CN105330579B - 依泽替米贝及其衍生物在治疗或预防癫痫中的应用 - Google Patents
依泽替米贝及其衍生物在治疗或预防癫痫中的应用 Download PDFInfo
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Abstract
本发明属于医药技术领域，具体涉及依泽替米贝及其衍生物在制备治疗或预防癫痫药物中的应用，这些衍生物从依泽替米贝的结构式中衍生而来，结构通式为。经过戊四唑（PTZ）诱导斑马鱼癫痫模型筛选化合物，依泽替米贝可显著降低斑马鱼快速运动距离（V>20mm/sec），但对总的运动距离无影响，说明其在无毒浓度下，依泽替米贝在治疗或预防癫痫疾病中表现出较好的药效。
Description
技术领域
本发明涉及依泽替米贝及其衍生物的用途，这些衍生物从依泽替米贝的结构式中衍生而来，它们在治疗或预防癫痫疾病中表现出较好的药效。
背景技术
癫痫（epilepsy）是慢性反复发作性短暂脑功能失调综合征，以脑神经元异常放电引起反复痫性发作为特征。癫痫发作可表现运动、感觉、意识、精神、行为和自主神经等功能异常。癫痫是神经系统常见疾病之一，全球约5000万患者，发病率约1%。癫痫可发生在任何年龄段，与性别、社会经济地位无关。癫痫死亡率约为20%，给家庭、社会造成很大的负担。目前，经过现有的抗癫痫药物治疗，仍有大约30%的患者耐药不能控制[1]。药物不能控制的癫痫即难治性癫痫的死亡率更高，可达50%，尽管对于药物不能控制的癫痫目前多采用手术治疗，但适合手术的患者只占一小部分。因此，寻找有效和安全的抗癫痫治疗药物是生物医学重要的目标之一[2,3]。
啮齿类癫痫模型包括最大电休克模型，点燃模型，戊四唑(PTZ)、海仁酸、青霉素等诱导癫痫模型，这些模型对于癫痫药物研发及机制研究起到了重要的作用。但是，现阶段癫痫依然是一个棘手的医学问题，其机制尚不清楚。斑马鱼是一种脊椎动物，与人类基因同源性高达85%，其信号传导通路与人类基本近似，生物结构和生理功能与哺乳动物高度相似，具有体积小（可用微孔板分析）、发育周期短、体外受精、透明（可直接用肉眼和解剖显微镜观察）、单次产卵数较高等特点[4]。斑马鱼模型既具有体外实验快速、高效、低廉、用药量小等优势，又具有哺乳类动物实验预测性强、可比度高、可观察多个器官等优点，近年已在化合物药效、毒性评价中得到广泛应用[5,6]。在斑马鱼胚胎发育早期多巴胺系统、5羟色胺系统和γ-氨基丁酸（简称GABA）系统发育，并且具有相应的功能。这为斑马鱼在神经系统疾病中的应用提供了可能[7]。PTZ是γ-氨基丁酸受体拮抗剂，而GABA是脑内重要的抑制性神经递质，抑制GABA后可诱导癫痫发作。按照这种诱导机制，PTZ诱导大鼠癫痫模型可筛选各种作用机制的抗癫痫药物[8,9]，癫痫的分级参照1972年Racine制定的标准可以分为：一级：静止不动伴随有嘴部／面部运动；二级：点头及落水狗样抖动；三级：前肢阵挛；四级：站立及双侧前肢阵挛；五级：持续站立及跌倒；六级：狂奔及尖叫[10]。Baraban and Berghmans应用同样的机制，建立了PTZ诱导斑马鱼癫痫模型，可模拟哺乳动物癫痫状态，斑马鱼表现为运动增加、快速的“涡旋行为”及阵挛发作[11,12]。多位研究者利用PTZ诱导斑马鱼癫痫模型验证了多种作用机制的抗癫痫药物在此模型中均能表现出一定的药效，包括钠离子阻滞剂：卡马西平、苯妥因纳；钙通道阻滞剂：加巴喷丁；抑制囊泡释放药：左乙拉西坦；GABA受体激动剂：地西泮；改变GABA再摄取和分解的药物：丙戊酸钠等[9,13]。2012年国外研究者利用斑马鱼癫痫模型从2000个已知活性的化合物中，筛选出46个有潜在抗癫痫作用的化合物。这些化合物包括甾族化合物，抗真菌、抗炎、抗氧化、血管扩张剂、杀虫剂、除草剂，相关药物进入后续研发阶段[14]。因此，斑马鱼癫痫模型可以高通量高效筛选抗癫痫药物，这对癫痫药物的研发意义重大。
综上所述，利用斑马鱼癫痫模型高通量筛选抗癫痫药物，寻求新的化合物来有效治疗或预防癫痫已成为研发热点，开发具有自主知识产权的抗癫痫药物迫在眉睫。
依泽替米贝（英文名称：Ezetimibe；中文名称：依泽替米贝；中文别名：依替米贝、伊折麦布；化学名称：1-(4-Fluorophenyl)-3-[3-(4-fluorophenyl)-3-hydroxy-propyl]-4-(4-hydroxyphenyl)-azetidin-2-one；分子式：C24H21F2NO3，分子量：409.43）是一类新型的选择性胆固醇吸收抑制剂，通过与小肠刷状缘膜小囊泡上膜蛋白（相对分子质量145X103）结合，抑制小肠对饮食中和经胆汁输送到肠道中的胆固醇的吸收，降低血清和肝脏中的胆固醇含量。
依泽替米贝的化学结构式如下：
。
至今尚未见有关依泽替米贝抗癫痫活性及抗癫痫靶点活性的相关报道。
发明内容
本发明的目的在于提供依泽替米贝及其衍生物在制备治疗或预防癫痫药物中的新用途。
本发明的目的是通过以下技术方案实现的：现有化合物依泽替米贝及其衍生物，其结构式通式如下：
（Ⅰ）
其特征在于：R为F、Cl、Br等卤素以及羟基，甲氧基等。
R1为OH,OMe,COOMe,CO2H,CH2CO2Me,CH2CO2H,CH2CONEt2,(CH2)2CO2Me,(CH2)2CO2H,(CH2)2CONEt2,等。
以上市化合物依泽替米贝为代表，进行抗癫痫药效评价实验。依泽替米贝结构式为：
。
本发明利用PTZ诱导斑马鱼癫痫模型进行依泽替米贝及其衍生物治疗或预防癫痫药效学实验。PTZ是γ-氨基丁酸（简称GABA）受体拮抗剂，PTZ诱导后，早期即刻基因c-fos上调，斑马鱼立即表现出运动增加、快速的“涡旋行为”及阵挛发作，这与人类癫痫发作症状相似。利用View Point行为学分析仪记录斑马鱼运动轨迹，利用该软件分析快速运动距离，筛选抗癫痫药物。该方法简单，高效，高通量。本发明提供了依泽替米贝及其衍生物在PTZ诱导斑马鱼癫痫模型中，可以显著的降低斑马鱼快速运动距离（相当于哺乳动物的癫痫状态），对总的运动距离无影响，说明在无毒浓度下，依泽替米贝表现出较好的抗癫痫药效，这表明依泽替米贝可以治疗或预防癫痫的发生。
本发明依泽替米贝价格低廉、安全性高、原料来源广泛，辅以药学上可接受的辅料，采用常规制剂技术即可制成各种口服、注射，具有良好的开发前景。
附图说明
图1 PTZ诱导斑马鱼癫痫模型轨迹图。红色代表V>20mm/sec的运动轨迹，绿色代表4 mm/sec < V <20mm/sec的运动轨迹，黑色代表V <4mm/sec的运动轨迹。苯妥英钠在300 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达67%（p < 0.01）；筛选的化合物Cmp5为依泽替米贝，单浓素筛选中100 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达94%（p < 0.01）。
图2 依泽替米贝抗癫痫药效柱状图。空白组快速运动距离为100 mm，溶剂组和模型组与空白组相比，快速运动距离显著增加（p < 0.01,p < 0.01），表明PTZ诱导斑马鱼癫痫模型成功，且表现为快速运动距离增加。模型组与溶剂组相比，快速运动距离无差异，表明溶剂DMSO1.0%对斑马鱼行为无影响。苯妥英钠在300 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达67%（p < 0.01）；依泽替米贝单浓素筛选中100 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达94%（p < 0.01）。
图3依泽替米贝在10、30、100 μM浓度的对正常斑马鱼总的运动距离抑制率为-12%、-32%、0%，均无统计学差异，结果表明其在此浓度下，斑马鱼无毒性反应。
图4 在PTZ诱导癫痫模型中，依泽替米贝在10、30、100 μM浓度抗癫痫抑制率分别为9%、60%（p<0.01)、76% （p<0.01)，量效关系明确，表现出较好的抗癫痫效果。
图5 依泽替米贝在10、30、100 μM浓度对PTZ诱导斑马鱼癫痫模型轨迹图。红色代表V>20mm/sec的运动轨迹，绿色代表4 mm/sec < V <20mm/sec的运动轨迹，黑色代表V <4mm/sec的运动轨迹。
具体实施方式
下面结合附图和实施例对本发明作进一步阐述，但本发明的保护范围并不限于此。
实施例一 定量观察单浓度依泽替米贝在斑马鱼癫痫模型中的药效
斑马鱼：
本实施例使用的斑马鱼为野生型斑马鱼，饲养和使用标准严格参照美国实验动物管理和使用委员会(IACUC)的要求进行。
养鱼水(Fish water)：
配置方法：1L反渗透水(reverse osmosis (RO) water)加入0.3g海盐(InstantOcean salts)。
二甲基亚砜(DMSO，分析纯)：
购买于阿拉丁(货号#1095515，批号#30573)。1% DMSO溶液(阴性对照)配置：使用时，用养鱼水配置成浓度为1.0%的工作液，现配现用。
PTZ(戊四唑，诱导剂)：
购自美国sigma公司，批号069K1245。使用时，用0.1% DMSO溶液配置成实验所需的浓度，本实验中PTZ的使用浓度为10mM。
苯妥英纳（阳性对照药）：
购自北京百灵威科技有限公司，批号GF01-FD。使用时，用1.0% DMSO溶液配置成实验所需的浓度，本实验中苯妥因纳的使用浓度为300 μM。
依泽替米贝(Ezetimibe)：
购买于sigma公司，批号078235。使用时，用1.0% DMSO溶液配置成100 μM进行实验。
实验方法：
(1) 实验分组及幼鱼处理：取96尾发育良好的斑马鱼幼鱼，幼鱼发育时相为受精后6 dpf (day-postfertilization，dpf) ，随机分为12组(模型组、溶剂组、阳性药物组、药物组)，每组胚胎数量为8只。操作时将幼鱼随机分配至96孔细胞培养平板(Greiner，德国)中，每孔1尾幼鱼，每孔饲养用水200 μL。
(2) 药物预处理：用移液器(量程100～1000µL，Eppendorf)迅速将预先配置好的药液加入96孔细胞培养平板对应的孔中，每孔180 μL。加药液之前，用移液器(量程10～1000µL，Eppendorf)将96孔板中孵育胚胎的饲养用水尽力移出，此操作需在短时间内预先完成，以防止胚胎干燥。实验环境温度控制在28.5℃左右，相对湿度40～70%。然后用锡箔纸将96孔板包裹好，做好实验标记，迅速放置于斑马鱼培养箱中继续培养1 h(培养箱温度控制在28.5 ± 0.5℃)。
(3) PTZ诱导：药物预处理1 h后，加入PTZ 20 μL，使终浓度为10 mM。稳定10分钟后，利用View Point行为学分析仪记录60分钟内斑马鱼的运动情况。
(4) 对斑马鱼的快速运动（V>20 mm/sec）距离（D）进行定量分析，计算药物的抗癫痫治疗效率，公式如下：
（5）同时，在轨迹记录结束后，在解剖显微镜下观察鱼的状态，包括：心率变化、是否侧翻、是否有触碰反应等。
（6）统计学处理结果以表示，多组间比较采用方差分析，两组间比较采用Dunnett’s T-检验进行统计学处理，p<0.05为差异性显著。
实验结果见表1， 图1、图2。在96孔板中可以一次单浓度筛选8个化合物，每组8尾鱼，空白组快速运动距离为100 mm，溶剂组和模型组与空白组相比，快速运动距离显著增加（p < 0.01, p < 0.01），表明PTZ诱导斑马鱼癫痫模型成功，且表现为快速运动距离增加。模型组与溶剂组相比，快速运动距离无差异，表明溶剂DMSO1.0%对斑马鱼行为无影响。苯妥英钠是一个钠离子通道阻滞剂，是一传统的抗癫痫药物。本实验中，苯妥英钠在300 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达67%（p < 0.01）；筛选的化合物Cmp5为依泽替米贝，单浓素筛选中100 μM浓度下，能显著降低快速运动距离，癫痫治疗效率达94%（p <0.01）。同时在轨迹记录结束后，在解剖显微镜下观察鱼的状态，依泽替米贝给药组，触碰后均有逃避反应，未见其它异常表型，表明依泽替米贝100μM浓度下未见毒性反应，快速运动距离下降亦是一种抗癫痫药效表现。
表1 依泽替米贝在PTZ诱导斑马鱼癫痫模型中的治疗效率（mean±sem）
与空白组相比，#,p<0.01；与溶剂组相比，*，p<0.01
实施例二定量观察多浓度依泽替米贝在斑马鱼癫痫模型中的药效及安全性
实验材料和实验方法同实施例一。通过软件分析总的运动距离，来考察10、30、100μM浓度的依泽替米贝对正常斑马鱼运动的影响。及在PTZ诱导癫痫模型中，考察10、30、100μM浓度的依泽替米贝对斑马鱼快速运动距离的影响，判断其有无抗癫痫药效。
结果见图3、4、5。依泽替米贝在10、30、100 μM浓度的对正常斑马鱼总的运动距离抑制率为-12%、-32%、0%，均无统计学差异，结果表明其在此浓度下，斑马鱼无毒性反应。在PTZ诱导癫痫模型中，依泽替米贝在10、30、100 μM浓度抗癫痫抑制率分别为9%、60%（p<0.01)、76% （p<0.01)，量效关系明确，表现出较好的抗癫痫效果。
参考文献
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Claims (3)
1.依泽替米贝在制备预防或治疗癫痫药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述药物为口服给药剂型、注射给药剂型、粘膜给药剂型或经皮给药剂型。
3.根据权利要求1所述的应用，其特征在于，所述药物为片剂、胶囊剂、颗粒剂、口服液、注射液、贴剂或凝胶剂。
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| CN101679236A (zh) * | 2007-01-24 | 2010-03-24 | 克尔克公司 | 依泽替米贝的制备方法和其的衍生物 |
| CN101796032A (zh) * | 2007-06-28 | 2010-08-04 | 英特维特国际股份有限公司 | 作为cb1拮抗剂的取代哌嗪 |
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| CN101679236A (zh) * | 2007-01-24 | 2010-03-24 | 克尔克公司 | 依泽替米贝的制备方法和其的衍生物 |
| CN101796032A (zh) * | 2007-06-28 | 2010-08-04 | 英特维特国际股份有限公司 | 作为cb1拮抗剂的取代哌嗪 |
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