CN117088942A - 改善肝纤维化的多肽化合物及其用途 - Google Patents
改善肝纤维化的多肽化合物及其用途 Download PDFInfo
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Abstract
本发明公开了一类多肽化合物及其用途,该多肽化合物含有以下氨基酸序列表示的母体肽:NH2‑Thr‑Arg‑Pro‑Ala‑Ser‑Phe‑Trp‑Glu‑Thr‑Ser‑COOH(SEQID NO.1);或在母体肽中的氨基酸序列经过氨基酸取代和/或NH2经乙酰化,且具有预防或治疗器官纤维化或器官疾病伴随的纤维化活性的由所述母体肽衍生的多肽化合物。本发明通过一系列体外细胞实验和体内动物实验证实该多肽化合物可用于治疗或改善器官纤维化及器官疾病伴随的纤维化病症,尤其适用于治疗或改善肝纤维化及器官疾病伴随的纤维化病症。
Description
技术领域
本发明属于生物化学技术领域,具体而言,本发明涉及一类能改善肝纤维化的多肽化合物,该多肽化合物可用于治疗或预防器官纤维化及器官疾病伴随的纤维化病症的药物中的用途;特别是肝纤维化及肝脏疾病伴随的纤维化病症。
背景技术
肝纤维化(hepatic fibrosis,HF)是一个病理生理过程,是由细胞外基质蛋白(主要是Ⅰ型胶原和Ⅲ型胶原)的积累形成纤维瘢痕沉积而代替正常组织导致的(Friedman SL.Journal of hepatology,2003,38:38-53.)。HF源于两种类型的慢性肝损伤:肝毒性损伤和胆汁淤积性肝损伤。导致肝毒性损伤的原因有病毒感染、酒精、包括非酒精脂肪性肝炎(Nonalcoholic steatohepatitis,NASH)在内的代谢综合征等(Bataller R,Brenner DA.The Journal of clinical investigation,2005,115(2):209-218)。而胆汁淤积性损伤是由胆汁排泄障碍引起的,由于不能主动经胆小管排泄,继而在肝内淤积,引起一系列器质性损伤和功能紊乱。两者造成肝纤维化的机制为:当肝脏长期受到损伤时,中性粒细胞和巨噬细胞等炎症细胞会募集到受损的肝脏,进而触发炎症反应,产生细胞因子和趋化因子来诱导肝星状细胞(hepatic stellate cell,HSCs)激活,进一步分化成产生I型胶原的肌成纤维细胞(myofibroblasts,MF),最后分泌细胞外基质(extracellular matrix,ECM),产生纤维瘢痕(Iredale J P.The Journal of clinical investigation,2007,117(3):539-548.)。
HF是慢性肝病重要的病理特征,也是进一步向肝硬化发展的主要中间缓解。根据目前的实验和临床研究,肝硬化是不可逆转的慢性进行性肝疾病,但是HF却是可逆转的。损伤后的修复反应会引起很多ECM微环境的改变,ECM的生成是HF产生的直接原因,它可直接暴露主要反应细胞和纤维化的下游效应器,即激活的HSCs和MF。而ECM的合成与降解之间的不平衡就会导致ECM的大量积累进而形成纤维化,而纤维化的发生会进一步的加重HSCs的激活,使得纤维化不断地加重。影响肝纤维化的原因有很多,病因主要有病毒感染、胆汁淤积、酗酒、有毒的药物毒物代谢物、代谢疾病、肥胖、自身免疫失衡、肠道菌群等(Weiskirchen R,Weiskirchen S,Tacke F.F1000Research,2018,7.)。
肝脏纤维化如果不加以控制和治疗,就会发展为肝硬化,最终将会导致肝功能受损和肝坏死的发生。而一旦发展为肝硬化,患者则处于极高危的患肝细胞癌的风险中(A.H.Ali,K.D.Lindor,Expert opinion on pharmacotherapy2016,17,1809-1815.),给患者带来巨大的痛苦甚至威胁其生命,从而必须依靠肝脏移植来治疗。所以可见肝纤维化严重威胁着我国以及世界人民的健康,亟待有效的药物来治疗!
然而迄今为止,目前暂无针对治疗HF的药物获批上市。多种治疗HF的药物正经历临床试验阶段,但都因药效不显著和不良反应严重等原因止步于临床试验阶段。因此研发更少副作用、更强针对性的治疗纤维化的药物显得尤为重要。
综上所述,目前临床研究的药物在治疗纤维化疾病方面仍存在较大的不足和安全风险,抗纤维化领域更加安全、有效的新靶点药物研发仍是一项艰巨的科学任务,而相应的药物分子的设计合成更是迫在眉睫!
发明内容
本发明的目的在于提供一类多肽化合物及其用途,该多肽化合为能改善器官纤维化及器官疾病伴随的纤维化病症的新型多肽化合物。本发明经过大量的实验研究证明该多肽化合物,没有不良反应发生,可用于治疗或改善器官纤维化及器官疾病伴随的纤维化病症,尤其适用于治疗或改善肝纤维化及器官疾病伴随的纤维化病症。
本发明的另一个目的在于提供上述新型多肽化合物在治疗或预防器官纤维化及器官疾病伴随的纤维化病症的用途,尤其是该新型多肽化合物可作为新一代治疗肝纤维化及肝脏疾病伴随的纤维化病症的药物。
为达上述目的,本发明提供的多肽化合物,其含有以下氨基酸序列表示的母体肽:
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH(SEQ ID NO.1);或
在所述母体肽中的氨基酸序列经过氨基酸取代和/或-NH2经乙酰化,且具有预防或治疗器官纤维化或器官疾病伴随的纤维化活性的由所述母体肽衍生的多肽化合物。在一实施例中,本发明的所述母体肽的-NH2经乙酰化,所述多肽化合物的氨基酸序列为:
化合物1(AC-M10)(SEQ ID NO.2):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH。
在一实施例中,本发明的所述母体肽中的氨基酸序列经过Ala取代。
优选地,所述多肽化合物的氨基酸序列选自以下任一种:
化合物2(T1A)(SEQ ID NO.3):
NH2-Ala-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物3(R2A)(SEQ ID NO.4):
NH2-Thr-Ala-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物4(P3A)(SEQ ID NO.5):
NH2-Thr-Arg-Ala-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物5(S5A)(SEQ ID NO.6):
NH2-Thr-Arg-Pro-Ala-Ala-Phe-Trp-Glu-Thr-Ser-COOH;
化合物6(F6A)(SEQ ID NO.7):
NH2-Thr-Arg-Pro-Ala-Ser-Ala-Trp-Glu-Thr-Ser-COOH;
化合物7(W7A)(SEQ ID NO.8):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Ala-Glu-Thr-Ser-COOH;
化合物8(E8A)(SEQ ID NO.9):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Ala-Thr-Ser-COOH;
化合物9(T9A)(SEQ ID NO.10):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Ala-Ser-COOH;或
化合物10(S10A)(SEQ ID NO.11):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ala-COOH。
在一实施例中,本发明的所述母体肽中的氨基酸序列第5位氨基酸Ser经过氨基酸取代且-NH2经乙酰化。
优选地,所述第5位氨基酸Ser经过Asp、D-Ser、Lys、Asn、Arg或Tyr取代。
优选地,所述多肽化合物的氨基酸序列选自以下任一种:
化合物11(S5D)(SEQ ID NO.12):
CH3-CO-NH-Thr-Arg-Pro-Ala-Asp-Phe-Trp-Glu-Thr-Ser-COOH;
化合物12(S5D-S)(SEQ ID NO.13):
CH3-CO-NH-Thr-Arg-Pro-Ala-(D-Ser)-Phe-Trp-Glu-Thr-Ser-COOH;
化合物13(S5K)(SEQ ID NO.14):
CH3-CO-NH-Thr-Arg-Pro-Ala-Lys-Phe-Trp-Glu-Thr-Ser-COOH;
化合物14(S5N)(SEQ ID NO.15):
CH3-CO-NH-Thr-Arg-Pro-Ala-Asn-Phe-Trp-Glu-Thr-Ser-COOH;
化合物15(S5R)(SEQ ID NO.16):
CH3-CO-NH-Thr-Arg-Pro-Ala-Arg-Phe-Trp-Glu-Thr-Ser-COOH;或
化合物16(S5Y)(SEQ ID NO.17):
CH3-CO-NH-Thr-Arg-Pro-Ala-Tyr-Phe-Trp-Glu-Thr-Ser-COOH。
在一实施例中,本发明的所述母体肽中的氨基酸序列第6位氨基酸Phe经过氨基酸取代且-NH2经乙酰化。
优选地,所述第6位氨基酸Phe经过Asp、D-Phe、Lys、Asn、Ser或Tyr取代。
优选地,多肽化合物的氨基酸序列选自以下任一种:
化合物17(F6D)(SEQ ID NO.18):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Asp-Trp-Glu-Thr-Ser-COOH;
化合物18(F6D-F)(SEQ ID NO.19):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-(D-Phe)-Trp-Glu-Thr-Ser-COOH;
化合物19(F6K)(SEQ ID NO.20):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Lys-Trp-Glu-Thr-Ser-COOH;
化合物20(F6N)(SEQ ID NO.21):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Asn-Trp-Glu-Thr-Ser-COOH;
化合物21(F6S)(SEQ ID NO.22):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Ser-Trp-Glu-Thr-Ser-COOH;或
化合物22(F6Y)(SEQ ID NO.23):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Tyr-Trp-Glu-Thr-Ser-COOH。
本发明还提供一种上述多肽化合物在制备用于预防或治疗器官纤维化及器官疾病伴随的纤维化病症的药物中的用途。
优选地,所述器官纤维化及器官疾病伴随的纤维化病症为肝纤维化及肝脏疾病伴随的纤维化病症。
通过体外细胞模型活性筛选和体内动物模型活性筛选,对系列活性多肽进行药效学评价,结果显示,本发明的多肽化合物没有不良反应发生,可用于预防或治疗器官纤维化及器官疾病伴随的纤维化病症。
本发明中提到的新型多肽化合物中的母体肽为同源性多肽。本发明中的同源性多肽是指,多肽本来具有天然母体肽的氨基酸序列,但其中一个氨基酸残基己被不同的氨基酸残基取代或经修饰,这些氨基酸残基彼此之间是保守的,所得到的多肽可用于实施本发明。
本发明的优点:
1)本发明的多肽化合物与其他肽相比,具有更好的生物学活性;
2)本发明的多肽化合物与其他药物相比,具有更好的预防或治疗器官纤维化及器官疾病伴随的纤维化病症,尤其是预防或治疗肝纤维化及肝脏疾病伴随的纤维化病症,本发明的多肽化合物势必在治疗肝纤维化药物研发方面提供了新的思路及科学研究方向;
3)本发明的多肽化合物与其他药物相比,在药物的血清稳定性实验中显示出更显著的药物活性;
4)本发明的多肽化合物合成产率高,稳定性好,易于放大生产,成本低。
本发明中所用缩写具体含义如下:
Fmoc为芴甲氧羰基,AC为乙酰基,resin为树脂,TFA为三氟乙酸,BSA为牛血清白蛋白,HPLC为高效液相色谱,DMF为N,N-二甲基甲酰胺,DAB为二氨基联苯胺,Thr为苏氨酸,Arg为精氨酸,Phe为苯丙氨酸;D-Phe为D-型苯丙氨酸;Trp为色氨酸;Ser为丝氨酸,D-Ser为D-型丝氨酸,Ala为丙氨酸,Asp为天冬氨酸,Pro为脯氨酸,Glu为谷氨酸,Ala为丙氨酸,Lys为赖氨酸,Asn为天冬酰胺,Tyr为酪氨酸。
附图说明
图1为本发明的实施例2中,在LX2细胞模型中,COL1A1表达情况的Western Blot图和统计柱状图(*:表示置信度>95%,两者差别有显著意义(P<0.05);**:表示置信度>99%,两者差别有非常显著意义(P<0.01);***:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图2为本发明的实施例2中,在LX2细胞模型中,α-SMA表达情况的Western Blot图和统计柱状图(*:表示置信度>95%,两者差别有显著意义(P<0.05);**:表示置信度>99%,两者差别有非常显著意义(P<0.01);***:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图3为本发明的实施例3中,小鼠血清ALT和AST以及肝组织羟脯氨酸的含量结果柱状图(*/#:表示置信度>95%,两者差别有显著意义(P<0.05);**/##:表示置信度>99%,两者差别有非常显著意义(P<0.01);***/###:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****/####:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图4为本发明的实施例3中,小鼠肝脏H&E染色病理切片图(*/#:表示置信度>95%,两者差别有显著意义(P<0.05);**/##:表示置信度>99%,两者差别有非常显著意义(P<0.01);***/###:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****/####:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图5为本发明的实施例3中,小鼠肝脏天狼星红染色病理切片图(*/#:表示置信度>95%,两者差别有显著意义(P<0.05);**/##:表示置信度>99%,两者差别有非常显著意义(P<0.01);***/###:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****/####:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图6为本发明的实施例3中,小鼠肝脏α-SMA免疫组化病理切片图(*/#:表示置信度>95%,两者差别有显著意义(P<0.05);**/##:表示置信度>99%,两者差别有非常显著意义(P<0.01);***/###:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****/####:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
图7为本发明的实施例3中,小鼠肝脏COL1A1免疫组化病理切片图(*/#:表示置信度>95%,两者差别有显著意义(P<0.05);**/##:表示置信度>99%,两者差别有非常显著意义(P<0.01);***/###:表示置信度>99.9%,两者差别有及其显著意义(P<0.001);****/####:表示置信度>99.99%,两者差别有及其显著意义(P<0.001))。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
为方便阐述,将母体肽命名为M10,以下简称“母体肽M10”或“M10”。
实施例1多肽化合物的合成
为方便阐述,本实施例以母体肽和化合物18的合成为例。
材料:
所有的氨基酸和树脂购自上海吉尔生化公司,树脂是Rink Amide MBHA(loading=0.53mmol/g)。除用于半制备高效液相色谱和高效液相色谱-质谱联用外,其他所有试剂均为分析纯,购自上海安耐吉有限公司。Phenomenex Luna C18制备柱(21.2mm×250mm)用来纯化多肽。高效液相色谱仪为Thermofisher公司产品,型号为Ultimate 3000。质谱采用Agilent质谱仪,型号为1260-6120进行测定。
方法:
1.母体肽的合成:
氨基酸序列为:
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH
(1)第一步:树脂溶胀
将1.5g Rink amide MBHA树脂在二氯甲烷(DCM)中溶胀,用二氯甲烷溶胀树脂2次,每次15min;
(2)第二步:氨基酸偶联
以Rink Amide MBHA树脂为载体,以1-羟基苯并三唑(3x)和N,N-二异丙基碳二亚胺(3x)为偶联剂,以N,N-二甲基甲酰胺为溶剂,进行程序反应,依次进行缩合反应连接保护的氨基酸得到:
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-Rink Amide MBHA肽树脂,其中每次缩合反应中Fmoc保护氨基酸的投料量与树脂用量的物质的量比为3:1,每次缩合反应中1-羟基苯并三唑和N,N-二异丙基碳二亚胺与Fmoc保护氨基酸用量的物质的量比为1:1,脱保护溶液为20%哌啶的DMF溶液。偶联完后,用纯甲醇收缩2次,每次15min,真空抽干,得到肽树脂。
(3)第三步:多肽切割与脱保护
将肽树脂:
加入切割液TFA/TIS/H2O=95/2.5/2.5(v/v/v)15mL于多肽反应器中充入氩气保护反应,摇床上反应240分钟。反应结束后,将含有多肽的切割剂注入含有冰乙醚中的圆底烧瓶中。静置至沉淀完全,离心,倾去上清液,沉淀用真空冻干机干燥,得到母体肽的肽序为:
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH。
(4)第四步:纯化
将上述第三步中所得粗品,用乙腈:H2O(含0.1%TFA)=5:95(体积比)的溶液10mL超声溶解,溶解澄清后用0.22μm一次性针式过滤器过滤,滤液通过20mm反相C18的填充的21.2mm x 250mm柱上进行2次半制备型HPLC而纯化。用40-60%乙腈-0.1%三氟乙酸/H2O梯度以10mL/min将该柱洗脱60.0分钟,收集含有母体肽的组分,沉淀用真空冻干机干燥。得到HPLC纯度为95%的纯品。用液质联用分析分离出的产物,用液质联用分析分离出的产物,发现质子化分子离子峰的m/2z值为:590.9,理论值为1180.5。
2.多肽化合物18的合成:
氨基酸序列为
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-(D-Phe)-Trp-Glu-Thr-Ser-COOH
(1)第一步:树脂溶胀
将1.5g Rink amide MBHA树脂在二氯甲烷(DCM)中溶胀,用二氯甲烷溶胀树脂2次,每次15min;
(2)第二步:氨基酸偶联
以Rink Amide MBHA树脂为载体,以1-羟基苯并三唑(3x)和O-苯并三氮唑-四甲基脲六氟磷酸盐(3x)为偶联剂,以N,N-二甲基甲酰胺为溶剂,进行程序反应,依次进行缩合反应连接保护的氨基酸得CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-(D-Phe)-Trp-Glu-Thr-Ser-Rink Amide MBHA线性肽树脂。其中每次缩合反应中N-Fmoc保护氨基酸的投料量与树脂用量的物质的量比为3:1,每次缩合反应中1-羟基苯并三唑和O-苯并三氮唑-四甲基脲六氟磷酸盐与N-Fmoc保护氨基酸用量的物质的量比为3:1,脱保护溶液为20%哌啶的DMF溶液。
(3)第三步:乙酰化反应
将取吡啶310.6μL、醋酸酐182μL溶于15mL的DMF中并投入到反应器中,通入氩气,反应30min DMF(10mL*3)洗涤,抽干。
(4)第四步:多肽切割与保护基的脱除
将(3)中所得树脂加入切割液TFA/TIS/H2O=95/2.5/2.5(v/v/v)15mL于多肽反应器中充入氩气保护反应,摇床上反应240分钟。反应结束后,将含有多肽的切割剂注入含有冰乙醚中的圆底烧瓶中。静置至沉淀完全,离心,倾去上清液,沉淀用真空冻干机干燥,得到化合物18的肽序为:
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-(D-Phe)-Trp-Glu-Thr-Ser-COOH
(5)第五步:纯化:
将上述第四步中所得粗品,用乙腈:H2O(含0.1%TFA)=5:95(体积比)的溶液10mL超声溶解,溶解澄清后用0.22μm一次性针式过滤器过滤,滤液通过20mm反相C18的填充的21.2mm x 250mm柱上进行2次半制备型HPLC而纯化。用40-60%乙腈-0.1%三氟乙酸/H2O梯度以10mL/min将该柱洗脱60.0分钟,收集含有母体肽的组分,沉淀用真空冻干机干燥。得到HPLC纯度为95%的纯品。用液质联用分析分离出的产物,用液质联用分析分离出的产物,发现质子化分子离子峰的m/2z值为:612.5,理论值为1223.3。
基于上述步骤合成的多肽化合物1-22,如下表1所示:
表1.本发明的多肽化合物
实施例2在LX-2细胞模型中,对化合物1-22进行活性的评价
在LX-2细胞模型中(因为LX-2细胞是人肝活化星状细胞,所以可以直接作为体外肝脏纤维化细胞评价模型,LX-2细胞由中山大学药学院黄志纾教授课题组惠赠),对多肽化合物1-10进行活性的抗纤维化活性进行初筛,检测COL1A1蛋白的表达情况,对多肽化合物11-22进行活性的抗纤维化活性进行第二次筛选,检测α-SMA蛋白的表达情况,多肽化合物1-22在200μM下进行细胞孵育48h。对照组给予同样体积的Dulbecco’s改良Eagle培养基(DMEM)(购自Gibco公司,美国),GAPDH(购自中国中杉金桥生物工程有限公司)作为内参。
实验结果如图1和2所示,从实验结果的统计图可以明显看出,与GAPDH内参相比,本发明的多肽化合物都能够不同程度地降低COL1A1或α-SMA的表达。实验结果也表明:本发明的多肽化合物在细胞层面能抑制纤维化的生成,同时也提示:该类新型的多肽化合物可潜在的用于肝纤维化及肝脏疾病伴随的纤维化病症的研究。
实施例3多肽化合物在四氯化碳(CCl4)诱导的小鼠肝纤维化模型上的药效学评价
本实施例以母体肽M10和多肽化合物18为例,对本发明的多肽化合物进行药效学评价。
1.受试药物:多肽化合物类似物。保存条件-20℃。
造模方法:将20只雄性C57BL/6J小鼠,雄性,体重18-22g,周龄8周,由北京维通利华实验动物技术有限公司提供,随机分为4组,分别为:
1)普通对照组:油(Oil)+生理盐水,腹腔注射,n=5;第1-6周给予小鼠Oil三天一次,其中第4-6周给予小鼠生理盐水每天一次。
2)模型对照组:CCl4+生理盐水,腹腔注射,n=5;第1-6周给予小鼠CCl4三天一次,其中第4-6周给予小鼠生理盐水每天一次;
3)给药组1:CCl4+1mg/kg母体肽M10,腹腔注射,n=5;第1-3周给予小鼠CCl4三天一次;第4-6周给予母体肽M10每天一次。
4)给药组2:CCl4+1mg/kg多肽化合物18,腹腔注射,n=5;第1-3周给予小鼠CCl4三天一次;第4-6周给予多肽化合物18每天一次。
其中给予小鼠20%的CCl4,按照小鼠体重5.0μL/g的给药体积,普通对照组小鼠给予相同体积和频率的注射玉米油,CCl4和Oil购自于上海阿拉丁生化科技股份有限公司。
2.药效评价:
在CCl4诱导的肝脏纤维化模型中,其表现特征为:中央静脉区域周围有炎性细胞浸润,肝细胞水肿变性,汇管区及肝小叶间隔有大量胶原纤维沉积等。在给药三周之后,处理小鼠并进行取材,通过眼球后静脉取血进行血清学指标检测,并取肝脏组织用于病理学分析。
3.实验方法:
苏木素-伊红(H&E)染色:取石蜡包埋的组织切片,60℃烘1h。脱蜡水化:二甲苯20分钟→二甲苯20分钟→无水酒精15分钟→无水酒精15分钟→95%酒精10分钟→90%酒精5分钟→80%酒精5分钟。染色:苏木精7分钟→自来水冲洗干净→1%盐酸乙醇分化1s→自来水冲洗→伊红染色15s-20s→自来水冲洗。脱水透明:75%酒精1s→85%酒精1s→95%酒精1s→100%酒精1s→二甲苯1s→二甲苯1s。封片晾干30分钟,树胶封片。
天狼腥红染色:烘片、脱蜡;双蒸水中静置5.0分钟;暗室中天狼腥红染色60-80分钟;0.5%冰醋酸涮洗5s;脱水透明,封片,拍照。
免疫组化:烘片、脱蜡,之后放入双蒸水中浸泡5分钟。抗原修复:将放病理切片的架子放于盛有柠檬酸的缓冲液(PH=6.0)烧杯中,高温高压15分钟;取出烧杯,静置在室温待温度下降至室温,然后取出片子,放入3.0%双氧水中10分钟,以阻断内源性过氧化物酶活性,之后用PBS洗涤3次,每次5分钟。利用1.0%BSA对组织进行封闭1h。去除1.0%BSA,按说明书推荐比例滴加抗体于组织上,4℃过夜;第二天取出病理切片,待恢复室温之后加入连有辣根过氧化物酶的二抗,37℃孵育60分钟,然后用DAB(TH&Ermo Scientific,USA)显色;用苏木素染液复染,用自来水冲洗5分钟,1%盐酸乙醇分化,再用自来水洗5分钟,脱水,风干,封片,拍照。
H&E染色液和天狼腥红染色液购自于上海生工生物工程有限公司,COL1A1和α-SMA抗体购自于CST公司。
4.结果分析:
图3为本实施例,小鼠血清的谷丙转氨酶(ALT)、谷草转氨酶(AST)以及肝组织羟脯氨酸的含量结果柱状图。从图3的结果可以看出:小鼠在腹腔注射给予CCl4后,出现严重的肝脏损伤,经母体肽M10和多肽化合物18药物治疗后,肝脏损伤程度减轻。
图4为本实施例中,小鼠肝脏H&E染色病理切片图;图5为本实施例中,小鼠肝脏天狼星红染色病理切片图。从图4和图5的结果可以看出:小鼠在腹腔注射给予CCl4后,肝脏出现胶原沉积以及纤维化在给予母体肽M10和多肽化合物18治疗后,小鼠肝脏炎性细胞聚集和肝脏纤维化均得到了显著的改善。由此可以证明,本发明的多肽化合物能够明显地治疗改善胶原沉积,可以很好的抑制ECM的积累,治疗改善肝脏纤维化。
图6为本实施例中,小鼠肝脏α-SMA免疫组化病理切片图;图7为本实施例中,小鼠肝脏COL1A1免疫组化病理切片图。从图6和图7的结果可以看出:经母体肽M10和多肽化合物18给药治疗后,小鼠肝脏COL1A1和α-SMA的表达量均出现了明显的治疗改善。由此可以证明,本发明的多肽化合物能够抑制肝脏COL1A1和α-SMA的表达。
综合上述实验结果表明,本发明的多肽化合物抑制了ECM的积累以及肺组织α-SMA、COL1A1的表达,可用于治疗肝纤维化及肝脏疾病伴随的纤维化病症。
以上实施例是为了详细说明本发明的技术方案而列举的典型范例,本发明以权利要求及发明内容的保护范围为准,不受所述实施方案的限制,对本发明进行简单替换或变动仍处于本发明创造的保护范围之中。
序列表
<110> 中山大学
<120> 改善肝纤维化的多肽化合物及其用途
<130> GD1899-21P126156
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<170> PatentIn version 3.5
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Claims (12)
1.多肽化合物,其含有以下氨基酸序列表示的母体肽:
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH(SEQ ID NO.1);或
在所述母体肽中的氨基酸序列经过氨基酸取代和/或-NH2经乙酰化,且具有预防或治疗器官纤维化或器官疾病伴随的纤维化活性的由所述母体肽衍生的多肽化合物。
2.根据权利要求1所述的多肽化合物,其特征在于,所述母体肽的-NH2经乙酰化,所述多肽化合物的氨基酸序列为:
化合物1(SEQ ID NO.2):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH。
3.根据权利要求1所述的多肽化合物,其特征在于,所述母体肽中的氨基酸序列经过Ala取代。
4.根据权利要求3所述的多肽化合物,其特征在于,所述多肽化合物的氨基酸序列选自以下任一种:
化合物2(SEQ ID NO.3):
NH2-Ala-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物3(SEQ ID NO.4):
NH2-Thr-Ala-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物4(SEQ ID NO.5):
NH2-Thr-Arg-Ala-Ala-Ser-Phe-Trp-Glu-Thr-Ser-COOH;
化合物5(SEQ ID NO.6):
NH2-Thr-Arg-Pro-Ala-Ala-Phe-Trp-Glu-Thr-Ser-COOH;
化合物6(SEQ ID NO.7):
NH2-Thr-Arg-Pro-Ala-Ser-Ala-Trp-Glu-Thr-Ser-COOH;
化合物7(SEQ ID NO.8):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Ala-Glu-Thr-Ser-COOH;
化合物8(SEQ ID NO.9):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Ala-Thr-Ser-COOH;
化合物9(SEQ ID NO.10):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Ala-Ser-COOH;或
化合物10(SEQ ID NO.11):
NH2-Thr-Arg-Pro-Ala-Ser-Phe-Trp-Glu-Thr-Ala-COOH。
5.根据权利要求1所述的多肽化合物,其特征在于,所述母体肽中的氨基酸序列第5位氨基酸Ser经过氨基酸取代且-NH2经乙酰化。
6.根据权利要求5所述的多肽化合物,其特征在于,所述第5位氨基酸Ser经过Asp、D-Ser、Lys、Asn、Arg或Tyr取代。
7.根据权利要求6所述的多肽化合物,其特征在于,所述多肽化合物的氨基酸序列选自以下任一种:
化合物11(SEQ ID NO.12):
CH3-CO-NH-Thr-Arg-Pro-Ala-Asp-Phe-Trp-Glu-Thr-Ser-COOH;
化合物12(SEQ ID NO.13):
CH3-CO-NH-Thr-Arg-Pro-Ala-(D-Ser)-Phe-Trp-Glu-Thr-Ser-COOH;
化合物13(SEQ ID NO.14):
CH3-CO-NH-Thr-Arg-Pro-Ala-Lys-Phe-Trp-Glu-Thr-Ser-COOH;
化合物14(SEQ ID NO.15):
CH3-CO-NH-Thr-Arg-Pro-Ala-Asn-Phe-Trp-Glu-Thr-Ser-COOH;
化合物15(SEQ ID NO.16):
CH3-CO-NH-Thr-Arg-Pro-Ala-Arg-Phe-Trp-Glu-Thr-Ser-COOH;或
化合物16(SEQ ID NO.17):
CH3-CO-NH-Thr-Arg-Pro-Ala-Tyr-Phe-Trp-Glu-Thr-Ser-COOH。
8.根据权利要求1所述的多肽化合物,其特征在于,所述母体肽中的氨基酸序列第6位氨基酸Phe经过氨基酸取代且-NH2经乙酰化。
9.根据权利要求8所述的多肽化合物,其特征在于,所述第6位氨基酸Phe经过Asp、D-Phe、Lys、Asn、Ser或Tyr取代。
10.根据权利要求9所述的多肽化合物,其特征在于,所述多肽化合物的氨基酸序列选自以下任一种:
化合物17(SEQ ID NO.18):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Asp-Trp-Glu-Thr-Ser-COOH;
化合物18(SEQ ID NO.19):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-(D-Phe)-Trp-Glu-Thr-Ser-COOH;
化合物19(SEQ ID NO.20):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Lys-Trp-Glu-Thr-Ser-COOH;
化合物20(SEQ ID NO.21):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Asn-Trp-Glu-Thr-Ser-COOH;
化合物21(SEQ ID NO.22):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Ser-Trp-Glu-Thr-Ser-COOH;或
化合物22(SEQ ID NO.23):
CH3-CO-NH-Thr-Arg-Pro-Ala-Ser-Tyr-Trp-Glu-Thr-Ser-COOH。
11.权利要求1-10任一项所述多肽化合物在制备用于预防或治疗器官纤维化及器官疾病伴随的纤维化病症的药物中的用途。
12.根据权利要求11所述的用途,其特征在于,所述器官纤维化及器官疾病伴随的纤维化病症为肝纤维化及肝脏疾病伴随的纤维化病症。
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