CN108341880B - 含有胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽及其用途 - Google Patents
含有胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽及其用途 Download PDFInfo
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Abstract
本发明提供一种包含胰高血糖素样肽‑1和胰高血糖素的片段类似物的嵌合多肽，所述多肽的序列如以下通式Ⅰ所示：通式Ⅰ：HX2EGTFTSDYSKYLX15X16X17AA X20X21FX23X24WLVKX29X30X31X32。本发明还提供了所述嵌合多肽的缀合物和聚合物，其中，所述聚合物是由上述嵌合多肽通过Cys残基共价连接而成或通过接头共价连接而成。本发明还提供了一种药物组合物，所述药物组合物包含上述嵌合多肽、上述嵌合多肽的缀合物、聚合物。本发明提供的嵌合多肽具有显著的降血糖作用和减轻体重作用，其药效和长效性优于阳性对照药，而且溶解性和稳定性好，更适于制剂，可用于制备糖尿病和肥胖等代谢性疾病的治疗药物。
Description
本申请要求2017年1月23日提交的名称为“含有胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽及其用途”、申请号为201710050234.0的中国发明专利申请的优先权。
技术领域
本发明属于医药生物技术领域，具体涉及一种含有胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽、其缀合物、聚合物，以及所述嵌合多肽、其缀合物、聚合物在制备用于预防和/或治疗糖尿病、肥胖和/或代谢综合征的药物中的用途。
背景技术
肥胖和2型糖尿病(T2DM)是困扰现代社会的主要公共健康问题之一。肥胖及其伴随的胰岛素抵抗是2型糖尿病发病的关键因素，据调查，80-90％的2型糖尿病患者超重或肥胖(邹大进等，上海医学，2014，37(9)，729～734)。因此，有效控制血糖和体重始终是有关研究领域的焦点课题。
在过去的十余年里，肠道激素尤其是作用于胰高血糖素样肽-1(GLP-1)通路的治疗药物受到业界的广泛认可。胰高血糖素样肽-1(GLP-1)是一种肠源性激素，主要由位于末端空肠、回肠和结肠的L细胞合成，在进餐反应中释放到循环系统。GLP-1(7-36，7-37)是体循环中GLP-1的主要活性形式，通过复杂的机制控制血糖，包括胰岛素和胰高血糖素的分泌，胃的排空以及外周胰岛素的调节。GLP-1(7-36，7-37)降血糖作用是葡萄糖依赖性的，可避免低血糖发生，而且具有抑制胰岛β-细胞的凋亡，促进胰岛β-细胞增生的作用，可逆转病情发展。但天然GLP-1的血浆半衰期仅为1-2分钟，从而限制了其作为药物的应用。研究表明体内的二肽激肽酶(DPPIV)特异性识别并降解GLP-1结构中受体结合活性部位N端His-Ala二肽片段，而使其快速失活，同时其他蛋白水解酶如内肽酶等也参与肾脏滤过清除过程。
基于人源GLP-1进行的药物研发基本是通过结构改造以提高代谢稳定性，延长血浆半衰期为目的，公开的专利技术可归结为：1)针对酶降解关键位点的结构改造(如中国专利申请CN00806548.9、CN99814187.9、CN200410017667.9等所公开的)；2)母体肽链共价偶联脂肪酰基团，提高与血浆蛋白结合力以避免体内快速清除(如中国专利CN201210513145.2、CN200810124641.2、CN20118000352.1等所公开的)；3)蛋白偶联/融合技术；4)PEG修饰等。分子量小于5000道尔顿的多肽从体内的主要消除途径为肾小球滤过作用，因此通过修饰以增加分子量和体积是延长GLP-1受体激动剂类多肽药物延长体内半衰期的主要技术手段。而采取这些结构修饰措施时高活性母体多肽的修饰物更具成药性优势。
前胰高血糖素来源的胰高血糖素(GC)、GLP-1、GLP-2等肠道激素的受体均是G蛋白偶联受体，而这些配体都是以α-螺旋结构的形式与受体结合。研究表明，α-螺旋结构的倾向与hGLP-1R的结合能力呈正相关(Biochemistry,46,5830-5840)，因此，除了结构中受体激动必要片段之外，支持其与受体结合的α-螺旋结构片段也是影响配体多肽生物活性强度的重要因素。人源GLP-1结构包括N-端不规则卷曲的7-13片段以及由柔性的Gly22连接的不连续的两段α-螺旋结构(13-20,24-36)。Exenatide(Exendin-4)是最早上市的GLP-1受体激动剂，是来源于美洲毒蜥蜴唾液的多肽，因其结构中连续的α螺旋的片段(11-27)以及有利于形成和稳定α螺旋的C-端，相较人源的GLP-1具有更强的受体激动活性(Biochemistry,46,5830-5840)。无论是短效产品还是每周给药1次的长效制剂，用药剂量都小于基于人源GLP-1序列的改构品种，如利拉鲁肽(0.8-1.8mg vs 15μg Exenatide)。但Exenatide与人源GLP-1只有53％的序列同源性而具有免疫原性，在给药30周后会使41％的受试病人体内出现抗体(Diabets Care,2004,27,2628-2635)。
胰高血糖素(GC)是由对应于前胰高血糖素(pro-glucagon)的33至61位的氨基酸组成的含29个氨基酸的衍生肽，其氨基酸序列为：HSQGTFTSDYSKYLDSRRAQDFVQWLMNT。GC三聚体晶体的X-射线衍射结构研究表明，其序列中10-25片段呈连续的α-螺旋二级结构(Nature 1975,257,751-757)。
然而，当前基于人源GLP-1研发的药物在活性、稳定性以及免疫原性等方面均有待于改善。
发明内容
基于现有技术的不足，本发明人意外地发现，以GC结构中的特定片段替换GLP-1序列的特定片段，并进行特定的氨基酸置换获得的嵌合多肽具有选择性GLP-1受体激动活性，其活性强于内源性GLP-1，而且还显示了良好的降血糖作用和酶代谢稳定性，可以作为用于有效控制血糖和减轻体重的更具有开发潜力的治疗剂。
因此，本发明的目的是提供一种胰高血糖素样肽-1和胰高血糖素片段类似物的嵌合多肽、其缀合物、聚合物。本发明的嵌合多肽是选择性GLP-1受体激动剂，其活性强于内源性GLP-1，具有更好的代谢稳定性，从而更适合长效化修饰，而且本发明提供的嵌合多肽的序列组成与内源性GLP-1和GC具有高度同源性(＞80％)，可以减少免疫原性，更具应用潜力。
一方面，本发明提供一种包含胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽，其中，所述嵌合多肽的序列如以下通式Ⅰ所示：
通式Ⅰ：
HX2EGTFTSDYSKYLX15X16X17AAX20X21FX23X24WLVKX29X30X31X32
其中：
X2为D-Ala、Gly或Aib(2-氨基异丁酸)；
X15为Asp或Glu；
X16为Glu或Aib；
X17为Arg或Gln；
X20为Gln或Lys；
X21为Glu或Asp；
X23为Ile或Val；
X24为Ala，Gln或Cys；
X29为Gly或Aib；
X30为Arg或Cys；
X31为Gly、Cys、-NH2或不存在；
X32为Cys、-NH2或不存在；
并且，在通式Ⅰ所示的序列中，有且只有一个Cys残基；当通式Ⅰ所示的序列含Aib残基时，X2、X16和X29三个位点中只有一个位点为Aib。
优选地，在通式Ⅰ所示的序列中，X2为D-Ala或Aib，且X16为Glu或Aib；更优选地，在通式Ⅰ所示的序列中，X2为Aib，且X16为Glu；
优选地，所述嵌合多肽的序列如SEQ ID NO:1-42中任一条所示；
进一步优选地，所述嵌合多肽的序列如SEQ ID NO:16-36中任一条所示；
另一方面，本发明提供了所述嵌合多肽的缀合物，优选地，所述缀合物为所述嵌合多肽的聚乙二醇修饰物，在所述嵌合多肽的Cys残基的侧链上共价连接聚乙二醇；更优选地，所述聚乙二醇的平均分子量为5-50KD；更优选地为20-45KD；优选地，所述聚乙二醇为直链或支链聚乙二醇；更优选地，所述聚乙二醇为分子量40-45KDa的支链型聚乙二醇。
另一方面，本发明还提供了所述嵌合多肽的聚合物，优选地，所述聚合物为所述嵌合多肽的二聚体，所述二聚体是所述嵌合多肽通过Cys残基共价连接而成或通过接头共价连接而成；
优选地，所述二聚体通过连有双官能胺交联基团的接头与多肽的巯基反应形成；
优选地，所述接头为双马来酰亚胺-聚乙二醇(Mal-PEG-Mal)；
更优选地，所述聚乙二醇平均分子量范围为3-20KD；
优选地，所述二聚体为同源二聚体；
再一方面，本发明还提供了一种药物组合物，所述药物组合物包含上述嵌合多肽、其缀合物、聚合物。
优选地，所述药物组合物还包含药学上可接受的载体和/或辅料。
更优选地，所述载体和/或辅料选自水溶性填充剂、pH调节剂、稳定剂、注射用水或渗透压调节剂中的一种或多种；
优选地，所述水溶性填充剂选自甘露醇、低分子右旋糖酐、山梨醇、聚乙二醇、葡萄糖、乳糖或半乳糖中的一种或多种；所述pH调节剂选自生理上可接受的有机酸或无机酸，例如枸橼酸、磷酸、乳酸、酒石酸、盐酸，或者生理上可接受的无机碱，例如氢氧化钾、氢氧化钠、氢氧化铵、碳酸钠、碳酸钾、碳酸铵、碳酸氢钾、碳酸氢钠、碳酸氢铵盐中的一种或多种；所述稳定剂选自EDTA-2Na、硫代硫酸钠、焦亚硫酸钠、亚硫酸钠、磷酸氢二钾、碳酸氢钠、碳酸钠、精氨酸、赖氨酸、谷氨酸、天冬氨酸、聚乙二醇、聚乙烯醇、聚乙烯吡咯烷酮、羧基/羟基纤维素或其衍生物如HPC、HPC-SL、HPC-L或HPMC、环糊精、十二烷基硫酸钠或三羟甲基氨基甲烷中的一种或多种；所述渗透压调节剂为氯化钠和/或氯化钾。
再另一方面，本发明提供了包含上述嵌合多肽、其缀合物、聚合物、或上述药物组合物在制备用于治疗糖尿病、肥胖和/或代谢综合征的药物中的用途。
优选地，本发明所述的组合物可以为静脉、肌肉或皮下注射剂形式，或口服、直肠、鼻腔给药的形式。剂量范围可以为5μg-10mg/次，这取决于治疗对象、给药方式、适应症以及其他因素等。
为了更好地理解本发明，详细说明如下：
胰高血糖素样肽-1和胰高血糖素片段嵌合多肽的基本结构
内源性GLP-1受体激动剂是GLP-1(7-36/37)，其多肽氨基酸序列为HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR(7-36)或HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(7-37)；而内源性胰高血糖素的多肽序列为：HSQGTFTSDYSKYLDSRRAQDFVQWLMNT(1-29)。
在本发明的实施方案中，嵌合多肽是将GLP-1(7-36/37)序列的12-26片段以GC序列的6-24的不同长短片段替换而得。GLP-1(7-36/37)原序列中N-端容易被二肽基肽酶(DPP-IV)识别，降解去除His-Ala的二肽而失去活性。本发明人发现，将嵌合多肽的X2位替换为D-Ala或Aib，极大地提高了对DPP-IV降解的抗性。本发明人还发现，通过对来源于GC的(16-20)片段进行适当改变，如将Ser16替换为Glu，Arg18替换为Ala，大大地提高了GLP-1受体的结合活性。本发明人进一步地发现，将本申请序列中的C-末端羧基酰胺化有助于提高多肽稳定性。
通过以上的结构设计，本发明提供的如通式Ⅰ所示序列的多肽与内源性GLP-1(7-36/37)相比，具有以下的优点：
1)本发明的多肽更容易在内环境中形成连续的α-螺旋结构，从而进一步增强了其在体内的活性；
2)本发明的多肽不容易被DPP-IV识别，从而避免了被DPP-IV酶降解失活；
3)本发明的多肽不仅对GLP-1受体具有增强的激动活性，而且是选择性GLP-1受体激动剂，于此同时体内稳定性得以提高，更有利于长效化修饰。
4)本发明提供的多肽序列中24位或C-末端替换或引入Cys残基不影响多肽的活性，可以选择作为修饰位点。而本领域技术人员已知的是，C-末端羧基酰胺化有助于多肽系列稳定，因此，本发明还进一步提供了特定序列的C-末端羧基形成酰胺。
聚乙二醇化
多肽类激素在机体内主要通过酶降解和肾清除途径代谢，其中肾清除占主导，是影响多肽类药物体内半衰期的主要因素。多肽序列中一些氨基酸残基侧链上的结构修饰特别是烷基化或聚乙二醇等大分子基团的缀合可以延缓肾清除，有效延长生物半衰期。
本发明的特定实施方案中，具有通式Ⅰ序列多肽，其X24、X30、X31、X32位点中的一个位点可以被替换为Cys，在Cys的巯基侧链上定点共价缀合聚乙二醇(PEG)基团。所述PEG是指平均分子量范围20-50KD的直链或支链聚乙二醇，本发明实施方案中优选直链单甲氧基PEG。
本发明所述的聚乙二醇是通过多种途径可以得到，包括商业途径获得或者根据本领域中已知的方法自行制备。本发明的实施方案中用于修饰的支链型聚乙二醇的通式为Mal-(mPEG)2，优选但不限于选自如下结构：
本发明所述PEG修饰可以通过本领域公知的任何方法实现，包括经由酰化、还原性烷化、Michael加成、硫醇烷化或通过PEG部分的活性基团(例如醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基)的其他化学选择性缀合方法。在本发明的特定实施方案中，通过Michael加成反应，即用马来酰亚胺活化的PEG修饰Cys的巯基侧链，产生硫醚键连接的聚乙二醇化多肽。在某些实施方案中，通过亲核取代反应，即用卤代乙酰基活化的PEG修饰硫醇，得到硫醚键连接的聚乙二醇化多肽。
二聚体
多肽的二聚体或多聚体形式也是延长多肽体内半衰期的有效手段。本发明还提供了所述多肽的二聚体形式。包括通过序列中24位Cys残基上的巯基侧链以分子间二硫键形成的同源二聚体或经由接头共价结合的二聚体形式。这些多肽二聚体是通过形成空间位阻有效屏蔽肽链中的酶切位点而延缓活性多肽降解。
在特定实施方案中，通式Ⅰ序列结构的单链多肽通过结构中半胱氨酸残基的侧链巯基氧化形成分子间二硫键。二硫键形成方法可采用本技术领域公知的常规技术，包括空气氧化、谷胱甘肽、K3Fe(CN)6，I2，DMSO氧化法等。
在一些特定实施方案中所述二聚体是通过连有双官能胺交联基团的接头与单体肽链的巯基反应形成。所述接头是但不限于双马来酰亚胺-聚乙二醇(Mal-PEG-Mal)，所述聚乙二醇平均分子量范围是但不限于3000-20000道尔顿。
活性
在本发明实施例中，通过对GLP-1、GC受体的体外激动活性试验证实了本发明提供胰高血糖素样肽-1和胰高血糖素片段类似物嵌合多肽是GLP-1受体的选择性激动剂，对GC受体没有影响，而且对GLP-1受体的激动活性强于内源配体GLP-1(7-36)。经过PEG长效化修饰的多肽在体外受体活性评价中显示了与内源性配体相当的受体激动活性强度，说明本发明提供的前体多肽序列更有利于保障长效化修饰体的生物活性。
用途
针对目前治疗糖尿病、肥胖、代谢综合征等现有药物治疗的局限性，本发明提供一种新型治疗方法，所述方法涉及给予包含本发明的嵌合肽、其缀合物、聚合物或其盐的药物组合物。包含本发明的嵌合多肽、其缀合物、聚合物或其盐的药物组合物在有效地降低血糖的同时具有促进减重和防止增重作用，与现有药物相比具有意想不到的有益作用。
药物组合物
再一方面，本发明还提供了一种药物组合物，所述药物组合物包含上述嵌合多肽或其盐、其缀合物、聚合物。
优选地，所述药物组合物还包含药学上可接受的载体和/或辅料。
更优选地，所述载体和/或辅料包括水溶性填充剂、pH调节剂、稳定剂、注射用水、渗透压调节剂中的一种或多种。
优选地，所述水溶性填充剂包括但不限于甘露醇、低分子右旋糖酐、山梨醇、聚乙二醇、葡萄糖、乳糖、半乳糖等；所述pH调节剂包括但不限于枸橼酸、磷酸、乳酸、酒石酸、盐酸等有机或无机酸，以及氢氧化钾、氢氧化钠、氢氧化铵、碳酸钠、碳酸钾、碳酸铵、碳酸氢钾、碳酸氢钠、碳酸氢铵盐等生理上可接受的无机碱或盐；所述稳定剂包括但不限于EDTA-2Na、硫代硫酸钠、焦亚硫酸钠、亚硫酸钠、磷酸氢二钾、碳酸氢钠、碳酸钠、精氨酸、赖氨酸、谷氨酸、天冬氨酸、聚乙二醇、聚乙烯醇、聚乙烯吡咯烷酮、羧基/羟基纤维素或其衍生物如HPC、HPC-SL、HPC-L或HPMC、环糊精、十二烷基硫酸钠或三羟甲基氨基甲烷等；所述渗透压调节剂包括但不限于氯化钠或氯化钾。
再另一方面，本发明提供了上述嵌合多肽在制备用于治疗糖尿病、肥胖和/或代谢综合征的药物中的用途。
优选地，本发明所述的组合物可以静脉、肌肉或皮下注射剂的形式或口服、直肠、鼻腔给药。剂量范围可以为5μg-10mg/次，这取决于治疗对象、给药方式、适应症以及其他因素等。
合成
在本发明提供的具有通式Ⅰ结构的嵌合多肽的基本肽链是通过本领域公知的方法制备得到：
1)通过常规固相或液相方法逐步或通过片段组装合成；
2)在宿主细胞中表达编码多肽的核酸构建体，并从宿主细胞培养物回收表达产物；
3)影响编码多肽的核酸构建体的无细胞体外表达，并回收表达产物；
或通过方法1)、2)或3)的任意组合来获得肽片段，随后连接这些片段以获得目标肽。
优选地，使用Fmoc固相合成方法制备得到目标肽。
优选地，通过以下方法完成目标多肽的聚乙二醇化修饰：将经过活化的PEG与本发明的多肽在pH5.0-7.0下反应，其中PEG与肽的摩尔比值为1-10，反应时间为0.5-12小时，反应温度为4-37℃。
缀合反应之后，可以通过本领域公知的合适方法将目标产物分离。适用的方法包括但不限于超滤法、透析法或色谱法等。
优选地，本发明所述多肽的二聚体形式是由通式Ⅰ表示的序列多肽如SEQ ID NO:1-42中的任一单体通过结构中半胱氨酸残基的侧链巯基氧化形成分子间二硫键的方式连接而成。二硫键形成方法可采用本技术领域公知的常规技术，包括空气氧化、谷胱甘肽、K3Fe(CN)6、I2、DMSO氧化法等。
活性评价
本发明的实施方案中通过GLP-1/GC受体介导的体外cAMP产生的影响评价了所述多肽对GLP-1/GC受体的作用。
本发明的实施方案中采用小鼠糖耐量试验，以艾塞那肽为阳性对照药，评价了本发明提供多肽的降血糖活性。
本发明的另一个实施方案中采用高脂饮食肥胖小鼠(Dio)模型评价了所述PEG修饰多肽的降血糖作用和对体重的影响，结果表明本发明提供的聚乙二醇修饰多肽具有显著的降血糖作用和减轻体重作用，其药效和长效性优于阳性对照药，而且溶解性和稳定性好，更适于制剂，可用于制备肥胖等代谢性疾病的控制和糖尿病的治疗药物。
附图说明
以下，结合附图来说明本发明的实施例，其中
图1为实施例8中如SEQ ID NO:18、40所示的多肽序列的PEG40KD缀合物给药两周对Dio小鼠体重影响的评价结果。
图2为实施例9中如SEQ ID NO:18、40所示的多肽序列的PEG40KD缀合物给药两周对Dio小鼠血糖影响的评价结果。
图3为实施例11中支链型(Y)PEG修饰多肽pg016、pg019对Dio小鼠体重影响的评价结果。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。本实施例仅为解释本发明，不意味以任何方式限制本发明内容。
氨基酸缩写的说明：
Gly：甘氨酸(G)
Ala：丙氨酸(A)
Val：缬氨酸(V)
Leu：亮氨酸(L)
Phe：苯丙氨酸(F)
Trp：色氨酸(W)
Ser：丝氨酸(S)
Thr：苏氨酸(T)
Glu：谷氨酸(E)
Gln：谷氨酰胺(Q)
Asp：天冬氨酸(D)
Asn：天冬酰胺(N)
Tyr：苯丙氨酸(Y)
Arg：精氨酸(R)
Lys：赖氨酸(K)
His：组氨酸(H)
Aib：2-氨基异丁酸
试剂缩写的说明
Boc:叔丁氧基羰基
Tert-Bu:叔丁基
DCM:二氯甲烷
DIC：二异丙基碳二亚胺
Fmoc:9-芴甲氧基羰基
HoBt:1-羟基苯并三唑
HBTU:2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基-脲鎓六氟磷酸酯
HATU:O-(7-氮杂苯并三唑-1-基)-N,N,N’,N’-四甲基-脲鎓六氟磷酸酯
Mtt:4-甲基三苯甲基
NMP:N-甲基吡咯烷酮
DMF:二甲基甲酰胺
Pbf:2,2,4,6,7-五甲基二氢苯并呋喃
Trt:三苯基甲基
EDT:乙二硫醇
TFA:三氟乙酸
TIS:三异丙基硅烷
FBS:胎牛血清
实施例1
通过以下的步骤制备通式Ⅰ所示的多肽
1)合成：采用Fmoc策略，用CS 336型多肽合成仪(CS Bio)，按照如下步骤逐步合成：
a)在活化剂系统存在下由树脂固相载体和Fmoc保护的C-端氨基酸偶联得到Fmoc-氨基酸-树脂；其中，合成C-端酰胺化多肽采用氨基树脂，如Rink Amide AM，Rink Amide，Rink MBHA等。
b)肽链的延长：通过固相合成法按照肽序列氨基酸顺序连接氨基酸，得到N-端和侧链保护的肽-树脂偶联物；带侧链氨基酸采取如下保护措施：色氨酸用Boc，谷氨酸用OtBu，赖氨酸用Boc，谷氨酰胺用Trt，酪氨酸用tBu，丝氨酸用Trt或tBu，天冬氨酸用OtBu，苏氨酸用tBu，半胱氨酸用Trt，组氨酸用Trt或Boc，精氨酸用Pbf保护。使用的偶联活化剂为HOBT/HBTU/DIEA和HOBT/HATU/DIEA，茚三酮法检测反应效率。
c)树脂上多肽的裂解：TFA/EDT/TIS/H2O(92.5:2.5:2.5:2.5v/v)溶液，置室温下反应90min，脱保护和脱树脂。抽滤得滤液，用过量乙醚沉淀粗多肽，离心，收集沉淀，再用少量乙醚洗涤沉淀，真空下干燥，得粗品多肽。同时脱除保护基和树脂，得到粗品胰高血糖素样肽-1和胰高血糖素片段类似物嵌合多肽；
2)纯化：将粗品胰高血糖素样肽-1和胰高血糖素片段类似物嵌合多肽溶解于水或10-15％乙腈(10-50mg/ml)，加入50-100mM的二硫基苏糖醇DTT或β-巯基乙醇进行变性，采用制备型HPLC法，C18色谱柱，乙腈－水－三氟乙酸系统分离纯化，浓缩，冻干，得巯基游离的纯品多肽。
以上述方法制备得到以下SEQ ID NO:1-42所示的多肽。
SEQ ID NO:1H(d-A)EGTFTSDYSKYLDERAAQEFICWLVKGRNH2
SEQ ID NO:2H(d-A)EGTFTSDYSKYLDERAAQDFVCWLVKGRG
SEQ ID NO:3H(d-A)EGTFTSDYSKYLEAibRAAQEFIAWLVKGRCNH2
SEQ ID NO:4H(d-A)EGTFTSDYSKYLEERAAQEFIAWLVKGRGC
SEQ ID NO:5H(d-A)EGTFTSDYSKYLEAibRAAQEFVCWLVKGRNH2
SEQ ID NO:6H(d-A)EGTFTSDYSKYLDAibRAAQEFVQWLVKGRGC
SEQ ID NO:7H(d-A)EGTFTSDYSKYLEAibRAAQEFIAWLVKGCNH2
SEQ ID NO:8H(d-A)EGTFTSDYSKYLDERAAQEFICWLVKAibRNH2
SEQ ID NO:9HGEGTFTSDYSKYLDERAAQEFICWLVKAibRG
SEQ ID NO:10HGEGTFTSDYSKYLEERAAQEFIAWLVKGRGC
SEQ ID NO:11HGEGTFTSDYSKYLDAibRAAQEFIAWLVKGRGCSEQ ID NO:12HGEGTFTSDYSKYLEERAAQEFIAWLVKAibRCNH2
SEQ ID NO:13HGEGTFTSDYSKYLDAibRAAQEFIAWLVKGRCNH2
SEQ ID NO:14HGEGTFTSDYSKYLDAibRAAQEFICWLVKGR NH2
SEQ ID NO:15HGEGTFTSDYSKYLEAibRAAQEFICWLVKGRNH2
SEQ ID NO:16HAibEGTFTSDYSKYLDERAAQEFICWLVKGRNH2
SEQ ID NO:17HAibEGTFTSDYSKYLEERAAQEFIAWLVKGRCNH2
SEQ ID NO:18HAibEGTFTSDYSKYLDERAAQEFIAWLVKGRCNH2
SEQ ID NO:19HAibEGTFTSDYSKYLEERAAQEFICWLVKGRNH2
SEQ ID NO:20HAibEGTFTSDYSKYLDERAAQEFIAWLVKGCNH2
SEQ ID NO:21HAibEGTFTSDYSKYLEERAAQEFICWLVKGRG
SEQ ID NO:22HAibEGTFTSDYSKYLDERAAQEFIAWLVKGRGC
SEQ ID NO:23HAibEGTFTSDYSKYLEERAAQDFVQWLVKGRCNH2
SEQ ID NO:24HAibEGTFTSDYSKYLDERAAQEFVQWLVKGRCNH2
SEQ ID NO:25HAibEGTFTSDYSKYLDERAAQDFVQWLVKGCNH2
SEQ ID NO:26HAibEGTFTSDYSKYLEERAAQEFVCWLVKGRNH2
SEQ ID NO:27HAibEGTFTSDYSKYLDERAAQEFVQWLVKGRGC
SEQ ID NO:28HAibEGTFTSDYSKYLDERAAQEFVCWLVKGRNH2
SEQ ID NO:29HAibEGTFTSDYSKYLEERAAQDFVCWLVKGRNH2
SEQ ID NO:30HAibEGTFTSDYSKYLDERAAQDFVCWLVKGRNH2
SEQ ID NO:31HAibEGTFTSDYSKYLEERAAQEFVCWLVKGRG
SEQ ID NO:32HAibEGTFTSDYSKYLDERAAQEFVCWLVKGRG
SEQ ID NO:33HAibEGTFTSDYSKYLEERAAQDFVCWLVKGRG
SEQ ID NO:34HAibEGTFTSDYSKYLDERAAQDFVCWLVKGRG
SEQ ID NO:35HAibEGTFTSDYSKYLEERAAQDFVQWLVKGRGC
SEQ ID NO:36HAibEGTFTSDYSKYLEERAAQEFVQWLVKGRGC
SEQ ID NO:37HAibEGTFTSDYSKYLDEQAAKEFICWLVKGRNH2
SEQ ID NO:38HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRCNH2
SEQ ID NO:39HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGCNH2
SEQ ID NO:40HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRGC
SEQ ID NO:41HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRCNH2
SEQ ID NO:42HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRGCNH2
实施例2
多肽的聚乙二醇修饰物的制备
1)连接：
马来酰基功能化：将如SEQ ID NO:3所示多肽溶解于含有5mM EDTA的pH6的50mM磷酸钠缓冲溶液中，浓度为2mg/mL。加入1.2-1.5倍摩尔量的固态的PEG-马来酰亚胺，搅拌溶解，于室温反应2hr。反应以HPLC监控，以5mMβ-巯基乙醇终止反应，置室温30min后纯化。
碘乙酰基功能化：通过肽前体和碘乙酰基功能化的mPEG(1:1)在7M脲/50mM Tris缓冲液(pH7.5-8.5)中室温搅拌反应45分钟完成聚乙二醇修饰，以在PEG和肽链上的Cys间形成共价硫醚键。
2)纯化:采用制备型离子交换柱层析，以SP SepharoseHP填料，以0-500mM氯化钠pH6磷酸盐缓冲液溶液线性梯度洗脱。流出液以HPLC和SDS-电泳检测，收集PEG-多肽流分，超滤浓缩，冷冻干燥而得。
3)纯品多肽经MILD-Q-TOF进行全分子量扫描，确定平均分子量。
采用上述方法制备了下表所示的多肽聚乙二醇修饰物。
表1多肽聚乙二醇修饰物
实施例3
二聚体(二硫键连接)的制备
将GLP-1类似物单体以适当浓度(1.5-2mmol/L)溶解于去离子水中，根据碳酸氢铵法或DMSO法形成二聚体，按实施例1中纯化方法纯化，得到GLP-1类似物二聚体纯品。
根据此方法制备了如下二聚体。
SEQ ID NO:1的同源二聚体
SEQ ID NO:3的同源二聚体
SEQ ID NO:13的同源二聚体
SEQ ID NO:15的同源二聚体
SEQ ID NO:17的同源二聚体
SEQ ID NO:18的同源二聚体
SEQ ID NO:19的同源二聚体
SEQ ID NO:22的同源二聚体
SEQ ID NO:26的同源二聚体
SEQ ID NO:28的同源二聚体
SEQ ID NO:32的同源二聚体
SEQ ID NO:36的同源二聚体
SEQ ID NO:38的同源二聚体
SEQ ID NO:40的同源二聚体
SEQ ID NO:41的同源二聚体
实施例4
Cys-双马来酰亚胺二聚体的制备
将0.05mmol的SEQ ID NO:1单体多肽和0.15-0.2mol的平均分子量3500的双马来酰亚胺聚乙二醇溶解于pH6的含有0.01mM EDTA的磷酸盐缓冲液中，室温下搅拌16小时后，继续在反应体系中加入0.05mmol的单体多肽，继续搅拌反应16-20小时，用HPLC监控反应，以5mMβ-巯基乙醇终止反应，置室温30min。采用制备型离子交换柱层析，以SP SepharoseHP填料，以0-500mM氯化钠溶液线性梯度洗脱。流出液以HPLC和SDS-电泳检测，收集PEG-多肽流分，超滤浓缩，进一步用制备型HPLC纯化，相应流分浓缩，冷冻干燥，得目标二聚体。同法制备得到SEQ ID NO:15、18、24、28、32、40、42通过双马来酰亚胺连接的同源二聚体。
实施例5
本发明的多肽在体外对DPP-Ⅳ降解的稳定性
受试样品于37℃在50mmol/L TEA-HCl(pH7.8)中与纯化的猪DPP-Ⅳ(5毫单位)共孵0、2、4、8小时。
HPLC法(色谱柱：Aeris widepore XB-C18 3.6μm，4.6×150mm；流动相：A:0.05％TFA，B:95％乙腈；检测波长：214nm)测定各时间点溶液中的残留样品峰面积，计算样品消除率。结果见表2。
表2体外对DPP-Ⅳ降解的稳定性
| 肽 | 半衰期(h) |
| GLP-1 | 3.8±0.21 |
| GC | 2.4±0.16 |
| SEQ ID NO:1 | ＞8 |
| SEQ ID NO:8 | ＞8 |
| SEQ ID NO:9 | ＞8 |
| SEQ ID NO:18 | ＞8 |
| SEQ ID NO:24 | ＞8 |
| SEQ ID NO:41 | ＞8 |
| SEQ ID NO:42 | ＞8 |
实施例6
对GLP-1/GC受体的作用
通过对GLP-1/GC受体介导的体外cAMP产生的影响评价所述多肽对GLP-1/GC受体的作用。
将转染有人GLP-1受体的中国豚鼠肺细胞和转染GC受体的HEK293细胞接种到96孔培养板，(200000个/孔)，以Hanks’平衡盐缓冲液洗涤后，与不同浓度的受试多肽样品(10-5-10-12mol/L)，在200μmol/L 3-异丁基-1-甲基茜草黄素存在下于37℃共孵20min。去除介质，溶解细胞，测定cAMP值，测定方法参照分析试剂盒说明。用Origin软件计算50％有效浓度。结果见表3。
表3多肽对cAMP的诱导产生作用
实施例7
多肽的聚乙二醇修饰物对GLP-1受体的激动作用
通过对GLP-1受体介导的体外cAMP产生的影响评价所述多肽对GLP-1受体的作用。
将转染有人GLP-1受体的中国豚鼠肺细胞接种到96孔培养板(200000个/孔)，以Hanks’平衡盐缓冲液洗涤后，与不同浓度的受试多肽样品(10-5-10-12mol/L)，在200μmol/L3-异丁基-1-甲基茜草黄素存在下于37℃共孵20min。去除介质，溶解细胞，测定cAMP值，测定方法参照分析试剂盒说明。用Origin软件计算50％有效浓度。结果见表4。
表4支链型PEG修饰多肽对GLP-1受体的激动作用
结论：支链型聚乙二醇修饰的本发明多肽序列样品对受体的激动活性相当于内源性配体(hGLP-1(7-37)-NH2)，人源GLP-1序列经PEG修饰活性明显减弱，说明多肽的聚乙二醇修饰物的活性强度取决于前体多肽。PEG修饰一般使多肽活性减弱或消失，但是前体多肽活性越强PEG修饰体的活性保留越多。
实施例8
前体多肽的降血糖作用评价
采用正常小鼠葡萄糖负荷实验评价了本发明的多肽的降血糖作用。动物(n＝8)于实验前禁食过夜，皮下注射生理盐水(10mL/kg)作为对照组；皮下注射艾塞那肽(5μg，每日2次)作为阳性对照药组；其余受试组分别为SEQ ID NO:9、18、24、40。
称取适量受试品(≥98％)溶于生理盐水，配制成50μg/ml的样品溶液。试验组小鼠，每只皮下注射200μl受试品溶液；
给药前测定血糖，葡萄糖(4.5g/kg)腹腔注射后即刻给与受试样品，尾尖取血测定药后30min的血糖值。
结果见表5。
所试4个样品均显示了不同程度的降血糖活性，其中SEQ ID NO:9，24药效与阳性药艾塞那肽相当，SEQ ID NO:18和40强于阳性药。
表5.本发明的多肽的降血糖作用
| 分组 | 动物数 | 血糖值 |
| 生理盐水 | 8 | 15.7±5.81 |
| 阳性对照 | 8 | 6.06±3.74 |
| SEQ ID NO:9 | 8 | 6.53±2.62 |
| SEQ ID NO:18 | 8 | 5.73±3.52 |
| SEQ ID NO:24 | 8 | 6.21±2.69 |
| SEQ ID NO:40 | 8 | 5.69±3.18 |
实施例9
PEG修饰多肽对Dio小鼠体重和血糖的影响
平均体重52.7g的Dio小鼠，4组，每组10只，空白组给与生理盐水，阳性对照组给与艾赛那肽25nmol/kg，每日2次，受试组分别给与实施例2中所示SEQ ID NO:18、40多肽的PEG修饰物(分别简称dp-1、dp-2，)100nmol/kg，每周一次，给药2周。在注射后0、2、4、6、8、10、12、14天测量体重变化，检测任意血糖水平。结果见图1、图2。
受试药物组，即dp-1、dp-2每周给药1次，连续给药2周，显示了与艾塞那肽相似或略好的降血糖作用，而且相较阳性对照药更为显著地减轻体重，给药14天减轻幅度可达到7-8％。
实施例10
支链型(Y)PEG修饰多肽降血糖作用和长效性
采用正常小鼠葡萄糖负荷试验评价了本发明多肽的降血糖作用以及长效性。
动物(n＝8)于实验前禁食过夜，皮下注射生理盐水(10mL/kg)作为对照组；皮下注射利拉鲁肽(100nmol/kg，每日1次)作为阳性对照药组；其余受试组分别给与实施例7的PEG修饰多肽pg016、pg019、pg029，给药剂量100nmol/kg。
给药前测定血糖，给与受试样品，分别于给药后不同时间段腹腔注射葡萄糖(2.5g/kg)后，尾尖取血测定给糖后30min的血糖值，计算血糖抑制率。
结果见表6。
表6.支链型(Y)PEG修饰多肽样品降血糖作用时效性
结论：阳性药利拉鲁肽降血糖作用持续至28小时，而受试样品pg016、pg019、pg029均可平稳持续至100小时，自124小时开始减弱。根据小鼠和人体代谢差异推测，受试样品在人体药效可以持续至1周以上。
实施例11
支链型(Y)PEG修饰多肽对模型动物体重和糖耐量的影响
平均体重45.9g的Dio小鼠，4组，每组10只，空白组给与生理盐水，阳性对照组给与利拉鲁肽100nmol/kg，每日1次，受试组分别给与实施例7的PEG修饰多肽pg016、pg019，剂量100nmol/kg，4日一次，给药4周。在给药后0、8、16、24、28天测量体重变化，第28天测定糖耐量。结果见图3、表7。
结果：阳性对照药利拉鲁肽给药4周体重下降12.3％，支链型PEG修饰的多肽样品pg016、pg019受试组体重分别减轻20.1％、17.4％，优于利拉鲁肽。
表7给药后第28天糖耐量测定-血糖AUC值(n＝5)
| 组别 | AUC(mmol/L) |
| 模型组 | 19.8 |
| 利拉鲁肽 | 9.1 |
| pg016 | 9.6 |
| pg019 | 8.2 |
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。
序列表
<110> 天津药物研究院有限公司
<120> 含有胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽及其用途
<130> DIC16110047
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 1
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 2
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 2
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 3
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 3
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 4
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 4
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 5
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 5
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 6
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 6
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 7
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 7
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 8
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为D型丙氨酸
<220>
<221> MUTAGEN
<222> (29)..(29)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 8
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 9
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (29)..(29)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 10
<211> 32
<212> PRT
<213> artificial sequence
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 11
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 12
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (29)..(29)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 12
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 13
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 13
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 14
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 14
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 15
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 16
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 16
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 17
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 17
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 18
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 18
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 19
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 19
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 20
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 20
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 21
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 21
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 22
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 22
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 23
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 23
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 24
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 24
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 25
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 25
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Cys
20 25 30
<210> 26
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 26
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 27
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 27
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 28
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 28
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 29
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 29
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 30
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 30
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 31
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 31
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 32
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 32
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 33
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 33
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 34
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 34
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 35
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 35
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 36
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 36
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 37
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Arg的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 37
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 38
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 38
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 39
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (30)..(30)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 39
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 40
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 40
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 41
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (31)..(31)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 41
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 42
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223> Xaa为Aib，2-氨基异丁酸
<220>
<221> BINDING
<222> (32)..(32)
<223> Cys的C端连接NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 42
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
Claims (16)
1.一种包含胰高血糖素样肽-1和胰高血糖素的片段类似物的嵌合多肽，其特征在于，所述嵌合多肽的序列如SEQ ID NO:1、8-9、12、16-19、24-25、28和37-42中任一条所示。
2.如权利要求1所述的嵌合多肽的缀合物，其特征在于，所述缀合物为所述嵌合多肽的聚乙二醇修饰物，在所述嵌合多肽的Cys残基的侧链上共价连接聚乙二醇。
3.如权利要求2所述的缀合物，其特征在于，所述聚乙二醇的平均分子量为5-50KD。
4.如权利要求2所述的缀合物，其特征在于，所述聚乙二醇的平均分子量为20-45KD。
5.如权利要求2所述的缀合物，其特征在于，所述聚乙二醇的平均分子量为40-45KD。
6.如权利要求2所述的缀合物，其特征在于，所述聚乙二醇为直链或支链聚乙二醇。
7.如权利要求2所述的缀合物，其特征在于，所述聚乙二醇为平均分子量40-45KD的支链聚乙二醇。
8.如权利要求1所述的嵌合多肽的聚合物，其特征在于，所述聚合物为所述嵌合多肽的二聚体，所述二聚体是所述嵌合多肽通过Cys残基共价连接而成或通过接头共价连接而成的。
9.一种药物组合物，所述药物组合物包含如权利要求1所述的嵌合多肽、如权利要求2-7中任一项所述的缀合物或如权利要求8所述的聚合物。
10.如权利要求9所述的药物组合物，其特征在于，所述药物组合物还包含药学上可接受的载体和/或辅料。
11.如权利要求10所述的药物组合物，其特征在于，所述载体和/或辅料选自水溶性填充剂、pH调节剂、稳定剂、注射用水或渗透压调节剂中的一种或多种。
12.如权利要求11所述的药物组合物，其特征在于，所述水溶性填充剂选自甘露醇、低分子右旋糖酐、山梨醇、聚乙二醇、葡萄糖、乳糖或半乳糖中的一种或多种；
所述pH调节剂选自生理上可接受的有机酸或无机酸，或者生理上可接受的无机碱中的一种或多种；
所述稳定剂选自EDTA-2Na、硫代硫酸钠、焦亚硫酸钠、亚硫酸钠、磷酸氢二钾、碳酸氢钠、碳酸钠、精氨酸、赖氨酸、谷氨酸、天冬氨酸、聚乙二醇、聚乙烯醇、聚乙烯吡咯烷酮、羧基/羟基纤维素或其衍生物、环糊精、十二烷基硫酸钠或三羟甲基氨基甲烷中的一种或多种，其中所述羧基/羟基纤维素或其衍生物为HPC、HPC-SL、HPC-L或HPMC；
所述渗透压调节剂为氯化钠和/或氯化钾。
13.如权利要求12所述的药物组合物，其特征在于，所述生理上可接受的有机酸或无机酸为枸橼酸、磷酸、乳酸、酒石酸或盐酸。
14.如权利要求12所述的药物组合物，其特征在于，所述生理上可接受的无机碱为氢氧化钾、氢氧化钠、氢氧化铵、碳酸钠、碳酸钾、碳酸铵、碳酸氢钾、碳酸氢钠或碳酸氢铵盐。
15.如权利要求1所述的嵌合多肽、如权利要求2-7中任一项所述的缀合物或如权利要求8所述的聚合物在制备用于治疗糖尿病和/或肥胖的药物中的用途。
16.如权利要求9-14中任一项所述的药物组合物在制备用于治疗糖尿病和/或肥胖的药物中的用途。
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| CN111349155B (zh) * | 2018-12-24 | 2022-04-05 | 浙江和泽医药科技股份有限公司 | 一种胰高血糖素类似物及其制备方法和用途 |
| CN114478744A (zh) * | 2020-11-13 | 2022-05-13 | 成都奥达生物科技有限公司 | 一种长效glp-1拮抗剂 |
| CN115463209B (zh) * | 2021-10-09 | 2025-05-23 | 上海海赜生物科技有限公司 | 一种口服降糖组合物及其应用 |
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