In summary, fangchinoline inhibits NRF2, thereby downregulating SLC7A11 expression and ultimately triggering ferroptosis in STAG2 mutant AML cell lines.

[0117] Example 10

[0118] This example conducted a preliminary evaluation of the anti-leukemia effect in vivo based on the fangchinoline liposomes prepared in Example 1, specifically:

[0119] 1. Materials and reagents: liposome-containing tetrandrine, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, and a cell counting chamber.

[0120] 2. Operation steps: SKM-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted. 6-8 week old NSG mice were inoculated with 5×10 6 Cells / mouse. Three days later, fangchinoline liposomes were intravenously injected at a controlled dose of 3 mg / kg every three days. The behavior, body weight, and survival status of the mice were observed daily. A normal saline control group was set up for comparison using the same method.

[0121] 3. Experimental results: Figure 19 As shown, the average survival of NSG mice in the fangchinoline liposome-injected group was 64.5 days, significantly longer than the 35.5 days in the saline control group (P=0.006). Combined with the experimental results of the aforementioned examples, it was demonstrated that fangchinoline liposomes can inhibit the proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1, and U-937, induce apoptosis, cell cycle arrest, and cell differentiation in these cells, thereby exerting an anti-AML effect.

[0122] Unless otherwise specified, all percentages used in the present invention are by mass.

[0123] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of fangchinoline in the preparation of therapeutic drugs for STAG2 mutation acute myeloid leukemia.

2. The use of fangchinoline according to claim 1 in preparing a drug for treating STAG2 mutation acute myeloid leukemia, characterized in that: The drug for treating STAG2 mutation acute myeloid leukemia uses fangchinoline or its salt as an effective ingredient or main effective ingredient.

3. The use of fangchinoline according to claim 1 in preparing a drug for treating STAG2 mutation acute myeloid leukemia, characterized in that: The drug for treating STAG2 mutation acute myeloid leukemia is a pharmaceutical composition, which uses fangchinoline or its salt as an active ingredient or a main active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

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