CN106692150B - 尼达尼布在制备预防和治疗肝纤维化与肝硬化的药物中的用途 - Google Patents
尼达尼布在制备预防和治疗肝纤维化与肝硬化的药物中的用途 Download PDFInfo
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- CN106692150B CN106692150B CN201611182863.0A CN201611182863A CN106692150B CN 106692150 B CN106692150 B CN 106692150B CN 201611182863 A CN201611182863 A CN 201611182863A CN 106692150 B CN106692150 B CN 106692150B
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- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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Abstract
本发明涉及尼达尼布在制备预防和治疗肝纤维化与肝硬化的药物中的用途。本发明证明了尼达尼布可以明显改善肝纤维化或肝硬化的程度,减轻肝损伤,并且无明显的肝细胞毒性,具备良好的应用前景。
Description
技术领域
本发明涉及尼达尼布的医药用途,尤其是尼达尼布在制备预防和治疗肝纤维化和肝硬化的药物中的用途。
背景技术
肝硬化是严重危害人类健康的常见慢性病之一。肝纤维化是肝硬化的病理学基础,多种病因(如病毒性肝炎、酒精性肝病、非酒精性肝病、药物及化学因素损伤等)会引起肝脏损害和炎症,导致肝纤维化,最终发展为肝硬化。肝纤维化是肝脏在各种慢性损伤的刺激下出现以胶原为主的细胞外基质过度沉积,并继发肝小叶结构和肝脏功能改变的病理过程。目前认为,肝纤维化是一个可逆的过程,因此,抗纤维化治疗可阻止甚至逆转肝纤维化,进而能防止肝硬化的发生,故一直受到高度重视。
大量研究证实,肝星状细胞(hepatic stellate cells,HSC)的活化是肝纤维化发生的核心环节。HSC主要位于肝细胞与肝窦内皮细胞之间的窦间隙中,约占肝脏细胞总量的5%-10%,胞内含大量维生素A脂滴,生理状态下HSC仅合成少量细胞外基质(extracellular matricx,ECM)用以维系肝组织的正常构架及生理功能,当肝脏受到炎症等慢性损伤刺激时,来自于炎性细胞(Kupffer细胞)、受损肝细胞、肝窦内皮细胞的多种细胞因子可以激活HSC,促进其分化为肌成纤维母细,在细胞形态、增殖、代谢、迁徙等功能均发生显著改变,开始表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),并大量合成ECM,肝纤维化随即开始启动。已有大量研究应用α-SMA作为检测活化HSCs和肌成纤维细胞的生物标志。肝组织中α-SMA的表达量与肝损伤区域和纤维化区域中HSCs的活化程度成正相关,是检测肝纤维化的金标准。此外,活化的HSC还大量分泌TGF-β1和PDGF等细胞因子,以自分泌或旁分泌的方式作用于自身或其他静止状态的HSC,使得HSC活化呈现级联放大效应,从而大大促进肝纤维化的进展。综上述,活化的HSC几乎是肝纤维化中异常ECM的唯一来源和核心促进因素,而以HSC作为靶点的进行干预也自然也是逆转肝纤维化最为重要的策略之一。
在本发明中,尼达尼布(nintedanib)的分子式为C31H33N5O4,具有下式分子结构:
尼达尼布Nintedanib(BIBF 1120)是一种有效的三重血管激酶抑制剂,作用于VEGFR1/2/3,FGFR1/2/3和PDGFRα/β,目前在在临床上用于治疗特发性肺纤维化(IPF),具体来说尼达尼布针对在肺纤维化病理机制中具有潜在影响的生长因子受体发挥作用,其中最为重要的就是血小板源性生长因子受体(PDGFR)、成纤维细胞生长因子受体(FGFR)和血管内皮生长因子受体(VEGFR)。尼达尼布通过阻断这些参与肺纤维化进程的信号转导通路,从而减少肺功能下降速度、减缓IPF疾病进展。此外,尼达尼布已被发现可以用于治疗多种癌症,包括:非小细胞肺癌,前列腺癌,卵巢癌,及结肠直肠癌,目前处于二期临床实验阶段。
现有技术中没有揭示尼达尼布具有抗肝硬化和抗肝纤维化的作用。
发明内容
本发明的一个目的是提供尼达尼布的制药用途,具体来说本发明要解决的技术问题是提供一种尼达尼布在制备预防和治疗肝纤维化和肝硬化的药物中的应用,将尼达尼布作为药物的主要活性成分,其疗效好、起效快、安全有效、毒副作用小,是预防和治疗肝纤维化和肝硬化的理想药物。
为了实现上述目的,本发明采用了如下技术方案:
本发明提供了尼达尼布在制备抑制肝星状细胞活化的药物中的应用。
进一步,所述肝星状细胞活化包括细胞src激酶激活、细胞产生α-SMA增多。
本发明提供了尼达尼布在制备抑制肝星状细胞增殖的药物中的应用。
本发明提供了尼达尼布在制备预防和/或治疗肝功能损伤的药物中的应用。
进一步,所述肝功能损伤包括化学诱导性肝功能损伤、物理诱导性肝功能损伤、药物诱导性肝功能损伤、毒素诱导性肝功能损伤、或感染诱导性肝功能损伤。在本发明的具体方案中,构建了化学诱导性肝功能损伤模型。
本发明的肝功能损伤包括肝纤维组织增生、肝纤维化、或肝硬化。
本发明的抑制肝星状细胞活化的药物,预防和/或治疗肝功能损伤的药物均可以包括作为有效成分的尼达尼布以及药学上可接受的成分。
进一步,本发明的所述药物中作为有效成分的尼达尼布占比为0.1-99%w/w,药学上可接受的成分的占比为0.1-99%w/w。
进一步,本发明的所述药物的剂型是药剂学上可以接受的任何药物剂型。包括但不限于片剂(包括分散片、肠溶片、咀嚼片、口腔崩解片、泡腾片等)、硬胶囊剂(包括肠溶胶囊)、软胶囊剂、颗粒剂、丸剂、微丸剂、滴丸剂、干混悬剂、口服溶液剂、干糖浆剂、散剂、口服混悬液、以及口服的速释或缓释或控释等剂型,注射剂(包括注射用粉针剂(包括注射用无菌灌装粉末、冻干粉针剂)、水溶液注射剂)、软膏剂、凝胶剂、乳液剂、乳胶剂、贴剂、栓剂、凝胶剂等等。
本发明的药物可以经口服途径应用,也可以经静脉、肌肉、皮内或皮下注射途径给药。
本发明的药物可以单独使用,也可以与其他用于治疗肝功能损伤的其他药物联合使用,例如:秋水仙碱、UDCA、水飞蓟宾、Sho-saikoto、α-干扰素、γ-干扰素等。
本发明的药学上可接受的成分包括但不限于药学上可接受的载体、稀释剂、赋形剂、佐剂、缓冲剂、pH调节剂、防腐剂、抗氧化剂、抑菌剂、稳定剂、悬浮剂、增溶剂、表面活性剂(例如润湿剂)、着色剂和致等渗(isotonicizing)溶质(即其使制剂与目标患者的血液或其它相关的体液等渗)。适当的载体、稀释剂、赋形剂等可以在标准药学书籍中找到。参见,例如药物添加剂手册(Handbookof Pharmaceutical Additives),第二版(编者M.Ash和I.Ash),2001(SynapseInformation Resources,Inc.,Endicott,New York,USA);Remington′sPharmaceutical Science,第18版,Mack Publishing Company,Easton,Pa.,1990;以及药物赋形剂手册(Handbook ofPharmaceutical Excipients),第二版,1994。
文中使用的术语“药学上可接受的”涉及化合物、成分、材料、组合物、剂型等,它们在医学判断的合理范围内,适于与患者的组织接触,并未带来过度的不希望的毒性、刺激性、过敏反应或其它问题或并发症,并有合理的益处/风险比。
本发明的“患者”可以是动物、哺乳动物、胎盘哺乳动物、啮齿动物(例如豚鼠、仓鼠、大鼠、小鼠)、鼠科动物(例如小鼠)、兔类动物(例如兔)、犬科动物(例如狗)、猫科动物(例如猫)、马科动物(例如马)、猪类(例如猪)、羊类(例如羊)、牛类(例如奶牛)、灵长类动物、猿猴(例如猴子或者猿)、猴类(例如绒猴、狒狒)、猿类(例如大猩猩、黑猩猩、猩猩、长臂猿)或者人类。
本发明还提供了一种抑制肝星状细胞活化的方法,所述方法包括对肝星状细胞施用有效量的尼达尼布或其药用盐或酯。
进一步,所述肝星状细胞活化包括肝星状细胞产生α-SMA增多、肝星状细胞中src激酶激活。
本发明还提供了一种抑制肝星状细胞增殖的方法,所述方法包括对肝星状细胞施用有效量的尼达尼布或其药用盐或酯。
本发明的“尼达尼布”可以是尼达尼布,也可以是尼达尼布的药用衍生物,所述药用衍生物包括药学上可接受的盐或酯等形式。
本发明所述的“预防”,是指在可能的肝功能损伤因素的存在下,使用后防止或降低肝功能损伤的产生。
本发明所述的“治疗”,是指减轻肝功能损伤的程度,或者治愈肝功能损伤使之正常化,或者减缓肝功能损伤的进程。
本发明的优点和有益效果:
本发明提供了一种治疗或预防肝功能损伤的药物——尼达尼布,在治疗肝功能损伤方面,疗效显著,毒副作用少,使用安全。
附图说明
图1显示尼达尼布对人肝星状细胞系LX-2细胞中Src激酶活性的影响;
图2显示尼达尼布对TGF-β诱导下的人肝星状细胞系LX-2细胞产生α-SMA的影响,其中,A:western blot图;B:柱状统计图;
图3显示尼达尼布对TGF-β诱导的人肝星状细胞系LX-2细胞和大鼠肝星状细胞系HSC-T6细胞增殖活力的影响,其中,A:LX-2细胞;B:HSC-T6细胞;
图4显示尼达尼布对肝纤维化小鼠的谷丙转氨酶(ALT)水平的影响;
图5显示尼达尼布对肝纤维化小鼠的透明质酸水平的影响;
图6显示尼达尼布对四氯化碳诱导的小鼠肝纤维化程度的影响,其中,A:正常对照组;B:肝纤维化模型组;C:低浓度给药治疗组;D:高浓度给药治疗组。
具体实施方式
通过参阅下述实施例可以更容易地了解本发明的内容,这些实施例只是为进一步说明本发明,并不意味着限定本发明的范围。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1尼达尼布对人肝星状细胞LX-2中Src激酶活性的影响
1.实验材料
LX-2细胞由首都医科大学提供;尼达尼布(Nintedanib)由南京正大天晴制药有限公司提供;Phospho-Src Family(Tyr416)一抗购自Cell Signaling Technology公司,货号为#6943;GAPDH一抗购自北京中杉金桥生物技术有限公司,货号为TA-08;TGF-β购自R&DSystems公司,货号为240-B-002。
2.实验方法
将LX-2细胞以5×105个/孔接种于6孔板中,培养过夜,然后换成无血清DMEM培养基,饥饿培养24小时(h)后,换成分别无血清稀释浓度分别为0μm、0.1μm、0.5μm、1μm、5μm的尼达尼布处理细胞4h,然后利用TGF-β刺激细胞:吸弃平皿的中液体,加入无血清培养基稀释的10ng/mL的TGF-β,37℃刺激10分钟(min),接着用遇冷的PBS洗两次,加入120μL含磷酸酶抑制剂的蛋白裂解液收取细胞,得到样品。将样品进行western blot实验检测Src激酶磷酸化水平。
3.实验结果
如图1所示,尼达尼布可以以浓度依赖的方式抑制Src的激活。
实施例2尼达尼布对人肝星状细胞LX-2产生α-SMA的影响
1.实验材料
Anti-α-SMA一抗购自Abcam公司,货号为ab5694。
2.实验方法
将LX-2细胞以5×105个/孔接种于6孔板中,培养过夜,然后换成无血清DMEM培养基,饥饿培养24h后,换成含2%血清、10ng/mL TGF-β及浓度分别为0μm、0.1μm、0.5μm、1μm、5μm尼达尼布的DMEM培养基孵育处理细胞,12h后用室温下的PBS洗两次,加入120μL的蛋白裂解液收取细胞,得到样品。将样品进行western blot检测α-SMA水平。
3.实验结果
如图2所示,尼达尼布可以以剂量依赖的方式抑制TGF-β诱导的人肝星状细胞系LX-2细胞产生α-SMA。
实施例3尼达尼布对人肝星状细胞LX-2和大鼠肝星状细胞HSC-T6细胞增殖活力的影响
1.实验材料
细胞计数试剂盒8(Cell Counting Kit-8,CCK-8)购自东仁化学科技有限公司,货号为CK04。
2.实验方法
将LX-2细胞和HSC-T6细胞以3000个/孔接种于96孔板中,培养过夜,然后换成无血清DMEM培养基,饥饿培养24h后,换成含2%血清、10ng/mL TGF-β及浓度分别为0μm、0.1μm、0.5μm、1μm、5μm尼达尼布的DMEM培养基孵育处理细胞,并留“对照”组用2%血清的DMEM培养基培养(不加TGF-β也不加尼达尼布处理)。分别在加药后的0h、24h、48h和72h,用CCK-8测细胞活力和密度:吸弃培养基,向每孔加入100μL用培养基10倍稀释的CCK-8溶液,将96孔板在培养箱内孵育1h,随后用分光光度计测量450nm下的吸光度以测量细胞密度。
3.实验结果
如图3所示,尼达尼布可以以剂量依赖的方式抑制TGF-β诱导的人肝星状细胞系LX-2细胞和大鼠肝星状细胞的增殖活力。
实施例4尼达尼布对肝纤维化小鼠模型的治疗作用
1.实验材料
实验动物:C57BL/6,SPF级,雄性,18~20g;来源及动物合格证:常州卡文斯实验动物有限公司SCXK(苏)2011-0003,NO.201607595
CCl4:批号20110212国药集团化学试剂有限公司
橄榄油:批号20130713国药集团化学试剂有限公司
EDTA·K2:批号F20091117国药集团化学试剂有限公司
0.9%氯化钠注射液(NS):lot:A14110401-1四川科伦药业股份有限公司
JM称重天平:余姚纪铭称重校验设备有限公司
Legend MACH离心机:Thermo仪器编号202309
连续分液器(Multipette plus):Eppendorf
85-2恒温磁力搅拌器:上海司乐仪器有限公司
注射器:1mL\2.5mL\5mL江苏客乐医用器械有限公司
a.试验试剂:苏木精、伊红和天狼星红购自国药集团化学试剂有限公司,饱和苦味酸缓冲液购自源叶生物科技有限公司,α-SMA抗体购自Sigma-Aldrich公司。
2.实验方法
(1)肝纤维化小鼠模型的建立
实验分组与各组动物注射剂量如表1所示。详细的:
将动物分为四个组,正常对照组、给药对照组、肝纤维化模型组、低浓度(15mg/kg)给药治疗组和高浓度(30mg/kg)给药治疗组,动物数量分别为:4只、4只、6只和10只,正常对照组不进行操作(表1)。
给药对照组:从第三周开始灌胃给药(每周五天,1次/天),给药30mg/kg,连续4周。
纤维化模型组:从第一周开始腹腔注射10%四氯化碳(橄榄油配制),注射体积2mL/kg,,每周3次,共6周。
低浓度给药治疗组:四氯化碳造模的同时,从第三周开始灌胃给药(每周五天,1次/天),给药15mg/kg,连续4周。
高浓度给药治疗组:四氯化碳造模的同时,从第三周开始灌胃给药(每周五天,1次/天),给药30mg/kg,连续4周。
在实验过程中根据检测指标、动物体重和状态的变化,快速建立肝纤维化模型,同时尽量避免或减少动物死亡。
表1分组和剂量
(2)血清和肝脏标本的制备
a.血清制备:6周后,处死小鼠。血液室温静置30min,3000转/分钟,20分钟,吸取上清,-80℃冻存。
b.肝脏处理:剪取左肝叶放入冻存管中,立即置于液氮中,然后放置于-80℃冰箱用于提取蛋白、RNA及细胞因子测定。剩余肝脏在PBS中清洗两次后,放入50ml离心管中用多聚甲醛溶液浸泡固定,用于石蜡切片。
(3)血清ALT和透明质酸的检测
将小鼠血清用生理盐水稀释5倍,送到302医院检验科检测ALT和透明质酸。
(4)Cordon-Sweet网状纤维染色
a)多聚甲醛固定后的大鼠肝脏组织块经脱水、透明、浸蜡后,用包埋机进行石蜡包埋,蜡块凝固后置4℃冰箱贮存,随后将组织蜡块制作成石蜡切片。
b)切片66℃烤箱中烤40min;
c)石蜡切片脱蜡至水二甲苯I、II、III各10min,无水乙醇、95%乙醇、90%乙醇各5min;
d)自来水冲洗1min;
e)入高锰酸钾溶液5min,去离子水洗,入草酸溶液5min,去离子水洗,入硫酸铁铵10min,去离子水洗,入氨银溶液1min,速洗,入甲醛,速洗;
f)脱水、封片:切片于75%、85%、95%、100%各级酒精溶液中各5分钟,1/2二甲苯5分钟,二甲苯I、II中各5分钟,最后用树胶封片。
3.实验结果
观察尼达尼布对肝纤维化小鼠生化指标和胶原沉积的影响。
如图4所示,结果表明,尼达尼布能降低肝纤维化小鼠的ALT水平,表明尼达尼布能减轻四氯化碳诱导的肝纤维化小鼠模型的肝细胞损伤,减轻四氯化碳诱导的肝纤维化小鼠模型的肝脏损伤的程度。而单独给药对照组与正常对照组对比,ALT水平没有明显的变化,这表明尼达尼布没有明显肝毒性。
如图5所示,结果表明,尼达尼布能降低肝纤维化小鼠的透明质酸的水平,表明尼达尼布能减轻四氯化碳诱导的肝纤维化小鼠模型的肝纤维化水平。而单独给药对照组与正常对照组对比,透明质酸水平没有明显的变化,这表明尼达尼布本身不会导致小鼠的肝纤维化。
如图6所示,网状纤维染色显示,肝纤维化模型组胶原纤维广泛存在于纤维增生区,纤维间隔较粗,包绕形成假小叶,而低浓度和高浓度给药治疗组纤维间隔减少,条索变细,这提示尼达尼布明显改善了四氯化碳诱导的肝纤维化小鼠模型的肝纤维化程度。
尽管已经示出和描述了本发明的实施例,本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。
Claims (6)
1.尼达尼布或其药用盐在制备预防和/或治疗肝纤维化的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物包括作为有效成分的尼达尼布或其药用盐以及药学上可接受的载体。
3.根据权利要求1所述的应用,其特征在于,所述药物的剂型是药剂学上可以接受的任何药物剂型。
4.一种体外抑制肝星状细胞活化的方法,其特征在于,所述方法包括对肝星状细胞施用有效量的尼达尼布或其药用盐。
5.根据权利要求4所述的方法,其特征在于,所述肝星状细胞活化包括肝星状细胞中src激酶激活、产生α-SMA增多。
6.一种体外抑制肝星状细胞增殖的方法,其特征在于,所述方法包括对肝星状细胞施用有效量的尼达尼布或其药用盐。
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