CN105012331A - 病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途 - Google Patents
病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途 Download PDFInfo
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Abstract
本发明公开了病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途。本研究发现病毒唑能够有效治疗急性淋巴细胞白血病，特别是对Ph+急性淋巴细胞白血病的疗效确切，可以制备成为治疗急性淋巴细胞白血病的药物，临床应用前景良好。
Description
技术领域
本发明涉及病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途。
背景技术
急性淋巴细胞白血病(ALL)是一种起源于淋巴细胞的B系或T系细胞在骨髓内异常增生的恶性肿瘤性疾病。异常增生的原始细胞可在骨髓聚集并抑制正常造血功能，同时也可侵及骨髓外的组织，如脑膜、淋巴结、性腺、肝等。我国曾进行过白血病发病情况调查，ALL发病率约为0.67/10万。在油田、污染区发病率明显高于全国发病率。ALL儿童期(0～9岁)为发病高峰，可占儿童白血病的70％以上。ALL在成人中占成人白血病的20％左右。
Ph染色体是由第9号和第22号染色体易位t(9；22)(q34；q11)，导致bcr-abl融合基因的形成，编码具有高酪氨酸激酶活性的BCR-ABL融合蛋白，通过激活下游多条生长信号通路，使造血细胞不依赖于造血刺激因子而生长、增殖，并且抵抗凋亡，最终发生恶性转化而发生白血病。Ph+急性淋巴细胞白血病(Ph+ALL)见于2％～5％儿童和20％～30％成人急性淋巴细胞白血病，年龄超过50岁者甚至高达50％。
BCR-ABL酪氨酸激酶抑制剂(TKI)是Ph+白血病治疗的里程牌，伊马替尼是首个获准上市的选择性BCR-ABL酪氨酸激酶抑制剂。TKI问世以前，Ph+ALL预后极差，尽管诱导化疗的完全缓解率CR达60％～90％，但CR持续时间仅为9～16个月，单纯化疗5年生存率仅10－20％。异基因造血干细胞移植是根治Ph+ALL的手段，但却明显受限于供体的获得及移植相关的高死亡率。虽然TKI显著改善了Ph+ALL的预后，TKI与化疗联合使初治Ph+ALL的完全缓解率达90％以上，但缓解持续时间短，复发率很高。而在复发、难治的Ph+ALL，伊马替尼仅能使19％的患者获得完全血液学缓解，疗效持续时间2-3月，中位生存时间4.9-9个月。耐药是伊马替尼在Ph+ALL中疗效不持久的最重要因素。第二代酪氨酸激酶抑制剂达沙替尼、尼罗替尼等虽然可以在一定程度上克服伊马替尼耐药，迅速改善患者的血液学、遗传学指标，完全缓解率达90％以上，但还是因为耐药出现使得缓解期也很短暂。
病毒唑，病毒唑又名利巴韦林、三氮唑核苷、尼斯可等，是广谱强效的抗病毒药物，目前广泛应用于病毒性疾病的防治，未见将其用于治疗急性淋巴细胞白血病的报道。
发明内容
本发明的目的在于提供病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途。
本发明提供了病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途。
其中，所述药物是以病毒唑为活性成分，加上药学上可接受的辅料制备而成的制剂。
其中，所述制剂为口服制剂。
其中，所述口服制剂是胶囊剂、片剂。
其中，所述治疗急性淋巴细胞白血病的药物是治疗Ph+急性淋巴细胞白血病的药物。
本发明还提供了一种治疗急性淋巴细胞白血病的药物，它是以病毒唑为活性成分，加上药学上可接受的辅料制备而成的制剂。
其中，所述制剂为口服制剂。
其中，所述口服制剂是胶囊剂、片剂。
本研究发现病毒唑能够有效治疗急性淋巴细胞白血病，特别是对Ph+急性淋巴细胞白血病的疗效确切，可以制备成为治疗急性淋巴细胞白血病的药物，为临床治疗急性淋巴细胞白血病提供了一种新的选择。
下面通过具体实施方式对本发明做进一步详细说明，但是并不是对本发明的限制，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
附图说明
图1病毒唑作用于Ph+淋巴细胞株的抗白血病作用的结果。A.不同浓度(30、60、90、120μM)的病毒唑分别作用于SUP-B15细胞持续24、48、72、96h，用MTT法检测细胞增殖，图中显示的是细胞的生存率。
图2病毒唑对SUP-B15细胞株的促凋亡作用的结果。A.60μM病毒唑分别作用于SUP-B15细胞24、48、72h，用流式分析annexinV-FITC和PI双染法检测细胞凋亡率。PBS作为阴性对照。
图3病毒唑可抑制SUP-B15细胞株的mTOR/eIF4E和MEK/ERK/Mnk1/eIF4E信号通路表达结果。A.SUP-B15细胞被不同浓度(30、60、90μM)的病毒唑作用48h后，提取蛋白，western bolt检测mTOR/eIF4E信号通路以及Mcl-1的表达。B.SUP-B15细胞被60μM病毒唑分别作用24、48、72h后，用western bolt检测mTOR/eIF4E信号通路以及Mcl-1的表达。
图4病毒唑单用可抑制SUP-B15细胞株eIF4F复合物的合成的结果。A.SUP-15细胞被病毒唑(30μM)作用48h。用500μl RIPA裂解液裂解1×107个细胞，离心所得总蛋白的一半上清液中加入7m-GTP-Sepharose beads(GEHealthcare)孵育1-2h，离心所得beads中加入50ul SDS-PAG上样缓冲液，煮沸7分钟，用SDS-聚丙烯酰胺凝胶电泳分离蛋白，用一抗eIF4G、eIF4E和4E-BP1进行免疫印迹。GAPDH作为内参水平。
具体实施方式
实验例1本发明药物治疗急性淋巴细胞白血病的作用
1.实验材料
1.1细胞株
SUP-B15人类Ph+急性淋巴细胞细胞株，表达P190BCR-ABL融合基因(the American Type Culture Collection,CRL-1929)
各类急性白血病及慢性粒细胞白血病原代细胞(Ph+ALL，Ph-ALL，AML，CML)：均来自四川大学华西医院血液科，采集外周血及骨髓标本前得到书面同意并获得医院伦理委员会的通过和批准。收集分离方法：选取符合纳入标准的急性白血病患者，于无菌条件下抽取外周血或骨髓3-4ml于肝素抗凝的无菌试管中。取15ml无菌离心管，将所取外周血加入等体积无菌PBS溶液中，按1:1比例稀释。另取一只15ml无菌离心管，预先加入等体积人淋巴细胞分离液，将稀释后的外周血沿管壁缓慢加入淋巴细胞分离液试管内，使二者形成清晰界面，以2000RPM(每秒2000转)离心25min。取出离心管，吸取中间层单个核细胞，置于另一离心管中，PBS洗涤细胞2次，1000RPM离心5min，弃去上清，用10％胎牛血清的RPMI-1640培养基重悬细胞。
1.2实验药物
病毒唑(RIB)：sigma公司
1.3实验试剂
小牛血清、青链霉素、RPMI 1640、IMDM培养基为hyclo公司产品；MTT为sigma公司产品；十二烷基硫酸钠(SDS)为Sigma公司产品，异丁醇、浓盐酸为天津天大化学试剂厂产品；BCA蛋白定量试剂盒为Thermo公司产品；RIPA buffer为碧云天公司产品；人淋巴细胞分离液(Ficoll液)为天津灏阳公司产品；一抗：P-AKT(S473)、p-mTOR(S2448)、p-4EBP1(T37/46)、p-eIF4E(S209)、p-cRaf(Ser338)、p-MEK1/2(Ser217/221),p-ERK1/2(Thr202/Tyr204)、p-Mnk1(Thr197/202)、p-Lyn(Tyr396)、Mcl-1、AKT、mTOR、4E-BP1、eIF4E、MEK、Mnk1、Cyclin D1(美国Cell SignalingTechnology公司)，GAPDH和辣根过氧化物酶标记的二抗购自碧云天公司。
1.4实验仪器
超净工作台：苏州净化设备公司
CO2恒温培养箱：Thermo HEPA CLASS 100，芬兰
低速自动平衡离心机：SORVALL LEGEND MACH 1.6R
普通光学显微镜：OLYMPUS，日本
96孔板：Corning公司，美国
-80℃冰箱：Heraeus，美国
凝胶成像仪：Bio-rad公司，美国
全自动酶标仪：Thermo Multiscan Spectrum，芬兰
-20℃冰箱BCD-287F为青岛海尔公司产品
15ml离心管为美国BD公司产品
2.实验方法
2.1病毒唑作用于Ph+淋巴细胞株的抗白血病作用
细胞株每个孔加5×104个细胞，原代细胞每个孔5×105个细胞，按实验分组加入不同浓度药物：1、5、10、50、75、100、250、500μM；
然后，每孔加入5mg/mL的MTT溶液20μl，再置于37℃、5％CO2孵箱中继续孵育4小时，然后加入MTT三联溶解液100μl，继续在37℃、5％CO2孵箱孵育过夜。第二天用酶标仪测定每孔在570nm处的吸光度值，计算抑制率。生长抑制率＝1-实验孔OD值/对照孔平均OD值，绘制生长曲线。根据药物对细胞的抑制与药物浓度对数成正比的原理，利用SPSS17.0中的Probitanalys模型，计算出药物对细胞作用的半数抑制浓度(IC50)。实验重复3次。
2.2病毒唑对SUP-B15细胞株的促凋亡作用
细胞株每个孔加5×104个细胞，按实验分组加入不同浓度药物：用30、60、90μM病毒唑作用于SUP-B15，24、48、72小时后，用Annexin V-FITC和propidium iodide(PI)双染法染色，流式细胞仪分析细胞凋亡率。
2.3病毒唑可抑制SUP-B15细胞株的mTOR/eIF4E和MEK/ERK/Mnk1/eIF4E信号通路表达
细胞株每个孔加5×104个细胞，按实验分组加入不同浓度药物：
SUP-B15细胞被不同浓度(30、60、90μM)的病毒唑作用24、48、72h，不同浓度的U0126或CGP57380单独作用于SUP-B15细胞，60μM病毒唑单用或联合10μM U0126作用于SUP-B15细胞6、24、48、72h；
取处理后的细胞1×107个，用RIPA裂解液裂解，得到的蛋白用BCA法测定蛋白浓度。取30-120ug总蛋白用SDS-聚丙烯酰胺凝胶电泳分离蛋白，使用以下一抗P-AKT(S473)、p-mTOR(S2448)、p-4EBP1(T37/46)、p-eIF4E(S209)、p-cRaf(Ser338)、p-MEK1/2(Ser217/221),p-ERK1/2(Thr202/Tyr204)、p-Mnk1(Thr197/202)、p-Lyn(Tyr396)、Mcl-1、AKT、mTOR、4E-BP1、eIF4E、MEK、Mnk1、Cyclin D1(Cell SignallingTechnology,Danvers,MA,USA)，GAPDH和辣根过氧化物酶标记的二抗进行免疫印迹分析。
2.4病毒唑抑制SUP-B15细胞株eIF4F复合物的合成
细胞株每个孔加5×104个细胞，按实验分组加入不同浓度药物：SUP-15细胞分别被病毒唑(30μM)作用48h；
取处理后的细胞1×107个，提取总蛋白，离心所得上清液中加入50ul7m-GTP-Sepharose beads(GE Healthcare)，4℃孵育1-2小时后珠子用1mlPBS洗三次，50ul上样缓冲液加入到珠子中，煮沸7分钟。用SDS-聚丙烯酰胺凝胶电泳分离蛋白，用一抗4E-BP1，eIF4E进行免疫印迹分析。
2.5统计方法
每个实验都重复三次，取平均值加减标准差做为最后的实验数据发表。各个样本之间采用t检验、配对t检验或者卡方检验进行分析，所有数据均使用SPSS 17.0进行分析。P值小于0.05说明差异具有统计学意义。
3.实验结果
3.1病毒唑对Ph+淋巴细胞株的抗白血病作用
结果如图1和表1所示：
表1不同浓度RIB对SUP-B15的抑制率
| 浓度(μM) | 1 | 5 | 10 | 50 | 75 | 100 | 250 | 500 |
| 抑制率(﹪) | 4.3099 | 9.3131 | 11.9246 | 46.9582 | 60.5503 | 67.0541 | 83.2683 | 92.3431 |
可以看出，病毒唑可以有效抑制SUP-B15细胞株的细胞增殖，病毒唑对SUP-B15细胞株的半数抑制浓度(IC50)为65.177umol/L。
实验结果说明，病毒唑可以治疗急性淋巴细胞白血病。
3.2病毒对SUP-B15细胞株的促凋亡作用
结果如图2与表1所示：
表4RIB对SUP-B15促凋亡作用(48h)
| 组别 | 凋亡抑制率(％) |
| Con | 2.3 |
| 30RIB | 10.36 |
可以看出，病毒唑可促进SUP-B15细胞株的凋亡发生，可以治疗急性淋巴细胞白血病。
3.3病毒唑可抑制SUP-B15细胞株的mTOR/eIF4E和MEK/ERK/Mnk1/eIF4E信号通路表达
结果如图3所示，Western Blot分析显示病毒唑可以下调SUP-B15细胞株mTOR/eIF4E信号通路蛋白Akt、mTOR、4EBP1、eIF4E磷酸化，ERK/Mnk1/eIF4E轴中MEK、ERK、Mnk1、eIF4E蛋白的磷酸化表达水平，以及下调下游靶蛋白Mcl-1的表达。
3.4病毒唑可抑制SUP-B15细胞株eIF4F复合物的合成
结果如图4所示，7-甲基鸟嘌呤帽亲和分析法显示病毒唑单药可增加eIF4E与4EBP1的结合，减少与eIF4G的结合，从而抑制eIF4F翻译起始复合物的合成。
4、实验结论
上述研究结果证实，病毒唑通过抑制mTOR/eIF4E、ERK/Mnk1/eIF4E两条通路来影响P-eIF4E的表达水平和eIF4F翻译起始复合物的组装，减少Mcl-1下游癌蛋白翻译，促进细胞凋亡，抑制Ph+急性淋巴细胞白血病细胞的增殖，能治疗急性淋巴细胞白血病，特别是Ph+急性淋巴细胞白血病，可以制备成为治疗急性淋巴细胞白血病的药物。
Claims (8)
1.病毒唑在制备治疗急性淋巴细胞白血病的药物中的用途。
2.根据权利要求1所述的用途，其特征在于：所述药物是以病毒唑为活性成分，加上药学上可接受的辅料制备而成的制剂。
3.根据权利要求1所述的用途，其特征在于：所述制剂为口服制剂。
4.根据权利要求3所述的用途，其特征在于：所述口服制剂是胶囊剂、片剂。
5.权利要求1～4任意一项所述的用途，其特征在于：所述治疗急性淋巴细胞白血病的药物是治疗Ph+急性淋巴细胞白血病的药物。
6.一种治疗急性淋巴细胞白血病的药物，其特征在于：它是以病毒唑为活性成分，加上药学上可接受的辅料制备而成的制剂。
7.根据权利要求1所述的用途，其特征在于：所述制剂为口服制剂。
8.根据权利要求7所述的用途，其特征在于：所述口服制剂是胶囊剂、片剂。
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