CN108186642A - 一种协同起作用治疗肺癌的药物组合物 - Google Patents
一种协同起作用治疗肺癌的药物组合物 Download PDFInfo
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Abstract
本发明公开了一种协同起作用治疗肺癌的药物组合物。所述的药物组合物含有治疗上有效剂量的Bcl‑xl抑制剂A‑1155463和荧光探针NA‑2a。该药物组合物中，荧光探针NA‑2a和Bcl‑xl抑制剂A‑1155463协同作用，通过荧光探针NA‑2a把细胞通透性打开，在不破坏Bcl‑xl抑制剂A‑1155463的功能并保证细胞完整性的前提下，帮助Bcl‑xl抑制剂A‑1155463进入细胞中发挥作用以达到更为显著的治疗肺癌效果。体外实验结果表明，对同一肺癌细胞株，荧光探针NA‑2a和Bcl‑xl抑制剂A‑1155463协同作用时对细胞株生长的抑制率较单一使用它们中的一种最高可提高40％左右。
Description
技术领域
本发明涉及治疗肺癌的药物组合物，具体涉及一种协同起作用治疗肺癌的药物组合物。
背景技术
癌症是严重威胁人类健康的重大疾病之一，癌症的总体发病率和死亡率呈上升趋势，癌症防治形势十分严峻。已有研究表明细胞凋亡程序失控与肿瘤的发生、发展及预后密不可分，Bcl-2蛋白家族是细胞凋亡过程中重要的调控分子，Bcl-xl蛋白是Bcl-2家族的重要成员，在调节细胞程序性死亡中起到重要作用。有研究显示，Bcl-xl在急性白血病细胞及结肠癌、直肠癌、骨肉瘤、食管鳞癌、卵巢癌、胃癌、膀胱癌、胰腺癌、肺癌等多种组织中表达上调。Bcl-xl的过度表达可抑制细胞凋亡，从而使细胞增殖和凋亡不平衡；另一方面，当机体细胞DNA受损害时，通过自身的修复机制对其进行修复，若得不到修复，则启动凋亡机制使其凋亡，若Bcl-xl过度表达，就会使这些具有遗传改变而又得不到修复的细胞免于凋亡，进而导致肿瘤发生(建明,赵俊.Bcl-xL蛋白与肿瘤[J].国际呼吸杂志,2008,28(18):1124-1127.)。此外，也有研究显示，Bcl-xl的过度表达与肿瘤耐药性相关(周珊珊,周文,等.抗凋亡因子Bcl-2/Bcl-X_L蛋白小分子抑制剂研究进展[J].中国药物化学杂志,2011(02):155-164)。
Bcl-xl抑制剂能抑制Bcl-xl在细胞中的抗凋亡功能，通过修复肿瘤细胞的正常凋亡途径，克服抗凋亡蛋白对凋亡的抵抗。但某些抑制剂存在结构稳定性差，细胞膜通透性、选择性不够理想等不足。
下述式(I)所示结构的荧光探针NA-2a是本申请人之前开发的一种具有线粒体靶向效应的荧光探针，该荧光探针对线粒体具有直接靶向效应但对细胞无明显抑制作用，具体请见公开为CN106977499A的发明专利。Bcl-xl抑制剂A-1155463(结构如下式(II)所示)是一种高度有效、选择性的Bcl-xl抑制剂，其对Bcl-xl的亲和力属于皮摩尔级别；
目前尚未见有将上述荧光探针和Bcl-xl抑制剂A-1155463联合使用用于治疗肺癌的相关报道。
发明内容
本发明要解决的技术问题是提供一种协同起作用治疗肺癌的药物组合物，该药物组合物对肺癌的治疗效果显著优于单一Bcl-xl抑制剂A-1155463的作用效果和单一荧光探针NA-2a的作用效果。
本发明所述的协同起作用治疗肺癌的药物组合物，含有治疗上有效剂量的Bcl-xl抑制剂A-1155463和下述式(I)所示结构的荧光探针NA-2a；
所述的“治疗上有效剂量”是指药物组合物中含有的Bcl-xl抑制剂A-1155463的量能有效地抑制肺癌肿瘤株的活性，达到治疗肺癌的效果。给药对象的不同，该有效剂量也不同，具体可以根据本领域技术人员的公知常识及现有技术进行确定。
本发明所述的协同起作用治疗肺癌的药物组合物，优选是由单一制剂Bcl-xl抑制剂A-1155463以及单一制剂荧光探针NA-2a(即将荧光探针NA-2a做成一个制剂)组成。所述单一制剂Bcl-xl抑制剂A-1155463可直接从市场上购买获得，而单一制剂荧光探针NA-2a则可将荧光探针NA-2a按现有常规技术制备成单一制剂。
在具体用药时，可以将单一制剂Bcl-xl抑制剂A-1155463以及单一制剂荧光探针NA-2a同时服用，也可以先服用单一制剂Bcl-xl抑制剂A-1155463再服用单一制剂荧光探针NA-2a，还可先服用单一制剂荧光探针NA-2a再服用单一制剂Bcl-xl抑制剂A-1155463。在采用先后服用单一制剂Bcl-xl抑制剂A-1155463和单一制剂荧光探针NA-2a时，优选是在先服用一个制剂12h后再服用另一个制剂。
本发明还提供下述式(I)所示结构的荧光探针NA-2a在制备治疗肺癌的药物中的应用；
具体的，是式(I)所示结构的荧光探针NA-2a在增强Bcl-xl抑制剂A-1155463对肺癌的治疗效果中的应用。
与现有技术相比，本发明所述药物组合物中，荧光探针NA-2a和Bcl-xl抑制剂A-1155463协同作用，通过荧光探针NA-2a把细胞通透性打开，在不破坏Bcl-xl抑制剂A-1155463的功能并保证细胞完整性的前提下，帮助Bcl-xl抑制剂A-1155463进入细胞中发挥作用以达到更为显著的治疗肺癌效果。本申请人的体外实验结果表明，对同一肺癌细胞株，荧光探针NA-2a和Bcl-xl抑制剂A-1155463协同作用时对细胞株生长的抑制率较单一使用Bcl-xl抑制剂A-1155463或单一使用荧光探针NA-2a最高可提高40％左右。
具体实施方式
下面结合具体实施例对本发明作进一步的详述,以更好地理解本发明的内容,但本发明并不限于以下实施例。
实施例1：荧光探针NA-2a制备
1)称取4-溴-1,8-萘二甲酸酐溶于150mL乙醇中，搅拌并加入3,4-亚甲二氧苯乙胺(4-溴-1,8-萘二甲酸酐和3,4-亚甲二氧苯乙胺的摩尔比为1：1.2)，反应温度80℃，反应进程使用薄层色谱进行跟踪监测。反应结束后，将反应体系冷却至室温，过滤并保留滤饼，使用乙醇淋洗滤饼，得到黄色固体NA-1a，产率约为90.47％；1H NMR(500MHz,DMSO-d6)δ:8.53(ddd,J＝9.5,7.9,1.0Hz,2H),8.31(d,J＝7.9Hz,1H),8.20(d,J＝7.9Hz,1H),7.99–7.96(m,1H),6.85(d,J＝1.6Hz,1H),6.80(s,1H),6.69(dd,J＝7.9,1.7Hz,1H),5.97(s,2H),4.20–4.15(m,2H),2.84(t,J＝7.5Hz,2H)；13C NMR(126MHz,DMSO-d6)δ:162.86,162.82,147.36,145.78,132.78,132.54,131.72,131.50,131.11,129.90,129.30,128.94,128.36,122.79,122.01,121.61,109.09,108.33,100.82,41.39,33.19.
2)称取NA-1a溶于二甲基亚砜中，加入甘氨酸乙酯和三乙胺(NA-1a、甘氨酸乙酯和三乙胺的摩尔比为1：1.5：2)，反应温度135℃，反应进程使用薄层色谱进行跟踪监测。反应结束后，反应体系冷却至室温，倒入冰水中，有黄色固体析出，使用乙酸乙酯萃取三次，收集合并酯层并用无水MgSO4干燥，减压除去溶剂，所得粗产物进行硅胶柱层析分离纯化(V石油醚：V乙酸乙酯＝20：1～5：1)，得到亮黄色固体2.9g，产率为65％。
对所得亮黄色固体进行核磁共振氢谱、核磁共振碳谱、电喷雾质谱分析，具体波谱特性如下：
(1)核磁共振氢谱：
1H NMR(400MHz,DMSO-d6)δ:8.66(d,J＝8.4Hz,1H),8.46(d,J＝7.2Hz,1H),8.26(d,J＝8.5Hz,1H),8.14(s,1H),7.74(t,J＝7.8Hz,1H),6.90–6.75(m,2H),6.67(dd,J＝10.7,9.0Hz,2H),5.97(s,2H),4.29(d,J＝5.5Hz,2H),4.17(q,J＝7.1Hz,4H),2.90–2.74(m,2H),1.23(s,3H)。
(2)核磁共振碳谱：
13C NMR(101MHz,DMSO-d6)δ:170.38,164.07,163.26,150.81,147.72,146.10,134.38,133.18,131.25,130.11,129.68,128.86,125.23,122.45,121.93,120.68,109.44,109.31,108.66,104.88,101.16,61.26,40.63,40.42,40.21,40.00,39.79,39.58,39.37,33.84,14.59。
(3)电喷雾质谱：ESI-MSm/z：445.14[M-H]-
因此，可确定上述亮黄色固体产物即为4-(乙氧羰基甲氨基)-N-(3，4-亚甲二氧苯乙基)-1，8-萘二甲酰亚胺，其化学结构式如下式(I)所示：
实施例2：荧光探针NA-2a和Bcl-xl抑制剂A-1155463联合用药对肺癌A549细胞株增殖的影响
本实验选用人肺癌细胞A549细胞株。所用细胞株培养在含10wt％小牛血清、100U/mL青霉素、100U/mL链霉素的1640培养液内，置37℃含体积浓度5％CO2孵箱中培养。所用的NA-2a按上述实施例1所述方法进行制备、纯化所得，纯度≥95％。所用Bcl-xl抑制剂A-1155463购于Selleck.cn。实验时以DMSO为溶剂配2mM的NA-2a原药，以及以DMSO为溶剂配1mM的Bcl-xl抑制剂A-1155463，均用培养基稀释到一定浓度之后加入96孔板中，实验分组情况为：
实验组，包括3个小组，具体如下：
实验1组：即2a(12h)+xl组，先使用荧光探针NA-2a处理12h,再使用Bcl-xl抑制剂A-1155463处理48h；
实验2组：即xl(12h)+2a组，先使用Bcl-xl抑制剂A-1155463处理12h，再使用荧光探针NA-2a处理48h；
实验3组：即xl+2a[48h]组，同时加入Bcl-xl抑制剂A-1155463和荧光探针NA-2a处理，处理时间为48h；
空白组：即CON组，除不加探针和Bcl-xl抑制剂A-1155463外，其他条件均与实验组相同；
对照1组：即2a(60h)组，不使用Bcl-xl抑制剂A-1155463处理，仅使用荧光探针NA-2a在同等实验条件下处理60h；
对照2组：即2a(48h)组，不使用Bcl-xl抑制剂A-1155463处理，仅使用荧光探针NA-2a在同等实验条件下处理48h；
对照3组：即xl(60h)，不用荧光探针NA-2a处理，仅使用Bcl-xl抑制剂A-1155463在同等实验条件下处理60h；
对照4组：即xl(48h)，不用荧光探针NA-2a处理，仅使用Bcl-xl抑制剂A-1155463在同等实验条件下处理48h。
(一)首次加药
取对数生长期的肺癌细胞A549接种于96孔板中，经胰蛋白酶消化后，用含10％小牛血清的培养液配制成浓度为5000个/mL的细胞悬液，以每孔180μL接种于96孔培养板中，使待测细胞密度至1000～10000个/孔(边缘孔用无菌PBS填充)；5％CO2，37℃孵育24h，至细胞单层铺满孔底，进行首次加药。2a(12h)+xl组和2a(60h)组加入20μL NA-2a稀释液，使其在培养液中的终浓度为5μmol/L，xl(12h)+2a组和xl(60h)组加入20μL Bcl-xl抑制剂A-1155463抑制剂稀释液，使其在培养液中的终浓度为10μmol/L，除xl+2a[48h]组之外的其它组补足相应体积培养液，于5％CO2，37℃孵育12h，设置5个复孔。
(二)再次给药
首次给药结束后，进行再次给药。给药时，2a(12h)+xl组和xl(48h)组加入20μLBcl-xl抑制剂A-1155463稀释液，使其在培养液中的终浓度为10μmol/L；xl(12h)+2a组和2a(48h)组加入20μL NA-2a稀释液，使其在培养液中的终浓度为5μmol/L；xl+2a[48h]组加入20μL Bcl-xl抑制剂A-1155463稀释液，使其在培养液中的终浓度为10μmol/L，然后再加入20μL NA-2a稀释液，使其在培养液中的终浓度为5μmol/L；其它组补足相应体积培养液，于5％CO2，37℃孵育48h。
(三)活力检测
再次给药结束后，每孔加入10μL的MTT溶液(5mg/mL PBS，即0.5％MTT)，继续培养4h；终止培养，小心吸去孔内培养液，每孔加入100μL DMSO充分溶解甲瓒沉淀，振荡器混匀后，在酶标仪用波长为570nm，参比波长为630nm测定各孔的光密度值；利用下述公式计算各组对细胞株生长的抑制率，其结果如下述表1所示：
实施例3：荧光探针NA-2a和Bcl-xl抑制剂A-1155463联合用药对肺癌细胞株NCI-H460细胞增殖的影响
重复实施例2，不同的是，所采用的细胞株是肺癌细胞株NCI-H460，且分组中没有xl+2a[48h]组。
各组对细胞株生长的抑制率如下述表1所示。
实施例4：荧光探针NA-2a和Bcl-xl抑制剂A-1155463联合用药对肺癌细胞株H1299细胞增殖的影响
重复实施例2，不同的是，所采用的细胞株是肺癌细胞株H1299。
各组对细胞株生长的抑制率如下述表1所示。
实施例5：荧光探针NA-2a和Bcl-xl抑制剂A-1155463联合用药对肺癌细胞株HCC-827细胞增殖的影响
重复实施例2，不同的是，所采用的细胞株是肺癌细胞株HCC-827，且分组中没有xl+2a[48h]组。
各组对细胞株生长的抑制率如下述表1所示。
表1：
/表示未做此实验。
由表1可知，在肺癌细胞株中，2a(12h)+xl组、xl(12h)+2a和xl+2a[48h]组的细胞活力低于CON组、低于xl(60h)和xl(48h)组，低于2a(60h)组和2a(48h)组。可见，在光探针NA-2a的帮助下，Bcl-xl抑制剂A-1155463对细胞的抑制率有显著提高。
综上所述，本发明所述的药物组合物可以增强对肺癌的治疗效果，表现出显著的药理学协同作用，有望用于所述探针与其他抑制剂的联合用药中，有望用于所述探针与所述抑制剂于其他肿瘤细胞株中的联合用药。
Claims (3)
1.一种协同起作用治疗肺癌的药物组合物，其特征在于：含有治疗上有效剂量的Bcl-xl抑制剂A-1155463和下述式(I)所示结构的荧光探针NA-2a；
2.下述式(I)所示结构的荧光探针NA-2a在制备治疗肺癌的药物中的应用；
3.根据权利要求2所述的应用，其特征在于：式(I)所示结构的荧光探针NA-2a在增强Bcl-xl抑制剂A-1155463对肺癌的治疗效果中的应用。
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