CN110354124A - 奥美拉唑Omeprazole在制备治疗多发性硬化疾病药物中的用途 - Google Patents
奥美拉唑Omeprazole在制备治疗多发性硬化疾病药物中的用途 Download PDFInfo
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- CN110354124A CN110354124A CN201810321915.0A CN201810321915A CN110354124A CN 110354124 A CN110354124 A CN 110354124A CN 201810321915 A CN201810321915 A CN 201810321915A CN 110354124 A CN110354124 A CN 110354124A
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Abstract
本发明属医药技术领域,涉及质子泵抑制剂奥美拉唑omeprazole的新的药用用途,特别涉及Omeprazole用于制备治疗多发性硬化疾病的药物中的用途。本发明所述的Omeprazole具有式(1)的结构,经生物信息学大数据挖掘、动物在体实验及细胞离体实验验证,证实Omeprazole可以直接促进离体培养的OPC分化成熟,在多发性硬化动物模型中促进髓鞘再生并改善动物行为学表现,可进一步用于制备治疗多发性硬化疾病的药物,具有极大的临床应用前景。
Description
技术领域
本发明属医药技术领域,涉及质子泵抑制剂奥美拉唑omeprazole的新的药用用途,特别涉及Omeprazole用于制备治疗多发性硬化疾病的药物中的用途。
背景技术
现有技术研究提示,多发性硬化(Multiple Sclerosis,MS)是由免疫介导的中枢神经系统(Central Nervous System,CNS)脱髓鞘疾病,其常见的临床症状为视力障碍、感觉异常、肢体无力、共济失调、中痛性痉挛等,严重的影响患者的日常生活、自理以及工作,给患者及其家庭带来极大痛苦。由于该疾病常首发于20-40岁的中青年人,是导致中青年残障的最重要疾病之一,可造成终身残疾及日常生活能力障碍,许多患者常年饱受该疾病的痛苦,常出现抑郁倾向及自杀倾向,因此有着极大的危害性。据统计,目前全球约有超过250万的MS患者,欧美发达国家的发病率较高,在我国,由于MS症状多样且起病较隐蔽,不少患者常被误诊为脑中风,而近年来随着人们对MS疾病认识的加深以及诊疗水平的提高,MS确诊率呈现出明显的逐年上升趋势[非专利文献1]。
MS的发病机制目前尚未阐明,有研究认为MS的发病与遗传易感性、基因突变、环境因素以及饮食等因素有关。MS是由免疫介导的CNS脱髓鞘疾病,主要有以下特征:1)人体免疫系统针对CNS髓鞘抗原产生大量自身反应性淋巴细胞,包括T淋巴细胞和B淋巴细胞等;2)外周免疫细胞透过血脑屏障进入CNS,引起CNS炎症反应;3)少突胶质细胞数量大量坏死,CNS髓鞘降解、脱落,产生大量髓鞘碎片并伴随轴突损伤;4)CNS胶质细胞的大量激活。研究证实,CNS髓鞘是由少突胶质细胞包裹轴突形成的重要结构,起到保护轴突、维持动作电位“跳跃式”传导的作用[非专利文献2];在MS病人中,免疫应答参与了CNS的髓鞘脱失,而髓鞘的脱失以及继发的神经元轴突损伤是MS病人出现临床神经功能受损的根本原因[非专利文献3]。
目前,关于MS的治疗,国际主流推荐采用的是疾病修饰疗法(Disease ModifyingTherapy,DMT),即针对免疫反应过程中的不同靶点,运用各类免疫调节制剂,预防疾病的发作、延缓疾病的复发,包括急性发作期的对症治疗和慢性迁延期的预防复发治疗。临床上常用的DMT有甲氨蝶呤、环磷酰胺、硫唑嘌呤以及糖皮质激素类药物等,尽管上述药物能在一定程度上延缓疾病发作,但有较大的不良反应和副作用[非专利文献4]。据了解,目前较新研制的MS治疗方法还有β-干扰素疗法和T细胞免疫疗法,但其仍需进一步临床试验评估其疗效及安全性,且其价格昂贵,普通民众难以承受[非专利文献5]。此外,更为重要的是,上述疗法主要针对的是免疫调节,仅能延缓疾病的发作和恶化,无法从根本上改善髓鞘及轴突损伤,因而无法帮助患者恢复受损的神经功能,而促进髓鞘的再生、恢复髓鞘功能是改善MS患者临床症状的关键。因此,寻找能促进髓鞘再生的疗法,成为了当前多发性硬化相关领域研究的新热点,国内外的研究人员均正在挖掘能够促进髓鞘再生的潜在疗法及药物[非专利文献6]。
Omeprazole为质子泵抑制剂,在临床实践中广泛用于治疗胃-食管反流病、消化道溃疡性疾病和佐格林-埃利森综合征(Zollinger Ellison syndrome);且Omeprazole不良反应及副作用小、价格低廉,易被患者接受[非专利文献7]。
综观国内外的研究,均未见Omeprazole及其他质子泵抑制剂在防治多发性硬化及促进髓鞘再生方面的药效及其应用报道。
基于现有技术的研究基础,本申请的发明人拟提供质子泵抑制剂奥美拉唑omeprazole新的药用用途,尤其是Omeprazole用于制备治疗多发性硬化疾病的药物中的用途。
与本发明有关的现有技术有:
[非专利文献1]Filippi M.Nat Rev Neurol.7(2):74-5,2011.
[非专利文献2]Mallucci G,et al.Prog Neurobiol.0:1-22,2015.
[非专利文献3]Verden D,et al.J Neurosci Res.00:00-00,2016.
[非专利文献4]Winkelmann A,et al.Nat Rev Neurol.12(4):217-33,2016.
[非专利文献5]O’Brein K,et al.Immunotherapy.2(1):99-115,2010.
[非专利文献6]Harlow DE,et al.FrontNeurol.6:257,2015.
[非专利文献7]Walan A.Scand J Gastroenterol Suppl.166:140-4,1989.。
发明内容:
本发明的目的在于,基于现有技术的研究基础,提供质子泵抑制剂奥美拉唑omeprazole新的药用用途,尤其是Omeprazole用于制备治疗多发性硬化疾病的药物中的用途。
本发明利用生物信息学大数据分析,结合基因测序技术,将髓鞘再生时的基因表达谱与Connectivity-Map药物-基因表达谱数据库进行匹配,研究结果显示Omeprazole具有潜在的促进髓鞘再生的作用,可进一步用于制备治疗多发性硬化疾病的药物中的用途。
本发明所述Omeprazole为质子泵抑制剂,化学名称为:5-甲氧基-2-[[(4-甲氧基-3,5-二甲基-2-吡啶基)甲基]亚磺酰基]-1H-苯并咪唑,分子式:C17H19N3O3S,分子量:345.42,具有式(1)的结构:
本发明基于生物信息学大数据分析所得结果,进行了离体细胞实验及整体动物模型实验,结果表明所述的Omeprazole可以促进少突胶质细胞系的分化成熟,在脱髓鞘动物模型中能促进髓鞘再生并且改善脱髓鞘病变的神经行为功能,在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型中改善小鼠临床神经功能评分。
本发明进行了如下实验:
1)(细胞实验)Omeprazole用于促进少突胶质细胞系分化、成熟的实验:
按常规方法,从SD新生鼠中分离培养少突胶质前体细胞(OPC),以DMSO处理作为对照组,以Omeprazole(浓度:2.5/5/10/20μM)处理作为实验组;每隔1天进行换液及给药,培养6天后,采用免疫细胞荧光染色、western blot及实时定量PCR技术观察少突胶质细胞分化成熟及髓鞘相关蛋白和基因的表达变化;
结果表明,Omeprazole可以促进少突胶质细胞系的分化成熟;
2)(整体实验)Omeprazole用于脱髓鞘动物模型中促进髓鞘再生的实验:
按常规方法,选取5-6周大的C57BL/6雄性小鼠,喂以含有0.2%Cuprizone药粉的饲料5周,造成小鼠CNS广泛的脱髓鞘;再以正常饲料喂养1周,令其自然髓鞘再生;总共持续6周时间;Omeprazole(5mg/kg,10mg/kg,腹腔注射;对照组采用生理盐水注射)治疗从第4周结束后开始,每天注射1次,持续2周,直至第6周结束;每周进行神经功能评定(平衡木实验);实验结束时,采用免疫组织荧光染色、western blot和透射电镜观察胼胝体部位髓鞘超微结构以及髓鞘相关蛋白的表达;
结果表明,Omeprazole在脱髓鞘动物模型中能促进髓鞘的再生并且改善脱髓鞘病变的神经行为功能;
3)(整体实验)Omeprazole在实验性自身免疫性脑脊髓炎(EAE)动物模型中改善临床神经功能的实验:
按常规方法,选取雌性C57BL/6小鼠,以少突胶质细胞糖蛋白相关肽(MOG35-55)免疫,建立EAE模型,Omeprazole从造模当天开始腹腔注射给药,每天进行模型评分,观察临床神经功能表现;
结果表明,Omeprazole在EAE模型中能显著改善小鼠的临床神经功能评分,对EAE模型小鼠有显著防治作用。
上述细胞实验和整体实验表明,本发明的质子泵抑制剂Omeprazole能促进少突胶质细胞系的分化成熟,在脱髓鞘动物模型中能够发挥治疗作用,促进髓鞘的再生,改善脱髓鞘小鼠的协调运动功能及EAE小鼠的临床神经功能。所述的Omeprazole可进一步用于制备治疗多发性硬化疾病的药物。
为了便于理解,下面通过附图和具体实施例方式对发明的Omeprazole在多发性硬化疾病中促进髓鞘再生的应用进行详细的描述。需要特别指出的是,附图和具体实施例仅是为了说明,显然本领域的技术人员可以根据本文说明,对本发明进行各种修正或改变,这些修正和改变也将纳入本发明范围之内。
附图说明
图1为DMSO处理和不同浓度Omeprazole处理OPC 6天后对OPC分化的影响,其中,
A.与DMSO对照组相比,Omeprazole能显著促进OPC向成熟少突胶质细胞的分化;B.Omeprazole促进OPC分化的效应与给药浓度呈正相关,10μM和20μM浓度给药差异具有显著性;C.单一浓度(10μM)Omeprazole给药,随着处理时间累加,OPC分化的程度也逐渐提高。红色标记的是髓鞘碱性蛋白(Myelin Basic Protein,MBP),是成熟少突胶质细胞及髓鞘的标志物;蓝色标记的是DAPI,是细胞核的标志物。
图2为DMSO处理和Omeprazole处理6天后对少突胶质细胞成熟的影响,其中,
A.不同放大倍数下免疫荧光染色所呈现的细胞形态,可见10μM Omeprazole处理后少突胶质细胞的网状分枝显著增多;B.为A中免疫荧光染色的统计,Omeprazole处理后MBP阳性的细胞占比显著增多;C,D根据细胞网状分枝形态,将少突胶质细胞分为成熟早期、中等成熟期和成熟期三期,Omeprazole治疗显著增加中等成熟期和成熟期少突胶质细胞的比例。
图3为DMSO和Omeprazole分别处理OPC 6天后,其中,
A.Omeprazole显著促进MBP的表达;B.Omeprazole显著提升髓鞘相关基因的表达。
图4为Cuprizone诱导的小鼠脱髓鞘模型中神经功能的行为学评估,采用平衡木实验观察小鼠的协调运动功能,其中,
与生理盐水对照组相比,Omeprazole治疗能改善小鼠协调运动功能,且高浓度(10mg/kg)给药的治疗效果优于低浓度(5mg/kg)给药。
图5免疫荧光染色(A,B)和Western Blot可见与生理盐水对照组相比,Omeprazole能改善脱髓鞘小鼠髓鞘结构完整性,提高小鼠胼胝体部位MBP的表达。
图6透射电镜观察脱髓鞘小鼠胼胝体部位髓鞘超微结构,其中,
生理盐水对照组中可见许多脱髓鞘的轴突及坏死水肿的轴突;Omeprazole治疗显著提升有髓轴突比例,坏死轴突数量明显减少。
图7为实验性自身免疫性脑脊髓炎(EAE)动物模型神经功能评分,
其中显示Omeprazole治疗可以显著抑制EAE小鼠的发病。
具体实施方式
实施例1:
1.细胞来源:原代细胞取自刚出生1日的SD新生鼠大脑皮质,共5只;
2.收集培养大脑皮质细胞:
将新生鼠置于冰上低温麻醉、酒精消毒后,于超净台下断头,取出大脑置含有HBSS的细胞培养皿内,于解剖显微镜下剥离脑膜,分离海马、丘脑等组织后,将剩下的大脑皮质移至含有DMEM的细胞培养皿内;待所有新生鼠大脑皮层取材结束后,利用巴氏吸管吹打至均质;通过细胞筛网滤过后离心收集细胞,重悬于DMEM20S培养基内(含4mM谷氨酸盐、1mM丙酮酸钠、20%胎牛血清和双抗的DMEM),以1×105细胞密度种植于T75细胞培养瓶内,并将其置于37°细胞培养箱内培养,每隔3天换液;
3.分离OPC细胞:
原代大脑皮层细胞培养10天后,将T75细胞培养瓶置于细胞摇床上,以200rpm的转速在37°下震荡摇脱2-4h,吸取细胞培养液,以去除小胶质细胞;换液后继续在细胞摇床上震荡摇脱过夜(约18h),离心收集细胞,加入OPC培养液(含有4mM谷氨酸盐、1mM丙酮酸钠、0.1%牛血清蛋白、50ug/ml脱铁运铁蛋白、5ug/ml胰岛素、30nM亚硒酸钠、10nM生物素D、10nM氢化可的松、10ng/ml PDGF-AA和10ng/ml bFGF的DMEM),以1×105细胞密度种植于细胞培养皿内;
4.分组和给药:
1)对照组:将DMSO加入到OPC培养基中处理细胞,每隔1天处理1次,持续6天;
2)Omeprazole处理组:将不同浓度Omeprazole(2.5/5/10/20μM)加入到OPC培养基中处理细胞,每隔1天处理1次,持续6天;
5.实验后处理:
1)免疫细胞荧光:
细胞处理结束后,固定细胞,封闭后孵育一抗(MBP、DAPI)后,充分洗去一抗后标记二抗(红色荧光标记MBP、蓝色荧光标记DAPI),于显微镜下观察OPC细胞分化成熟情况;
2)检测髓鞘主要成分蛋白(MBP)表达:
处理细胞6天后,收集细胞,提取细胞蛋白,进行Western Blot实验检测所得细胞MBP的表达;
3)检测髓鞘相关基因表达:
处理细胞6天后,收集细胞,提取细胞RNA,用Real-Time PCR检测细胞内髓鞘相关基因的表达;
6.数据分析:
采用Graphad Prism 5.0软件进行分析,若P<0.05认为差异有显著性;
7.结果显示:
1)MBP是成熟少突胶质胶质细胞的标志物,与DMSO对照组相比,Omeprazole处理后MBP阳性的细胞数显著增多,提示Omeprazole可以促进OPC的分化,其作用呈浓度依赖性,10μM和20μM浓度Omeprazole处理与对照组相比差异具有显著性(***P<0.001,与DMSO对照组相比),单一浓度Omeprazole处理细胞可出现时间累加效应(如图1所示);依据少突胶质细胞的网状分枝形态,可将少突胶质细胞分为成熟早期、成熟中期和成熟期,Omeprazole处理显著提高成熟中期和成熟期少突胶质细胞的比例(如图2所示;*P<0.05,***P<0.001,与DMSO对照组相比),提示Omeprazole可以促进少突胶质细胞的成熟;
2)Western Blot结果提示,Omeprazole明显提升髓鞘成分蛋白(MBP)的表达(如图3所示);
3)Real-Time PCR结果提示,Omeprazole明显促进髓鞘相关基因MBP、CNP、PLP和MAG的表达(如图3所示);
实验结果表明:Omeprazole可以促进少突胶质细胞系的分化成熟。
实施例2:
1.实验动物:采用SPF级5-6周大的C57/BL6雄性小鼠48只,体重16-18g,来源于中科院实验动物中心;
2.Cuprizone脱髓鞘模型制备:从南通特罗菲饲料有限公司订购粉末状饲料,加0.2%Cuprizone药粉(C9012,Sigma),加80%的双蒸水,充分混合饲料后,制作成圆柱状。用含有Cuprizone的饲料喂养小鼠5周时间,第5周结束后改用普通饲料(不含Cuprizone)再喂养小鼠1周;
3.分组和给药:
1)正常组(Normal);
2)模型对照组(Cuprizone+溶剂对照组):溶剂对照为含有0.1%DMSO的丙三醇溶液(因Omeprazole难溶于水),从Cuprizone饲料喂养4周结束后(造模后第29天)开始腹腔注射给药,0.2ml/只/天,共给药2周;
3)模型+低剂量Omeprazole(5mg/kg):从Cuprizone饲料喂养4周结束后(造模后第29天)开始腹腔注射给药,0.2ml/只/天,共治疗2周;
4)模型+高剂量Omeprazole(10mg/kg):从Cuprizone饲料喂养4周结束后(造模后第29天)开始腹腔注射给药,0.2ml/只/天,共治疗2周;
4.实验后处理:
1)平衡木实验:
将一根长1米、宽1.5厘米的木条悬空置于离水平台面60厘米高的架子上,架子的一端放有一个密闭的纸盒,内含木屑垫料,称之为安全盒;检测小鼠从木条的一端穿过木条到达安全盒端的时间;为尽量减少小鼠行为的个体差异,仅记录小鼠通过木条中间80厘米的用时,并且每次检测前先将小鼠放入到安全盒内适应环境1分钟后再进行实验。每周进行一次实验,每只小鼠测试2次,取平均值;
2)免疫组织化学染色:
灌流固定后将小鼠大脑取出,制备冰冻切片,通过封闭、一抗孵育、洗涤、二抗孵育、洗涤等步骤后,利用激光共聚焦显微镜进行镜下观察;
3)检测髓鞘主要成分蛋白(MBP)表达:
实验结束后,用戊巴比妥钠(75mg/kg,腹腔注射)麻醉小鼠,分离胼胝体组织,提取组织蛋白,进行Western Blot实验检测组织内MBP的表达;
4)透射电镜:
实验结束后,用戊巴比妥钠(75mg/kg,腹腔注射)麻醉小鼠,分离胼胝体组织,放入电镜固定液中固定,经过漂洗、后固定、漂洗、脱水、临界点干燥、镀膜等步骤后,放入扫描电镜观察;
5.数据分析:
采用Graphad Prism 5.0软件进行分析,若P<0.05认为差异有显著性。
6.结果显示:
1)平衡木实验提示,2周的Omeprazole腹腔注射可以改善小鼠的运动协调行为功能,高剂量(10mg/kg)给药的疗效与对照组相比差异具有显著性(如图4所示;每组n=10-12,***P<0.001,与模型对照组相比);
2)免疫荧光染色结果提示,10mg/kg Omeprazole腹腔注射治疗2周可以提高胼胝体内MBP的表达(如图5所示;每组n=3-4,**P<0.01,与模型对照组相比);Western Blot结果提示10mg/kg Omeprazole治疗可以提高MBP的蛋白表达;
3)透射电镜结果提示:Omeprazole治疗能提高有髓轴突的数量,减少轴突坏死、水肿(如图6所示);
实验结果表明:Omeprazole在脱髓鞘动物模型中能促进髓鞘的再生并且改善脱髓鞘病变的神经行为功能。
实施例3:
1.实验动物:采用SPF级雌性C57BL/6小鼠,共32只,8周龄,体重18-20g(中科院实验动物中心),饲养条件为清洁级,3-4只/笼,任意供水;试验期间3级以上瘫痪及濒危小鼠予以单笼饲养,鼠粮用饲水浸湿后喂给;实验动物用完全随机法分组;
2.EAE模型制备:
1)乳化剂准备:将少突胶质细胞糖蛋白多肽片段-MOG35-55(上海华大天源公司人工合成,HPLC级别,纯度95%),完全弗氏佐剂(Complete Freund’s Adjuvant,CFA,Sigma公司,F5881),卡介苗(60mg/支,北京生物制品研究所)充分乳化配成白色黏稠的油包水剂;每只老鼠需要0.2ml乳化剂,其中含有300μl MOG35-55+CFA 5mg/ml+10mg/ml卡介苗;
2)小鼠麻醉:开放吸入5%~8%异氟烷麻醉,注射乳化剂过程中通过面罩吸入异氟烷维持麻醉;
3)模型诱导:第0天,用0.1ml注射器吸取弗氏完全佐剂(CFA)乳化的抗原0.2ml,背部皮下分4点注射,每点注射50μl,同时腹腔注射200μl用PBS溶解的500ng Pertussistoxin(PTX,百日咳毒素,Sigma公司,P7208),间隔48h再注射一次PTX;
3.分组和给药:
32只小鼠随机分为4组,每组8只:
1)正常组(N,Normal);
2)模型组(EAE+溶剂对照):溶剂对照为含有0.1%DMSO的丙三醇溶液(因Omeprazole难溶于水),从造模当天开始每天腹腔注射给药,0.2ml/只/天;
3)模型+低剂量Omeprazole(5mg/kg):从造模当天开始每天腹腔注射给药,0.2ml/只/天;
4)模型+高剂量Omeprazole(10mg/kg):从造模当天开始每天腹腔注射给药,0.2ml/只/天;
4.EAE模型临床神经功能评分标准:
临床分级 | 临床表现 |
0 | 无症状 |
1 | 鼠尾张力障碍 |
2 | 单侧后肢的瘫痪 |
3 | 双后肢瘫痪伴有前肢无力 |
4 | 四肢瘫痪 |
5 | 濒死状态或死亡 |
死亡鼠在发现死亡当日记为5分,次日起不再计分;介于两个分级间的临床表现可以允许取两个分级的平均值;
5.数据分析:
采用Graphpad Prism 5.0软件进行分析,若P<0.05认为差异有显著性;
6.结果显示:
模型组小鼠最早于诱导后第11天出现症状,大部分以尾部远端张力下降、爬行时尾尖下垂为首发症状,发病进展集中在免疫后第14-20天,疾病高峰期为诱导后第17天,发病率为100%;Omeprazole低剂量组疾病高峰期为第20天,发病率为50%;Omeprazole高剂量组疾病高峰期为第19天,发病率为57.1%。在疾病进展最为明显的14-20天内,Omeprazole治疗显著降低EAE小鼠临床神经功能评分,且高剂量组疗效更为明显(如图7所示,*,**,***分别表示Omeprazole高剂量组与EAE组相比,P<0.05,0.01,0.001;##,###分别表示Omeprazole低剂量组与EAE组相比,P<0.01,0.001);疾病高峰期时,Omeprazole高剂量组的临床神经功能评分显著低于模型组(图7;*,P<0.05,与EAE组相比)(本实验过程中,正常组中有1只小鼠于饲养过程中逃逸,Omeprazole高剂量组中有1只小鼠临床表现出现异常,这2只小鼠未纳入结果统计之中);
实验结果表明,Omeprazole在EAE模型中能显著改善小鼠的临床神经功能,对EAE模型小鼠有显著防治作用。
Claims (6)
1.式(1)结构的奥美拉唑Omeprazole在制备治疗多发性硬化疾病药物中的用途,
所述的Omeprazole,化学名称为:5-甲氧基-2-[[(4-甲氧基-3,5-二甲基-2-吡啶基)甲基]亚磺酰基]-1H-苯并咪唑,分子式:C17H19N3O3S,分子量:345.42
2.按权利要求1所述的用途,其特征在于,所述的Omeprazole为质子泵抑制剂。
3.按权利要求1所述的用途,其特征在于,所述的多发性硬化疾病为自身免疫性介导的、以中枢神经系统广泛脱髓鞘为主要病理特征的脑脊髓炎。
4.按权利要求1或3所述的用途,其特征在于,所述的Omeprazole促进少突胶质细胞系的分化成熟。
5.按权利要求1或3所述的用途,其特征在于,所述的Omeprazole促进髓鞘再生。
6.按权利要求1或3所述的用途,其特征在于,所述的Omeprazole改善脱髓鞘病变的神经行为功能以及临床神经功能。
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