CN112438972B - 组织蛋白酶抑制剂的医药用途 - Google Patents
组织蛋白酶抑制剂的医药用途 Download PDFInfo
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Abstract
本发明属于医药技术领域,涉及组织蛋白酶抑制剂的医药用途,具体涉及一些组织蛋白酶抑制剂在制备预防或治疗纤维化疾病的药物中的用途。所述的组织蛋白酶抑制剂的结构如下:其中R1,R2如权利要求和说明书所述。所述的纤维化疾病包括肺纤维化、肝纤维化、肾纤维化、心脏纤维化、子宫内膜纤维化、眼纤维化、胰腺纤维化、脾纤维化、骨髓纤维化疾病或由纤维化诱发的疾病。本发明中,所述的组织蛋白酶抑制剂单独使用或者和其他药物联合使用。
Description
技术领域:
本发明属于医药技术领域,涉及组织蛋白酶抑制剂的医药用途,具体涉及一些组织蛋白酶抑制剂在制备预防或治疗纤维化疾病的药物中的用途。
背景技术:
纤维化(Fibrosis)是指由于炎症导致器官实质细胞发生坏死,组织内细胞外基质异常增多和过度沉积的病理过程。本质上纤维化是组织遭受损伤后的修复反应,以保护组织器官的相对完整性。增生的纤维结缔组织虽然修复了缺损,但却不具备原来器官实质细胞的结构和功能。如果这种修复反应过度、过强和失控时,就会引起器官的纤维化和导致器官的功能下降。
纤维化可发生于多种器官,包括肺、肝脏、肾脏和心脏等重要脏器。主要病理改变为器官组织内肌成纤维细胞病灶的形成、纤维结缔组织增多以及实质细胞减少,持续进展可致器官结构破坏和功能减退,乃至衰竭,严重威胁人类健康和生命。在全世界范围内,组织纤维化是许多疾病致残、致死的主要原因,据美国有关统计资料证明,该国因各种疾病而致死的病人中,接近45%可以归于组织纤维增生疾病。
目前,已达成共识的是上皮细胞-间充质细胞转换(EMT)在纤维化发生发展过程中发挥了十分关键的作用。抑制EMT过程是治疗纤维化疾病最为有效的策略之一。EMT与大量的分子过程密切相关,诸如:转录因子的活化、激活某些信号通路、细胞表面蛋白的表达的改变,以及一些细胞外基质降解酶的持续升高等。
组织蛋白酶(cathepsins)是代表性的具有降解细胞外基质的降解酶。是溶酶体内部系统的酶系之一,参与细胞外基质重铸过程而与大部分疾病都有密切关联。组织蛋白酶与EMT也关系密切。由此可见,针对cathepsin进行小分子干预,对于纤维化疾病具有治疗效果。尤其是目前临床仅有两种药物治疗肺纤维化疾病,不能满足日益增长的纤维化患者的用药需求。因此,拓宽此类治疗药物的作用类型,对于此类药物的开发具有十分重要的意义。
发明内容:
本发明要解决的技术问题是针对缺乏有效的预防和治疗纤维化疾病的药物不足的现状,丰富此类疾病治疗药物的类型。提供几个组织蛋白酶抑制剂的新用途,即在制备用于有效的预防或治疗纤维化疾病药物中的应用。
本发明解决上述技术问题所采用的技术方案是:
组织蛋白酶抑制剂在制备预防或治疗纤维化疾病的药物中的用途。
所述的组织蛋白酶抑制剂的结构为:
其中,R1、R2为5-10元芳基甲酰基、5-10元芳基磺酰基、5-10元杂环芳基甲酰基,
优选为5-6元芳基甲酰基、5-6元芳基磺酰基、5-6元杂环芳基甲酰基;更优选苯甲酰基、苯磺酰基、咪唑甲酰基、吡啶甲酰基、三氮唑基甲酰基;
所述芳基和杂环芳基可以任选被1-3个相同R3取代或2-3个不同的R4,R5,R6取代,所述杂环芳基可以任选含有N、O和S的杂原子;
R3为氢、C2-C6直链或支链的烷基、C5-C6环烷基、5-10元芳基、5-10元杂环芳基、5-10元杂环基、羟基、氨基、羧基及氟、氯、溴或碘原子、氰基、甲氧基、苄氧基、三氟甲基、茚满基、硝基、偶氮基;
R4,R5,R6互不相同,为氢、C2-C6直链或支链的烷基、C5-C6环烷基、5-10元芳基、5-10元杂环芳基、5-10元杂环基、羟基、氨基、羧基及氟、氯、溴或碘原子、氰基、甲氧基、苄氧基、三氟甲基、茚满基、硝基、偶氮基。
所述的组织蛋白酶抑制剂选自:(R)-N-(3-溴苯磺酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯、(R)-N-苯甲酰基苯丙氨酸-(S)-2-(4-三氟甲基苯磺酰胺基)-3-苯丙酯、(R)-N-苯甲酰基苯丙氨酸-(R)-2-(4-溴苯磺酰胺基)-3-苯丙酯、(R)-N-(咪唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯、(R)-N-(1,2,4-三氮唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯、(R)-N-(4-吡啶甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯、(R)-N-(2-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯、(R)-N-(3-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯。
进一步地,所述的纤维化疾病包括肺纤维化、肝纤维化、肾纤维化、心脏纤维化、子宫内膜纤维化、眼纤维化、胰腺纤维化、脾纤维化、骨髓纤维化疾病或由纤维化诱发的疾病。
所述的肺纤维化疾病是药物性肺纤维化,特发性肺纤维化,结节病、尘肺、过敏性肺炎或放射线诱发的肺纤维化,其他病因未明的肺纤维化,以及由肺纤维化诱发的疾病;
所述的肝纤维化是病毒性肝炎、酒精性肝炎、自身免疫性疾病、脂肪肝、营养不良、慢性充血性心力衰竭或药物引起的肝脏纤维化,其他病因未明的肝脏纤维化,以及由肝纤维化诱发的疾病;
所述的肾纤维化是高血压、肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥、肾盂肾炎、肾结石、高血脂、糖尿病、高尿酸尿症、高钙尿症引起的肾脏纤维化,其他病因未明的肾脏纤维化,以及由肾纤维化诱发的疾病;
所述的心纤维化是缺血性心脏疾病、高血压、病毒性心肌炎、代谢性心肌病、克山病、扩张性心肌病、肥厚性心肌病、限制性心肌病或心律失常引起的心脏纤维化、心脏重构和心肌肥厚,其他病因未明的心纤维化,以及由心纤维化诱发的疾病;
所述的子宫内膜纤维化是不同原因引起的子宫内膜纤维化病变,以及由子宫内膜纤维化诱发的疾病;
所述的眼纤维化是眼睛外伤、眼睛手术或糖尿病引起的眼视网膜纤维增生疾病,以及由眼纤维化诱发的疾病。
本发明中所述的组织蛋白酶抑制剂单独使用或者和其他药物联合使用。
附图说明
图1为体外可抑制TGF-β1刺激的肺纤维化、肾纤维化、心脏纤维化和肝纤维化标志蛋白α-SMA表达的上调。
其中A、B、C和D依次为预先用30μM化合物6或溶媒(Control)处理的肺上皮细胞A549细胞、肾小管上皮细胞CD3、原代心成纤维细胞和肝星状细胞LX2经10ng/mL的TGF-β1刺激后纤维化标志蛋白α-SMA表达的变化。
##P<0.01,与control组比较;**P<0.01,与TGF-β1组比较。
图2为化合物6体外可阻断TGF-β1刺激的肺纤维化、肾纤维化、心脏纤维化和肝纤维化效应细胞表面硬度的改变。
其中A、B、C和D依次为预先用30μM化合物6或溶媒(Control)处理的肺上皮细胞A549细胞、肾小管上皮细胞CD3、原代心成纤维细胞和肝星状细胞LX2经10ng/mL的TGF-β1刺激后细胞表面硬度的变化。
##P<0.01,与control组比较;**P<0.01,与TGF-β1组比较。
图3为化合物6腹腔注射给药对博莱霉素诱导的小鼠肺纤维化模型的病理学检查结果。
A为HE染色实验结果,B为Masson染色检测胶原表达的结果,C为免疫组织化学法检测α-SMA表达的结果。
具体实施方式
实施例1:组织蛋白酶抑制活性测试
1.实验材料:组织蛋白酶L和S在37℃的温度下、5%CO2饱和湿度的培养箱内培养。各化合物在无菌条件下用二甲基亚砜(DMSO)溶解后,用RPMI-1640培养液稀释至所需浓度,DMSO最终浓度小于0.5%。RPMI 1640购于Gibco(Grand Island,USA)、胎牛血清(FBS)购于Biological Industries、青霉素-链霉素购于HyClone公司、胰蛋白酶(Trypsin,1:250)购于Biosharp公司、二甲基亚砜(DMSO)购于Sigma Chemical公司、单丹磺酰戊二胺(MDC)购于南京凯基生物技术股份有限公司。
2.仪器:二氧化碳培养箱(日本SANYO公司,型号:MCO-5AC)、倒置显微镜(日本OLYMPUS公司,型号:CKX41)、酶联免疫分析仪(Tecan,Austria)、细胞培养板(美国Costar公司)、PH测试仪(上海梅特勒-托利多仪器有限公司,型号:DELTA-320)。
3.实验方法(荧光染色法):组织蛋白酶L或S:PH=5.5的MES-NaOH缓冲溶液,其中含2.5mmol/L的EDTA,2.5mmol/L的DTT和10%的DMSO。以20μmol/L Z-Phe-Arg-AMC为荧光底物。用上述缓冲液将组织蛋白酶稀释至4nM备用,再用上述缓冲液将Cathepsin的底物稀释至40μM备用。将体积为50μL(空白)或49μL的酶分别与不加抑制剂或1uL不同浓度的抑制剂混合均匀。在37℃恒温培养箱培育30min后向其中加入荧光底物50μL,混匀后立即进行时间依赖效应的荧光强度检测(激发波长380nm,发射波长460nm),该过程的检测使用的是酶标仪。抑制剂浓度分别为0、0.01、0.1、1、10、100μM。测试时间为10min,每1min扫描一次。得到的值做荧光强度随时间变化的直线,该直线的斜率即表明组织蛋白酶分解底物的初速度,而酶反应的初速度既可理解为酶的活性,通过与不加抑制剂的组织蛋白酶分解底物的速度比较(斜率),就可看出抑制效果的好坏。将三个对照组所得的斜率求平均值,认为此值即是酶活力为100%,将其余不同浓度的斜率值分别除以上述平均值得到对应的酶的残余活力。用1减去残余活力即为酶的抑制率,通过SPSS软件分析即可得到每个抑制剂的IC50值。化合物1-8的结构如下:试验结果见表1,
(R)-N-(3-溴苯磺酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物1)
(R)-N-苯甲酰基苯丙氨酸-(S)-2-(4-三氟甲基苯磺酰胺基)-3-苯丙酯(化合物2)
(R)-N-苯甲酰基苯丙氨酸-(R)-2-(4-溴苯磺酰胺基)-3-苯丙酯(化合物3)
(R)-N-(咪唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物4)
(R)-N-(1,2,4-三氮唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物5)
(R)-N-(4-吡啶甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物6)
(R)-N-(2-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物7)
(R)-N-(3-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯(化合物8)
表1代表性化合物对组织蛋白酶两种亚型cathepsin L and S的IC50值
实施例2:基于纤维化标志蛋白α-SMA的化合物6体外抗肺纤维化、肝纤维化、肾纤维化和心纤维化作用研究。
以酶活性最优的化合物6为例:
肺上皮细胞A549细胞、肝星状细胞LX-2、肾小管上皮细胞CD3和原代心成纤维细胞预先用300μM化合物6或溶媒处理,然后经10ng/mL的TGF-β1刺激上述四种细胞向肌成纤维细胞转型而构建体外肺、肝、肾和心纤维化模型,以纤维化标志蛋白α-SMA表征纤维化的程度。如图1所示,10ng/ml的TGF-β1刺激人肺上皮细胞A549、肝星状细胞LX-2、肾小管上皮细胞CD3和原代小鼠心成纤维细胞48h后,纤维化标志蛋白α-SMA的明显上调(P<0.01=证明经典的肺、肝、肾和心纤维化模型的成功构建)。300μM的化合物6可明显阻断TGF-β1诱导的纤维化标志蛋白α-SMA的上调,表明了化合物6对肺、肝、肾和心纤维化病理进程具有抑制作用。
实施例3:细胞硬度为纤维化标志物考察化合物6体外抗肺纤维化、肝纤维化、肾纤维化和心纤维化作用。
前期研究结果表明细胞硬度可作为表征纤维化程度的的生物标志物。肺上皮细胞A549细胞、肝星状细胞LX-2、肾小管上皮细胞CD3和原代心成纤维细胞预先用300μM化合物6或溶媒处理,然后经10ng/mL的TGF-β1刺激上述四种细胞向肌成纤维细胞转型而构建体外肺、肝、肾和心纤维化模型,原子力显微镜检测上述四种细胞经TGF-β1刺激后细胞硬度的变化,以细胞硬度(杨氏模量)为标志物表征纤维化的程度。结果表明(图2),在上述肺纤维化、肝纤维化、肾纤维化和心纤维化模型中,化合物6可明显阻断TGF-β1诱导的细胞硬度的增加,进一步证明了化合物6体外可产生抗肺纤维化、肝纤维化、肾纤维化和心纤维化作用。
实施例4:化合物6体内抗纤维化作用研究。
1.模型制备及给药:
选用体重20-24g之间的成年雄性C57小鼠,分别设定空白组(生理盐水)、模型组、受试药组(化合物6,1.4g/kg,灌胃给药),每组10只动物。博莱霉素按3mg/kg经气管灌注给药制备小鼠肺纤维化模型;博莱霉素造模后第7天给予不同受试药物,每日给药一次,连续给药21天。
2、病理组织取材:
于给药后第28日,小鼠腹腔注射0.1ml/10g水合氯醛麻醉,打开胸腔,剪开左心房,用预冷的生理盐水从右心室缓慢注入心脏,经肺循环灌注至肺脏发白为止,剪下左肺叶放入4%多聚甲醛固定24h,石蜡包埋后切片,其余肺组织贮藏在液氮中备用。样本用等渗生理盐水解冻,并制成匀浆并于-20℃贮藏。
3、小鼠肺脏组织病理切片制备及纤维化评价指标:上述组织病理学样本采用4%多聚甲醛浸泡24h,乙醇脱水,石蜡包埋,曙红苏木紫染色,Masson三色染色,光镜下进行组织学评价。评价指标包括:肺泡腔体积、肺泡壁厚度、中性粒细胞和胶原含量等。同时,对上述组织病理学样本使用免疫组化技术检测小鼠肺组织中肺纤维化标志蛋白α-SMA表达量的变化。
4、化合物6的体内抗肺纤维化作用评价结果:
实验结果见图3。HE染色实验结果显示,空白对照组动物肺组织内结构清晰,未见纤维化表现。博来霉素模型组小鼠肺组织切片严重纤维化,表现为肺泡结构破坏或消失,纤维结缔组织填充,并伴有炎症细胞浸润;肺泡间质明显水肿增宽,大量成纤维细胞和胶原组织沉积。化合物6给药组小鼠肺组织结构较模型组相对完整,肺泡内未见明显的炎症细胞,存在少量的纤维灶,纤维化程度明显减轻。Masson染色结果显示,空白对照组动物肺组织内有少量胶原分布。博来霉素模型组支气管壁周围和肺泡隔区域,可见蓝色胶原纤维明显增多。化合物6给药组小鼠与模型组相比,胶原纤维沉积明显减少。免疫组织化学检测结果显示,空白对照组动物肺组织肺泡间隔细胞偶见α-SMA阳性表达。博来霉素模型组肺组织增厚的肺泡间隔和塌缩的肺泡结构内均可见α-SMA表达量显著升高,大量阳性表达细胞形成典型的成纤维细胞灶。化合物6给药组肺泡间隔中仍可见散落的成纤维细胞灶存在,但α-SMA阳性表达细胞较博来霉素组明显减少。以上病理组织切片结果显示,化合物6体内产生明显的抗纤维化作用。
Claims (10)
2.如权利要求1所述的用途,其特征在于,所述的组织蛋白酶抑制剂为:
(R)-N-(3-溴苯磺酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
(R)-N-苯甲酰基苯丙氨酸-(S)-2-(4-三氟甲基苯磺酰胺基)-3-苯丙酯
(R)-N-苯甲酰基苯丙氨酸-(R)-2-(4-溴苯磺酰胺基)-3-苯丙酯
(R)-N-(咪唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
(R)-N-(1,2,4-三氮唑-1-基甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
(R)-N-(4-吡啶甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
(R)-N-(2-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
(R)-N-(3-羟基苯甲酰基)-苯丙氨酸-(S)-2-苯甲酰胺基-3-苯丙酯
3.如权利要求1或2所述的用途,其特征在于,所述的纤维化疾病包括肺纤维化、肝纤维化、肾纤维化、心纤维化、子宫内膜纤维化、眼纤维化、胰腺纤维化、脾纤维化、骨髓纤维化疾病。
4.如权利要求3所述的用途,其特征在于,肺纤维化疾病是药物性肺纤维化,特发性肺纤维化或者由结节病、尘肺、过敏性肺炎、放射线诱发的肺纤维化或者其他病因未明的肺纤维化。
5.如权利要求3所述的用途,其特征在于,肝纤维化疾病是由病毒性肝炎、酒精性肝炎、自身免疫性疾病、脂肪肝、营养不良、慢性充血性心力衰竭或药物引起的肝纤维化或者其他病因未明的肝纤维化。
6.如权利要求3所述的用途,其特征在于,肾纤维化疾病是由高血压、肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥、肾盂肾炎、肾结石、高血脂、糖尿病、高尿酸尿症、高钙尿症引起的肾纤维化或者其他病因未明的肾纤维化。
7.如权利要求3所述的用途,其特征在于,心纤维化疾病是由缺血性心脏疾病、高血压、病毒性心肌炎、代谢性心肌病、克山病、扩张性心肌病、肥厚性心肌病、限制性心肌病或心律失常引起的心纤维化或者其他病因未明的心纤维化。
8.如权利要求3所述的用途,其特征在于,子宫内膜纤维化疾病是不同原因引起的子宫内膜纤维化病变;眼纤维化疾病是由眼睛外伤、眼睛手术或糖尿病引起的眼视网膜纤维增生疾病。
9.根据权利要求1-2、4-8中任何一项所述的用途,其特征在于,所述的组织蛋白酶抑制剂单独使用或者和其他药物联合使用。
10.根据权利要求3所述的用途,其特征在于,所述的组织蛋白酶抑制剂单独使用或者和其他药物联合使用。
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