CN116919979A - 一种治疗癫痫的药物复合物及用途 - Google Patents
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Abstract
本发明提供了一种治疗癫痫的药物复合物，它是由四面体框架核酸和星型胶质细胞靶向肽AS1复合而成；所述星型胶质细胞靶向肽AS1的氨基酸序列如SEQ ID NO.5所示。本发明能够降低星型胶质细胞内的腺苷激酶，增加腺苷含量，减少海马区自发性的癫痫样放电；并通过减少苔藓样纤维的增生和异常的突触连接，减轻癫痫的发作。经实验证明：本发明在降低癫痫发作方面效果显著，具有良好的临床应用前景。
Description
技术领域
本发明属于生物医药领域，具体涉及一种治疗癫痫的药物复合物及用途。
背景技术
癫痫是一类以神经元异常放电为主要特征的神经系统疾病，患者常常表现为全身或局部肢体不自主抽搐或扭动，严重者常伴有意识的丧失。大多数患者急性发作之后可转为慢性癫痫，患者常常因为癫痫反复发作，严重影响其日常工作、生活和社会交际。目前癫痫的治疗主要以药物为主，但是现有的抗癫痫药物主要以调节神经元膜上离心通道、膜上兴奋性谷氨酸受体的抑制以及参与神经递质水平的调节为主。例如临床上常用的丙戊酸钠、卡马西平、氯硝西泮等等，然而约1/3的患者对于现有的抗癫痫药物治疗耐受，患者常常经历数年的抗癫痫药物治疗后癫痫仍得不到有效控制，最终需要进行手术干预。这个过程中，患者的不得不面临长期服用抗癫痫药物带来的副作用，以及大量的经济负担和社会、心理层面的煎熬。因而，寻找新的更有效的抗癫痫药物是极为必要的。
腺苷(Adenosine)是一种内源性的抗癫痫剂，具有降低脑内神经元兴奋性的作用，在正常脑内，腺苷被星型胶质细胞内的腺苷激酶所代谢，并被维持在一个相对稳定的水平；但是在癫痫患者脑内，由于星型胶质细胞内的腺苷激酶大量增加，腺苷被过度代谢，造成癫痫脑内腺苷水平大量减少，神经元便处于兴奋状态。因而，通过降低癫痫脑内星型胶质细胞内的腺苷激酶的水平，增加内源性腺苷的水平，将有助于通过发挥腺苷的自身抗癫痫作用减轻癫痫发作。
专利CN114159458A公开了一种对癫痫具有治疗作用的四面体框架核酸，但疗效有限，目前还没有基于四面体框架核酸的靶向星型胶质细胞以降低其中腺苷激酶水平，从而达到抗癫痫作用的药物研究报道。
发明内容
本发明的目的在于提供一种能通过降低星型胶质细胞内腺苷激酶水平，从而减轻癫痫发作的药物。
本发明提供了一种治疗癫痫的药物复合物，它是由四面体框架核酸和星型胶质细胞靶向肽AS1复合而成；所述星型胶质细胞靶向肽AS1的氨基酸序列如SEQ ID NO.5所示；
所述四面体框架核酸和星型胶质细胞靶向肽AS1的摩尔比为1～5：200～250。
进一步地，所述四面体框架核酸和星型胶质细胞靶向肽AS1的摩尔比为1：216。
进一步地，所述四面体框架核酸连接有腺苷激酶的反义寡核苷酸。
更进一步地，所述四面体框架核酸的四条DNA单链序列如SEQ ID NO.1～4所示。
本发明还提供了一种前述药物复合物的制备方法，它包括如下步骤：
取四面体框架核酸和星型胶质细胞靶向肽AS1按所述摩尔比混合孵育，即得。
进一步地，所述孵育的温度为20～30℃，时间0.5～3.5h。
更进一步地，所述孵育的温度为25℃，时间1h。
进一步地，所述四面体框架核酸的合成方法为：
将等摩尔比的四条DNA单链加入到TM缓冲液中，95℃维持10min，之后快速降温到4℃维持20min以上，即得。
本发明最后提供了一种前述的药物复合物在制备治疗癫痫的药物中的用途。
本发明在前期研究成果CN114159458A的基础上，意外发现将腺苷激酶的反义寡核苷酸连接在四面体框架核酸特定的单链上，与特定的星型胶质细胞靶向肽AS1复合，能够实现靶向星型胶质细胞，并显著降低星型胶质细胞内的腺苷激酶，增加腺苷含量，减少海马区自发性的癫痫样放电；并通过减少苔藓样纤维的增生和异常的突触连接，减轻癫痫的发作。经实验证明：本发明在降低癫痫发作方面效果显著，具有良好的临床应用前景。
显然，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。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1是基于四面体框架核酸及其单链构建的tFNA-ADKASO和结合星型胶质细胞靶向肽AS1构建的tFNA-ADKASO@AS1药物的PAGE电泳图。
图2是tFNA-ADKASO和tFNA-ADKASO@AS1药物的原子力显微镜图。
图3是tFNA-ADKASO@AS1药物的Zeta电位图。
图4是tFNA-ADKASO@AS1药物的粒径大小图。
图5是通过免疫荧光染色的方法证实AS1赋予了tFNA-ADKASO靶向进入星型胶质细胞的能力(AS1将tFNA-ADKASO将更好地靶向到星型胶质细胞内，实现了tFNA-ADKASO@AS1药物靶向的作用；A-C显示静脉注射tFNA-ADKASO后，tFNA-ADKASO更多地进入到神经元内；D-F显示静脉注射tFNA-ADKASO@AS1后，tFNA-ADKASO@AS1更多地进入到了星型胶质细胞内。G、H为统计对比图；I为tFNA-ADKASO@AS1靶向进入星型胶质细胞的示意图)。
图6各组小鼠海马区星型胶质细胞内腺苷激酶表达结果(A-D显示各组小鼠海马内星型胶质细胞内ADK的荧光表达情况和WB表达情况分析，以及对比统计图；E，F显示LC/MS测量的各组小鼠海马内腺苷E的水平和其代谢产物腺苷激酶F的水平变化；Ctrl：正常组；KA：造模组；KA+tFNA：四面体基础结构处理组；KA+tFNA-ADKASO：携带ADKASO的四面体核酸结构处理组；KA+tFNA-ADKASO@AS1：治疗组)。
图7是各组小鼠海马区自发性癫痫样放电的脑电图和频率统计分析图(A，B各组小鼠海马区自发性癫痫样放电的脑电示意图和统计分析图；Ctrl：正常组；KA：造模组；KA+tFNA：四面体基础结构处理组；KA+tFNA-ADKASO：携带ADKASO的四面体核酸结构处理组；KA+tFNA-ADKASO@AS1：治疗组)。
图8是各组小鼠海马区苔藓纤维染色的荧光图和其长度统计分析图(Ctrl：正常组；KA：造模组；KA+tFNA：四面体基础结构处理组；KA+tFNA-ADKASO：携带ADKASO的四面体核酸结构处理组；KA+tFNA-ADKASO@AS1：治疗组)。
图9是各组小鼠海马区异常突触的电镜图和其数量变化的统计分析图(Ctrl：正常组；KA：造模组；KA+tFNA：四面体基础结构处理组；KA+tFNA-ADKASO：携带ADKASO的四面体核酸结构处理组；KA+tFNA-ADKASO@AS1：治疗组)。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1、tFNA-ADKASO@AS1药物的合成
将四条DNA单链(S1、S2、ADKASO-S3、S4)溶解于TM Buffer(10mM Tris-HCl,50mMMgCl2,pH＝8.0)中，四条DNA单链的终浓度为1μM，充分混合，迅速加热至95℃保持10分钟，之后迅速降温至4℃并维持20分钟以上，即可得到携带有腺苷激酶反义寡核苷酸的四面体框架核酸结构tFNA-ADKASO(经PAGE证实，图1)。
四条单链的序列(SEQ ID NO.1～4)如下：
再与AS1靶向肽(SEQ ID NO.5序列：C-LNSSQPS-C)于室温下孵育1小时(其中四面体框架核酸与AS1靶向肽的摩尔比为：1：216)，而后得到tFNA-ADKASO@AS1药物。合成后的tFNA-ADKASO@AS1药物，原子力显微镜(图2)和粒径(图4)显示其大小约为300nm，可认为tFNA-ADKASO@AS1药物已成功合成，Zeta电位约为0.3mV(图3)。
以下通过实验例证明本发明的有益效果。
实验例1、tFNA-ADKASO@AS1药物对癫痫的治疗效果。
1、实验方法
模型动物：采用kainic acid(KA)诱导ICR小鼠制备癫痫模型，探究tFNA-ADKASO@AS1药物调节星型胶质细胞内腺苷激酶和腺苷的水平治疗癫痫的作用。于KA诱导小鼠癫痫发作后1周，开始经尾静脉注射tFNA-ADKASO@AS1药物(剂量500nM/天)，每间隔24小时注射一次，连续注射14天。对照组分别给予等量生理盐水、单纯四面体(tFNA)以及携带腺苷激酶反义寡核苷酸的四面体(tFNA-ADKASO)进行对照。
相关指标检测：于tFNA-ADKASO@AS1药物注射后14天，取小鼠脑组织切片进行免疫荧光染色，用GFAP标记海马区星型胶质细胞，并用ADK标记星型胶质细胞内的腺苷激酶，统计并分析ADK荧光信号的变化。WB检测小鼠海马内ADK水平的变化。同时，利用高效液相的方法检测海马组织内腺苷和其代谢产物单磷酸腺苷水平的变化，并通过脑电图检测的方式记录并分析比较各组小鼠海马区癫痫波的频率，最后我们分析了小鼠海马内苔藓纤维的增生和异常突触连接的变化。
2、实验结果
(1)免疫荧光染色证实，AS1能更好地将tFNA-ADKASO靶向到星型胶质细胞内，从而实现腺苷激酶的反义寡核苷酸更好进入到星型胶质细胞内的作用(图5)。
(1)免疫荧光和WB结果发现造模组小鼠海马区星型胶质细胞内ADK水平明增高，而治疗组对比其变化，ADK的表达明显减少，说明tFNA-ADKASO@AS1药物降低了海马区星型胶质细胞内的ADK水平。高效液相结果发现tFNA-ADKASO@AS1药物有效增加了海马内腺苷的含量，减少了其代谢产物腺苷激酶的水平(图6)。
(2)EEG记录发现造模组小鼠海马自发性癫痫样放电增多，而治疗组对比其变化，有明显减少的自发性癫痫样放电频率，说明tFNA-ADKASO@AS1药物减少了自发性癫痫样放电的频率，具有很好的减轻癫痫发作的作用(图7)。
(3)免疫荧光发现造模组小鼠海马区苔藓纤维明显的增多，而治疗组对比其变化，有明显减少的苔藓纤维，说明tFNA-ADKASO@AS1药物减轻了海马区苔藓纤维的增生(图8)。
(4)电镜检测发现，小鼠海马组织内异常的突触连接数量明显增多，而治疗组对比其变化，有明显减少的异常突触连接的数量，说明tFNA-ADKASO@AS1药物减轻了海马区异常的突触连接(图9)。
综上，本发明基于四面体框架核酸构建的tFNA-ADKASO@AS1，能够靶向星型胶质细胞，并显著降低星型胶质细胞内的腺苷激酶，增加腺苷含量，减少海马区自发性的癫痫样放电，同时通过减少苔藓样纤维的增生和异常的突触连接，减轻癫痫的发作。在治疗癫痫方面效果优于四面体框架核酸以及携带有腺苷激酶反义寡核苷酸的四面体框架核酸，具备实际推广应用价值。
Claims (9)
1.一种治疗癫痫的药物复合物，其特征在于：它是由四面体框架核酸和星型胶质细胞靶向肽AS1复合而成；所述星型胶质细胞靶向肽AS1的氨基酸序列如SEQ ID NO.5所示；
所述四面体框架核酸和星型胶质细胞靶向肽AS1的摩尔比为1～5：200～250。
2.根据权利要求1所述的药物复合物，其特征在于：所述四面体框架核酸和星型胶质细胞靶向肽AS1的摩尔比为1：216。
3.根据权利要求1或2所述的药物复合物，其特征在于：所述四面体框架核酸连接有腺苷激酶的反义寡核苷酸。
4.根据权利要求3所述的药物复合物，其特征在于：所述四面体框架核酸的四条DNA单链序列如SEQ ID NO.1～4所示。
5.一种权利要求1～4任一项所述药物复合物的制备方法，其特征在于：它包括如下步骤：
取四面体框架核酸和星型胶质细胞靶向肽AS1按所述摩尔比混合孵育，即得。
6.根据权利要求5所述的制备方法，其特征在于：所述孵育的温度为20～30℃，时间0.5～3.5h。
7.根据权利要求6所述的制备方法，其特征在于：所述孵育的温度为25℃，时间1h。
8.根据权利要求7所述的制备方法，其特征在于：所述四面体框架核酸的合成方法为：
将等摩尔比的四条DNA单链加入到TM缓冲液中，95℃维持10min，之后快速降温到4℃维持20min以上，即得。
9.权利要求1～4任一项所述的药物复合物在制备治疗癫痫的药物中的用途。
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