CN109481427A - 化合物fc-99在制备预防或治疗急性肝衰竭药物中的应用 - Google Patents
化合物fc-99在制备预防或治疗急性肝衰竭药物中的应用 Download PDFInfo
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Abstract
本发明公开了如式(I)所示的FC‑99在制备预防或治疗急性肝衰竭药物中的应用,具体涉及FC‑99通过诱导Let‑7a(microRNA Let‑7A)的表达从而抑制肝衰竭的发生发展,以至于预防或治疗肝衰竭,属于生物医药领域。本发明通过建立D‑氨基半乳糖联合脂多糖诱导的急性肝衰竭小鼠动物模型,FC‑99对爆发性肝功能衰竭进行干预,结果显示其不仅明显降低转氨酶、抑制炎症因子释放、减少肝细胞凋亡、维持肝脏显微结构稳定,而且可以显著性降低急性肝衰竭小鼠的死亡率;利用生物信息学分析和分子生物学检测发现,FC‑99是通过诱导Let‑7a高表达,靶向抑制IL‑6、IRF4、CD86和SOCS1等基因表达来实现的。因此,本申请专利的研究结果表明,化合物FC‑99可在制备预防或治疗急性肝衰竭药物中得到应用。在上述应用中,其注射、口服使用剂量范围是0.01mg~1000mg/天,也可根据病情的轻重或剂型的不同偏离此范围。
Description
技术领域
发明涉及FC-99抑制肝衰竭的发生发展从而预防或治疗肝衰竭病症;具体涉及FC-99通过诱导Let-7a 的表达,从而预防或治疗肝衰竭病症。
技术背景
急性肝衰竭(Acute liver failure,缩写为“ALF”)是一类由多种因素引起的严重肝脏损害,短时间内肝细胞大量死亡,进而导致肝脏本身的合成、解毒、排泄和生物转化等功能发生严重障碍的危重临床综合征;其病因复杂,病情发展迅速,病死率高达约50%,严重威胁到患者生命安全(Mark J.W.McPhail.et al.,2015, doi:10.1097/MOG.0000000000000174)。我国是世界人口大国,每年由病毒、酒精、药物等原因造成的急性肝衰竭可达数十万人。
ALF的致病因素广泛,这极大地影响了对ALF治疗方法的有效管理。最近,一些研究发现对病人的血液进行大量置换对ALF具有潜在的治疗功效。该治疗方法阐明了血浆中有害的炎性介质能延长炎症进程以及延缓肝损伤修复。另外,肝脏辅助设备也对肝衰竭具有有效的治疗效果,但是对ALF的治疗缺乏有力的证据支持。临床上,肝移植的开展使急性肝衰竭的整体生存率明显升高(超过60%),但供肝稀缺、费用昂贵、移植后排斥会发生反应,使其成为绝大多数肝衰竭患者可望而不可及的治疗方法(Lee WM.,2012, doi:10.1055/s-0032-1301733)。因此,我国急性肝衰竭的病死率仍居高不下,寻找有效的治疗药物及药物作用靶点已成为肝病研究的重要方向。
有文献报道,一种非编码单链的小核糖核酸Let-7a-5p(MicroRNA Let-7a-5p,缩写为“let-7a”)的表达增加会抑制小鼠肝脏中IL-6的产生,从而缓解刀豆蛋白导致的肝脏炎性损伤(Yingying Zhang.et al.,2012, doi:10.1007/s10875-012-9840-7);LPS处理人肝星状细胞,let-7a的表达出现减少。另外,酒精性肝病小鼠的血浆中let-7a的表达也是减少(Kelly McDaniel.et al.,2017,doi:10.1074/jbc.M116.773291)。提示:Let-7a 的低表达与肝衰竭有关。在肝损伤中,肝细胞的CD86基因表达增加将导致CD4+T细胞中细胞因子的增多和活化,从而改变其稳态和功能,最终促进肝损伤(Jiaren Sun.etal.,2005,79(16):10730-9)。另有研究发现,干扰素调节因子(Interferon regulatory factor 4,缩写为“IRF4”)作为IRF家族的一种转录因子,具有活化T细胞和B细胞的作用,它在急性损伤后的肝移植手术中表达增加。IRF4可作为一个全新的药物靶点,从免疫抑制的角度来治疗急性肝衰竭(Tengqian Tang.et al.,2015,doi:10.1016/j.intimp.2015.06.014);细胞因子信号1抑制因子(Suppressors of cytokine signaling1,缩写为“SOCS1”)负性调节细胞因子信号,能够抑制组织损伤(Chang Cheng.et al.,2014,doi:10.1016/j.toxlet.2013.12.023),干扰SOCS1基因表达则会促进肝纤维化的发展(TakafumiYoshida.et al.,2004,199(12):1701-7)。此外,促炎因子IL-6也能促进肝炎的发展(Yingying Zhang.et al.,2013,doi:10.1007/s10875-012-9840-7)。Liu报道IL-6是let-7a-5p的分子靶点 (Xiao-yan Liu.et al.,2013,doi:10.7150/ijbs.5425)。综上所述,IL-6、IRF4、CD86和SOCS1等基因对肝脏炎症和免疫调节产生重要影响,进而对急性肝衰竭的发生发展进程中发挥必不可少的调控作用。
FC-99(N1-[(4-methoxy)methyl]-4-methyl-1,2-Benzenediamine)是我们设计合成的小分子化合物,前期研究发现,FC-99能够有效缓解脓毒症引起的急性肺损伤和肾脏等器官组织形态改变(Wei Gong.et al.,2014, doi:10.1111/bph.12797;Huan Dou.et al.,2014,doi:10.1165/rcmb.2013-0411OC);FC-99通过抑制DC细胞的磷酸化来治疗系统性红斑狼疮小鼠,改善狼疮性肾炎、脾脏等多器官损伤(Jianjian Ji.et al.,2015,doi:10.1016/j.imlet.2015.10.017);其对葡聚糖硫酸钠引起的结肠炎也有显著的治疗作用(Liu Yang.et al.,2015, doi:10.1016/j.lfs.2015)。目前没有关于应用FC-99治疗急性肝衰竭的报道。本发明人通过大量的研究发现 FC-99诱导Let-7a靶向抑制IL-6、IRF4、CD86和SOCS1等基因,从而抑制肝脏中炎症因子的产生和免疫细胞的过度活化,减少肝细胞大量死亡,最终改善急性肝衰竭症状。
发明内容
本发明提供了FC-99用于制备预防和治疗肝衰竭的药物中的应用。
本发明提供了FC-99在诱导肝衰竭Let-7a中的药物中的应用。
本发明为了实现上述目的,通过D-GalN/LPS诱导建立急性肝衰竭小鼠模型,然后给予肝衰竭小鼠腹腔注射不同浓度的FC-99溶液(对照组用等量的生理盐水溶液)处理,观察了在使用FC-99处理后,对肝衰竭小鼠的血浆天冬氨酸转氨酶(ALT)、丙氨酸转氨酶(AST)、肿瘤坏死因子-α(TNF-α)和白介素-6 (IL-6)等炎症因子、肝细胞凋亡、肝组织病理结构,小鼠死亡率等肝衰竭相关指标的影响。评估了FC-99 溶液对肝衰竭症小鼠的治疗效果。研究结果提示,FC-99能明显降低ALT、AST、抑制炎症因子TNF-α和 IL-6的释放、减少肝细胞凋亡、维持肝脏显微结构稳定,并且显著性降低急性肝衰竭小鼠的死亡率,减缓肝衰竭发生后肝脏功能的损害。因此,使用FC-99可有效的改善肝衰竭病症,临床应用前景良好。
本发明提供的FC-99可以添加药学上可接受的载体制成常见的药用制剂,如片剂、胶囊、粉剂、糖浆、液剂、悬浮剂、针剂,可以加入香料、甜味剂、液体或固体填料或稀释剂等常用药用辅料。
本发明提供的FC-99在临床上的给药方式可以采用口服、注射等方式。
本发明提供的FC-99在用于肝衰竭时,其口服、注射使用剂量范围是0.01mg~1000mg/天,也可根据病情的轻重或剂型的不同偏离此范围。
附图说明
图1是FC-99对D-GalN/LPS急性肝衰竭小鼠生存率的影响(mean±SD,n=8)
图2是FC-99抑制D-GalN/LPS急性肝衰竭小鼠血清转氨酶的产生(mean±SD,n=5)
图3是FC-99抑制D-GalN/LPS急性肝衰竭小鼠血清炎性因子的产生(mean±SD,n=5)
图4是FC-99对D-GalN/LPS急性肝衰竭小鼠肝组织形态改变(n=5)
图5是FC-99对D-GalN/LPS急性肝衰竭小鼠肝细胞凋亡改变(n=5)
图6是FC-99促进D-GalN/LPS急性肝衰竭小鼠肝脏中let-7a的表达(mean±SD,n=5)
图7是FC-99抑制D-GalN/LPS急性肝衰竭小鼠肝脏中IL-6、IRF4、CD86和SOCS1等基因的表达(mean ±SD,n=5)
具体实施方式
本文中公开了本发明的详细实施方案;然而,应当理解所公开的实施方案仅是说明可以按多种形式体现的本发明。本文中所给出的与本发明的多个实施方案相关的每个实施例意在是示意性的并且非限制性的。本文中附图不必然地按照比例,可以放大一些特征以显示特定组分的细节。此外,图中所显示的任何量值、说明等意在是示意性的并且非限制性的。故,本文中公开的特定结构和功能细节不得理解为限制性的,而仅应理解为指导本领域相关技术人员以多种方式使用本发明的代表性基础。如果不脱离本发明的精神和范围,对本发明进行修改或等同替换的,均应涵盖在本发明的权利要求的保护范围之中。
实施例一 FC-99溶液的配制
化合物FC-99,采用DMSO促溶后配制成的化合物FC-99的储备液,于注射前以无菌生理盐水稀释配成化合物FC-99的工作液。
实施例二 FC-99对D-GalN/LPS急性肝衰竭小鼠生存率的影响
4~6周C57BL/6小鼠,雄性(购于南京大学-南京生物医药研究院)。模型严格参照文献(Sainan Zhang,Sci Rep,2016,doi:10.1038/srep33206)介绍的方法。D-GalN和LPS(购于Sigma-Aldrich公司)。每只小鼠腹腔注射500μl的包含20mg D-GalN和10μg/kg LPS的无菌生理盐水,终浓度即分别为D-GalN 1μg/kg和LPS 10μg/kg。
实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组(100mg/kg)、模型组(LPS/D-GalN 组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组(100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2 小时后,LPS/D-GalN(10μg/kg LPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;化合物FC-99组以等体积的生理盐水代替LPS/D-GalN;正常对照组不做处理以等体积的生理盐水代替LPS/D-GalN和FC-99工作液。每隔24h记录小鼠死亡情况,连续观察7天。
结果表明,化合物FC-99可以显著抑制LPS/D-GalN诱导的小鼠死亡,且FC-99的抑制作用与剂量呈较好的依赖关系。结果见图1。图中与LPS/D-GalN组比较,*p<0.05,**p<0.01。
实施例三 FC-99抑制D-GalN/LPS诱导急性肝衰竭小鼠血清转氨酶升高
4~6周C57BL/6小鼠,雄性。实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组 (100mg/kg)、模型组(LPS/D-GalN组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组 (100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2小时后,LPS/D-GalN(10μg/kg LPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;正常对照组不做处理以等体积的生理盐水代替 LPS/D-GalN和FC-99工作液。24h后摘眼球取血,置于5ml无菌肝素抗凝管中。室温静置30min后3000 rpm、20℃离心10min,将上层血清取出至500ul无酶离心管中,12000rpm、4℃离心20min。自动生化分析仪(购于德国Beckman Coulter公司)检测小鼠血清中与肝损伤相关的转氨酶ALT和AST的表达。
结果表明,FC-99可以显著抑制D-GalN/LPS诱导的血浆转氨酶ALT和AST的表达,且FC-99的抑制作用与剂量呈较好的依赖关系。结果见图2。图中与NS组比较,##p<0.01;与D-GalN/LPS组比较,*p<0.05, **p<0.01。
实施例四 FC-99降低D-GalN/LPS诱导急性肝衰竭小鼠血清炎性因子产生
4~6周C57BL/6小鼠,雄性。实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组 (100mg/kg)、模型组(LPS/D-GalN组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组 (100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2小时后,LPS/D-GalN(10μg/kg LPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;正常对照组不做处理以等体积的生理盐水代替 LPS/D-GalN和FC-99工作液。24h后摘眼球取血,置于5ml无菌肝素抗凝管中。室温静置30min后3000 rpm、20℃离心10min,将上层血清取出至500ul无酶离心管中,12000rpm、4℃离心20min。ELISA法检测小鼠血清炎性因子TNF-α和IL-6的表达。
结果表明,FC-99可以显著抑制D-GalN/LPS诱导的血浆炎性因子TNF-a和IL-6的表达,且FC-99的抑制作用与剂量呈较好的依赖关系。结果见图3。图中与NS组比较,##p<0.01,###p<0.001;与D-GalN/LPS 组比较,*p<0.05,**p<0.01,***p<0.001。
实施例五 FC-99对急性肝衰竭小鼠肝组织病变的改善作用
4~6周C57BL/6小鼠,雄性。实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组 (100mg/kg)、模型组(LPS/D-GalN组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组 (100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2小时后,LPS/D-GalN(10μg/kg LPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;正常对照组不做处理以等体积的生理盐水代替 LPS/D-GalN和FC-99工作液。24h后采取颈椎脱臼处死小鼠。将小鼠肝取出浸泡于4%中性甲醛溶液做 HE染色。
结果表明,化合物FC-99可显著改善D-GalN/LPS诱导的小鼠肝组织损伤。结果见图4。
实施例六 FC-99抑制急性肝衰竭小鼠肝细胞凋亡
4~6周C57BL/6小鼠,雄性。实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组 (100mg/kg)、模型组(LPS/D-GalN组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组 (100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2小时后,LPS/D-GalN(10μg/kgLPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;正常对照组不做处理以等体积的生理盐水代替 LPS/D-GalN和FC-99工作液。24h后采取颈椎脱臼处死小鼠。将小鼠肝取出浸泡于OTC做冰冻切片,采用TUNEL凋亡检测试剂盒(购于Roche公司)检测肝组织中肝细胞凋亡情况。
结果表明,化合物FC-99可显著改善D-GalN/LPS诱导的小鼠急性肝衰竭引起的肝细胞大量凋亡。结果见图5。
从实施例二、三、四、五和六可以得出以下结论:本发明化合物FC-99具有体内抗炎活性,可以有效抑制D-GalN/LPS诱导的急性肝衰竭发病进程。
实施例七 分子生物学检测FC-99对let-7a的影响
4~6周C57BL/6小鼠,雄性。实验分组及处理:随机分为正常对照组(NS组)、化合物FC-99组 (100mg/kg)、模型组(LPS/D-GalN组)、治疗低剂量组(30mg/kg FC-99+LPS/D-GalN组)、治疗高剂量组 (100mg/kg FC-99+LPS/D-GalN组)。治疗低剂量组和治疗高剂量组分别按30mg/kg和100mg/kg的剂量进行化合物FC-99工作液的腹腔注射,2小时后,LPS/D-GalN(10μg/kg LPS+1000mg/kg D-GalN)腹腔注射造模;模型组以等体积的生理盐水代替化合物FC-99工作液;正常对照组不做处理以等体积的生理盐水代替 LPS/D-GalN和FC-99工作液。24h后采取颈椎脱臼处死小鼠。将小鼠肝取出,Trizol法提取肝脏中总RNA, Taqman探针法检测let-7a的表达。
结果表明,化合物FC-99可显著提高D-GalN/LPS诱导的小鼠急性肝衰竭疾病状态下let-7a的表达,有剂量梯度差异。结果见图6。图中与NS组比较,###p<0.005;与D-GalN/LPS组比较,**p<0.01,***p<0.005。
实施例八 FC-99通过let-7a-5p靶向抑制IL-6、IRF4、CD86和SOCS1等基因
利用不同的microRNA及靶基因在线分析软件(http://www.targetscan.org/,http://microrna.sanger.ac.uk/targets/v5/)分别对IL-6、IRF4、CD86和SOCS1进行分析和比较,判断上述基因与let-7a之间是否存在可能的调控关系,并用QPCR方法进行验证检测。
通过生物信息学分析:IL-6等四个基因3’-UTR序列中均含有一个let-7a可能的结合位点,提示炎症相关基因IL-6、IRF4、CD86和SOCS1等为let-7a-5p的靶点基因。分子生物学检测结果显示,FC-99处理组的小鼠肝脏中IL-6、IRF4、CD86和SOCS1等基因表达均下调。
结果表明,FC-99能显著抑制IL-6、IRF4、CD86和SOCS1等基因表达。结果见图7。
从实施例七和八得出以下结论:本发明化合物FC-99通过诱导let-7a-5p高表达,靶向抑制IL-6、IRF4、 CD86和SOCS1等基因,从而有效改善急性肝衰竭损伤。
Claims (7)
1.本发明提供了FC-99在制备预防或治疗治疗肝衰竭的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,制备预防或治疗各种原因引起的肝衰竭的药物中的应用。
3.根据权利要求1所述的应用,其特征在于,所述FC-99诱导Let-7a的表达来预防或治疗肝衰竭。
4.根据权利要求1所述的应用,其特征在于,所述FC-99诱导Let-7a、靶向抑制IL-6基因的表达来预防或治疗肝衰竭。
5.根据权利要求1所述的应用,其特征在于,所述FC-99诱导Let-7a、靶向抑制IRF4基因的表达来预防或治疗肝衰竭。
6.根据权利要求1所述的应用,其特征在于,所述FC-99诱导Let-7a、靶向抑制CD86基因的表达来预防或治疗肝衰竭。
7.根据权利要求1所述的应用,其特征在于,所述FC-99诱导Let-7a、靶向抑制SOCS1基因的表达来预防或治疗肝衰竭。
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WO2019200216A1 (en) * | 2018-04-12 | 2019-10-17 | The Methodist Hospital System | Modulation of irf-4 and uses thereof |
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