CN108743577B - 一种抗他莫昔芬耐药乳腺癌的药物组合物 - Google Patents
一种抗他莫昔芬耐药乳腺癌的药物组合物 Download PDFInfo
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Abstract
本发明公开了一种抗他莫昔芬耐药乳腺癌的药物组合物，该药物组合物包括多西他赛和肽酰精氨酸脱亚氨酶抑制剂。本发明在现有技术的基础上，通过大量实验研究，将PADs抑制剂CL‑amidine和多西他赛进行联合用药，可以起到协同增效的作用。该药物组合物显著抑制他莫昔芬耐药乳腺癌细胞的细胞增殖与细胞周期，并提高肿瘤细胞的细胞凋亡率。可减少多西他赛的用量，增加药物疗效，降低药物毒副作用，从而达到更好治疗他莫昔芬耐药乳腺癌的作用。
Description
技术领域
本发明涉及一种药物组合物，具体涉及抗他莫昔芬耐药乳腺癌的药物组合物。
背景技术
他莫昔芬是一种重要的内分泌治疗药物，主要用于治疗雌激素受体阳性的乳腺癌或卵巢癌患者。然而无论雌激素受体状态变得如何，很多患者最终会出现他莫昔芬耐药。他莫昔芬耐药目前已成为导致乳腺癌复发的关键因素，由于其发生耐药的机制尚不清楚，因此成为困扰临床肿瘤治疗的一个重要问题。
多西他赛又名多西紫杉醇，是一种人工合成的紫杉烷类抗肿瘤药物。多西他赛通过促进微管蛋白聚合并抑制其解聚来形成非功能性微管束，从而抑制肿瘤细胞生长并促进其死亡。多西他赛因其较高的抗肿瘤活性被FDA批准用于多种临床癌症的治疗，如非小细胞肺癌、头颈部癌、胃癌、胰腺癌、前列腺癌，尤其是对卵巢癌和晚期乳腺癌有十分显著的疗效。但目前尚无研究报道多西他赛对他莫昔芬耐药乳腺癌患者的治疗效果。由于临床长时间使用多西他赛会导致副反应的增加，如过敏反应、肝功能异常、体液潴留以及中性粒细胞减少等，因此寻求一种方式能够降低药物的毒副反应成为了提升多西他赛在临床癌症治疗方面潜能的关键。近年来有研究发现，小分子靶向抑制剂与临床化疗药物多西他赛联用能够在既增强化疗药物杀伤癌细胞的效果同时，又降低了药物的浓度，这为未来临床化疗药物的使用提供了新的思路。
肽酰精氨酸脱亚氨酶(peptidylarginine deiminases，PADs)是一种钙离子依赖的蛋白修饰酶，能将蛋白质多肽链中的精氨酸残基转变为瓜氨酸残基，此反应也称为脱亚氨基作用。PADs是参与蛋白质翻译后修饰的一种重要酶类，在许多生理和病理过程中参与作用，如炎症、凋亡及衰老过程。CL-amidine是PADs家族广泛抑制剂，其与PADs蛋白底物肽酰精氨酸结构相似并且能够使PADs蛋白酶的活性位点的半胱氨酸(Cys645)发生共价修饰，从而抑制PADs蛋白酶的活性。
发明内容
发明目的：本发明的目的是为了克服现有临床治疗的缺陷，提供一种协同抗他莫昔芬耐药乳腺癌的药物组合。该药物组合通过将PADs抑制剂CL-amidine和多西他赛进行联合用药，以他莫昔芬耐药的乳腺癌细胞作为研究对象。该药物组合显著抑制他莫昔芬耐药乳腺癌细胞的细胞增殖与细胞周期，并提高肿瘤细胞的细胞凋亡率。
技术方案：为了实现以上目的，本发明所采取的技术方案如下：
一种抗他莫昔芬耐药乳腺癌的药物组合物，该药物组合物包括多西他赛和肽酰精氨酸脱亚氨酶抑制剂。
作为优选方案，以上所述的抗他莫昔芬耐药乳腺癌的药物组合物，肽酰精氨酸脱亚氨酶抑制剂为肽酰精氨酸脱亚氨酶抑制剂为CL-amidine，其结构式为。
多西他赛的结构式如下：
作为优选方案，以上所述的抗他莫昔芬耐药乳腺癌的药物组合物，肽酰精氨酸脱亚氨酶抑制剂和多西他赛的摩尔比为25～100:0.01～1。
作为优选方案，以上所述的抗他莫昔芬耐药乳腺癌的药物组合物，肽酰精氨酸脱亚氨酶抑制剂和多西他赛的摩尔比为250:1。
本发明所述的抗他莫昔芬耐药乳腺癌的药物组合物在制备防治抗他莫昔芬耐药乳腺癌药物中的应用。
本发明所述的抗他莫昔芬耐药乳腺癌的药物组合物，其特征在于，将药物组合物和药学上可接受的载体制备成片剂、胶囊剂、颗粒剂、丸剂、粉针剂或注射剂。
有益效果：本发明提供的中药组合物和现有技术相比具有以下优点：
本发明的药物组合物PADs抑制剂CL-amidine和多西他赛作为活性成分发挥抗他莫昔芬耐药乳腺癌的协同作用，发明人通过大量实验对这两种组分的用量进行了筛选，结果发现PADs抑制剂CL-amidine和多西他赛的摩尔比为(25～100):(0.01～1)的范围内时，其抗他莫昔芬耐药乳腺癌的增殖效果较好，体外实验中，最优选的PADs抑制剂CL-amidine与多西他赛的摩尔比为250:1。
本发明中的PADs抑制剂CL-amidine与多西他赛分别低于单用有效剂量。本发明提供的PADs抑制剂CL-amidine与多西他赛联合使用，可减少多西他赛的用量，增加药物疗效，降低药物毒副作用，从而达到更好治疗他莫昔芬耐药乳腺癌的作用。
附图说明
图1为CCK-8比色法检测多西他赛(Docetaxel)和CL-amidine分别处理TamR细胞72h时的IC50值的柱状图。
图2为分别用0μM多西他赛，0.1μM多西他赛，25μM CL-amidine，0.1μM多西他赛与25μM CL-amidine联合处理TamR细胞24，48，72，96，144h后CCK-8比色法评估细胞增殖的柱状图。
图3为不同重量比例的CL-amidine和多西他赛抗肿瘤实验结果。
图4为Graphpad Prism 6.0分别统计对照(PBS)，多西他赛(Doc)，CL-amidine，多西他赛与CL-amidine联合处理组TamR细胞G0/G1期，S期和G2/M期细胞数目。
图5为流式细胞仪检测对照(PBS)，多西他赛，CL-amidine，多西他赛(Doc)与CL-amidine联合处理组48h后TamR细胞凋亡变化。
图6为Graphpad Prism 6.0分别统计对照(PBS)，多西他赛(Doc)，CL-amidine，多西他赛与CL-amidine联合处理组TamR细胞总凋亡率的变化。
具体实施方式
下面结合具体实施例进一步阐明本发明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1
1.CCK-8比色法检测细胞生长抑制率：
1.1实验原理：Cell Counting Kit-8简称CCK-8,是一种基于WST-8(化学名：2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐)的广泛应用于细胞增殖和细胞毒性的快速高灵敏度检测试剂。在电子耦合试剂存在的情况下，可以被线粒体内的脱氢酶还原生成高度水溶性的橙黄色的甲臜产物(formazan)。颜色的深浅与细胞的增殖成正比，与细胞毒性成反比。使用酶标仪在450nm波长处测定OD值，间接反映活细胞数量。
1.2CCK-8比色法检测CL-amidine和多西他赛对TamR细胞生长抑制率：
实验步骤：他莫昔芬耐药乳腺癌(TamR)细胞以接种浓度1×104个/mL浓度接种于96孔板(100μL/孔)，同时设置空白调零孔(仅培养基孔)和对照孔(PBS处理孔)，5％CO2，37％培养过夜，加入已设置好浓度梯度的CL-amidine和多西他赛(Docetaxel)进行处理，每个样品5个复孔，5％CO2，37％培养72h后小心吸去培养基，每孔加入100μL无血清培养基配制的CCK-8溶液(即10％CCK-8)；继续培养3h后，用酶标仪检测450nm处每孔的吸光度值，用式(1)计算药物中含有CL-amidine和多西他赛对TamR细胞生长的抑制率，结果如图1所示。
细胞生长抑制率(％)＝(1-A样品组/A对照组)×100％，采用CCK-8法分别测定了CL-amidine和多西他赛对TamR细胞增殖的抑制活性。结果显示，多西他赛和CL-amidine对他莫昔芬耐药乳腺癌细胞的生长都具有较强的抑制作用。多西他赛和CL-amidine的IC50分别为66.57μM和69.96μM。
1.3CCK-8比色法检测CL-amidine和多西他赛联用对TamR细胞生长抑制率：
实验步骤：TamR细胞以接种浓度1×104个/mL浓度接种于96孔板(100μL/孔)，同时设置空白调零孔(仅培养基孔)和对照孔(PBS处理孔)，5％CO2，37％培养过夜，加入已设置好固定浓度的CL-amidine(25μM)与多西他赛(0.1μM)进行处理，每个样品5个复孔，5％CO2，37％分别培养0，24，48，72，96，144h后小心吸去培养基，每孔加入100μL无血清培养基配制的浓度为10％的CCK-8溶液，继续培养3h后，用酶标仪检测450nm处每孔的吸光度值，同样用式(1)计算对照处理，CL-amidine与多西他赛单独处理，CL-amidine与多西他赛联合处理对TamR细胞生长的抑制率，结果如图2所示。结果显示，CL-amidine与多西他赛联合处理96小时，可以显著抑制细胞的增殖。
发明人通过大量实验对这两种组分的用量进行了筛选，结果发现所述的PADs抑制剂CL-amidine和多西他赛的摩尔比为(25～100):(0.01～1)的范围内时特别是250:1时最优！筛选结果如图3所示。
2.流式细胞仪检测细胞周期：
实验原理：碘化丙啶(Propidium，PI)是一种双链DNA荧光染料，其嵌入双链DNA后可以产生荧光，并且荧光强度和双链DNA的含量成正比。细胞内的DNA被PI染色后，可以用流式细胞仪对细胞进行DNA含量测定，根据DNA含量的分布情况，进行细胞周期和细胞凋亡分析。
实验步骤：TamR细胞以1×106个/mL的浓度接种在6cm的培养皿中(4mL/皿),5％CO2，37％培养过夜，然后加入CL-amidine和多西他赛单药以及CL-amidine和多西他赛联合药物，与细胞共同孵育24h。之后吸去所有培养基，用预冷的PBS清洗细胞1次并收集到1.5mL离心管中；用0.25％胰酶(0.5mL/皿)消化细胞4min，收集细胞到1.5mL离心管中；用预冷的PBS清洗细胞2次，每次均4℃300g离心5min；吸去大部分上清，留少许PBS，细胞沉淀轻轻弹匀，至细胞悬液体积为50-100μL，涡旋使细胞处于悬浮状态，缓慢滴加1mL冰冷75％乙醇，4℃冰箱过夜；从4℃冰箱取出固定细胞样品，4℃1000g离心5min收集细胞，1mL冰冷的PBS清洗细胞1次；400μL冰冷PBS重悬细胞；加20μL RNase A Solution 37℃水浴放置30min；400目筛网过滤细胞；加入400μL PI染液，轻轻混匀后4℃冰箱孵育60min，流式细胞仪检测细胞周期，结果如图4所示。细胞周期阻滞在G2/M期。
3.流式细胞仪检测细胞凋亡：
实验原理：在正常细胞中，磷脂酰丝氨酸(PS)只分布在细胞膜脂质双层的内侧，而在细胞凋亡早期，细胞膜中的磷脂酰丝氨酸(PS)由脂膜内侧翻向外侧。Annexin V是一种分子量为35～36kD的Ca2+依赖性磷脂结合蛋白，与磷脂酰丝氨酸具有高度亲和力，故可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的细胞膜结合。因此Annexin V被公认为检测细胞早期凋亡的灵敏指标之一。将Annexin V进行绿色荧光(FITC或EGFP)标记，以标记了的Annexin V作为探针，利用荧光显微镜或流式细胞仪可检测细胞凋亡的发生。碘化丙啶(Propidium Iodide,PI)是一种核酸染料，它不能透过正常细胞或早期凋亡细胞的完整的细胞膜，但对凋亡中晚期的细胞和坏死细胞，PI能够透过细胞膜而使细胞核染红。因此将Annexin V与PI匹配使用，就可以将处于不同凋亡时期的细胞区分开来。
实验步骤：TamR细胞以1×106个/mL的浓度接种在6cm的培养皿中(4mL/皿),5％CO2，37％培养24h，加入CL-amidine和多西他赛单药以及CL-amidine和多西他赛联合药物，与细胞共同孵育48h。之后吸去所有培养基，用预冷的PBS清洗细胞1次并收集到1.5mL离心管中；用0.25％不含EDTA胰酶(0.5mL/皿)消化细胞4min，收集细胞到1.5mL离心管中；用预冷的PBS清洗细胞2次，每次均4℃300g离心5min；加入100μL的1×Binding Buffer重悬细胞；先加入5μL Annexin V-FITC染液染色5min，再加入5μL PI染液染色5min，轻轻混匀，避光、室温反应；加入600μL的1×Binding Buffer，轻轻混匀，1h内流式细胞仪检测细胞凋亡，结果如图5和图6所示。两种药物连用能显著增强TamR细胞的凋亡。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.药物组合物多西他赛和肽酰精氨酸脱亚氨酶抑制剂CL-amidine在制备防治抗他莫昔芬耐药乳腺癌药物中的应用；
CL-amidine结构式为：
肽酰精氨酸脱亚氨酶抑制剂和多西他赛的摩尔比为250:1。
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| CN103189063A (zh) * | 2010-11-01 | 2013-07-03 | 宾夕法尼亚州研究基金会 | 治疗组合物和方法 |
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