CN116284433B - 一种胰岛素和glp-1的缀合物及其应用 - Google Patents
一种胰岛素和glp-1的缀合物及其应用 Download PDFInfo
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- CN116284433B CN116284433B CN202211111896.1A CN202211111896A CN116284433B CN 116284433 B CN116284433 B CN 116284433B CN 202211111896 A CN202211111896 A CN 202211111896A CN 116284433 B CN116284433 B CN 116284433B
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Abstract
本发明涉及一种胰岛素和GLP‑1的缀合物及其应用,所述缀合物通过特定的Linker将胰岛素衍生物和GLP‑1衍生物连接而成,其同时具备胰岛素活性和GLP‑1活性。本发明提供的缀合物能够用于有效治疗糖尿病,且降低低血糖风险,同时能显著降低胰岛素使用导致的体重增加问题,具有良好的糖尿病治疗和血糖控制潜力。
Description
技术领域
本发明涉及多肽技术及其缀合物技术领域,尤其涉及一种胰岛素和GLP-1的缀合物及其应用。
背景技术
糖尿病是一组因胰岛素绝对或相对分泌不足和/或胰岛素利用障碍引起的碳水化合物、蛋白质、脂肪等代谢紊乱性疾病,以高血糖为主要标志,可由遗传和环境等多种因素引起。糖尿病是人类三大致死疾病之一,它的死亡率仅次于心脑血管疾病和癌症。
胰岛素是机体内唯一降低血糖的激素,同时促进糖原、脂肪、蛋白质合成,外源性胰岛素及胰岛素衍生物主要用来治疗糖尿病。胰岛素由A、B两个肽链组成,人胰岛素(Insulin Human)A链有11种21个氨基酸,B链有15种30个氨基酸,共51个氨基酸;其中A7(Cys)-B7(Cys)、A20(Cys)-B19(Cys)四个半胱氨酸中的巯基形成两个二硫键,使A、B两链连接起来,此外A链中A6(Cys)与A11(Cys)之间也存在一个二硫键。胰岛素由胰脏内的胰岛β细胞受内源性或外源性物质如葡萄糖、乳糖、核糖、精氨酸、胰高血糖素等的刺激而分泌。胰岛素在细胞水平的生物作用是通过与靶细胞膜上的特异受体结合而启动的;胰岛素受体为胰岛素起作用的靶细胞膜上特定部位,仅可与胰岛素或含有胰岛素分子的胰岛素原结合,具有高度的特异性。
胰岛素虽然是治疗糖尿病最有效的手段,但长期使用胰岛素也会存在一些副作用,比如引起肥胖。胰岛素除了能够调节体内糖代谢,还具备调节脂肪代谢的作用:胰岛素能够促进脂肪的合成与贮存,使血液中游离脂肪酸减少,同时抑制脂肪的分解氧化。这也是为什么会引发体重增加,尤其是腹部脂肪增加的原因。此外,胰岛素使用还存在一定的低血糖风险。
胰高血糖素样肽1(GLP-1)是一种由肠道L细胞分泌的促胰素,具有促进胰岛素分泌、抑制胰高血糖素的释放、刺激胰岛β细胞增殖、诱导胰岛β细胞再生、阻止胰岛β细胞凋亡、改善胰岛素敏感性和增加葡萄糖的利用等作用。因此,GLP-1及其类似物和衍生物在治疗I和II型糖尿病的发生、发展中起着重要作用。GLP-1类似物和胰高血糖素的氨基酸序列有近一半相同,同样具有葡萄糖依赖性的促胰岛素分泌和生物合成、抑制胰高血糖素分泌及抑制胃排空等多种功能,同时,GLP-1作为一种肠源性激素,其是在营养物质(特别是碳水化合物)的刺激下才释放入血的,其促胰岛素分泌作用呈葡萄糖浓度依赖性,能够在血糖升高时发挥降糖作用,抑制胰高糖素分泌,增加饱腹感,减少饥饿感,从而达到降低血糖作用。此外,GLP-1还可作用于中枢神经系统(特别是下丘脑)抑制食欲,减少进食量,从而使人体产生饱胀感和食欲下降,减少卡路里的摄入量。因此,GLP-1及其类似物优点在于其不仅能够有效降低血糖,且能够降低体重、调节血压血脂、使心血管获益、无低血糖风险。
胰岛素能够与胰岛素受体结合,GLP-1能够激活GLP-1受体,因此,若将胰岛素和GLP-1进行缀合修饰,则缀合物可以同时对胰岛素受体和GLP-1受体具有激动活性,兼备两者作用,有效调节血糖的情况下,降低低血糖风险,调节体重增加,并对心血管获益有所帮助。并且,GLP-1和胰岛素联用,能够使缀合物靶向至下丘脑,以降低食欲以及减少血液中葡萄糖浓度。另外的,GLP-1和胰岛素的缀合物可以靶向至β细胞以驱动增加胰岛β细胞生产胰岛素。
目前,鲜有关于胰岛素和GLP-1缀合物相关的研究和报道,因此,本发明提供了一种胰岛素和GLP-1的缀合物,其能够用于治疗糖尿病,同时不会导致患者使用药物产生增重或肥胖的风险,其有助于患者体重减轻。
发明内容
定义
本发明的术语“胰岛素”包括天然存在的胰岛素,例如人胰岛素,以及其胰岛素类似物。人胰岛素由两个多肽链组成,分别被称为人胰岛素A链和人胰岛素B链。
本文所用的术语“天然胰岛素”意在指定包含SEQ ID NO.1的A链和SEQ ID NO.2的B链的51个氨基酸的异源双链体。而天然胰岛素A链意在SEQ ID NO.1的21个氨基酸序列所示的天然胰岛素A链,天然胰岛素B链意在SEQ ID NO.2的30个氨基酸序列所示的天然胰岛素B链。
所述胰岛素类似物是对天然胰岛素进行修饰获得的胰岛素多肽,所述修饰包括去除和/或取代(置换)天然胰岛素中存在的一个或多个氨基酸残基,在天然胰岛素中添加(延长)一个或多个氨基酸残基,上述修饰具有形式上可以源自于天然存在的胰岛素(例如人胰岛素)的分子结构;所述修饰也包括将胰岛素A链和B链通过linker连接,由此制备得到单链胰岛素类似物。
本发明中的术语人胰岛素A链类似物和人胰岛素B链类似物,分别包括人胰岛素的A链和B链,并具有本发明所述的一个或多个氨基酸残基的取代、缺失和/或添加。
本发明中,对于胰岛素或胰岛素类似物,术语例如A1、A2或B1、B2等分别代表胰岛素或其类似物A链中的位置1、2(从N端计算);B链中的位置1、2。使用氨基酸的一个字母代码表示该位置的氨基酸类型,术语例如A21A、A21G和A21Q表示在A21位的氨基酸分别是A、G和Q。
本发明中,术语例如desB30分别表示胰岛素B链第30位氨基酸残基去除的胰岛素类似物。
本文中,胰岛素的命名按照以下原则进行:根据相对人胰岛素的突变和修饰(酰化)给予名称。就酰基部分的命名而言,按照IUPAC命名法进行命名,其它情况下按照肽命名法。例如:例如为“十八烷二酰-γGlu-AEEA-AEEA”或“17-羧基十七烷酰-γGlu-AEEA-AEEA”,其中:AEEA是氨基酸NH2(CH2)2O(CH2)2OCH2COOH的简化符号;γGlu是氨基酸γ谷氨酸的简化符号。
本发明中,术语GLP-1(7-37)类似物是指对人天然GLP-1(7-37)氨基酸进行修饰获得的多肽,所述修饰包括去除和/或取代(置换)和/或添加(延长)一个或多个氨基酸残基,所述氨基酸可以是天然存在的氨基酸,也可以是人工合成的氨基酸。本发明用一种简单的命名来描述所述类似物:例如[Val8]GLP-1(7-37)是指其中在位置8上天然存在的组氨酸已经由缬氨Val取代的GLP-1(7-37)类似物。
本发明中,有关肽(例如GLP-1或胰岛素)的术语“衍生物”表示经化学修饰(如共价修饰等)的肽或其类似物。典型的修饰是酰胺、糖类、烷基、酰基、酯等。GLP-1(7-37)衍生物的实例是Nε26-((4S)-4-(十六烷酰基氨基)-羧基-丁酰基)[Arg34,Lys26]GLP-1-(7-37)。
如本文所用,对GLP-1或胰岛素及其类似物的C端或N端的涉及,意指GLP-1或胰岛素的天然C端或N端,或分别相对于天然序列在其C端或N端添加、删除或置换一个或多个氨基酸后的C端或N端。
本发明中,术语接头,是能够与至少两个待连接的结构(例如多肽或蛋白)反应,将其连接为一个整体结构,或维持整体结构足够紧密以保持缔合状态的任何合适多肽或化合物。多肽接头可整合在连接得到的分子或结构中。
本发明中,术语“脂肪族二酸”包括直链或支链脂族二羧酸,其具有至少两个碳原子并为饱和或不饱和的。脂肪族二酸的非限定实例为琥珀酸、己二酸、辛二酸、癸二酸、十二烷二酸、十四烷二酸、十六烷二酸、十七烷二酸、十八烷二酸和二十烷二酸。
本发明中,术语“药学上可接受的盐”是指保留母体的生物活性的多肽或蛋白的盐。
术语“载体”是指可以将编码蛋白质或多肽的核苷酸片段可操作地插入其中以引起所述蛋白质或多肽表达的媒介物。载体可用于转化、转导或转染宿主细胞,以使其在宿主细胞内表达携带的遗传元件。载体的实例包括质粒、人工染色体、噬菌体、病毒颗粒等。载体可以含有多种用于控制表达的元件,包含启动子序列、转录起始序列、增强子序列、选择元件和报告基因。载体还可以包括有助于其进入细胞的材料,包括但不限于病毒颗粒、脂质体或蛋白质包膜。
载体可以是重组表达载体或克隆载体。本发明提供了载体(例如表达载体),其含有编码所述胰岛素和GLP-1缀合物的本发明提供的核酸序列。载体的实例包括但不限于逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如单纯疱疹病毒)、痘病毒、杆状病毒、乳头状瘤病毒、乳多空病毒(例如SV40)、λ噬菌体和M13噬菌体、质粒,如pcDNA3.3、pMD18-T、pOptivec、pCMV、pEGFP、pIRES、pQD-Hyg-GSeu、pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS10、pLexA、pACT2.2、pCMV-SCRIPT.RTM.、pCDM8、pCDNA1.1/amp、pcDNA3.1、pRc/RSV、PCR2.1、pEF-1、pFB、pSG5、pXT1、pCDEF3、pSVSPORT、pEF-Bos等。
本发明中术语“重组表达载体”是编码基因的核酸分子,其在宿主细胞中表达且含有控制所述基因的表达的必需元件。通常,表达载体包含转录启动子、目的基因和转录终止子。
本发明中宿主细胞是指可将包含编码目的蛋白质或多肽的核苷酸序列片段的载体引入到其中进行克隆或基因表达的细胞。适用于克隆或表达本文载体中的DNA的宿主细胞是原核生物、酵母或更高级真核生物细胞。
为了实现本发明的目的,本发明提供一种胰岛素和GLP-1的缀合物,所述缀合物兼具对胰岛素受体和GLP-1受体的激动活性。本发明的缀合物将两者缀合修饰,在具备完全的降糖效力的胰岛素基础上,引入GLP-1活性,能起到降低患者食欲、减轻体重的作用,解决传统使用胰岛素及其衍生物会引起患者体重增加的副作用。
本发明提供的缀合物中的胰岛素类似物可以是天然胰岛素或胰岛素类似物/衍生物或单链胰岛素类似物/衍生物,更优选为具备长效作用的单链胰岛素衍生物。本发明缀合物中的GLP-1可以是GLP-1衍生物,优选为具备长效作用的GLP-1衍生物,更优选地,为GLP-1(7-37)衍生物。优选地,本发明的缀合物在其胰岛素和/或GLP-1部分连接脂肪酸侧链,以延长其作用时间,达到长效效果。
第一方面,本发明提供的胰岛素和GLP-1的缀合物的氨基酸结构如下所示:
GLP-1(7-37)类似物-(GQAP)2-5-单链胰岛素类似物;
其中,
(1)所述单链胰岛素结构如下式所示:
胰岛素B链-Linker-胰岛素A链;
胰岛素B链选自B25H、desB30人胰岛素B链,或B16H、B25H、desB30人胰岛素B链,或B16E、B25H、desB30人胰岛素B链;
胰岛素A链为A14E人胰岛素A链;
Linker分别与胰岛素B链的N末端、胰岛素A链的C末端相连;
(2)所述GLP-1(7-37)类似物选自如下氨基酸序列所示类似物:
HIEGTFTSDVSSYLEEQAAREFIAWLVKRGG,
HTEGTFTSDVSSYLEEQAAREFIAWLVKRGG,
HIEGTFTSDVSSYLEEQAAREFIAWLVKGRG,
HTEGTFTSDVSSYLEEQAAREFIAWLVKGRG,
H-Aib-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG,
HVEGTFTSDVSSYLEEQAAREFIKWLVRRGG,
HIEGTFTSDVSSYLEEQAAREFIKWLVRRGG,
HTEGTFTSDVSSYLEEQAAREFIKWLVRRGG,
YVEGTFTSDVSSYLEEQAAREFIKWLVRGRG,
YVEGTFTSDVSSYLEEQAAREFIKWLVRGGR;
(3)(GQAP)2-5分别与胰岛素B链的C末端和GLP-1(7-37)类似物的N末端相连。其中所述(GQAP)2-5述优选为(GQAP)3。
优选地,所述Linker为VGLSSGQAP。
优选地,上述胰岛素B链为B25H、desB30人胰岛素B链,胰岛素A链为A14E人胰岛素A链,GLP-1(7-37)类似物为HIEGTFTSDVSSYLEEQAAREFIAWLVKRGG。
优选地,所述缀合物的氨基酸结构为HIEGTFTSDVSSYLEEQAAREFIAWLVKRGGGQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPKVGLSSGQAPGIVEQCCTSICSLEQLENYCN(SEQ IDNO.3)。本发明所述缀合物包含与所述GLP-1(7-37)类似物上的氨基酸K和单链胰岛素类似物上的氨基酸K上的ε氨基连接的脂肪酸侧链。所述脂肪酸侧链优选为HOOC(CH2)14-20CO-,最优选地,所述侧链为HOOC(CH2)16CO-或HOOC(CH2)18CO-。
优选地,本发明的脂肪酸侧链通过接头与氨基酸K连接,所述接头选自:
其中,m为0-6的整数,n为1-3的整数,s为0-3的整数,t为0-4的整数,p为1-23的整数。
更优选地,所述接头为:
其中,s和n均为1。
因此,本发明优选提供一种胰岛素和GLP-1的缀合物或其药学上可接受的盐,所述缀合物的氨基酸结构为:HIEGTFTSDVSSYLEEQAAREFIAWLVKRGGGQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPKVGLSSGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NO.3);其通过氨基酸序列上的两个Lys(一个Lys位于GLP-1序列段,另一个位于胰岛素B链序列段)的ε氨基经接头连接侧链HOOC(CH2)16CO-或HOOC(CH2)18CO-;所述接头为:
其中,s和n均为1。
另一方面,本发明提供了一种药物组合物,包括本发明公开的缀合物或其药学上可接受的盐,以及药学上可接受的辅料。
另一方面,本发明提供一种本发明所述的缀合物或其药学上可接受的盐、或其药物组合物在制备治疗代谢性疾病的药物中的应用。
本发明所述的代谢性疾病包括但不限于:糖尿病(I型糖尿病、II型糖尿病)、超重和肥胖、脂肪性肝炎(NASH、ASH)、心血管疾病、脂肪肝、肝硬化、非酒精性脂肪肝病、代谢综合征及各种糖尿病并发症。
另一方面,本发明提供一种所述缀合物氨基酸序列的制备方法,所述方法可以采用化学合成法,也可以采用制备重组基因工程菌发酵表达法。
另一方面,本发明提供一种能编码所述缀合物氨基酸序列的多核苷酸;其能够编码合成本发明的缀合物。
另一方面,本发明提供一种重组载体,所述载体包含本发明所述的多核苷酸。
另一方面,本发明提供一种重组宿主细胞,其包含本发明的载体,并能够分泌表达本发明的缀合物。
本发明实施例提供的技术方案与现有技术相比具有如下优点:
本发明提供的缀合物能够用于治疗胰岛素依赖性糖尿病,具有优异的治疗效果。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本发明的实施例,并与说明书一起用于解释本发明的原理。
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为本发明的胰岛素和GLP-1的缀合物对小鼠模型的血糖变化测试结果图;
图2为本发明的胰岛素和GLP-1的缀合物对DB/DB小鼠模型的减重实验结果图。
具体实施方式
本发明的缀合物的氨基酸序列可以通过化学合成法制备,也可以通过制备重组基因工程菌发酵表达的方法制备,本实施例以发酵表达为例进行介绍。
实施例1:胰岛素和GLP-1的缀合物构建表达
(1)缀合物蛋白的表达
将促包涵体序列(FKFEFKFE)、酶切序列(DDDDK)和本发明缀合物串联作为融合蛋白,融合蛋白氨基酸序列为:
FKFEFKFEDDDDKHIEGTFTSDVSSYLEEQAAREFIAWLVKRGGGQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPKVGLSSGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NO.4)。
构建上述融合蛋白的编码基因序列,并使用化学合成的方式获得基因片段为:
TTTAAATTTGAATTTAAATTCGAAGATGACGATGATAAACATATTGAAGGCACCTTTACGAGCGATGTGAGCAGCTATCTGGAAGAACAAGCGGCGCGCGAATTTATTGCGTGGCTGGTGAAACGCGGCGGTGGCCAAGCGCCGGGTCAAGCGCCGGGCCAAGCGCCGTTTGTTAATCAGCATCTGTGCGGCAGCCATCTGGTGGAAGCGCTGTATCTGGTGTGCGGCGAACGCGGCTTTCATTATACCCCGAAAGTGGGCCTGAGCAGCGGCCAAGCCCCGGGCATCGTGGAACAGTGCTGCACGAGCATTTGCAGCCTGGAACAGCTGGAAAACTATTGCAAC(SEQ ID NO.5);通过NdeI和XhoI位点,将上述片段插入原核表达质粒pET-30a(+)中并测序验证,得到的用于转化测定的表达质粒,称作pET-30a(+)-HS-GI-001。
将上述构建的表达质粒转化入表达细胞BL21(TransGenBiotech)构建重组工程菌,并-80℃保存。将保存的重组工程菌经过三级发酵培养收集获得菌体细胞浆。菌体细胞浆经过纯化、EK酶酶切、酶切后纯化和精纯得到纯度90%以上的胰岛素和GLP-1的缀合物蛋白,测序验证表达结果正确。
(2)胰岛素和GLP-1的缀合物的制备(以HS-GI-001为例)
脂肪酸修饰:将步骤(1)中制备的胰岛素和GLP-1的缀合物蛋白HS-GI-001加水,配制成0.5-10mg/mL溶解液,加入1M氢氧化钠调整pH至11.0-11.5,摇匀使蛋白完全溶解,UV定量多肽浓度;按多肽与十八烷二酸单叔丁酯-谷氨酸(1-叔丁酯)-AEEA-AEEA-OSU-摩尔比1:4称取脂肪酸粉末溶于乙腈中,将多肽样品与脂肪酸溶液混合,将混合液于4℃静置一小时,然后样品加水稀释5倍,用1M柠檬酸(或10%乙酸)调pH至4.8终止反应,放于4℃静置酸沉10min,酸沉后以13000g,4℃离心30min,然后将沉淀放于-80℃保存。
脂肪酸脱保护与纯化:向得到的沉淀中加入TFA至多肽终浓度约10mg/mL,震荡使沉淀溶解,置于室温静置脱保护30min,然后滴入4M NaOH调节pH至7.5-8.5终止反应。
用蛋白纯化层析系统(赛谱SDL100)将反应终止后的反应液按3mL/min流速,泵入事先用平衡液3(0.1%TFA,20%乙腈)平衡过的UniHybrid10-200(购自苏州纳微科技有限公司)进行浓缩,平衡液3淋洗后,再按0-100%洗脱液(0.1%TFA,80%乙腈)梯度洗脱,收集洗脱峰经RP-UPLC检测纯度约为90%。
洗脱峰用水稀释3倍,酸沉调整pH至4.80,4℃酸沉30min,离心后沉淀中加入PBST缓冲液(pH7.0)复溶后-80℃冻存;得到GLP-1和胰岛素缀合物HS-GI-001。
实施例2:胰岛素和GLP-1的缀合物细胞活性检测
细胞培养:选取培养状态良好的HEK293/Luc/GLP1R细胞,弃去瓶中培养液,用PBS液摇洗1次,加入0.05%TRYPSIN消化液消化,然后加入DMEM基础培养液终止消化,离心收集细胞,用DMEM培养液调整细胞密度为8×105个/mL,50μL/孔接种于96孔全白细胞培养板中,于37℃、5%CO2条件下培养过夜。
检测溶液配制:配制测定培养液,用测定培养液分步稀释样品至320nM,单次稀释倍数不超过10倍;之后在96孔板中按照进行5倍系列稀释,共8个梯度,每个稀释度做2个复孔。
体外活性检测:用Fire-Lumi荧光素酶检测试剂盒(金斯瑞)检测GLP-1类似物的衍生物的体外活性;从培养箱中取出培养好的细胞培养板,按照试剂盒使用方法,将稀释好的溶液加入细胞板中,每孔50μL,置于37℃、5%CO2条件下孵育6hr。从培养箱中取出样品板,平衡至RT;加入100μL Fire-Lumi检测液,反应5min,震荡10s,检测荧光强度。试验数据采用四参数回归计算法进行处理,可以计算出待测样品的EC50值,结果见表1:
表1
索马鲁肽 | 胰岛素缀合物HS-GI-001 | |
EC50(nm) | 0.934 | 2.445 |
由表1可知,本发明的胰岛素缀合物体外GLP-1活性略高于索马鲁肽的1/3,可见,本申请的缀合物具备预期的GLP-1活性。
实施例3:293-IR-B细胞进行Insulin类似物的衍生物体外活性测定
细胞培养:HEK-293-IR-B细胞用加压培养基(DMEM/HIGHGLUCOSE培养液添加10%FBS,按100μg/mL加入G418硫酸盐),按1:(3-4)传代培养,37℃、5%CO2培养箱中培养,待细胞处于对数生长期用于实验。取消化后HEK-293-IR-B细胞,用加压培养基重悬计数取约3×106个细胞于T75 cm2培养瓶中,加压培养基补至15mL,37℃、5%CO2培养箱培养44-52小时;用1-2mL 0.25%胰酶消化,按3×105个/mL完全培养液接种入96孔细胞板,每孔100μL,细胞充分分散后,置37℃、5%CO2培养箱继续培养24小时。弃上清,换成样品稀释液(取DMEM/HIGH GLUCOSE培养液用DMEM无葡萄糖培养基稀释至葡萄糖含量为0.8mg/mL),100μL/孔,37℃、5%CO2培养箱培养24小时。
样品溶液的制备:称取德谷胰岛素或长效周胰岛素(icodec序列)适量,用0.02mol/L的磷酸盐缓冲液(pH8.0)溶解稀释制成质量浓度约10mg/mL的供试品母液,取供试品母液22μL加样品稀释液278μL为溶液1,从溶液1开始于96孔板中做6倍倍比稀释,共8个稀释度,每个稀释度2复孔。
细胞活性检测:用葡萄糖含量检测试剂盒(德赛诊断)检测Insulin类似物的衍生物的体外活性;弃细胞培养上清50μL/孔,加入样品50μL/孔(每孔样品终浓度减半),37℃、5%CO2培养箱反应24小时。于新的96孔板中加入葡萄糖氧化酶200μL/孔,细胞板每孔取上清20μL加入此板,37℃反应10分钟。用酶标仪读取508nm处的OD值并记录数据。试验数据采用四参数回归计算法进行处理,可以计算出待测样品的EC50值,结果见表2:
表2
胰岛素体外活性检测结果如表2所示,由表2可知,相较于目前诺和诺德在研的周胰岛素衍生物(A14E,B16H,B25H,desB30人胰岛素,参见专利CN201380030897.6授权权利要求1中记载的胰岛素衍生物之一),本发明的胰岛素缀合物具有与之相当的胰岛素体外活性。
实施例4:小鼠降糖实验研究
(1)实验动物:
6-8周龄/雄性C57BL/6J小鼠,20只,体重在18-20g之间;
(2)实验方法:
造模:小鼠先在在适应性饲养两周,然后小鼠禁食16h后腹腔注射STZ(80mpk)诱导高血糖模型,诱导3天后检测血糖,随机血糖值在16.8mmol/L以上为造模成功。
分组与给药:按照表3内容对模型小鼠分组,并给药(阳性对照与阴性对照等摩尔给药);
表3
(3)结果统计
图1为血糖测试结果,由图可知,在等摩尔浓度及等摩尔注射量的情况下,本发明HS-Gl-001与Icodec降糖活性基本相同,与体外活性检测结果相符。
实施例5:在DB/DB小鼠上的减重药效实验
(1)实验动物:
实验动物为BKS-LeprREM/Gpt小鼠,数量20只,周龄为6周龄,雄性;
(2)实验方法:
造模:选取DB/DB小鼠,按照体重随机分组,每组包含5只小鼠,分为空白对照组、阳性对照组1(索马鲁肽),阳性对照组2(Icodec)和HS-GI-001组;阳性对照组1、2与HS-GI-001均为等摩尔给药。
给药方式:腹部皮下注射,给药频率为每天一次,连续给药2天,具体见表4:
表4
(3)结果统计
减重实验结果见图2,由图可知,在等摩尔给药情形下,本发明的胰岛素缀合物减重效果显著,不仅远远高于Icodec,更是显著优于索马鲁肽。因此,本发明的胰岛素缀合物不仅具备优异的胰岛素降糖活性,同时能显著抑制体重增加。
需要说明的是,在本文中,诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所述的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一种胰岛素和GLP-1的缀合物或其药学上可接受的盐,其特征在于,所述缀合物的氨基酸结构如下所示:
HIEGTFTSDVSSYLEEQAAREFIAWLVKRGGGQAPGQAPGQAP FVNQHLCGSHLVEALYLVCGERGFHYTPKVGLSSGQAPGIVEQCCT SICSLEQLENYCN;
所述缀合物通过与氨基酸序列上的两个K残基上的ε氨基连接侧链,所述侧链为COOH-(CH2)n-CO-,n为10-20的整数。
2.根据权利要求1所述的缀合物或其药学上可接受的盐,其特征在于,所述侧链通过接头与氨基酸K连接。
3.根据权利要求2所述的缀合物或其药学上可接受的盐,其特征在于,所述接头为γ-Glu-AEEA-AEEA,结构为:
其中,s和n均为1。
4.根据权利要求3所述的缀合物或其药学上可接受的盐,其特征在于,所述侧链为COOH-(CH2)16-CO-或COOH-(CH2)18-CO-。
5.一种药物组合物,所述药物组合物包含权利要求1-4中任一项所述的缀合物或其药学上可接受的盐,以及药学上可接受的辅料。
6.根据权利要求5所述的药物组合物,其特征在于,所述药物组合物为注射液制剂或口服制剂。
7.权利要求1-4中的任一项所述的缀合物或其药学上可接受的盐、权利要求5或6所述的药物组合物在制备治疗糖尿病的药物中的应用。
8.权利要求1-4中的任一项所述的缀合物或其药学上可接受的盐、权利要求5或6所述的药物组合物在制备治疗肥胖型II型糖尿病的药物中的应用。
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