技术领域Technical Field
本发明属于生物医药领域，具体涉及一种含青蒿琥酯与venetoclax和阿糖胞苷的药物组合物及在制备治疗急性髓细胞性白血病药物中的应用。The present invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition containing artesunate, venetoclax and cytarabine, and an application of the pharmaceutical composition in preparing a drug for treating acute myeloid leukemia.
背景技术Background Art
急性髓性白血病(AML)是最具侵袭性的血液系统恶性肿瘤之一，老年AML患者主要采用去甲基化药物(HMAS)或低剂量阿糖胞苷(LDAC)进行治疗，患者预后极差，5年总体生存率不超过10％。针对年轻的AML患者则一直沿用40年前的“7+3”(柔红霉素+阿糖胞苷)诱导方案进行治疗，该治疗方案的缓解率在30-80％之间，但由于复发原因，长期生存率和治愈率较低，治疗效果需要改善。Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancies. Elderly AML patients are mainly treated with hypomethylating drugs (HMAS) or low-dose cytarabine (LDAC), and the prognosis of patients is extremely poor, with a 5-year overall survival rate of no more than 10%. Young AML patients have been treated with the "7+3" (daunorubicin + cytarabine) induction regimen from 40 years ago. The remission rate of this treatment regimen is between 30-80%, but due to relapse, the long-term survival rate and cure rate are low, and the treatment effect needs to be improved.
凋亡阻滞为AML的共同特征，抗凋亡蛋白Bcl-2和Mcl-1的异常表达提高了AML细胞的凋亡阈值，是AML发病和药物耐受的主要原因之一，Venetoclax是目前唯一被批准用于CLL和AML治疗的Bcl-2家族抗凋亡蛋白小分子抑制剂，但单独应用在AML中的作用有限，临床研究中发现venetoclax改善了阿扎胞苷、地西他滨或低剂量阿糖胞苷对AML的治疗效果，该联合方案于2018年获批用于治疗老年不适宜强化治疗的AML患者，但该联合方案相比于阿糖胞苷单独用药，患者的中位生存期仅延长了4个月，最终由于耐药的出现导致治疗失败，治疗效果仍需进一步改善。Apoptosis arrest is a common feature of AML. Abnormal expression of anti-apoptotic proteins Bcl-2 and Mcl-1 increases the apoptosis threshold of AML cells and is one of the main causes of AML onset and drug resistance. Venetoclax is currently the only small molecule inhibitor of Bcl-2 family anti-apoptotic proteins approved for the treatment of CLL and AML, but its effect in AML is limited when used alone. Clinical studies have found that venetoclax improves the therapeutic effect of azacitidine, decitabine or low-dose cytarabine on AML. This combination regimen was approved in 2018 for the treatment of elderly AML patients who are not suitable for intensive treatment. However, compared with cytarabine alone, the median survival of patients with this combination regimen was only extended by 4 months. Ultimately, the treatment failed due to the emergence of drug resistance, and the treatment effect still needs to be further improved.
抗凋亡蛋白Mcl-1被认为是介导venetoclax耐受的主要原因，Venetoclax通过抑制Bcl-2释放Bim诱导细胞发生凋亡，但释放的Bim被Mcl-1扣押导致venetoclax的耐受。Mcl-1也可以转移到细胞核中与染色质结合来减少DNA损伤，并诱导p-Chk1启动DNA损伤修复，Mcl-1也参与阿糖胞苷的耐受过程。靶向抑制Mcl-1有望改善venetoclax与阿糖胞苷联合方案对AML的治疗效果。细胞内Mcl-1蛋白水平受Noxa和Bim调控，Noxa能够置换出Mcl-1中的Bim并诱导Mcl-1发生降解，提示诱导Noxa的药物可能通过降低Mcl-1的蛋白水平靶向抑制Mcl-1，增强venetoclax与阿糖胞苷联用治疗效果。The anti-apoptotic protein Mcl-1 is considered to be the main cause of venetoclax tolerance. Venetoclax induces cell apoptosis by inhibiting the release of Bim from Bcl-2, but the released Bim is seized by Mcl-1, leading to venetoclax tolerance. Mcl-1 can also transfer to the nucleus and bind to chromatin to reduce DNA damage, and induce p-Chk1 to initiate DNA damage repair. Mcl-1 is also involved in the tolerance process of cytarabine. Targeted inhibition of Mcl-1 is expected to improve the therapeutic effect of venetoclax combined with cytarabine on AML. The intracellular Mcl-1 protein level is regulated by Noxa and Bim. Noxa can displace Bim in Mcl-1 and induce Mcl-1 degradation, suggesting that drugs that induce Noxa may target and inhibit Mcl-1 by reducing the protein level of Mcl-1, thereby enhancing the therapeutic effect of venetoclax combined with cytarabine.
目前我国研究人员已总结出了青蒿琥酯与阿糖胞苷联用方案，同时临床已批准了venetoclax与阿糖胞苷联用方案，但这两种方案均由于其他抗凋亡蛋白的反馈性上调产生耐药，对AML的治疗效果有限。At present, Chinese researchers have summarized the combination of artesunate and cytarabine, and the combination of venetoclax and cytarabine has been clinically approved. However, both of these regimens have limited therapeutic effects on AML due to drug resistance caused by the feedback upregulation of other anti-apoptotic proteins.
发明内容Summary of the invention
为了克服现有的技术缺陷，本发明提供了一种含青蒿琥酯与venetoclax和阿糖胞苷的药物组合物及在制备治疗急性髓细胞性白血病药物中的应用。进一步的说组合物中青蒿琥酯通过诱导Noxa，与venetoclax联合协同诱导凋亡，降低Mcl-1蛋白，克服Mcl-1介导的阿糖胞苷耐受，增强阿糖胞苷诱导的DNA损伤作用，三种药物组合物能够诱导急性髓细胞白血病细胞凋亡及DNA损伤，改善venetoclax与阿糖胞苷联合方案对AML的治疗现状。In order to overcome the existing technical defects, the present invention provides a pharmaceutical composition containing artesunate, venetoclax and cytarabine and its use in the preparation of drugs for treating acute myeloid leukemia. Further, artesunate in the composition induces Noxa, synergistically induces apoptosis with venetoclax, reduces Mcl-1 protein, overcomes Mcl-1-mediated cytarabine tolerance, and enhances cytarabine-induced DNA damage. The three pharmaceutical compositions can induce apoptosis and DNA damage in acute myeloid leukemia cells, and improve the current status of the combined treatment of venetoclax and cytarabine for AML.
为实现上述目的，本发明采用技术方案为：To achieve the above purpose, the present invention adopts the following technical solution:
一种含青蒿素类衍生物的组合物，组合物为A组分、B组分和C组分按8-40:1-2:1-80比例混合；其中，A composition containing artemisinin derivatives, wherein the composition comprises component A, component B and component C mixed in a ratio of 8-40:1-2:1-80; wherein:
所述A组分为青蒿琥酯及其类似物中的一种或几种；类似物可为青蒿素、二氢青蒿素、蒿甲醚、蒿乙醚等；The component A is one or more of artesunate and its analogs; the analogs may be artemisinin, dihydroartemisinin, artemether, arteether, etc.;
所述B组分为venetoclax及其类似物中的一种或几种；类似物可为navitoclax等；The B component is one or more of venetoclax and its analogs; the analogs may be navitoclax, etc.;
所述C组分为阿糖胞苷及其类似物中的一种或几种；类似物可为阿扎胞苷、地西他滨、吉西他滨等；The C component is one or more of cytarabine and its analogs; the analogs may be azacitidine, decitabine, gemcitabine, etc.;
优选，所述A组分为青蒿琥酯；所述B组分为venetoclax；所述C组分为阿糖胞苷。Preferably, the component A is artesunate; the component B is venetoclax; and the component C is cytarabine.
青蒿琥酯结构式如Ⅰ所示。The structural formula of artesunate is shown in Ⅰ.
Bcl-2抑制剂venetoclax为式Ⅱ所示的化合物：The Bcl-2 inhibitor venetoclax is a compound represented by formula II:
阿糖胞苷为式Ⅲ所示的化合物：Cytarabine is a compound represented by formula III:
一种含青蒿素类衍生物的组合物的应用，所述组合物在制备治疗包括白血病和淋巴瘤在内的血液肿瘤药物中的应用。An application of a composition containing artemisinin derivatives, and application of the composition in the preparation of a drug for treating blood tumors including leukemia and lymphoma.
所述组合物相互协调诱导白血病细胞凋亡及DNA损伤制备治疗急性髓细胞性白血病药物中的应用。The composition is used in preparing a drug for treating acute myeloid leukemia by inducing leukemia cell apoptosis and DNA damage in a coordinated manner.
所述急性髓细胞性白血病为THP-1或MOLM-13细胞。The acute myeloid leukemia cells are THP-1 or MOLM-13 cells.
所述在制备治疗THP-1药物时，组合物A组分、B组分和C组分按8：1：5比例混合；When preparing the drug for treating THP-1, the components A, B and C of the composition are mixed in a ratio of 8:1:5;
所述在制备治疗MOLM-13药物时，组合物A组分、B组分和C组分按40：2：1比例混合。When preparing the drug for treating MOLM-13, component A, component B and component C of the composition are mixed in a ratio of 40:2:1.
一种含青蒿素类衍生物的组合物的制剂，所述制剂活性成分以及药物上接受的载体混合，其中，活性成分为权利要求1所述的组合物，活性成分占制剂质量的0.01-99％。A preparation of a composition containing artemisinin derivatives, wherein the active ingredient of the preparation is mixed with a pharmaceutically acceptable carrier, wherein the active ingredient is the composition according to claim 1, and the active ingredient accounts for 0.01-99% of the preparation mass.
所述制剂剂型为片剂、胶囊、颗粒。The preparation is in the form of tablets, capsules and granules.
所述制剂为将组合物中各组分混合后与药物上可接受到的载体混合制备制剂，或将组合物中各组分分别与药物上可接受到的载体制备制剂而后混合。The preparation is prepared by mixing the components in the composition and then mixing them with a pharmaceutically acceptable carrier, or by separately preparing the components in the composition with a pharmaceutically acceptable carrier and then mixing them.
进一步的说，在将本发明组合物制成同时给药的药剂的方案中，青蒿琥酯、Bcl-2抑制剂venetoclax和阿糖胞苷可以含在同一种药物制剂如片剂或胶囊中，患者按照说明书指示进行服用，或将上述组合物中的三种成分制成一种控释的制剂，先后释放组合物中的成分，患者只需服用该控释的组合物制剂；在将本发明组合物制备成交叉给药的制剂方案中，可以将青蒿琥酯、Bcl-2抑制剂venetoclax和阿糖胞苷分别做成不同的制剂，并采用本领域常规的方式将它们包装或结合在一起，然后患者按照药品说明书指示的交叉顺序服用，或者将该药物组合物制备成青蒿琥酯、Bcl-2抑制剂venetoclax和阿糖胞苷交叉释放的控释制剂。Furthermore, in the scheme of preparing the composition of the present invention into a medicament for simultaneous administration, artesunate, the Bcl-2 inhibitor venetoclax and cytarabine can be contained in the same pharmaceutical preparation such as tablets or capsules, and the patient takes it according to the instructions of the instructions, or the three components in the above-mentioned composition are prepared into a controlled-release preparation, and the components in the composition are released sequentially, and the patient only needs to take the controlled-release composition preparation; in the scheme of preparing the composition of the present invention into a preparation for cross-administration, artesunate, the Bcl-2 inhibitor venetoclax and cytarabine can be made into different preparations respectively, and they can be packaged or combined together in a conventional manner in the art, and then the patient takes it in the cross-order indicated in the drug instructions, or the pharmaceutical composition is prepared into a controlled-release preparation with cross-release of artesunate, the Bcl-2 inhibitor venetoclax and cytarabine.
本发明所具有的优点：The advantages of the present invention are:
本发明提供了青蒿琥酯、Bcl-2抑制剂venetoclax和阿糖胞苷组合物，并在机制上阐明青蒿琥酯能够通过诱导Noxa，与venetoclax协同诱导细胞凋亡，克服venetoclax的耐受，同时下调Mcl-1蛋白，克服阿糖胞苷诱导的p-Chk1反馈性激活和DNA损伤修复过程，增强阿糖胞苷对白血病细胞的杀伤作用。本发明克服了现有技术青蒿琥酯与阿糖胞苷联用方案和venetoclax与阿糖胞苷联用方案由于其他抗凋亡蛋白反馈性上调产生的耐药，显著改善了venetoclax与阿糖胞苷联合方案对AML的治疗效果。The present invention provides a composition of artesunate, Bcl-2 inhibitor venetoclax and cytarabine, and explains in terms of mechanism that artesunate can induce cell apoptosis by inducing Noxa and cooperating with venetoclax to overcome venetoclax tolerance, while downregulating Mcl-1 protein, overcoming the p-Chk1 feedback activation and DNA damage repair process induced by cytarabine, and enhancing the killing effect of cytarabine on leukemia cells. The present invention overcomes the drug resistance caused by the feedback upregulation of other anti-apoptotic proteins in the prior art combination of artesunate and cytarabine and the combination of venetoclax and cytarabine, and significantly improves the therapeutic effect of the combination of venetoclax and cytarabine on AML.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例提供的采用本发明组合物在THP-1和MOLM-13细胞中考察对AML细胞的生长抑制效果图；FIG1 is a graph showing the growth inhibition effect of the composition of the present invention on AML cells in THP-1 and MOLM-13 cells provided in an embodiment of the present invention;
图2为本发明实施例提供的采用本发明组合物在THP-1和MOLM-13细胞中考察对AML细胞的凋亡诱导效果图；FIG2 is a graph showing the apoptosis induction effect of the composition of the present invention on AML cells in THP-1 and MOLM-13 cells, provided in an embodiment of the present invention;
图3为本发明实施例提供的采用本发明组合物在THP-1和MOLM-13细胞中考察对AML细胞的杀伤效果图；FIG3 is a graph showing the killing effect of the composition of the present invention on AML cells in THP-1 and MOLM-13 cells provided in an embodiment of the present invention;
图4为本发明实施例提供的采用本发明组合物在THP-1和MOLM-13细胞中考察抑制集落形成效果图。FIG. 4 is a diagram showing the effect of inhibiting colony formation in THP-1 and MOLM-13 cells using the composition of the present invention provided in an embodiment of the present invention.
图5为本发明实施例提供的采用本发明组合物在NOD-SCID小鼠体内考察抑制肿瘤生长效果图，其中，A为小鼠肿瘤图片,B为小鼠瘤重,C为小鼠体重。FIG5 is a diagram showing the effect of using the composition of the present invention to inhibit tumor growth in NOD-SCID mice in vivo, provided in an embodiment of the present invention, wherein A is a picture of mouse tumor, B is mouse tumor weight, and C is mouse body weight.
图6为本发明实施例提供的采用本发明组合物在NOD-SCID小鼠体内考察延长异种移植小鼠生存期图。FIG6 is a graph showing the use of the composition of the present invention in an embodiment of the present invention to investigate the extension of the survival period of xenografted mice in NOD-SCID mice.
图7为本发明实施例提供的采用本发明组合物在THP-1和MOLM-13细胞中考察诱导促凋亡Noxa,下调抗凋亡Mcl-1,抑制复制应激检查点激酶p-Chk1,诱导DNA损伤蛋白□-H2A.X蛋白效果图。。FIG7 is a graph showing the effects of using the composition of the present invention in THP-1 and MOLM-13 cells to induce pro-apoptotic Noxa, downregulate anti-apoptotic Mcl-1, inhibit replication stress checkpoint kinase p-Chk1, and induce DNA damage protein □-H2A.X.
具体实施方式DETAILED DESCRIPTION
下面结合实施例进一步说明本发明的实质性内容，但并不以此限定本发明保护范围。The essential contents of the present invention are further illustrated below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.
本申请实施例中所用的细胞和药品如下：The cells and drugs used in the examples of this application are as follows:
细胞：人急性髓细胞性白血病细胞THP-1购自American Type CultureCollection(ATCC)。人急性髓细胞性白血病细胞Molm-13购自DSMZ-Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH(Braunschweig Germany)。药品：以下实施例中所用药物组合物均按下列方法所述来制备；青蒿琥酯购自Sigma，储存浓度10mM；venetoclax购自Selleck,储存浓度10mM；阿糖胞苷购自MedChemExpress，储存浓度20mM。Cells: Human acute myeloid leukemia THP-1 cells were purchased from American Type Culture Collection (ATCC). Human acute myeloid leukemia Molm-13 cells were purchased from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig Germany). Drugs: The pharmaceutical compositions used in the following examples were prepared according to the following methods; artesunate was purchased from Sigma, with a storage concentration of 10 mM; venetoclax was purchased from Selleck, with a storage concentration of 10 mM; cytarabine was purchased from MedChemExpress, with a storage concentration of 20 mM.
实施例1Example 1
试剂和方法：Reagents and methods:
准确称量青蒿琥酯和ABT-199，分别溶解于二甲基亚砜,各自配成10mM的贮存液,在-20℃下保存,使用时用乙醇稀释到适当的浓度。准确称量阿糖胞苷，溶于0.22μm滤膜过滤的PBS；而后再将青蒿琥酯、venetoclax和阿糖胞苷使用时用新鲜的培养基(1640培养基)稀释到合适的浓度。所有实验中，严格控制二甲基亚砜和乙醇的终浓度，二甲基亚砜终浓度不超过1‰，乙醇终浓度不超过1％，不影响细胞活性。Accurately weigh artesunate and ABT-199, dissolve them in dimethyl sulfoxide, prepare 10mM stock solutions, store them at -20℃, and dilute them with ethanol to the appropriate concentration when using. Accurately weigh cytarabine, dissolve it in PBS filtered by 0.22μm filter membrane; then dilute artesunate, venetoclax and cytarabine to the appropriate concentration with fresh culture medium (1640 culture medium) when using. In all experiments, the final concentrations of dimethyl sulfoxide and ethanol were strictly controlled, the final concentration of dimethyl sulfoxide did not exceed 1‰, and the final concentration of ethanol did not exceed 1%, which did not affect cell activity.
将THP-1细胞和Molm-13细胞培养于1640培养基，含10％灭活的胎牛血清，10mmol/L L-glutamine,100U/mL青霉素,and 100μg/mL链霉素，37℃、5％CO2饱和湿度条件下培养。THP-1 cells and Molm-13 cells were cultured in 1640 medium containing 10% inactivated fetal bovine serum, 10 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO2 in a saturated humidity environment.
将对数生长期细胞，按105密度接种于24孔板中，向细胞中加入不同的药物浓度处理(参见表1-6)。72h后，通过台盼蓝染色法观察死亡细胞。将混匀的细胞按1:1(v/v)与台盼蓝染料混合，染色后，用光学显微镜及血球计数板计数正常细胞和蓝染死细胞(参见图1)。依照如下公式计算细胞生长抑制率(GI)，并求得半数生长抑制浓度(GI50：使细胞生长抑制率达50％时的药物浓度)。Cells in logarithmic growth phase were seeded in 24-well plates at a density of 105 , and different drug concentrations were added to the cells for treatment (see Table 1-6). After 72 hours, dead cells were observed by trypan blue staining. The mixed cells were mixed with trypan blue dye at a ratio of 1:1 (v/v). After staining, normal cells and blue-stained dead cells were counted using an optical microscope and a blood cell counting plate (see Figure 1). The cell growth inhibition rate (GI) was calculated according to the following formula, and the half-maximal growth inhibition concentration (GI50 : the drug concentration that makes the cell growth inhibition rate reach 50%) was obtained.
GI＝(对照孔细胞数-加药孔细胞数)/对照孔细胞数×100％GI = (number of cells in control well - number of cells in drug-added well) / number of cells in control well × 100%
用Compusyn软件计算药物组合物的协同指数(CI)。The synergy index (CI) of the drug combination was calculated using Compusyn software.
表1不同浓度的青蒿琥酯和venetoclax的组合协同抑制THP-1细胞生长实验Table 1 Synergistic inhibition of THP-1 cell growth by combination of different concentrations of artesunate and venetoclax
由表1可见，急性髓细胞性白血病细胞株THP-1细胞用青蒿琥酯0.2-0.8μM，与venetoclax 0.1-1μM联合处理72h。青蒿琥酯和venetoclax单独用药时，抑制细胞生长比例均＜50％。青蒿琥酯的加入明显增强了venetoclax的抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，联合指数＞1表示两种化合物为拮抗作用，＜1为协同作用。青蒿琥酯与venetoclax的联合指数均＜0.6，显示两个化合物有协同作用。0.8μM的青蒿琥酯与0.1μM venetoclax联合应用，抑制THP-1细胞生长比例达到59％，CI＝0.12，显示强协同抑制细胞生长作用。As can be seen from Table 1, acute myeloid leukemia cell line THP-1 cells were treated with artesunate 0.2-0.8μM and venetoclax 0.1-1μM for 72h. When artesunate and venetoclax were used alone, the cell growth inhibition ratio was <50%. The addition of artesunate significantly enhanced the cell growth inhibition effect of venetoclax. The combination index of the two drugs was calculated using Compusyn software. A combination index of >1 indicates that the two compounds are antagonistic, and <1 indicates a synergistic effect. The combination index of artesunate and venetoclax is <0.6, indicating that the two compounds have a synergistic effect. The combination of 0.8μM artesunate and 0.1μM venetoclax inhibited the growth of THP-1 cells by 59%, CI = 0.12, showing a strong synergistic cell growth inhibition effect.
表2不同浓度的青蒿琥酯和venetoclax的组合协同抑制MOLM-13细胞生长实验Table 2 Synergistic inhibition of MOLM-13 cell growth by combination of different concentrations of artesunate and venetoclax
由表2可见，急性髓细胞性白血病细胞株MOLM-13细胞对青蒿琥酯和venetoclax的敏感性强于THP-1细胞，用青蒿琥酯0.1-0.4μM，与venetoclax0.0025-0.01μM联合处理72h。青蒿琥酯和venetoclax单独用药时，抑制细胞生长比例均＜50％。青蒿琥酯的加入明显增强了venetoclax的抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，青蒿琥酯与venetoclax的联合指数均＜0.2，显示两个化合物有非常强的协同作用。0.2μM的青蒿琥酯与0.01μM venetoclax联合应用，抑制MOLM-13细胞生长比例达到74％，CI＝0.08，显示强协同抑制细胞生长作用。As shown in Table 2, acute myeloid leukemia cell line MOLM-13 cells are more sensitive to artesunate and venetoclax than THP-1 cells. Artesunate 0.1-0.4μM, combined with venetoclax 0.0025-0.01μM, was treated for 72h. When artesunate and venetoclax were used alone, the cell growth inhibition ratio was <50%. The addition of artesunate significantly enhanced the cell growth inhibition effect of venetoclax. The combination index of the two drugs was calculated using Compusyn software. The combination index of artesunate and venetoclax was <0.2, indicating that the two compounds had a very strong synergistic effect. The combined application of 0.2μM artesunate and 0.01μM venetoclax inhibited the growth of MOLM-13 cells by 74%, CI = 0.08, showing a strong synergistic cell growth inhibition effect.
根据试验发现，青蒿琥酯与venetoclax通过协同诱导细胞发生凋亡，发挥协同杀伤AML细胞的作用，但在体内动物实验水平作用有限，因而需要寻找新的联合方案。According to experimental findings, artesunate and venetoclax synergistically kill AML cells by inducing cell apoptosis, but their effects are limited at the in vivo animal experiment level, so there is a need to find new combination regimens.
表3不同浓度的青蒿琥酯和阿糖胞苷的组合联合抑制THP-1细胞生长实验Table 3 Experiment on the combined inhibition of THP-1 cell growth by different concentrations of artesunate and cytarabine
由表3可见，THP-1细胞用青蒿琥酯0.2-0.8μM，与阿糖胞苷0.25-1μM联合处理72h。青蒿琥酯和阿糖胞苷单独用药时，抑制细胞生长比例均＜50％。青蒿琥酯不能明显增强阿糖胞苷抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，联合指数＞1，显示两个化合物有拮抗作用。0.8μM的青蒿琥酯与0.5μM venetoclax联合应用，抑制THP-1细胞生长比例为49％，CI＝1.23，显示拮抗作用。As shown in Table 3, THP-1 cells were treated with artesunate 0.2-0.8μM and cytarabine 0.25-1μM for 72h. When artesunate and cytarabine were used alone, the cell growth inhibition ratio was <50%. Artesunate could not significantly enhance the cell growth inhibition effect of cytarabine. The combination index of the two drugs was calculated by Compusyn software. The combination index was >1, indicating that the two compounds had an antagonistic effect. 0.8μM artesunate was combined with 0.5μM venetoclax to inhibit THP-1 cell growth by 49%, CI = 1.23, showing an antagonistic effect.
表4不同浓度的青蒿琥酯和阿糖胞苷的组合联合抑制MOLM-13细胞生长实验Table 4 Experiment on the combined inhibition of MOLM-13 cell growth by different concentrations of artesunate and cytarabine
由表4可见，MOLM-13细胞用青蒿琥酯0.1-0.4μM，与阿糖胞苷0.00125-0.005μM联合处理72h。青蒿琥酯和阿糖胞苷单独用药时，抑制细胞生长比例均＜50％。青蒿琥酯不能明显增强阿糖胞苷抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，联合指数＞0.7，部分剂量下协同指数>1，显示两个化合物没有明显的协同作用。0.2μM的青蒿琥酯与0.005μM阿糖胞苷联合应用，抑制THP-1细胞生长比例为45％，CI＝0.73，显示弱协同抑制细胞生长作用。As shown in Table 4, MOLM-13 cells were treated with artesunate 0.1-0.4μM and cytarabine 0.00125-0.005μM for 72h. When artesunate and cytarabine were used alone, the cell growth inhibition ratio was <50%. Artesunate could not significantly enhance the cell growth inhibition effect of cytarabine. The combination index of the two drugs was calculated by Compusyn software. The combination index was >0.7, and the synergistic index was >1 at some doses, indicating that the two compounds had no obvious synergistic effect. The combination of 0.2μM artesunate and 0.005μM cytarabine inhibited the growth of THP-1 cells by 45%, CI = 0.73, showing a weak synergistic cell growth inhibition effect.
由上述实验数据表明，青蒿琥酯与阿糖胞苷在AML细胞水平不能发挥明显的协同作用。The above experimental data indicate that artesunate and cytarabine cannot exert obvious synergistic effects at the AML cell level.
表5不同浓度的venetoclax和阿糖胞苷的组合协同抑制THP-1细胞生长实验Table 5 Synergistic inhibition of THP-1 cell growth by the combination of different concentrations of venetoclax and cytarabine
由表5可见，THP-1细胞用venetoclax 0.1-1μM，与阿糖胞苷0.25-1μM联合处理72h。Venetoclax和阿糖胞苷单独用药时，抑制细胞生长比例均＜50％。Venetoclax的加入能明显增强阿糖胞苷抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，联合指数<0.6，显示两个化合物有协同抑制细胞生长的作用。0.1μM的venetoclax与0.5μM阿糖胞苷联合应用，抑制THP-1细胞生长比例为50％，CI＝0.52，显示协同抑制细胞生长作用。As can be seen from Table 5, THP-1 cells were treated with venetoclax 0.1-1μM and cytarabine 0.25-1μM for 72h. When venetoclax and cytarabine were used alone, the cell growth inhibition ratio was <50%. The addition of venetoclax can significantly enhance the cell growth inhibition effect of cytarabine. The combination index of the two drugs was calculated using Compusyn software, and the combination index was <0.6, indicating that the two compounds have a synergistic cell growth inhibition effect. The combination of 0.1μM venetoclax and 0.5μM cytarabine inhibited THP-1 cell growth by 50%, CI = 0.52, showing a synergistic cell growth inhibition effect.
表6不同浓度的venetoclax和阿糖胞苷的组合协同抑制MOLM-13细胞生长实验Table 6 Synergistic inhibition of MOLM-13 cell growth by the combination of different concentrations of venetoclax and cytarabine
由表6可见，MOLM-13细胞用venetoclax 0.0025-0.01μM，与阿糖胞苷0.00125-0.005μM联合处理72h。Venetoclax和阿糖胞苷单独用药时，抑制细胞生长比例均＜50％。Venetoclax的加入能明显增强阿糖胞苷抑制细胞生长的作用，用Compusyn软件计算两药的联合指数，联合指数<0.3，显示两个化合物有较强的协同抑制细胞生长的作用。0.01μM的venetoclax与0.005μM阿糖胞苷联合应用，抑制THP-1细胞生长比例为79％，CI＝0.07，显示强协同抑制细胞生长作用。As can be seen from Table 6, MOLM-13 cells were treated with venetoclax 0.0025-0.01μM and cytarabine 0.00125-0.005μM for 72h. When venetoclax and cytarabine were used alone, the cell growth inhibition ratio was <50%. The addition of venetoclax can significantly enhance the cell growth inhibition effect of cytarabine. The combination index of the two drugs was calculated using Compusyn software, and the combination index was <0.3, indicating that the two compounds have a strong synergistic effect of inhibiting cell growth. The combination of 0.01μM venetoclax and 0.005μM cytarabine inhibited the growth of THP-1 cells by 79%, CI = 0.07, showing a strong synergistic cell growth inhibition effect.
虽然阿糖胞苷与venetoclax联合方案已经被批准应用于老年AML患者的治疗，但临床治疗作用有限，因而仍需要寻找新的联合方案。Although the combination of cytarabine and venetoclax has been approved for the treatment of elderly AML patients, its clinical therapeutic effect is limited, so there is still a need to find new combination regimens.
在两种AML细胞株中，青蒿琥酯与venetoclax联合和venetoclax与阿糖胞苷联合能协同抑制AML细胞生长，青蒿琥酯与阿糖胞苷联合不能发挥明显的协同作用。根据以上试验，进一步采用青蒿琥酯与venetoclax和阿糖胞苷三药浓度进行联合，考察对AML细胞的杀伤作用。In two AML cell lines, the combination of artesunate and venetoclax and venetoclax and cytarabine can synergistically inhibit the growth of AML cells, while the combination of artesunate and cytarabine cannot exert obvious synergistic effects. Based on the above experiments, artesunate, venetoclax and cytarabine were further combined at three drug concentrations to examine the killing effect on AML cells.
而后根据上述单独物质以及两两组合的效果设置各实验组，并按照上述方式对AML细胞的生长抑制进行检测，参见图1。Then, experimental groups were set up according to the effects of the above-mentioned single substances and the combinations of two of them, and the growth inhibition of AML cells was detected in the above-mentioned manner, see Figure 1.
对于THP-1细胞各实验分组如下：(1)空白对照组；(2)青蒿琥酯组(0.8μM)；(3)Venetoclax组(0.1μM)；(4)阿糖胞苷组(0.5μM)；(5)青蒿琥酯(0.8μM)+venetoclax(0.1μM)组；(6)青蒿琥酯(0.8μM)+阿糖胞苷(0.5μM)组；(7)Venetoclax(0.1μM)+阿糖胞苷(0.5μM)组；(8)青蒿琥酯(0.8μM)+venetoclax(0.1μM)+阿糖胞苷(0.5μM)组。The experimental groups for THP-1 cells are as follows: (1) blank control group; (2) artesunate group (0.8 μM); (3) venetoclax group (0.1 μM); (4) cytarabine group (0.5 μM); (5) artesunate (0.8 μM) + venetoclax (0.1 μM) group; (6) artesunate (0.8 μM) + cytarabine (0.5 μM) group; (7) venetoclax (0.1 μM) + cytarabine (0.5 μM) group; (8) artesunate (0.8 μM) + venetoclax (0.1 μM) + cytarabine (0.5 μM) group.
对于MOLM-13细胞各实验分组如下：(1)空白对照组；(2)青蒿琥酯组(0.2μM)；(3)Venetoclax组(0.01μM)；(4)阿糖胞苷组(0.005μM)；(5)青蒿琥酯(0.2μM)+venetoclax(0.01μM)组；(6)青蒿琥酯(0.2μM)+阿糖胞苷(0.005μM)组；(7)Venetoclax(0.01μM)+阿糖胞苷(0.005μM)组；(8)青蒿琥酯(0.2μM)+venetoclax(0.01μM)+阿糖胞苷(0.005μM)组。The experimental groups for MOLM-13 cells are as follows: (1) blank control group; (2) artesunate group (0.2 μM); (3) venetoclax group (0.01 μM); (4) cytarabine group (0.005 μM); (5) artesunate (0.2 μM) + venetoclax (0.01 μM) group; (6) artesunate (0.2 μM) + cytarabine (0.005 μM) group; (7) venetoclax (0.01 μM) + cytarabine (0.005 μM) group; (8) artesunate (0.2 μM) + venetoclax (0.01 μM) + cytarabine (0.005 μM) group.
由图1可见，在THP-1细胞中不同实验组对AML细胞的生长抑制作用，青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组能抑制70％和59％的细胞生长，活性好于任意单独用药组(青蒿琥酯25％，venetoclax31％，阿糖胞苷38％)，青蒿琥酯与阿糖胞苷联合抑制44％的细胞生长，没有明显增强阿糖胞苷或青蒿琥酯的生长抑制作用，而青蒿琥酯+venetoclax+阿糖胞苷组能抑制85％的细胞生长,活性好于任意两药联合组。As can be seen from Figure 1, in THP-1 cells, different experimental groups had an inhibitory effect on the growth of AML cells. The artesunate + venetoclax group and the venetoclax + cytarabine group could inhibit 70% and 59% of cell growth, respectively, which were better than any single drug group (artesunate 25%, venetoclax 31%, cytarabine 38%). The combination of artesunate and cytarabine inhibited 44% of cell growth, without significantly enhancing the growth inhibitory effect of cytarabine or artesunate. The artesunate + venetoclax + cytarabine group could inhibit 85% of cell growth, which was better than any combination of the two drugs.
由于MOLM-13细胞对青蒿琥酯、venetoclax和阿糖胞苷比THP-1细胞更敏感，各组效果分别为青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组能抑制71％和70％的细胞生长，活性好于任意单独用药组(青蒿琥酯17％，venetoclax 38％，阿糖胞苷26％)，青蒿琥酯与阿糖胞苷联合抑制35％的细胞生长，没有明显增强阿糖胞苷或青蒿琥酯的生长抑制作用，青蒿琥酯+venetoclax+阿糖胞苷组能抑制92％的细胞生长,活性好于任意两药联合组。提示青蒿琥酯能够明显增强venetoclax与阿糖胞苷联合方案对AML细胞的生长抑制作用。Since MOLM-13 cells are more sensitive to artesunate, venetoclax and cytarabine than THP-1 cells, the effects of each group are artesunate + venetoclax group and venetoclax + cytarabine group, which can inhibit 71% and 70% of cell growth, respectively, and the activity is better than any single drug group (artesunate 17%, venetoclax 38%, cytarabine 26%). Artesunate combined with cytarabine inhibits 35% of cell growth, and does not significantly enhance the growth inhibitory effect of cytarabine or artesunate. Artesunate + venetoclax + cytarabine group can inhibit 92% of cell growth, and the activity is better than any two-drug combination group. It suggests that artesunate can significantly enhance the growth inhibitory effect of venetoclax and cytarabine combination on AML cells.
实施例2Example 2
取对数生长期的THP-1细胞和Molm-13细胞，分别按105密度接种于6孔板中，再向不同THP-1和MOLM-13细胞中加入不同的药物单独或联合处理24h或12h。THP-1细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.8μM)；(3)Venetoclax组(0.1μM)；(4)阿糖胞苷组(8μM)；(5)青蒿琥酯(0.8μM)+venetoclax(0.1μM)组；(6)青蒿琥酯(0.8μM)+阿糖胞苷(8μM)组；(7)Venetoclax(0.1μM)+阿糖胞苷(8μM)组；(8)青蒿琥酯(0.8μM)+venetoclax(0.1μM)+阿糖胞苷(8μM)组。THP-1 cells and Molm-13 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 105 , and different drugs were added to different THP-1 and MOLM-13 cells for treatment alone or in combination for 24 h or 12 h. The THP-1 cell experimental groups were as follows: (1) blank control group; (2) artesunate group (0.8 μM); (3) venetoclax group (0.1 μM); (4) cytarabine group (8 μM); (5) artesunate (0.8 μM) + venetoclax (0.1 μM) group; (6) artesunate (0.8 μM) + cytarabine (8 μM) group; (7) venetoclax (0.1 μM) + cytarabine (8 μM) group; (8) artesunate (0.8 μM) + venetoclax (0.1 μM) + cytarabine (8 μM) group.
MOLM-13细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.2μM)；(3)Venetoclax组(0.01μM)；(4)阿糖胞苷组(0.08μM)；(5)青蒿琥酯(0.2μM)+venetoclax(0.01μM)组；(6)青蒿琥酯(0.2μM)+阿糖胞苷(0.08μM)组；(7)Venetoclax(0.01μM)+阿糖胞苷(0.08μM)组；(8)青蒿琥酯(0.2μM)+venetoclax(0.01μM)+阿糖胞苷(0.08μM)组。采用AV-PI试剂盒检测细胞凋亡情况(参见图2)；The MOLM-13 cell experimental groups are as follows: (1) blank control group; (2) artesunate group (0.2μM); (3) venetoclax group (0.01μM); (4) cytarabine group (0.08μM); (5) artesunate (0.2μM) + venetoclax (0.01μM) group; (6) artesunate (0.2μM) + cytarabine (0.08μM) group; (7) venetoclax (0.01μM) + cytarabine (0.08μM) group; (8) artesunate (0.2μM) + venetoclax (0.01μM) + cytarabine (0.08μM) group. The AV-PI kit was used to detect cell apoptosis (see Figure 2);
结果如图所示，在THP-1细胞中，青蒿琥酯+venetoclax组能诱导28％的细胞发生凋亡，活性好于任意单独用药组(青蒿琥酯4％，venetoclax 5％)，青蒿琥酯+阿糖胞苷(8％)和venetoclax+阿糖胞苷组(9％)不能明显诱导细胞发生凋亡，青蒿琥酯+venetoclax+阿糖胞苷组能诱导38％的细胞发生凋亡,活性好于任意两药联合组。在MOLM-13细胞中，venetoclax单独能诱导25％的细胞发生凋亡，与青蒿琥酯或阿糖胞苷联合凋亡率增加至43％或45％，活性好于任意单独用药组(青蒿琥酯11％，venetoclax 25％，阿糖胞苷14％)，青蒿琥酯与阿糖胞苷联合能够诱导15％的细胞凋亡，没有明显增强阿糖胞苷或青蒿琥酯的凋亡诱导作用，青蒿琥酯+venetoclax+阿糖胞苷组能诱导59％的细胞发生凋亡,活性好于任意两药联合组。提示青蒿琥酯能够明显增强venetoclax与阿糖胞苷联合方案对AML细胞的凋亡诱导作用。The results are shown in the figure. In THP-1 cells, the artesunate + venetoclax group can induce 28% of cells to undergo apoptosis, and its activity is better than any single drug group (artesunate 4%, venetoclax 5%). Artesunate + cytarabine (8%) and venetoclax + cytarabine groups (9%) cannot significantly induce cell apoptosis. Artesunate + venetoclax + cytarabine group can induce 38% of cells to undergo apoptosis, and its activity is better than any combination of the two drugs. In MOLM-13 cells, venetoclax alone can induce apoptosis in 25% of cells, and the apoptosis rate increased to 43% or 45% when combined with artesunate or cytarabine, which is better than any single drug group (artesunate 11%, venetoclax 25%, cytarabine 14%). Artesunate combined with cytarabine can induce apoptosis in 15% of cells, without significantly enhancing the apoptosis-inducing effect of cytarabine or artesunate. Artesunate + venetoclax + cytarabine group can induce apoptosis in 59% of cells, which is better than any two-drug combination group. This suggests that artesunate can significantly enhance the apoptosis-inducing effect of venetoclax and cytarabine combination on AML cells.
实施例3Example 3
取对数生长期的THP-1细胞和Molm-13细胞，分别按105密度接种于6孔板中，向THP-1和MOLM-13细胞中加入不同的药物单独或联合处理24h或12h。THP-1细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.8μM)；(3)Venetoclax组(0.1μM)；(4)阿糖胞苷组(8μM)；(5)青蒿琥酯(0.8μM)+venetoclax(0.1μM)组；(6)青蒿琥酯(0.8μM)+阿糖胞苷(8μM)组；(7)Venetoclax(0.1μM)+阿糖胞苷(8μM)组；(8)青蒿琥酯(0.8μM)+venetoclax(0.1μM)+阿糖胞苷(8μM)组。THP-1 cells and Molm-13 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 105 , and different drugs were added to THP-1 and MOLM-13 cells for treatment alone or in combination for 24 h or 12 h. The THP-1 cells were divided into the following experimental groups: (1) blank control group; (2) artesunate group (0.8 μM); (3) venetoclax group (0.1 μM); (4) cytarabine group (8 μM); (5) artesunate (0.8 μM) + venetoclax (0.1 μM) group; (6) artesunate (0.8 μM) + cytarabine (8 μM) group; (7) venetoclax (0.1 μM) + cytarabine (8 μM) group; (8) artesunate (0.8 μM) + venetoclax (0.1 μM) + cytarabine (8 μM) group.
MOLM-13细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.2μM)；(3)Venetoclax组(0.01μM)；(4)阿糖胞苷组(0.08μM)；(5)青蒿琥酯(0.2μM)+venetoclax(0.01μM)组；(6)青蒿琥酯(0.2μM)+阿糖胞苷(0.08μM)组；(7)Venetoclax(0.01μM)+阿糖胞苷(0.08μM)组；(8)青蒿琥酯(0.2μM)+venetoclax(0.01μM)+阿糖胞苷(0.08μM)组。采用PI单染法检测细胞周期变化(参见图3)。The MOLM-13 cell experimental groups are as follows: (1) blank control group; (2) artesunate group (0.2 μM); (3) venetoclax group (0.01 μM); (4) cytarabine group (0.08 μM); (5) artesunate (0.2 μM) + venetoclax (0.01 μM) group; (6) artesunate (0.2 μM) + cytarabine (0.08 μM) group; (7) venetoclax (0.01 μM) + cytarabine (0.08 μM) group; (8) artesunate (0.2 μM) + venetoclax (0.01 μM) + cytarabine (0.08 μM) group. PI single staining method was used to detect cell cycle changes (see Figure 3).
结果如图所示，在THP-1细胞中，青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组能诱导26％和19％的Sub-G1期细胞增加，活性好于任意单独用药组(青蒿琥酯4％，venetoclax 4％，阿糖胞苷8％)，青蒿琥酯+阿糖胞苷组不能明显诱导Sub-G1期细胞比例增加，青蒿琥酯+venetoclax+阿糖胞苷组能诱导32％的Sub-G1期细胞比例增加,活性好于任意两药联合组。在MOLM-13细胞中，venetoclax单独能诱导33％的Sub-G1期细胞比例增加，与青蒿琥酯和阿糖胞苷联合Sub-G1期细胞比例增加至45％和40％，活性好于任意单独用药组(青蒿琥酯9％，venetoclax 33％，阿糖胞苷11％)，青蒿琥酯与阿糖胞苷联合能够诱导14％的Sub-G1期细胞比例增加，没有明显增强阿糖胞苷或青蒿琥酯的作用，青蒿琥酯+venetoclax+阿糖胞苷组能诱导51％的Sub-G1期细胞比例增加,活性好于任意两药联合组。提示青蒿琥酯能够明显增强venetoclax与阿糖胞苷联合诱导AML细胞发生DNA断裂的作用。以上结果证实青蒿琥酯能够明显增强venetoclax与阿糖胞苷联合对AML细胞的杀伤作用。The results are shown in the figure. In THP-1 cells, the artesunate + venetoclax group and the venetoclax + cytarabine group can induce an increase of 26% and 19% of Sub-G1 phase cells, and the activity is better than any single drug group (artesunate 4%, venetoclax 4%, cytarabine 8%). The artesunate + cytarabine group cannot significantly induce an increase in the proportion of Sub-G1 phase cells. The artesunate + venetoclax + cytarabine group can induce an increase of 32% of Sub-G1 phase cells, and the activity is better than any two-drug combination group. In MOLM-13 cells, venetoclax alone can induce a 33% increase in the proportion of Sub-G1 cells, and combined with artesunate and cytarabine, the proportion of Sub-G1 cells increased to 45% and 40%, which is better than any single drug group (artesunate 9%, venetoclax 33%, cytarabine 11%). Artesunate combined with cytarabine can induce a 14% increase in the proportion of Sub-G1 cells, without significantly enhancing the effect of cytarabine or artesunate. The artesunate + venetoclax + cytarabine group can induce a 51% increase in the proportion of Sub-G1 cells, which is better than any two-drug combination group. This suggests that artesunate can significantly enhance the effect of venetoclax combined with cytarabine on inducing DNA breaks in AML cells. The above results confirm that artesunate can significantly enhance the killing effect of venetoclax combined with cytarabine on AML cells.
实施例4Example 4
在THP-1和MOLM-13细胞中采用软琼脂集落形成试验考察对单细胞增殖潜力的影响。The effect on the proliferation potential of single cells was investigated using soft agar colony formation assay in THP-1 and MOLM-13 cells.
取受试药物20μL混入软琼脂底层，分别取对数生长期的THP-1细胞和Molm-13细胞各5000个混入软琼脂上层，14天后在解刨显微镜下计数直径大于75μm(50个细胞以上)的集落(参见图4)。20 μL of the test drug was mixed into the soft agar bottom layer, and 5000 THP-1 cells and Molm-13 cells in the logarithmic growth phase were mixed into the soft agar upper layer. After 14 days, colonies with a diameter greater than 75 μm (more than 50 cells) were counted under a dissecting microscope (see Figure 4).
THP-1细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.8μM)；(3)Venetoclax组(0.1μM)；(4)阿糖胞苷组(0.5μM)；(5)青蒿琥酯(0.8μM)+venetoclax(0.1μM)组；(6)青蒿琥酯(0.8μM)+阿糖胞苷(0.5μM)组；(7)Venetoclax(0.1μM)+阿糖胞苷(0.5μM)组；(8)青蒿琥酯(0.8μM)+venetoclax(0.1μM)+阿糖胞苷(0.5μM)组。The THP-1 cell experimental groups were as follows: (1) blank control group; (2) artesunate group (0.8 μM); (3) venetoclax group (0.1 μM); (4) cytarabine group (0.5 μM); (5) artesunate (0.8 μM) + venetoclax (0.1 μM) group; (6) artesunate (0.8 μM) + cytarabine (0.5 μM) group; (7) venetoclax (0.1 μM) + cytarabine (0.5 μM) group; (8) artesunate (0.8 μM) + venetoclax (0.1 μM) + cytarabine (0.5 μM) group.
MOLM-13细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.2μM)；(3)Venetoclax组(0.01μM)；(4)阿糖胞苷组(0.005μM)；(5)青蒿琥酯(0.2μM)+venetoclax(0.01μM)组；(6)青蒿琥酯(0.2μM)+阿糖胞苷(0.005μM)组；(7)Venetoclax(0.01μM)+阿糖胞苷(0.005μM)组；(8)青蒿琥酯(0.2μM)+venetoclax(0.01μM)+阿糖胞苷(0.005μM)组。实验结果如图所示，青蒿琥酯单独应用不能抑制集落形成；Venetoclax和阿糖胞苷单独应用抑制不超过30％的集落形成，抑制作用有限；青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组约抑制70％的集落形成，作用好于任意单独用药组；青蒿琥酯与阿糖胞苷联合约抑制50％的集落形成，没有明显增强阿糖胞苷对集落形成的抑制作用；青蒿琥酯+venetoclax+阿糖胞苷组约抑制90％的集落形成，作用好于任意两药联合组。The MOLM-13 cell experimental groups were as follows: (1) blank control group; (2) artesunate group (0.2 μM); (3) venetoclax group (0.01 μM); (4) cytarabine group (0.005 μM); (5) artesunate (0.2 μM) + venetoclax (0.01 μM) group; (6) artesunate (0.2 μM) + cytarabine (0.005 μM) group; (7) venetoclax (0.01 μM) + cytarabine (0.005 μM) group; (8) artesunate (0.2 μM) + venetoclax (0.01 μM) + cytarabine (0.005 μM) group. The experimental results are shown in the figure. Artesunate alone cannot inhibit colony formation; Venetoclax and cytarabine alone inhibit no more than 30% of colony formation, and the inhibitory effect is limited; the artesunate + venetoclax group and the venetoclax + cytarabine group inhibited about 70% of colony formation, which was better than any single drug group; artesunate combined with cytarabine inhibited about 50% of colony formation, and did not significantly enhance the inhibitory effect of cytarabine on colony formation; artesunate + venetoclax + cytarabine group inhibited about 90% of colony formation, which was better than any two-drug combination group.
实施例5Example 5
为考察青蒿琥酯与venetoclax和阿糖胞苷联合的体内抗AML作用：To investigate the in vivo anti-AML effect of artesunate in combination with venetoclax and cytarabine:
40只雌性NOD-SCID小鼠右侧腋皮下接种MOLM-13细胞5×106个/只。当肿瘤体积达到100mm2左右，随机分为8组，(1)空白对照组；(2)青蒿琥酯组(100mg/kg)；(3)Venetoclax组(100mg/kg)；(4)阿糖胞苷组(50mg/kg)；(5)青蒿琥酯(100mg/kg)+venetoclax(100mg/kg)组；(6)青蒿琥酯(100mg/kg)+阿糖胞苷(50mg/kg)组；(7)Venetoclax(100mg/kg)+阿糖胞苷(50mg/kg)组；(8)青蒿琥酯(100mg/kg)+venetoclax(100mg/kg)+阿糖胞苷(50mg/kg)组。青蒿琥酯溶于10％(5％NaHCO3)+90％生理盐水，腹腔注射给药，每天给药1次，连续给药10天；Venetoclax溶于10％乙醇+30％PEG 400+60％Phosal 50PG，口服给药，每天给药1次，连续给药10天；阿糖胞苷溶于生理盐水，腹腔注射给药，每天给药1次，连续给药10天。40 female NOD-SCID mice were subcutaneously inoculated with 5×106 MOLM-13 cells/mouse in the right axilla. When the tumor volume reached about 100 mm2 , they were randomly divided into 8 groups: (1) blank control group; (2) artesunate group (100 mg/kg); (3) venetoclax group (100 mg/kg); (4) cytarabine group (50 mg/kg); (5) artesunate (100 mg/kg) + venetoclax (100 mg/kg) group; (6) artesunate (100 mg/kg) + cytarabine (50 mg/kg) group; (7) venetoclax (100 mg/kg) + cytarabine (50 mg/kg) group; (8) artesunate (100 mg/kg) + venetoclax (100 mg/kg) + cytarabine (50 mg/kg) group. Artesunate was dissolved in 10% (5% NaHCO3 ) + 90% saline and administered by intraperitoneal injection once a day for 10 consecutive days; Venetoclax was dissolved in 10% ethanol + 30% PEG 400 + 60% Phosal 50PG and administered orally once a day for 10 consecutive days; cytarabine was dissolved in saline and administered by intraperitoneal injection once a day for 10 consecutive days.
由图5可见，青蒿琥酯组抑瘤作用相对较弱，抑瘤率为22.9％。Venetoclax和阿糖胞苷能够有效抑制肿瘤生长，抑瘤率分别为64.2％和44.8％。青蒿琥酯+阿糖胞苷组的抑瘤率为44.8％，与阿糖胞苷组抑瘤率相同，提示青蒿琥酯不能增强阿糖胞苷的抑瘤作用。青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组的抑瘤率分别为68.2％和75％，均没有明显增强venetoclax的抑瘤作用。青蒿琥酯+venetoclax+阿糖胞苷组抑瘤率为92％，抑瘤作用优于任意两药联合组。青蒿琥酯+venetoclax+阿糖胞苷组小鼠体重在试验过程中没有明显改变。As can be seen from Figure 5, the tumor inhibition effect of the artesunate group is relatively weak, with a tumor inhibition rate of 22.9%. Venetoclax and cytarabine can effectively inhibit tumor growth, with tumor inhibition rates of 64.2% and 44.8%, respectively. The tumor inhibition rate of the artesunate + cytarabine group was 44.8%, which was the same as the tumor inhibition rate of the cytarabine group, indicating that artesunate could not enhance the tumor inhibition effect of cytarabine. The tumor inhibition rates of the artesunate + venetoclax group and the venetoclax + cytarabine group were 68.2% and 75%, respectively, and neither significantly enhanced the tumor inhibition effect of venetoclax. The tumor inhibition rate of the artesunate + venetoclax + cytarabine group was 92%, and the tumor inhibition effect was better than that of any two-drug combination group. The body weight of mice in the artesunate + venetoclax + cytarabine group did not change significantly during the experiment.
实施例6Example 6
为模拟真实AML模型，将35只雌性NOD-SCID小鼠经尾静脉接种MOLM-13细胞2×106个/只。三天后，将小鼠按体重随机分为5组，分别为(1)空白对照组；(2)Venetoclax(100mg/kg)组；(3)青蒿琥酯(100mg/kg)+venetoclax(100mg/kg)组；(4)Venetoclax(100mg/kg)+阿糖胞苷(50mg/kg)组；(5)青蒿琥酯(100mg/kg)+venetoclax(100mg/kg)+阿糖胞苷(50mg/kg)组。青蒿琥酯溶于10％(5％NaHCO3)+90％生理盐水，腹腔注射给药；Venetoclax溶于10％乙醇+30％PEG 400+60％Phosal50PG，口服给药；阿糖胞苷溶于生理盐水，腹腔注射给药。青蒿琥酯和venetoclax每周给药5天，连续给药4周；阿糖胞苷连续给药7天。记录小鼠的体重和存活时间，用Kaplan-Meier法绘制小鼠的存活曲线。THP-1细胞异种移植模型及给药方案与MOLM-13细胞方案相同(参见图6)。To simulate the real AML model, 35 female NOD-SCID mice were inoculated with 2×106 MOLM-13 cells/mouse via the tail vein. Three days later, the mice were randomly divided into 5 groups according to body weight, namely (1) blank control group; (2) venetoclax (100 mg/kg) group; (3) artesunate (100 mg/kg) + venetoclax (100 mg/kg) group; (4) venetoclax (100 mg/kg) + cytarabine (50 mg/kg) group; (5) artesunate (100 mg/kg) + venetoclax (100 mg/kg) + cytarabine (50 mg/kg) group. Artesunate was dissolved in 10% (5% NaHCO3 ) + 90% saline and administered by intraperitoneal injection; Venetoclax was dissolved in 10% ethanol + 30% PEG 400 + 60% Phosal50PG and administered orally; Cytarabine was dissolved in saline and administered by intraperitoneal injection. Artesunate and venetoclax were administered 5 days a week for 4 consecutive weeks; Cytarabine was administered for 7 consecutive days. The body weight and survival time of mice were recorded, and the survival curve of mice was drawn by Kaplan-Meier method. The THP-1 cell xenograft model and administration regimen were the same as those of MOLM-13 cells (see Figure 6).
由图6可见，Venetoclax组、青蒿琥酯+venetoclax组、venetoclax+阿糖胞苷组和青蒿琥酯+venetoclax+阿糖胞苷组与对照组相比均显著延长异种移植小鼠生存期，青蒿琥酯+venetoclax+阿糖胞苷组相比venetoclax组、青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组生存期进一步延长，ILS％达118.8％。在尾静脉异种移植THP-1细胞模型中，青蒿琥酯+venetoclax组、venetoclax+阿糖胞苷组和青蒿琥酯+venetoclax+阿糖胞苷组与对照组相比均显著延长异种移植小鼠生存期，青蒿琥酯+venetoclax+阿糖胞苷组相比venetoclax组、青蒿琥酯+venetoclax组和venetoclax+阿糖胞苷组生存期进一步延长，ILS％达45.1％。以上结果证实，在体内青蒿琥酯能够有效增强venetoclax和阿糖胞苷联合方案的抗AML作用。As can be seen from Figure 6, the Venetoclax group, artesunate + venetoclax group, venetoclax + cytarabine group and artesunate + venetoclax + cytarabine group all significantly prolonged the survival of xenotransplanted mice compared with the control group. The survival of the artesunate + venetoclax + cytarabine group was further prolonged compared with the venetoclax group, artesunate + venetoclax group and venetoclax + cytarabine group, and the ILS% reached 118.8%. In the tail vein xenograft THP-1 cell model, the artesunate + venetoclax group, venetoclax + cytarabine group, and artesunate + venetoclax + cytarabine group significantly prolonged the survival of xenograft mice compared with the control group. The artesunate + venetoclax + cytarabine group had a further prolonged survival compared with the venetoclax group, artesunate + venetoclax group, and venetoclax + cytarabine group, with an ILS% of 45.1%. The above results confirm that artesunate can effectively enhance the anti-AML effect of the venetoclax and cytarabine combination regimen in vivo.
实施例7Example 7
取对数生长期的THP-1细胞和Molm-13细胞，分别按105密度接种于6孔板中，向THP-1和MOLM-13细胞中加入不同的药物单独或联合处理24h或12h，THP-1细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.8μM)；(3)Venetoclax组(0.1μM)；(4)阿糖胞苷组(8μM)；(5)青蒿琥酯(0.8μM)+venetoclax(0.1μM)组；(6)青蒿琥酯(0.8μM)+阿糖胞苷(8μM)组；(7)Venetoclax(0.1μM)+阿糖胞苷(8μM)组；(8)青蒿琥酯(0.8μM)+venetoclax(0.1μM)+阿糖胞苷(8μM)组。MOLM-13细胞实验分组如下(1)空白对照组；(2)青蒿琥酯组(0.2μM)；(3)Venetoclax组(0.01μM)；(4)阿糖胞苷组(0.08μM)；(5)青蒿琥酯(0.2μM)+venetoclax(0.01μM)组；(6)青蒿琥酯(0.2μM)+阿糖胞苷(0.08μM)组；(7)Venetoclax(0.01μM)+阿糖胞苷(0.08μM)组；(8)青蒿琥酯(0.2μM)+venetoclax(0.01μM)+阿糖胞苷(0.08μM)组。采用western-blot技术对青蒿琥酯+venetoclax+阿糖胞苷三药联合进行机制研究，在THP-1和MOLM-13细胞中考察了凋亡相关蛋白、DNA损伤蛋白和复制应激检查点激酶(参见图7)。THP-1 cells and Molm-13 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 105 , respectively. Different drugs were added to THP-1 and MOLM-13 cells for treatment alone or in combination for 24 h or 12 h. The THP-1 cells were divided into the following experimental groups: (1) blank control group; (2) artesunate group (0.8 μM); (3) venetoclax group (0.1 μM); (4) cytarabine group (8 μM); (5) artesunate (0.8 μM) + venetoclax (0.1 μM) group; (6) artesunate (0.8 μM) + cytarabine (8 μM) group; (7) venetoclax (0.1 μM) + cytarabine (8 μM) group; (8) artesunate (0.8 μM) + venetoclax (0.1 μM) + cytarabine (8 μM) group. The MOLM-13 cell experimental groups were as follows: (1) blank control group; (2) artesunate group (0.2 μM); (3) venetoclax group (0.01 μM); (4) cytarabine group (0.08 μM); (5) artesunate (0.2 μM) + venetoclax (0.01 μM) group; (6) artesunate (0.2 μM) + cytarabine (0.08 μM) group; (7) venetoclax (0.01 μM) + cytarabine (0.08 μM) group; (8) artesunate (0.2 μM) + venetoclax (0.01 μM) + cytarabine (0.08 μM) group. Western-blot technology was used to study the mechanism of the triple drug combination of artesunate + venetoclax + cytarabine, and apoptosis-related proteins, DNA damage proteins and replication stress checkpoint kinases were investigated in THP-1 and MOLM-13 cells (see Figure 7).
实验结果如图所示，以青蒿琥酯为基础的联合方案诱导了Noxa的上调，Noxa能够通过与Mcl-1结合发挥间接抑制Mcl-1的作用，而单独Noxa的上调并不足以诱导细胞发生凋亡以及DNA损伤，需同时抑制Bcl-2，Venetoclax与青蒿琥酯联合能协同诱导PARP和caspase-3的裂解以及γ-H2A.X的上调，在青蒿琥酯+venetoclax+阿糖胞苷组中进一步增加，并伴随Mcl-1下调。阿糖胞苷是AML治疗的基石，阿糖胞苷能够掺入DNA，抑制DNA的合成，使细胞发生周期阻滞并诱导DNA损伤发挥抗肿瘤作用，但阿糖胞苷处理会同时诱导复制应激检查点激酶Chk的磷酸化激活，启动DNA损伤修复过程，进而以阿糖胞苷为基础的联合方案诱导p-Chk1的上调，青蒿琥酯和venetoclax联合诱导caspase以依赖的Mcl-1的下调，从而克服了阿糖胞苷诱导的p-Chk1的上调，与阿糖胞苷组、青蒿琥酯+阿糖胞苷组和venetoclax+阿糖胞苷组相比，青蒿琥酯+venetoclax+阿糖胞苷组的p-Chk1和Mcl-1水平更低。以上结果证实，青蒿琥酯通过诱导Noxa上调，增强venetoclax的凋亡诱导作用，同时降低Mcl-1的蛋白水平，进而克服阿糖胞苷诱导的p-Chk1的激活，增强阿糖胞苷DNA损伤作用。The experimental results are shown in the figure. The artesunate-based combination regimen induced the upregulation of Noxa. Noxa can indirectly inhibit Mcl-1 by binding to Mcl-1. However, the upregulation of Noxa alone is not sufficient to induce cell apoptosis and DNA damage, and Bcl-2 must be inhibited at the same time. The combination of venetoclax and artesunate can synergistically induce the cleavage of PARP and caspase-3 and the upregulation of γ-H2A.X, which was further increased in the artesunate + venetoclax + cytarabine group, accompanied by Mcl-1 downregulation. Cytarabine is the cornerstone of AML treatment. Cytarabine can be incorporated into DNA, inhibit DNA synthesis, cause cell cycle arrest and induce DNA damage to exert anti-tumor effects. However, cytarabine treatment will also induce phosphorylation activation of replication stress checkpoint kinase Chk, initiate the DNA damage repair process, and then the cytarabine-based combination regimen induces upregulation of p-Chk1. Artesunate and venetoclax combined induce caspase-dependent Mcl-1 downregulation, thereby overcoming the cytarabine-induced p-Chk1 upregulation. Compared with the cytarabine group, artesunate + cytarabine group and venetoclax + cytarabine group, the artesunate + venetoclax + cytarabine group had lower p-Chk1 and Mcl-1 levels. The above results confirmed that artesunate enhanced the apoptosis-inducing effect of venetoclax by inducing Noxa upregulation, while reducing the protein level of Mcl-1, thereby overcoming the activation of p-Chk1 induced by cytarabine and enhancing the DNA damage effect of cytarabine.
本发明THP-1和MOLM-13细胞的基础抗凋亡蛋白表达水平不同，MOLM-13细胞高表达Bcl-2同时低表达Mcl-1和Bim，对venetoclax和阿糖胞苷敏感；THP-1细胞高表达Bcl-2、Mcl-1和Bim，对venetoclax和阿糖胞苷相对耐受，因此，本发明选择不同浓度的青蒿琥酯、venetoclax和阿糖胞苷处理两种细胞，在两种细胞中均发现三药联合产生了比任意两药联合更强的杀伤AML细胞的作用。The basic anti-apoptotic protein expression levels of THP-1 and MOLM-13 cells of the present invention are different. MOLM-13 cells highly express Bcl-2 while lowly express Mcl-1 and Bim, and are sensitive to venetoclax and cytarabine; THP-1 cells highly express Bcl-2, Mcl-1 and Bim, and are relatively tolerant to venetoclax and cytarabine. Therefore, the present invention selects different concentrations of artesunate, venetoclax and cytarabine to treat the two cells, and it is found in both cells that the combination of the three drugs produces a stronger killing effect on AML cells than the combination of any two drugs.
综上所述，本发明将三药组合联用与单独的青蒿琥酯、venetoclax、阿糖胞苷或任意两种药物组合相比，青蒿琥酯既通过诱导Noxa，协同venetoclax诱导凋亡，又下调Mcl-1的蛋白水平克服了阿糖胞苷的p-Chk1耐受机制，使三药联合产生了比任意两药联合更强的协同杀伤AML细胞的作用。In summary, compared with artesunate, venetoclax, cytarabine or any combination of two drugs alone, artesunate not only induces Noxa and cooperates with venetoclax to induce apoptosis, but also downregulates the protein level of Mcl-1 to overcome the p-Chk1 resistance mechanism of cytarabine, so that the three-drug combination produces a stronger synergistic killing effect on AML cells than the combination of any two drugs.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210085636.5ACN116531395B (en) | 2022-01-25 | 2022-01-25 | Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210085636.5ACN116531395B (en) | 2022-01-25 | 2022-01-25 | Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia |
| Publication Number | Publication Date |
|---|---|
| CN116531395A CN116531395A (en) | 2023-08-04 |
| CN116531395Btrue CN116531395B (en) | 2024-11-05 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210085636.5AActiveCN116531395B (en) | 2022-01-25 | 2022-01-25 | Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia |
| Country | Link |
|---|---|
| CN (1) | CN116531395B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9918972B2 (en)* | 2014-12-23 | 2018-03-20 | The University Of Maryland, Baltimore | Treatment of leukemia with artemisinin derivatives and combinations with other antineoplastic agents |
| WO2016192680A1 (en)* | 2015-06-03 | 2016-12-08 | Triastek, Inc. | Dosage forms and use thereof |
| WO2017214491A1 (en)* | 2016-06-09 | 2017-12-14 | Blinkbio, Inc. | Silanol based therapeutic payloads |
| CN107050014A (en)* | 2017-05-26 | 2017-08-18 | 遵义医学院附属医院 | Artesunate as anti-leukocythemia tumour medicine application |
| US11554105B2 (en)* | 2018-10-31 | 2023-01-17 | University Of Maryland, Baltimore | Combinations of artemisinins, BCL-2 inhibitors, and kinase inhibitors for cancer treatment |
| WO2020171727A2 (en)* | 2019-02-19 | 2020-08-27 | Bluepharma - Industria Farmacêutica, S.A. | Mucoadhesive compositions and uses thereof |
| CN110151775B (en)* | 2019-07-01 | 2022-06-03 | 中国医科大学附属盛京医院 | Application of 17-DMAG in the preparation of drugs for inhibiting childhood acute lymphoblastic leukemia |
| Publication number | Publication date |
|---|---|
| CN116531395A (en) | 2023-08-04 |
| Publication | Publication Date | Title |
|---|---|---|
| KR101891505B1 (en) | Use of anhydroicaritin in preparing medicine for preventing or treating decrease in blood cells | |
| US11524009B2 (en) | Combination comprising at least one spliceosome modulator and at least one inhibitor chosen from BCL2 inhibitors, BCL2/BCLxL inhibitors, and BCLxL inhibitors and methods of use | |
| AU2016209737A1 (en) | Anti-senescence compounds and uses thereof | |
| JP2018517759A (en) | Compositions and uses comprising carboplatin | |
| WO2023227112A1 (en) | USE OF PHARMACEUTICAL COMBINATION OF β-ELEMENE AND OSIMERTINIB IN PREPARATION OF ANTI-LUNG CANCER MEDICAMENT | |
| JP2021063014A (en) | Leukemia therapeutic agent | |
| CN112704679B (en) | Pharmaceutical composition containing homoharringtonine and application thereof | |
| CN116531395B (en) | Composition containing artemisinin derivatives and application of composition in preparation of medicines for treating leukemia | |
| Zhou et al. | Coronarin D suppresses proliferation, invasion and migration of glioma cells via activating JNK signaling pathway | |
| TW202435896A (en) | Antineoplastic drug combination comprising azvudine and a chemotherapy agent | |
| CN101167741B (en) | Sulforaphane and platinum medicine anti-cancer combination preparation | |
| JP7307732B2 (en) | Use of ginsenoside M1 for the manufacture of a medicament for treating oral cancer | |
| KR20200050761A (en) | Anti-cancer composition comprising disulfiram | |
| CN111393278B (en) | A kind of Usnea longiflora derivative and its use in preparing medicine for treating gallbladder cancer | |
| AU2020255063B2 (en) | Combined use of A-nor-5α androstane compound drug and anticancer drug | |
| CN114748630B (en) | Platinum anti-cancer medicine composition with improving effect and application thereof | |
| CN116509865B (en) | Application of nitrone compounds in preparation of medicines for preventing or treating bone marrow suppression | |
| JP7414230B2 (en) | Antihematologic malignant tumor drug | |
| JP7217875B2 (en) | Preventive and/or therapeutic agent for blood cancer | |
| CN101199534A (en) | Antitumor application of a kind of protopanaxadiol derivative | |
| WO2025056747A1 (en) | Combination of a wee1 inhibitor and a pkmyt1 inhibitor | |
| CN118059234A (en) | Application of anti-PD-1 monoclonal antibody combined mitoxantrone liposome in preparation of medicines for treating tumor heart complications | |
| TW202421113A (en) | A composition for the treatment of liver cell carcinoma comprising ovatodiolide and antrocin | |
| WO2022256467A1 (en) | Compositions and methods comprising deoxy-pentitols | |
| Farrell | Inhibiting fatty acid binding proteins decreases multiple myeloma cell proliferation and increases efficacy of dexamethasone |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |