CN116726005A - 一种治疗运动损伤的药物及筛选方法和应用 - Google Patents
一种治疗运动损伤的药物及筛选方法和应用 Download PDFInfo
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Abstract
本发明涉及一种治疗运动损伤的药物及其应用,一种肌腱损伤修复动物模型的构建方法,所述治疗运动损伤是治疗急性损伤和慢性损伤中的至少一种,优选的,所述治疗运动损伤是治疗肌腱损伤,更优选的,所述治疗肌腱损伤是促进肌腱愈合和/或抑制肌腱粘连形成。本发明的优点(1)药物筛选效果好。(2)筛选的药物能够防治肌腱粘连并且对腱愈合影响较小。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种治疗运动损伤的药物及其应用。
背景技术
运动损伤在日常生活及运动领域十分常见,尤其是在运动医学领域。其中,最为常见的为肌腱损伤。肌腱粘连是肌腱损伤及其修复术后的常见并发症,发病率可达60%,严重影响患者的肢体活动,甚至导致终生残疾。肌腱损伤后,如何减术后的粘连一直是临床上一个重要问题。
目前,肌腱粘连的治疗常常需要通过手术松解、切除粘连组织,但是术后仍有再次粘连的可能,进而形成“粘连、松解、再粘连”的恶性循环。一般认为,肌腱愈合包括内源性愈合和外源性愈合两种途径。治疗中,常希望促进内源性愈合,抑制外源性愈合。在松解治疗过程中,应用生物材料制成的防粘连膜包裹手术修复的肌腱,可以起到物理阻隔的作用,防治肌腱粘连;但是目前的生物材料防粘连膜作用单一、疗效较差且易引发炎症,限制了其临床应用。而且,临床缺乏相关的防粘连治疗药物。在肌腱损伤后愈合过程中,外源性愈合主要是腱周组织长入肌腱或者在肌腱表面,形成肉芽组织,帮助肌腱修复;内源性愈合是在充分的滑液营养下,肌腱细胞/肌腱干细胞本身的增生,并通过合成和分泌胶原蛋白进行自我修复。其中外源性愈合是导致肌腱粘连的主要原因。现有的防粘连膜和药物都存在其局限性,因此迫切需要特异性强的新药物以及可以发现新药物的有效手段。
在药物的应用上,目前应用于肌腱抗粘连的药物主要有三种。一是抗炎药物,例如布洛芬、吲哚美辛和透明质酸。这些药物作用于肌腱愈合的炎症期。二是抗肿瘤药,如甘露糖-6-磷酸和5-氟尿嘧啶。三是抗纤维化药物,如胰蛋白酶抑制剂。后面两种药物具有抑制成纤维细胞成长分化的作用,从而减轻肌腱粘连。
目前对于既能抑制外源性愈合又不会抑制内源性愈合的药物的需求仍然是巨大的,而且一直缺乏一种有效对这种理想药物进行寻找的方式。
发明内容
本发明的目的在于提供一种能治疗运动损伤的药物,所述治疗运动损伤是治疗急性损伤和慢性损伤中的至少一种,优选的,所述治疗运动损伤是治疗肌腱损伤,更优选的,所述治疗肌腱损伤是促进肌腱愈合和/或抑制肌腱粘连形成。
为实现上述目的,本发明采用的技术方案为:
式1化合物或或其药学上可接受的盐的用途,其特征在于,用于制备治疗促进肌腱愈合且能够抑制肌腱粘连形成的药物组合物或制剂中的用途。
R1-R4独立的选自取代或非取代的H、卤素、CN、OH、NH2、烷基、烷烯基、烷炔基、烷氧基、环烷基、杂环烷基、芳基、杂芳基,
R5、R7独立的选自取代或非取代的H、卤素、CN、OH、NH2、C1-C6的烷基,R5、R7任选可被一个或多个任选如下取代基取代烷基、烷烯基、烷炔基、烷氧基、环烷基,杂环烷基、芳基、杂芳基,
R6选自取代或非取代的烷基、烷烯基、烷炔基、-(CH)n-COOH、-(CH)n-COO-R10、-(CH)n-CO-R10,-(CH)n-CO-NH-R10、-(CH)n-O-CO-NH-R10、-(CH)n-CO-NR10R11、-(CH)n-O-CO-NR10R11、-(CH)n-O-CO-R10、-(CH)n-O-CO-NR10R11、-(CH)n-O-COO-R10、-C(=O)SR10、-C(=S)-R10,
R8-R9独立的选自C、N、O、S,
n选自1-12,
R10、R11独立的选自取代或非取代的C1-C10的烷基、取代或非取代的C3-C10的环烷基、杂环烷基、芳基、杂芳基,
上述取代基可被一个或多个任选如下取代基取代烷基、烷烯基、烷炔基、烷氧基、环烷基,杂环烷基、芳基、杂芳基。
所述式I化合物为
所述奥兰替尼的英文名为Orantinib,别名为苏6668,SU 6668,SU6668,TSU-68,CAS号为210644-62-5。
另一个技术方案,噻克索酮或其药学上可接受的盐的用途,其特征在于,用于制备治疗运动损伤制备治疗运动损伤药物组合物或制剂中的用途。
所述噻克索酮的英文名为tioxolone,CAS号为4991-65-5。
进一步的,所述治疗运动损伤是治疗急性损伤和慢性损伤中的至少一种,优选的,所述治疗运动损伤是治疗肌腱损伤,更优选的,所述治疗肌腱损伤是促进肌腱愈合和/或抑制肌腱粘连形成。
所述的药物组合物或药剂中含有0.001-99wt%的式I化合物或其药学上可接受的盐,按组合物的总重量计。
所述的药物组合物或药剂的剂型为口服剂型、或注射剂。
所述口服剂型包括片剂、胶囊剂、膜剂、颗粒剂。
所述的药物组合物或药剂中中还含有药学上可接受的载体。
所述的药物组合物中还含有其他促进肌腱愈合和/或能够抑制肌腱粘连形成的活性成分。
本发明基于肌腱损伤修复动物模型,利用原代细胞培养和流式细胞仪技术提取肌腱干细胞和肌成纤维细胞、并用于药物筛选,所筛选的噻克索酮和奥兰替尼能够防治肌腱粘连并且对腱愈合影响较小。
附图说明
图1是本发明提供的大鼠肌腱粘连的构建示意图。
图2是本发明提供的流式分选肌成纤维细胞结果。
图3是本发明提供的肌腱干细胞的鉴定结果。
图4是药物的筛选结果。
图5是小鼠造模后14天,未治疗(wt)与治疗组(medicine)HE染色结果。
图6是小鼠造模后14天,未治疗(wt)与治疗组(medicine)一型胶原免疫荧光染色结果。
图7是小鼠造模后14天,未治疗(wt)与治疗组(medicine)scx免疫荧光染色结果。
图8是小鼠造模后14天,未治疗(wt)与治疗组(medicine)三型胶原免疫荧光染色结果。
图9是小鼠造模后14天,未治疗(wt)与治疗组(medicine)sma免疫荧光染色结果。
具体实施方式
为使本领域的技术人员更好地理解本发明的技术方案,以下实施例对本发明的作进一步详细描述,以下实施例仅用于说明发明,但不用来限制本发明的范围。
实施例1构建大鼠肌腱损伤修复动物模型
所有动物研究方案均由上海交通大学机构审查委员会批准(SYXK(Hu)2016-0020)。250g~300g的大鼠腹腔注射戊巴比妥麻醉,然后固定并备皮。在跟腱处做一纵行切口,暴露跟腱。分离腱周组织,横向切断跟腱。用6-0缝线,使用改良Kessler缝合法缝合跟腱。4-0缝线缝合皮肤并消毒。
如图1A所示,经过麻醉并备皮、暴露并分离跟腱、剪断跟腱、缝合跟腱和缝皮并消毒五个步骤,构建大鼠跟腱粘连的模型。并在术后10天切开跟腱处皮肤,可见跟腱周围附着的肉芽组织和粘连组织,并需要用锐器才可与腱周分离(图1B)。
实施例2肌腱干细胞和肌成纤维细胞用于药物筛选
术后十天处死大鼠并获取其后肢。75%酒精消毒1h后,在超净台内切开跟腱处皮肤并暴露跟腱。小心的剪下跟腱周围新生的粘连组织,置于3mg/ml的Ⅰ型胶原酶内在37°摇床内消化3h。消化结束后离心弃去上层液体,将下层细胞重悬接种于10cm培养皿中,置于37℃培养箱内培养,二氧化碳浓度5%。
在细胞处于对数生长期后,利用流式分选筛选出α-SMA阳性的细胞并继续培养。简而言之,吸弃原培养基,1ml PBS冲洗细胞一次。加入胰酶消化,离心弃上清。加入2ml PBS重悬细胞,离心弃上清。加入500μl染色缓冲液重悬细胞,进行细胞计数,将细胞浓度调整至107/ml。加入α-SMA流式抗体,吹打混匀细胞悬液,避光孵育15min。加入2ml PBS离心5min,重复洗涤两次。上机,利用流式细胞仪分选出α-SMA阳性的细胞。将肌成纤维细胞接种于10cm培养皿中,置于37°培养箱内培养,二氧化碳浓度5%。
获取正常大鼠后肢,75%酒精消毒1h。在超净台内切开跟腱处皮肤并暴露跟腱,小心的将跟腱取下并剪碎至1mm*1mm*1mm大小,注意不要将肌肉和腱周组织混入。将剪碎的肌腱组织置于4mg/ml的Ⅰ型胶原酶内在37℃摇床内消化4h。消化结束后离心弃去上层液体,将下层细胞重悬接种于10cm培养皿中,置于37℃培养箱内培养,二氧化碳浓度5%。
利用免疫荧光对肌腱干细胞进行鉴定。PBS浸洗细胞3次,每次3min。用4%的多聚甲醛固定15min,PBS浸洗3次,每次3min。0.5%Triton X-100室温通透20min,PBS浸洗3次,每次3min。在玻片上滴加封闭液,室温封闭30min。吸掉封闭液,每张玻片滴加足够量的稀释好的ColⅠ和Scx一抗并放入湿盒,4℃孵育过夜。第二天PBST浸洗3次,每次3min,滴加稀释好的荧光二抗,湿盒中37℃孵育1h,PBST浸洗3次,每次3min。滴加DAPI避光孵育5min,PBST浸洗3次。用含抗荧光淬灭剂的封片液进行封片,然后在荧光显微镜下观察采集图像。
利用流式分选仪对预染抗体的原代粘连组织细胞进行分选。可见α-SMA阳性的细胞,即肌成纤维细胞,占总粘连组织细胞的比例为1.30%(图2)。将目标细胞收集、培养和扩增,供后续药物筛选使用。
用肌腱干细胞的特异性标志物Scx和ColⅠ对提取的原代肌腱干细胞进行鉴定。DAPI为蓝色荧光,Scx为绿色荧光,ColⅠ为红色荧光,可见Scx和ColⅠ均在胞内有表达(图3)。代表从肌腱中提取的原代细胞确实为肌腱干细胞,可用作后续药物筛选实验。
实施例3药物筛选及药物筛选分析
利用Multidrop自动分液器将生长状态良好的肌腱干细胞和肌成纤维细胞均匀地种到透明384孔U底板中,每个孔中细胞数为400个,每个孔中为100μL的完全DMEM培养基。接着在种好细胞的384孔板中,用JANUS自动化移液工作站加入化合物库中的小分子。控制筛选的化合物终浓度为10μM。完成加药之后将细胞板放入37℃二氧化碳细胞培养箱中,孵育3天。药物处理完毕后加入cell proof溶液,显色,EnSpire酶标仪检测结果。
药物筛选的部分结果以热图的形式表示(图4)。以加入了DMSO的细胞进行对照,相对抑制率高于50%用红色绘制,相对抑制率低于50%用蓝色绘制。可见L-抗坏血酸对两种细胞的相对抑制率均低于50%,而另外四种药物WAY-600、GSK1070916、伊斯平斯和SRT1720,对于两种细胞的相对抑制率均高于50%。其中噻克索酮对于肌成纤维细胞的相对抑制率高于50%,而对肌腱干细胞的相对抑制率低于50%,可作为一种潜在的理想药物,其中奥兰替尼对于肌成纤维细胞的相对抑制率为57%,而对肌腱干细胞的相对增值率是15%,因此,奥兰替尼可作为一种潜在的理想药物,既可抑制肌腱粘连的形成又不影响肌腱的愈合。
实施例4经典小鼠粘连模型的构建
小鼠2.5%avertin0.2ml麻醉,将其趾深屈肌腱近端剪断,背侧朝上将其脚底显示在体视显微镜的视野下,正中切开皮肤,钝性分离并完全暴露趾深屈肌腱,切断肌腱后使用8-0缝线、kessler缝合术缝合肌腱,用6-0缝线缝合皮肤,自由饲养,不外加固定。
实施例5利用经典小鼠粘连模型实验
我们对细胞筛选结果更优的奥兰替尼,进行了经典小鼠粘连模型实验。
如图5所示,小鼠造模后14天,未治疗(wt)与治疗组(medicine)HE染色。▲表示粘连组织,虚线圈画的部分为造模所在区域,T代表肌腱。根据粘连半定量评分,治疗后肌腱周围粘连减少,t=5.814df=6,**P<0.01。标尺:1cm。肌腱整体的连续性也优于未治疗组。
如图6所示,小鼠造模后14天,未治疗(wt)与治疗组(medicine)一型胶原免疫荧光染色。T代表肌腱。可见治疗组中肌腱的一型胶原表达更多,但没有统计学意义。标尺:500um
如图7所示,小鼠造模后14天,未治疗(wt)与治疗组(medicine)scx免疫荧光染色。T代表肌腱。可见治疗组中肌腱的SCX表达更多,但没有统计学意义。标尺:500um
如图8所示,小鼠造模后14天,未治疗(wt)与治疗组(medicine)三型胶原免疫荧光染色。T代表肌腱,M代表肌肉。治疗组三型胶原表达减少,但没有统计学意义。标尺:500um
如图9所示,小鼠造模后14天,未治疗(wt)与治疗组(medicine)sma免疫荧光染色。T代表肌腱,M代表肌肉。治疗组sma表达减少,但没有统计学意义。标尺:500um
结果表明,奥兰替尼是一种非常理想的治疗肌腱损伤的药物,其能够促进肌腱愈合的同时抑制肌腱粘连形成。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种变换,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征和步骤,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (10)
1.式1化合物或或其药学上可接受的盐的用途,其特征在于,用于制备治疗运动损伤组合物或制剂中的用途,
R1-R4独立的选自取代或非取代的H、卤素、CN、OH、NH2、烷基、烷烯基、烷炔基、烷氧基、环烷基、杂环烷基、芳基、杂芳基,
R5、R7独立的选自取代或非取代的H、卤素、CN、OH、NH2、C1-C6的烷基,R5、R7任选可被一个或多个任选如下取代基取代烷基、烷烯基、烷炔基、烷氧基、环烷基,杂环烷基、芳基、杂芳基,
R6选自取代或非取代的烷基、烷烯基、烷炔基、-(CH)n-COOH、-(CH)n-COO-R10、-(CH)n-CO-R10,-(CH)n-CO-NH-R10、-(CH)n-O-CO-NH-R10、-(CH)n-CO-NR10R11、-(CH)n-O-CO-NR10R11、-(CH)n-O-CO-R10、-(CH)n-O-CO-NR10R11、-(CH)n-O-COO-R10、-C(=O)SR10、-C(=S)-R10,
R8-R9独立的选自C、N、O、S,
n选自1-12,
R10、R11独立的选自取代或非取代的C1-C10的烷基、取代或非取代的C3-C10的环烷基、杂环烷基、芳基、杂芳基,
上述取代基可被一个或多个任选如下取代基取代烷基、烷烯基、烷炔基、烷氧基、环烷基,杂环烷基、芳基、杂芳基。
2.如权利要求1所述用途,所述式I化合物为奥兰替尼,其结构如下式所示:
3.如权利要求1或2所述用途,其中药物组合物或药剂中含有0.001-99wt%的式I化合物或其药学上可接受的盐,按组合物的总重量计。
4.噻克索酮或其药学上可接受的盐的用途,其特征在于,用于制备治疗运动损伤制备治疗运动损伤药物组合物或制剂中的用途。
5.如权利要求1至4任意一项所述用途,其特征在于所述治疗运动损伤是治疗急性损伤和慢性损伤中的至少一种,优选的,所述治疗运动损伤是治疗肌腱损伤,更优选的,所述治疗肌腱损伤是促进肌腱愈合和/或抑制肌腱粘连形成。
6.如权利要求1至5任意一项所述用途,其中所述的药物组合物或药剂的剂型为口服剂型、或注射剂,优选的所述口服剂型包括片剂、胶囊剂、膜剂、颗粒剂。
7.如权利要求6所述用途,其中所述的药物组合物或药剂中中还含有药学上可接受的载体。
8.如权利要求7所述用途,其中所述的药物组合物中还含有其他促进肌腱愈合和/或能够抑制肌腱粘连形成的活性成分。
9.肌腱干细胞和肌成纤维细胞在筛选肌腱修复和/或防粘连药物中的用途。
10.如权利要求9所述用途,其中所述用途为筛选肌腱修复和防粘连药物,其中待筛选的药物对于肌成纤维细胞的相对抑制率高于50%,而对肌腱干细胞的相对抑制率低于50%,可作为一种潜在的理想药物。
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