CN115304666A - 一种治疗代谢疾病的胰高血糖素类似物 - Google Patents
一种治疗代谢疾病的胰高血糖素类似物 Download PDFInfo
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- CN115304666A CN115304666A CN202210863408.6A CN202210863408A CN115304666A CN 115304666 A CN115304666 A CN 115304666A CN 202210863408 A CN202210863408 A CN 202210863408A CN 115304666 A CN115304666 A CN 115304666A
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- glucagon
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Abstract
本发明涉及生物药物领域,特别是涉及一种治疗代谢疾病的胰高血糖素类似物,结构式为:H‑X2‑X3‑GTFTSD‑X10‑SKYLD‑X16‑X17‑AAQ‑DFVQWLMN‑X29‑Xz或H‑S‑Q‑GTFTSD‑Y‑SKYLD‑X16‑X17‑AAQ‑DFVQWLMN‑X29‑Xz‑NH2。本发明的胰高血糖素类似物,具有GLP‑1/GCG/GIP三受体激动活性,以及更好的耐酶稳定性,包括对中性内肽酶(NEP)以及二肽基肽酶‑4(DPP‑4),与天然胰高血糖素,GLP‑1,GIP相比具有更长的体内半衰期和持续作用时间。
Description
技术领域
本发明涉及生物药物领域,特别是涉及一种治疗代谢疾病的胰高血糖素类似物及其制备方法和用途。
背景技术
糖尿病是一种严重的慢性病,当胰腺产生不了足够的胰岛素或者人体无法有效地利用所产生的胰岛素时,就会出现糖尿病。目前上市的蛋白类糖尿病药物主要是GLP-1受体(GLP-1R)激动剂,如杜拉鲁肽(Dulaglutide,商品名:)、阿必鲁肽(Albiglutide,商品名)、利拉鲁肽(Liraglutide,商品名及分别用于治疗肥胖和糖尿病)、艾塞那肽(Exenatide,商品名)、利西拉肽(Lixisenatide,商品名)及可能即将上市的索马鲁肽(Semaglutide)等。杜拉鲁肽、阿必鲁肽、利拉鲁肽及索马鲁肽都是天然胰高血糖素样肽-1(GLP-1)的类似物,在经过GLP-1序列突变后分别与IgG的FC片段、人白蛋白及脂肪酸融合或交联,获得活性高且稳定的GLP-1R激动剂。而艾塞那肽(Exenatide,即Exendin-4)则是一种来源于蜥蜴(Heloderma suspectum)唾腺的 39个氨基酸小肽。Exendin-4虽然是GLP-1R的强效激动剂,但活性比天然GLP-1及GLP-1 类似物都高。这些GLP-1R的激动剂虽然都能有效地降低血糖,控制食欲,但是对于减重的效果并不是太显著。其中利拉鲁肽(商品名)虽然被获批用于治疗肥胖,然而实际上其体重减轻大概只有5.6公斤。目前用于肥胖的药物减重一般在5–10%左右(与安慰剂相比),即整体上平均减重的比例不超过患者体重的10%(Rudolph L.Leibel等,BiologicResponses to Weight Loss and Weight Regain:Report From an American DiabetesAssociation Research Symposium,Diabetes,64(7):2299-2309,2015)。
减肥手术(Bariatric surgery)可以显著改善肥胖症和治疗糖尿病,然而其应用并不广泛,因为大部分的患者出于手术风险及长期后遗症的考虑,并不愿意接受这种手术(Obesity and Diabetes,New Surgical and Nonsurgical Approaches,Springer出版社,2015)。研究发现,经外科减肥手术的患者肠降血糖素(Incretin)分泌会激增(Obesity andDiabetes,New Surgical and Nonsurgical Approaches,Springer出版社,2015)。临床前及临床研究也发现,同时给病人输注GLP-1/胰高血糖素(Glucagon,GCG)(Tricia M.Tanetc.,DIABETES,VOL. 62:1131-1138,2013),或者GLP-1,胃泌酸调节素(Oxyntomodulin,OXM)和PYY,对于促进能量代谢,抑制食欲,控制体重(Tricia Tan etc.,J ClinEndocrinol Metab,2017,102(7):2364–2372) 都有明显效果。从临床需要而言,可以简单地把这些多肽直接混合用于临床。但是由于这些不同种类的多肽的体内稳定性及降解速度的差异,导致最终体内药效不可控,实际是很难简单将这些多肽混合后作为复方药物使用的。因此,目前新一代的糖尿病药物开发主要是设法把这些激动剂活性集中于一个分子中,如GLP-1R/GIPR和GLP-1R/GCGR双效激动剂,甚至 GLP-1R/GIPR/GCGR三效激动剂(Chakradhar,Shraddha.All in one:researchers create combination drugs fordiabetes and obesity.Nature Medicine,22(7):694-695,2016)。
目前这类药物的设计开发主要有以下几种方式:1.基于具有GLP-1/GCG双重活性的人体內源多肽胃泌酸调节素(Oxyntomodulin,OXM)的修饰开发。OXM是一种人体天然具有GLP-1 与GCG双重活性的多肽(Diabetes,2005,54:2390–2395)。但是OXM的活性并不高(具有约10%的GCG活性和约1%的GLP-1活性),而且体内稳定性和半衰期都很差,因此OXM 本身无法直接用于临床,往往需要通过引入非天然氨基酸,各种修饰等来提高其体内活性和稳定性。如OPKO Biologics的Mod-6030就是N端进行了可降解PEG修饰的长效OXM(OrenHershkovitz:Presentation Number:SAT-787。The Endocrine Society's 95th AnnualMeeting and Expo,June 15–18,2013-San Francisco)。TT401(LY2944876)则是另一种PEG修饰的OXM 类似物(Chakradhar,Shraddha."All in one:researchers createcombination drugs for diabetes and obesity."Nature Medicine,vol.22,no.7,2016:694-5)。PSA-OXM则是用多聚唾液酸(polysialic acid)修饰的OXM类似物(Vorobiev I等,Biochimie,2013,95(2):264-70)。但是限于OXM 的低活性、稳定性差等原因,其临床效果并不好,大部分研究已经被放弃。2.利用肠降血糖素(incretin)序列上的同源性,在OXM、GLP-1和GCG等结构基础上通过多重突变,修饰,甚至引进非天然氨基酸来获得多重活性的稳定杂合肽(Matthias H.等,Unimolecular Polypharmacy for Treatment ofDiabetes and Obesity,24:51–62,2016)。Matthias H.等的综述文章里详尽地介绍了目前处于临床或临床前的各种杂合肽形式。如Alessandro Pocai 等报道的基于OXM的双效GLP-1R/GCGR激动剂(Glucagon-Like Peptide 1/Glucagon Receptor Dual AgonismReverses Obesity in Mice,Diabetes;58(10):2258-2266,2009),或者Richard D.DiMarchi等报道的基于GCG的双效GLP-1R/GCGR激动剂(US9018164 B2)甚至三效的 GLP-1R/GCG/GIPR激动剂(US9150632)。这些多特异性的杂合肽大多基于GLP-1或GCG,通过序列突变来提高活性和抵抗蛋白酶水解,比如将L型氨基酸突变为D型氨基酸(如 D-Ser)、或引入非天然氨基酸Aib来提高体内稳定性,同时进行脂肪酸链或聚乙二醇(PEG) 修饰等来延长半衰期,临床预期给药周期为一天一次(脂肪酸修饰)或一周一次(PEG修饰)。另外,Aisling M.Lynch等报道了将天然GCG的第二位Ser突变为D-Ser,并在C末端引入 Exendin-4的C末端肽的GCG类似物(D-Ser2-glucagon-exe),并在DIO体内进行了药效实验,每天两次给药(Novel DPP IV-resistant C-terminally extended glucagon analogue exhibitsweight-lowering and diabetes-protective effects in high-fat-fed mice mediatedthrough glucagon and GLP-1receptor activation,Diabetologia:57:1927–1936,2014),减重效果明显。
虽然研究开发具有GLP-1R、GCGR、GIPR多重激动活性的分子,是非常有临床前景的,但真正要获得一个理想的这类药物,实际上却是非常困难的。
首先是安全性问题,特别是免疫原性问题。降糖减肥类药物需要长期使用,对安全性要求极高。为了设计获得一个具有GLP-1、GCG及GIP高活性的并且体内稳定的多肽,现有的技术方案都往往都引入了较多的突变位点,并且经常引入非天然氨基酸及其他修饰。这些突变及非天然氨基酸的引入,都增加了潜在免疫原性的风险。一般情况下与人源序列具有越高的同源性,在人体中免疫原性风险就相对的越低。罗氏与益普生合作研发的GLP-1受体激动剂降糖药Taspoglutide(仅引入了2个非天然氨基酸Aib),抗体生成率达到了49%,目前已经暂停了所有临床Ⅲ期的研究(JULIO ROSENSTOCK等,The Fate of Taspoglutide,a Weekly GLP-1Receptor Agonist,Versus wice-Daily Exenatide for Type 2,DIABETES CARE,36:498-504, 2013)。PHIL AMBERY等(THE ENDOCRINOLOGIST,SPRING,2017:12-13)在GCG 的序列基础上筛选了500多个结构,才获得了一条候选肽MEDI0382。其中,为了保持较高的GLP-1与GCG双重活性和体内稳定性,与GCG相比,MEDI0382引进了9个突变位点,突变率达到了约30%;同样,Andreas Evers等(J Med Chem.2017May 25;60(10):4293-4303) 在Exendin-4的结构基础上引入了9个突变位点,突变率达到了约23%,并进行了脂肪酸链修饰,才获得了同时具有较高GLP-1与GCG双重活性的杂合肽;Brian Finan等(Brian Finan 等,Nat Med.21:27-36,2015)设计的GLP-1/GCG/GIP三活性肽是在GCG的C末端加入了 GPSSGAPPPS序列,并引入了7个突变氨基酸,包括第二位突变为非天然氨基酸Aib。因此,现有的技术方案往往都引入了较多的突变位点,并且经常引入非天然氨基酸及其他修饰,才能获得同时具有GLP-1、GCG及GIP高活性的多肽。这些突变,修饰及非天然氨基酸的引入,都增加了潜在免疫原性的风险。而对于治疗糖尿病,肥胖这类疾病的药物,安全性是极其重要的。因此,开发一种不含非天然氨基酸,并且包含尽量少的突变氨基酸的高活性多效 GLP-1/GCG多重激动剂,是非常有意义的。
另一方面,现有研究对如何组合这些肠降血糖素的活性以及活性之间的合适比例,始终还未有公论。如PCT申请WO2015155139A1、WO2015155140A1及WO201515541A1公开了以Exendin-4为基础改造的GLP-1R/GCGR双效激动肽或GLP-1R/GCG/GIPR三效激动肽。其中WO201515541A1公开了一种具有GLP-1R/GCGR/GIPR三效激动活性的杂合肽,研究认为该三效激动活性的肽具有很好的降糖减肥效果;但是WO2015155139A1和WO2015155140A1 则是为了避免GIPR激动活性导致的低血糖风险,制备成GLP-1R/GCGR双效激动肽,反而具有更好的降糖减肥效果。A.Seth等研究也认为引入GIP活性并不能增强GLP-1对血糖控制效果(A.Seth等,Co-administration of a lipidated GIPR agonist with a GLP-1analogueprovides no additional benefit on HbA1c%over GLP1 analogue in db/db mice,EASD virtual meeting,2015)。
再次,对于GLP-1、Exendin-4、GCG这类序列高度同源,受体同属于GPCR家族,肽链长度只有30-40个氨基酸左右的小肽而言,单个位点突变或者若干个位点同时突变之后对不同受体的活性变化是非常难以预测的,因此要想获得理想的多重激动活性杂合肽也是极其困难的。例如Joseph Chabenne等报道(Joseph Chabenne等,A glucagon analogchemically stabilized for immediate treatment of life-threateninghypoglycemia,Molecular Metabolism,3:293-300, 2014)在对GCG进行丙氨酸扫描(Alascan),GCG的各个位点独立地被丙氨酸取代后,相对残留活性保留跨度从0.2%-100%,并表示GCG的第1、2、3、4、6-12、14、15、22、 23、25-27、29位突变都会使GCGR激动活性大幅减弱(文章中的表4)。然而我们也可以在其他的报道中看到在上述这些位点中取单个或若干个位点同时进行突变,用其他的氨基酸取代时,活性的变化并不总是与丙氨酸扫描的结果一致。如Jonathan W Day等(Jonathan W Day 等,A new glucagon and GLP-1co-agonisteliminates obesity in rodents,Nature Chemical Biology, 5:749-757,2009)报道,对GCG的16位进行16S→G、16S→T、16S→H、16S→E等不同突变,其GCGR激动活性反而是提高的,这与Joseph Chabenne的丙氨酸扫描结果完全矛盾。其次,Joseph Chabenne研究认为在23位用丙氨酸取代,将导致GCGR激动活性几乎完全丧失(仅保留1.1%);但是Jonathan WDay等将23位突变成Ile,其GCG活性并没有下降。又比如丙氨酸扫描结果认为第二位S对保留GCG活性是非常重要的(突变为Ala时活性仅保留了1/3),但是Brian Finan等报道(FinanB等,A rationally designed monomeric peptide triagonist corrects obesity anddiabetes in rodents.Nat Med.2015;21:27-36.),对GCG第二位氨基酸分别进行2S→Aib、2S→dSer、2S→G、2S→dAla替代突变,再组合其他位点的突变后,GCGR的相对激动活性反而提高为200%—640%。在我们的研究中也发现,把一些有利于提高GLP-1,GCG或GIP活性的突变组合引入时,很多时候其效果与单个位点突变时是完全不一致的。并且,GLP-1、Exendin-4、GCG或GIP这类多肽,在N及C末端增加或减少氨基酸都会影响其生物学活性。如N端去掉一个或两个氨基酸,GLP-1、GCG等的激动活性就会完全丧失。如Oxyntomodulin只比Glucagon的C末端多了KRNRNNIA这8个氨基酸,其GCGR激动活性就丧失了90%左右(Alessandro Pocai等,Glucagon-Like Peptide 1/Glucagon Receptor Dual AgonismReverses Obesity in Mice,Diabetes;58(10):2258-2266,2009;Henderson SJ等,Robustanti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptideagonist in rodents and non-human primates,Diabetes Obes Metab,2016)。
又如Joseph R.Chabenne和Richard D.DiMarchi等曾报道,在Glucagon的C末端增加一段Exendin-4的C末端小肽cex(SEQ ID NO.67,GPSSGAPPPS)后,使得其GLP-1R激动活性从0.7%增至1.6%,提高了约2倍左右(Optimization of the Native Glucagon Sequencefor Medicinal Purposes,J Diabetes Sci Technol.4(6):1322–1331,2010及专利US9018164 B2),并且还损失了约50%的GCG活性。Evers A等也报道(Evers A,Design ofNovel Exendin-Based Dual Glucagon-like Peptide-1(GLP-1)/Glucagon ReceptorAgonists,J Med Chem.;60(10):4293-4303. 2017)在GCG类似物的C末端加上cex序列后,GLP-1R激动活性反而下降了2/3左右,但是GCG的激动活性更是损失了90%以上(文章中表2,肽7和8)。因此,对于GLP-1,Glucagon 这类30个氨基酸长度的小肽,序列的改变对其活性变化是极其敏感的;而对于双重活性多肽,由于涉及对两个不同受体的激动,其变化就更加复杂,根本无法预测任何一个氨基酸改变后对GLP-1R和GCGR激动活性会是什么样的后果。
GCGR、GLP-1R等GPCR受体下游信号传导的复杂性,也增加了设计一个理想的多重活性杂合肽的难度。GCGR、GLP-1R的受体在胞内存在多条信号传导通道,包括G蛋白(Gαs、 Gαi、Gαq等)和抑制蛋白(β-arrestin-1和β-arrestin-2)等下游信号因子形成了多条不同的信号传导通道,不同通道的激活其生理效应是不一样的,甚至有些通道活性与生理学功能的关系并未明了。例如在GLP-1序列中引入不同的突变或者不同的氨基酸序列,就能得到不同偏向性激动(biased-agonist)的结构,从而产生不同的生理学效应(Marlies V.等,J AmChem Soc., 138(45):14970-14979,2016;Hongkai Zhang等,Nat Commun.,6:8918,2015)。
因此,虽然理论上说设计获得一条同时具有高GLP-1、Glucagon及GIP活性的多肽是非常有临床意义的,但是实际上也是非常困难的。如果能够设计一种活性平衡,但是又尽量少引入突变位点和非天然氨基酸,尽量接近天然序列使得其潜在免疫原性更低,并且稳定性提高,具有很好的血糖控制和体重控制的多重活性杂合肽,是非常有临床意义的。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供一种胰高血糖素类似物,该胰高血糖素类似物表现出GLP-1R/GCGR/GIPR三受体激动活性。
天然GCG具有相对于天然GLP-1大约1%的GLP-1R激动活性,但并没有GIPR激动活性。而本发明的胰高血糖素类似物,可表现GLP-1R/GCGR/GIPR三受体激动活性。
为实现上述目的及其他相关目的,本发明的第一方面提供一种胰高血糖素类似物(GCG类似物),所述胰高血糖素类似物的结构中含有如式I或式II所示的结构,式I所示结构为: HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz(SEQ ID NO.1),式 II所示结构为:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2(SEQ IDNO.2),其中,X10选自Y,K或L之任一,X16选自S、E或A之任一,X17选自Q、E、A或R之任一,X20选自Q、R或K之任一;X21选自D、L或E之任一;X23选自V或I之任一;X29为T或缺失,Xz选自GGPSSGAPPPS(SEQ ID NO.65)、GGPSSGAPPS(SEQ ID NO.66)、 GPSSGAPPPS(SEQ ID NO.67)、GPSSGAPPS(SEQ ID NO.68)、PSSGAPPPS(SEQ ID NO.69)、 PSSGAPPS(SEQ ID NO.70)、SSGAPPPS(SEQ ID NO.71)或SSGAPPS(SEQ ID NO.72) 之任一。
进一步地,当所述胰高血糖素类似物的结构式为:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2(SEQ ID NO.2)时,是指所述胰高血糖素类似物的C端进行了酰胺化修饰。
如本发明的一些实施方式所列举,本发明胰高血糖素类似物的氨基酸序列如SEQID NO.6-28及SEQ ID NO.47-53之任一所示。
进一步地,在一个优选的实施方案中,所述胰高血糖素类似物的结构式为:
HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz(SEQ ID NO.3)
或HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz-NH2(SEQ ID NO.4)
其中,X16选自S或E之任一,X17选自Q或E之任一,X29为T或缺失,Xz选自GGPSSGAPPPS(SEQ ID NO.65)、GGPSSGAPPS(SEQ ID NO.66)、GPSSGAPPPS(SEQ ID NO.67)、GPSSGAPPS(SEQ ID NO.68)、PSSGAPPPS(SEQ ID NO.69)、PSSGAPPS (SEQ ID NO.70)、SSGAPPPS(SEQ ID NO.71)或SSGAPPS(SEQ ID NO.72)之任一。
进一步地,当所述胰高血糖素类似物的结构式为:
HSQGTFTSD-Y-SKYLD-X16-X17-AAQDFVQWLMN-X29-Xz-NH2(SEQ ID NO.4)时,是指所述胰高血糖素类似物的C端进行酰胺化修饰。
上述胰高血糖素类似物具有相似于或优于天然胰高血糖素的GCGR激动活性,和具有相似于或优于天然GLP-1的GLP-1R激动活性,和额外增加的GIPR激动活性。
在本发明的其中一个实施例中,优选的胰高血糖素类似物在天然胰高血糖素基础上C末端加入GPSSGAPPPS,最少仅突变2-3个氨基酸且不引入非天然氨基酸,也无需任何修饰,就能保留或提高GLP-1R与GCGR激动活性,而且还具有额外增加的GIPR激动活性,产物本身具有很好的稳定性。更少的突变位点,且不进行后续修饰,尽量保持了天然结构,减少了潜在的免疫原性风险。
已有文献报道在糖尿病治疗中,高水平的GIP会引起频繁的低血糖症状(TMcLaughlin 等人,J Clin Endocrinol Metab,95,1851-1855,2010;A Hadji-Georgopoulos,Jclin Endocrinol Metab,56,648-652,1983)。但是在小鼠动物模型试验中,本发明提供的具有GIPR激动活性的胰高血糖素类似物,能够平稳的控制血糖,并无低血糖症状。另有文献报道称,为了减少日常摄食、减轻体重、提高胰岛素敏感性及能量消耗,拮抗GIPR也是可取的方法(Irwin等, Diabetologia 2007,50,1532-1540;Althage等,J BiolChem,2008,283(26):18365–18376)。而在小鼠动物模型试验中,本发明提供的GIP活性较高的胰高血糖素类似物,相对于GIP活性较低甚至无GIP活性的对比类似物,对肥胖小鼠的日常摄食控制、体重减轻及胰岛素敏感度提高都有更显著的效果。
本发明胰高血糖素类似物具有更好的耐酶稳定性,包括对中性内肽酶(NEP)以及二肽基肽酶-4(DPP-4);与天然胰高血糖素,GLP-1,GIP相比具有更长的体内半衰期和持续作用时间。
本发明的第二方面,提供一种分离的多核苷酸,所述分离的多核苷酸编码前述胰高血糖素类似物。
本发明的第三方面,提供一种重组表达载体,包含前述分离的多核苷酸。
本发明的第四方面,提供一种宿主细胞,所述细胞含有前述重组表达载体或基因组中整合有外源的前述分离的多核苷酸。
本发明的第五方面,提供前述胰高血糖素类似物的制备方法,选自以下之任一:
(1)利用化学合成方法合成所述胰高血糖素类似物;
(2)在合适的条件下培养前述宿主细胞,使之表达所述胰高血糖素类似物,而后分离及纯化获得所述胰高血糖素类似物。
具体地,本发明的胰高血糖素类似物可通过标准肽合成方法进行制备,例如,通过标准固相或液相方法,逐步或通过片段组装,并分离和纯化最终的肽化合物产物,或通过重组和合成方法任意组合。可优选通过固相或液相肽合成方法来合成本发明的胰高血糖素类似物。
本发明的第六方面,提供前述胰高血糖素类似物在制备治疗代谢相关疾病的药物中的用途。
本发明提供的胰高血糖素类似物可以用于治疗糖尿病相关的代谢综合征,如血脂失调,包括甘油三酯过高、低HDL胆固醇及高LDL胆固醇;胰岛素抗性或葡萄糖耐受不良等。
代谢综合征与冠心病和血管斑块积累相关的其他病症风险增加相关,例如中风和外周血管疾病,成为粥样动脉硬化心血管病(ASCVD)。患有代谢综合征的患者可自处于早期的胰岛素抗性状态发展成完全成熟的二型糖尿病,并且ASCVD的风险进一步增加。不受限于任何特定理论,胰岛素抗性、代谢综合征及血管疾病之间的关系可以涉及一种或多种共同发病机制,包括胰岛素刺激的血管舒张障碍、由氧化应力增强所致的胰岛素抗性相关性可用性降低,以及脂肪细胞衍生激素(诸如脂联素)异常(Lteif,Mather,Can.J.Cardiol.20(增刊B): 66B-76B,2004)。
本发明的胰高血糖素类似物还可用于治疗肥胖症。在一些方面中,本发明的胰高血糖素类似物通过降低食欲、减少食物摄取、降低患者体内脂肪水平、提高能量消耗等机制来治疗肥胖症。
在一些潜在的实施方案中,本发明的胰高血糖素类似物可用于治疗非酒精性脂肪肝病 (NAFLD)。NAFLD是指广谱肝脏疾病,范围自单纯脂肪肝(脂肪变性)至非酒精性脂肪变性肝炎(NASH)至肝硬化(肝脏的不可逆晚期瘢痕形成)。NAFLD的所有病期均表现出肝脏细胞中的脂肪累积。单纯脂肪肝为某类型的脂肪、甘油三酯在肝脏细胞中的异常积累,但无发炎或瘢痕形成。在NASH中,脂肪累积与不同程度的肝脏发炎(肝炎)和瘢痕形成(纤维化)相关。发炎性细胞可破坏肝脏细胞(肝细胞坏死)。在术语“脂肪变性肝炎”和“脂肪变性坏死”中,脂肪变性是指脂肪浸润,肝炎是指肝脏中的炎症,并且“坏死”是指经破坏的肝脏细胞。NASH可最终导致肝脏瘢痕形成(纤维化)且接着导致不可逆晚期瘢痕形成(肝硬化),由NASH导致的肝硬化为NAFLD谱内的最后且最严重的病期。
本发明的第七方面,提供一种治疗代谢相关疾病的方法,包括步骤:向对象施用前述胰高血糖素类似物。
在其中一个实施例中,本发明将所述胰高血糖素类似物用于治疗肥胖、代谢综合症及非酒精肝炎等。
本发明的研究人员已经发现本发明的胰高血糖素类似物在中性pH或微弱酸性的pH下具有足够的水溶性且具有改善的化学稳定性。在其中一个实施例中,进行了IPGTT实验。施用了本发明胰高血糖素类似物的小鼠在注射葡萄糖后,呈现出极平稳的血糖波动。此外,本发明的胰高血糖素类似物在DIO小鼠施用后,诱导了体重的显著降低。同时血脂相关的各种指标明显下降。
本发明进一步提供一种促进体重减少或者防止体重增加的方法,包括在对象中施用所述的胰高血糖素类似物。
本发明的第八方面,提供一种组合物,含有前述胰高血糖素类似物或前述宿主细胞的培养物,以及药学上可接受的载体。
本发明的第九方面,提供前述胰高血糖素类似物在制备融合蛋白中的用途。
本发明的第十方面,提供一种融合蛋白,所述融合蛋白的结构中含有前述胰高血糖素类似物。
进一步地,所述融合蛋白的结构中还含有长效单元。
进一步地,所述长效单元选自共价连接脂肪酸、聚乙二醇或其衍生物,白蛋白,转铁蛋白和免疫球蛋白及片段。
本发明的第十一方面,提供一种被修饰的多肽,其结构中含有前述胰高血糖素类似物。
进一步地,所述的胰高血糖素类似物被脂肪酸、聚乙二醇或其衍生物所修饰;所述的被修饰的多肽以共价或者非共价方式与白蛋白,转铁蛋白和免疫球蛋白及片段相结合。
本领域技术人员知晓,为了增加本发明胰高血糖素类似物的半衰期或稳定性可以对其进行修饰,例如可以将聚乙二醇或其衍生物、羟乙基淀粉衍生物或脂肪酸共价连接至本发明的胰高血糖素类似物。在一个具体的实施方案中,为了提高稳定性,可以在本发明的胰高血糖素类似物预期不会影响受体结合/活化的位置上引入赖氨酸残基,共价连接至γ-谷氨酸间隔物并在ε-氨基上加入棕榈酸进行修饰。
与现有技术相比,本发明具有如下有益效果:
本发明的胰高血糖素类似物,具有GLP-1/GCG/GIP三受体激动活性,以及更好的耐酶稳定性,包括对中性内肽酶(NEP)以及二肽基肽酶-4(DPP-4)的抵抗;因此与天然胰高血糖素,GLP-1,GIP相比具有更长的体内半衰期和持续作用时间。综上所述,目前现有报道的GCG类似物普遍采用(1)单分子杂合肽交联脂肪酸、PEG或FC等,施行一天一次或以上的频率给药(Matthias H.等,Unimolecular Polypharmacy for Treatment of Diabetesand Obesity,24:51–62,2016);或者(2)将天然GCG的第二位Ser突变为D-Ser等非天然氨基酸抵抗DPP-IV降解(Novel DPP IV-resistant C-terminally extended glucagonanalogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1receptor activation,Diabetologia:57:1927–1936,2014),每天给药两次。保持天然氨基酸组成且通过每天两次给药的多重活性多肽目前尚未见报道。而本发明则是提供一种足够稳定、高活性的多效GCG类似物,无须交联脂肪酸、PEG白蛋白或免疫球蛋白Fc片段,且无需将第二位Ser突变为非天然氨基酸,因此能最大程度地减低潜在的免疫原性风险,且省略繁琐的化学修饰/交联步骤,简化制备过程,提高产物一致性。
附图说明
图1:为编号为C381的多肽在pH 7.4水溶液中的HPLC谱图。
图2:为编号为C493的多肽在pH4.5水溶液中的HPLC谱图。
图3:为编号为C816的多肽在pH 7.4水溶液中的HPLC谱图。
图4:为编号为C002的多肽在pH 7.4水溶液中的HPLC谱图。
图5:为编号为C611的多肽在pH 4.5水溶液中的HPLC谱图。
图6:为编号为C611的多肽在pH 7.4水溶液中的HPLC谱图。
图7:为C239在pH 7.4水溶液中的HPLC谱图。
图8A:为残留活性随时间变化曲线图。
图8B:为残留活性随时间变化曲线图。
图9A:为示例性的几种胰高血糖素类似物对GLP-1R激动活性检测图。
图9B:为示例性的几种胰高血糖素类似物对GLP-1R激动活性检测图。
图9C:为示例性的几种胰高血糖素类似物对GCGR激动活性检测图。
图9D:为示例性的几种胰高血糖素类似物对GCGR激动活性检测图。
图9E:为不同浓度梯度下胰高血糖素类似物及对照刺激GIPR产生的cAMP含量。
图9F:为不同浓度梯度下胰高血糖素类似物及对照刺激GIPR产生的cAMP含量。
图9G:为不同浓度梯度下胰高血糖素类似物及对照刺激GIPR产生的cAMP含量。
图9H:为不同浓度梯度下胰高血糖素类似物及对照刺激GIPR产生的cAMP含量。
图10:为体外细胞胰岛素分泌测定结果。
图11A:为正常ICR小鼠的IPGTT实验血糖变化曲线图。
图11B:为正常ICR小鼠的IPGTT实验血糖变化曲线图。
图11C:为血糖曲线下面积(AUC)比较结果。
图12A:为饮食诱导肥胖(DIO)小鼠中体重变化(%)与时间(天)关系的图表。
图12B:为饮食诱导肥胖(DIO)小鼠中体重变化(%)与时间(天)关系的图表。
图12C:为饮食诱导肥胖(DIO)小鼠中体重变化(%)与时间(天)关系的图表。
图12D:为DIO小鼠中的体重减少情况比较图。
图13:为DIO小鼠中的体重减少情况比较图。
图14:为C495质谱分析图。
图15:为C382质谱分析图。
具体实施方式
除非下文另外定义,本发明所提及的所有技术和科学用语具有本发明所属领域的技术人员通常理解的意义。
胰高血糖素类似物:
本发明提供的胰高血糖素类似物将18位的精氨酸(R)突变为丙氨酸(A)。18位丙氨酸的突变会使GCGR激动活性下降约30%(Joseph Chabenne等,A Glucagon analogchemically stabilized for immediate treatment of life-threateninghypoglycemia,Molecular Metabolism,3: 293-300,2014)。然而,经过本发明人大量的组合筛选后发现,在一些特定位点进行特定的氨基酸突变,如与16及17位的特定氨基酸突变组合后,并在C末端加入CEX或类似序列,即使18位突变成A,其GCGR活性也并无明显降低。更重要的是,18位A突变使胰高血糖素类似物的GLP-1和GIPR激动活性显著提升,从而使本发明的胰高血糖素类似物成为有效的三特异性活性肽。
本发明的三特异性活性肽具有激动GLP-1R、GCGR及GIPR的效力,且各活性相对于GLP-1,GCG和GIP,其活性保留率极高。目前多数的三活性肽都在天然多肽的基础上引入多个氨基酸突变,甚至非天然氨基酸才能成为稳定的三效激动剂。在本发明的筛选过程中发现,多位点引入突变确实更容易获得GLP-1R、GCGR及GIPR活性较高的杂合多肽,另外非天然氨基酸的引入也更容易获得稳定性高的多肽。然而体外活性与稳定性的高低只是可以成为临床药物的一个前提条件,还需要关注安全性。引入过多的突变位点或非天然氨基酸,容易带来更高的免疫原性风险。
血清稳定性实验:
天然GLP-1、胰高血糖素Glucagon或胃泌酸调节素(Oxyntomodulin)都因血清稳定性太差及过短的体内半衰期而无法真正作为临床药物。而同样只有39个氨基酸的小肽艾塞那肽 (Exenatide,商品名)却能因为稳定性提高而成功上市。在本发明的其中一个实施例中,优选的胰高血糖素类似物呈现出了非常高的稳定性。
免疫原性实验:
在本发明的实施例6中,天然人源Glucagon(C001)在大鼠体内的免疫原性极低。胰高血糖素类似物相对于天然Glucagon突变不多于3个氨基酸时,抗体滴度均小于<1:200,而随着突变的氨基酸数量增加,抗体滴度也随着增加,表明潜在的免疫原性风险增高。对于治疗代谢相关疾病的药物,如糖尿病,肥胖等领域的药物,安全性的要求是极高的。而本发明获得的胰高血糖素类似物在引入不超过3个突变位点的情况下,具有较低的免疫原性,并达到了理想的活性与稳定性标准,这是从未有人报道过的。
动物体内药效学研究:
在本发明的一个实施例中,优选的胰高血糖素类似物具有良好的降低血糖、抑制脂肪组织形成,减低体重的效果。尽管GIP与GLP-1及Glucagon同属于肠降血糖素(Incretin)一族,但是却并没有被广泛开发成药物的趋势。究其原因,一个是由于部分二型糖尿病患者对 GIP失去敏感性,另一个原因是在啮齿类动物中,GIPR激动出现潜在的致肥胖现象(Miyawaki, K.等,Inhibition of gastric inhibitory polypeptide signalingprevents obesity.Nat.Med.8,738–742, 2002)。然而在本发明的实施例中,优选的GIPR激动活性较高的胰高血糖素类似物明显具有更显著的减重效果。
在本发明的另一个动物体内实施例9中,优选的GCG类似物与相同氨基酸序列且经脂肪酸修饰的相应GCG类似物呈现相似的减重效果。
术语:
术语“糖尿病”包括一型糖尿病、二型糖尿病、妊娠糖尿病以及引起高血糖症的其他症状。该术语用于指由于代谢紊乱,胰腺产生不了足够的胰岛素,或身体的细胞未能适当响应胰岛素,因此组织细胞吸收葡萄糖效率下降导致葡萄糖在血液中积累的症状。
一型糖尿病也称为胰岛素依赖性糖尿病和幼年发病型糖尿病,通过β细胞破坏引起,通常导致绝对胰岛素缺乏。
二型糖尿病也称为非胰岛素依赖性糖尿病和成年发病型糖尿病,普遍与胰岛素抗性相关。
术语“肥胖”意指脂肪组织的过量,当能量摄取超过能量消耗时,过量卡路里贮存于脂肪中,则导致肥胖。在本文中体重指数(BMI=体重(千克)除以身高(米)的平方)超过25的个体视为肥胖。
术语“受体激动剂”可以定义为与受体结合且引发天然配体的通常应答的多肽、蛋白或其他小分子。肠降血糖素是通过增强葡萄糖刺激的胰岛素分泌来调控血糖的胃肠激素(Drucker.D J,Nauck,MA,Lancet 368:1696-705,2006)。迄今为止有两种已知的肠降血糖素:胰高血糖素样肽-1(GLP-1)和葡萄糖依赖性促胰岛素多肽(GIP)。前胰高血糖素原(preproglucagon)是 158个氨基酸组成的前体多肽,其在组织中被差异性加工而形成多种结构上相关的胰高血糖素原衍生肽,包括胰高血糖素(Glucagon)、胰高血糖素样肽-1(GLP-1)、胰高血糖素样肽-2 (GLP-2)和胃泌酸调节素(Oxyntomodulin,OXM)。GIP是由133个氨基酸的前体 (pre-pro-GIP)通过蛋白水解加工得到的42个氨基酸的成熟肽,这些分子参与多种生物功能,包括葡萄糖体内平衡、胰岛素分泌、胃排空和肠生长以及食物摄取调节。
胰高血糖素样肽-1(GLP-1)是从肠L-细胞分泌的30或31个氨基酸的一种肠促胰岛素激素,有GLP-1(7-36)和GLP-1(7-37)两种活性形式。GLP-1在进餐后释放到循环中,并通过激活GLP-1受体发挥生物活性。GLP-1具有许多生物学作用,包括葡萄糖依赖性的促胰岛素分泌,抑制胰高血糖素生成,延缓胃排空和抑制食欲(Tharakan G,Tan T,BloomS.Emerging therapies in the treatment of‘diabesity’:beyond GLP-1.TrendsPharmacol Sci 2011;32(1):8-15.) 等。天然GLP-1由于能够被二肽基肽酶-4(DPP-4),中性肽链内切酶(NEP),血浆激肽释放酶或纤溶酶等快速降解因而限制了其治疗潜力。由于天然GLP-1在体内仅有大约2分钟的超短半衰期,因此,出现了通过利用化学修饰和/或制剂形式来改善功效以治疗糖尿病和肥胖症的方法(Lorenz M,Evers A,Wagner M.Recentprogress and future options in the development of GLP-1receptor agonists forthe treatment of diabesity.Bioorg Med Chem Lett 2013;23(14):4011-8.TomlinsonB,Hu M,Zhang Y,Chan P,Liu ZM.An overview of new GLP-1 receptor agonists fortype 2diabetes.Expert Opin Investig Drugs 2016;25(2):145-58)。
胃泌酸调节素(Oxyntomodulin)是37个氨基酸的小肽,其包含胰高血糖素完整的29个氨基酸序列。胃泌酸调节素是GLP-1R和GCGR的双重激动剂,在进餐后通过肠L-细胞与GLP-1一起分泌。与胰高血糖素类似,胃泌酸调节素在人和啮齿动物中产生显着的体重减轻。胃泌酸调节素的减肥活性已在肥胖小鼠中与等摩尔剂量的选择性GLP-1R激动剂进行比较。已经发现,与选择性的GLP-1R激动剂相比,胃泌酸调节素具有抗高血糖作用,能够显著的减轻体重和具有降脂活性(The glucagon receptor is involved in mediating thebody weight-lowering effects of oxyntomodulin,Kosinski JR等,Obesity(SilverSpring),20):1566-71, 2012)。在超重和肥胖患者中,皮下施用天然胃泌酸调节素在四周内减少体重1.7公斤。胃泌酸调节素也被证明可以减少人类的食物摄取和增加能量消耗(Subcutaneous oxyntomodulin reduces body weight in overweight and obesesubjects:a double-blind,randomized, controlled trial,Wynne K等,Diabetes,54:2390-5,2005;Oxyntomodulin increases energy expenditure in addition todecreasing energy intake in overweight and obese humans:a 14andomizedcontrolled trial;Wynne K等,Int J Obes(Lond),30:1729-36,2006)。但是同样由于分子量偏小及DPP-IV的降解,胃泌酸调节素具有较短的半衰期。目前GLP-1受体 (GLP-1R)和胰高血糖素受体(GCGR)的双效激动剂普遍都是基于胃泌酸调节素的,并且为了改善胃泌酸调节素的短效及酶解的缺陷而做了突变(胃泌酸调节素类似物),且大都采用第二位丝氨酸Ser突变为α-氨基异丁酸(Aib)或D-Ser的方法,通过引入非天然氨基酸来抵抗DPP-IV的酶解。胃泌酸调节素类似物虽然表现出了初步的降糖及减脂效果,但是其作用机制仍不确切,胃泌酸调节素受体一直没有发现,目前仅仅是通过GCGR或GLP-1R敲除的小鼠或者细胞试验验证胃泌酸调节素能与这2种受体结合而起作用。
胰高血糖素(Glucagon)是29个氨基酸的肽,其对应于前胰高血糖素原的53-81位氨基酸,序列如SEQ ID NO.5所示(C.G.Fanelli等,Nutrition,Metabolism&Cardiovascular Diseases(2006)16,S28-S34)。胰高血糖素受体激活已显示在啮齿类动物和人两者中增加能量消耗且减少食物摄入(Habegger K.M.等人,The metabolic actionsof glucagon revisited,Nat.Rev. Endocrinol.2010,6,689-697)并且这些效应在啮齿类动物中是稳定和持续的。胰高血糖素具有许多生理效应,例如通过刺激糖原分解和糖异生,增加低血糖状况下的血糖水平,调节肝酮生成,调节胆汁酸代谢和通过迷走神经的饱腹效应。胰高血糖素已经用于急性低血糖症,胰高血糖素受体激活减少食物摄取并促进动物和人的脂肪分解和体重减轻。
葡萄糖依赖性促胰岛素样肽(GIP)是具有42个氨基酸的多肽,其在食物摄取之后从小肠 K细胞释放,其主要作用为抑制胃酸分泌和加强葡萄糖刺激的胰岛素分泌,故称为抑胃肽 (gastric inhibitory peptide)/葡萄糖依赖性促胰岛素多肽(glucose-dependent insulinotropic peptide)。
“GLP-1受体(GLP-1R)激动剂”可以定义为与GLP-1R结合且能够引发与天然GLP-1相同或类似的特征性反应的多肽、蛋白或其他小分子。GLP-1R激动剂通过完全或部分激活GLP-1R,继而引起一系列细胞内的下游信号通路反应,产生相应的细胞活性:如β细胞分泌胰岛素;典型的GLP-1R激动剂包括天然GLP-1及其突变体、类似物,如艾塞那肽、利拉鲁肽(Liraglutide)等。
“胰高血糖素受体(GCGR)激动剂”可以定义为与GCGR结合且能够引发与天然胰高血糖素(glucagon)相同或类似特征性反应的多肽、蛋白或其他小分子。GCGR激动剂通过完全或部分激活GCGR,继而引起一系列细胞内的下游信号通路反应,产生相应的细胞活性:如肝细胞糖原分解、糖质新生、脂肪酸氧化及生酮作用等。
GLP-1R/GCGR双效激动剂:本发明的GLP-1R/GCGR双效激动剂包括能同时激动GLP-1R 和GCGR的蛋白或多肽。如Alessandro Pocai等报道的基于Oxyntomodulin的双效激动剂(Alessandro Pocai等,Glucagon-Like Peptide 1/Glucagon Receptor Dual AgonismReverses Obesity in Mice,Diabetes;58(10):2258-2266,2009),或者RichardD.DiMarchi等报道的基于glucagon的双效激动剂(US9018164 B2)。
GLP-1R/GCGR/GIPR三效激动剂:本发明的GLP-1R/GCGR/GIPR三效激动剂包括能同时激动GLP-1R、GCGR和GIPR的蛋白或多肽,或称为“三特异性激动剂”。
三特异性活性肽:本发明中的经优选的三特异性活性肽是指同时具有GLP-1R/GCGR/GIPR 激动活性的多肽,或称为“三效活性肽”。
EC50(concentration for 50%of maximal effect)是指某一药物或者物质在刺激其相应的生物学反应的50%时所需的浓度。EC50值越低,表明该药物或物质的刺激或激动能力越强,例如,更直观地可表现为引起的细胞内信号越强,从而诱导某激素产生的能力越佳。
低密度脂蛋白(LDL):属于血浆脂蛋白的一种,是血液中胆固醇的主要载体。其倾向于将胆固醇沉积在动脉壁上。白细胞试图消化低密度脂蛋白,但在这个过程中却将它们变成毒素。越来越多的白细胞被吸引到发生变化的区域,致使动脉壁发炎。随着时间的推移,这些斑块沉积物可以积聚在动脉壁上,使得通道变得非常窄和缺乏韧性。如果太多的斑块累积,则动脉可以完全阻塞。当LDL与胆固醇形成的复合物(LDL-C)在动脉壁上产生太多斑块时,血液将不能自由流过动脉。斑块随时可能会在动脉中突然塌陷,导致血管堵塞,最终引发心脏病。
高密度蛋白(HDL):有助于清除动脉上的LDL,起到清道夫的作用,将LDL从动脉上清走并回到肝脏。
甘油三酯(TG):是另一种类型的脂肪,用于储存饮食过多的能量。血液中高水平的甘油三酯与动脉粥样硬化有关。高甘油三酯可以由超重和肥胖,身体缺乏运动,吸烟,过量的酒精消耗和高碳水化合物(超过总卡路里的60%)摄入引起。有时基础疾病或遗传疾病是高甘油三酯的原因。具有高甘油三酯的人通常具有高的总胆固醇水平,包括高LDL胆固醇和低 HDL胆固醇,许多患有心脏病或糖尿病的人也具有高甘油三酯水平。
GPCR:G蛋白偶联受体(G Protein-Coupled Receptor),它是细胞信号传导中的重要蛋白质,其拓扑构象为7次跨膜的受体。当膜外的配体作用于该受体时,该受体的膜内部分与G 蛋白相互结合,激活G蛋白。G蛋白可通过两种途径,传递细胞外的信息:第一种方式是打开跨膜离子通道,让外界的离子进入;第二种方式是激活第二信使,如cAMP、IP3/DAG等。钙离子通常被认为是cAMP、IP3/DAG下游的第三信使。
缩写
COMU:1-[(1-(氰基-2-乙氧基-2-氧代亚乙基氨基氧基)二甲基氨基吗啉代亚甲基)]甲铵六氟磷酸盐
DCM:二氯甲烷
DMF:N,N-二甲基甲酰胺
DIPEA:二异丙基乙胺
EtOH:乙醇
Et2O:***
HATU:2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
MeCN;乙腈
NMP:N-甲基吡咯烷酮
TFA:三氟乙酸
TIS:三异丙基硅烷
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。
除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS INENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
实施例1:胰高血糖素类似物的一般制备及纯化方法
采用现有技术,例如现有文献(V.等人,Beilstein J.Org.Chem.,10:1197–1212(2014); Palomo,J.M.,RSC Adv.,4:32658-32672(2014);Behrendt,R.等人,J.Pept.Sci.,22:4-27(2015)) 中的多肽固相合成及修饰方法来制备本专利所涉及的各多肽。
具体地,可用标准Fmoc法在CEM Liberty肽合成仪上进行固相肽合成。
在使用之前将Rink Amide TentaGel S Ram树脂(0.25mmol/g,1g)在NMP(10mL)中溶胀,加入到固相合成装置中,树脂中加入哌啶/DMF(20%,10mL)反应30min进行脱Fmoc 保护,抽干,用DMF洗涤(5×10mL),抽干。加入第一个Fmoc-氨基酸溶液(0.2M, NMP/DMF/DCM,1:1:1,5mL)以及COMU/NMP(0.5M,2mL)和DIPEA/DMF(2.0M; 1mL),室温反应1h,茚三酮检测树脂无色透明,抽干,用DMF(5×10mL)洗涤。然后加入哌啶/DMF(20%,10mL)反应30min进行脱Fmoc保护,抽干,用DMF洗涤(5×10mL),抽干。重复上述加入Fmoc-氨基酸进行反应和哌啶/DMF进行脱Fmoc保护的步骤直至完成最后一个组氨酸的偶联。
将树脂用EtOH(3×10mL)和Et2O(3×10mL)洗涤并在室温下干燥至恒重。树脂加入TFA/TIS/苯酚/EDT/水(82.5/5/5/2.5/5,v/v,40mL)冰浴反应2h,将粗肽从树脂上切割下来,过滤,重复该步骤三次。将滤液合并,减压除去大部分TFA,用***沉淀,离心,沉淀用***洗涤三次,室温下干燥至恒重得到粗肽。使用配备有C-18柱和部分收集器的瓦里安SD-1型制备液相色谱仪,用流动相A(0.1%TFA,水溶液)和流动相B(0.1%TFA,90%MeCN,水溶液)的梯度洗脱,通过制备型反相HPLC将粗肽纯化至纯度大于97%,所得肽列于表1。其中,C端酰胺结尾的多肽采用上述方法进行合成;其余多肽采用Wangle Resin(0.4mmol/g, 1g)进行固相合成,树脂溶胀后直接加入Fmoc-氨基酸进行偶联反应,脱Fmoc保护,多肽切割,纯化,操作步骤与C端酰胺结尾多肽合成步骤相同。带有脂肪酸修饰的多肽合成与纯化为常规技术,并可参见Finan B等(Finan B等,A rationally designed monomeric peptidetriagonist corrects obesity and diabetes in rodents.Nat Med.2015;21:27-36.)或Chhabr等(Chhabr等, Appraisal of New Variants of Dde Amine Protecting Groupfor Solid Phase Peptide Synthesis. Tetrahedron Lett.1998,39(12),1603–1606)的文章所述。
经过纯化后的肽通过LC/MS分析,分析结果如表2。其中,图14、15分别为示例性的编号为C495和C382的胰高血糖素类似物质谱分析图。质谱条件如下:
仪器:Waters ZQ 2000
质谱(Probe):ESI
喷雾器流速(Nebulizer Gas Flow):1.5L/min
CDL:-20.0v
CDL温度:250℃
加热块温度(Block Temp):200℃
质谱电压(Probe Bias):+4.5kv
检测器(Detector):1.5kv
流动相流速(T.Flow):0.2ml/min
缓冲液浓度(B.Conc.):50%H2O/50%ACN
表1
注:表中的X为氨基异丁酸,K’表示此位置为赖氨酸残基并共价连接至脂肪酸,该脂肪酸结构如下式I所示:
表2
实施例2:稳定性研究
本实施例的目的是研究实施例1制备获得的各种胰高血糖素类似物在水溶液中的化学稳定性。
将待测多肽(胰高血糖素类似物)及对照品,配制于相应pH的20mM的磷酸盐缓冲液PB或醋酸缓冲液中,多肽终浓度为0.2mg/ml并用无菌过滤器(0.22μm,MilliporeSLGP033RB) 进行过滤除菌。将制备的多肽溶液在40℃放置7天。然后在4500rpm离心20分钟并使用 RP-HPLC-UV分析上清液(t7)。测定残余完整肽的量,并平行分析未保温处理的样品(t0)。比较在t0和t7时的目标化合物的峰面积,依照下列等式得到“残余肽%”:
残余肽含量%=[(肽峰面积t7)×100]/肽峰面积t0。
稳定性表示为“残余肽含量”。
检测方法
检测波长:214nm;
色谱柱:柱温40℃,Phenomenex Luna C8(2)5μm(150×4.6mm);
流动相:H2O+0.1%TFA:ACN+0.1%TFA(流速1.0ml/分钟);
梯度:95:5(0分钟)至0:100(30分钟);
实验结果分析:从表3实验数据可以得出本发明优选的胰高血糖素类似物(多肽)在中性及弱酸性水溶液中均具有较高的稳定性。
表3
图1-7示例性地展示C381等几种胰高血糖素类似物的液相HPLC分析谱图。
图1对应的积分数据:
图2对应的积分数据:
图3对应的原始数据:
图4对应的原始数据:
图5对应的原始数据:
图6对应的原始数据:
图7对应的原始数据:
实施例3:血清稳定性
(1)表1中相应多肽用5mM Tris-HCl,pH8.5,0.02%TWEEN80溶液配制成浓度为1.0mg/ml的溶液,除菌过滤(0.22μm,Millipore SLGP033RB)后,用大鼠血清稀释10倍,混匀,分装到无菌离心管中;
(2)上述样品各取3管于-20℃冻存作为对照,其余置37℃恒温箱,于不同时间点取样检测活性;
(3)采用实施例4所示方法,检测多肽GCGR激动活性。
相对活性:以0小时的活性值为100%,后续时间点测得的值与之相比而获得。实验结果分析:从表4和图8A和8B可以得出血清稳定性。
表4
注:N.D.表示低于检测下限
实施例4:细胞活性测定
(一)GLP-1R激动活性测定:
GLP-1R激动活性检测采用荧光素酶报告基因检测法(Jonathan W Day等:NatChem Biol.2009 Oct;5(10):749-57)。将人源GLP-1R基因克隆至哺乳动物细胞表达质粒pCDNA3.1中,构建成重组表达质粒pCDNA3.1-GLP-1R,同时荧光素酶(luciferase)全长基因克隆至pCRE质粒得到 pCRE-Luc重组质粒。pcDNA3.1-GLP-1R和pCRE-Luc质粒按摩尔比1:10的比例转染CHO细胞,筛选稳转表达株。
在9-cm细胞培养皿中用含10%FBS和300μg/ml G418的DMEM/F12培养基培养细胞,等汇合度至90%左右时,弃去培养上清,加入2ml胰酶消化3min后,加入2ml含10%FBS 和300μg/ml G418的DMEM/F12培养基中和,转移至15ml离心管中,1000rpm离心5min后,弃去上清,加入2ml含10%FBS和300μg/ml G418的DMEM/F12培养基重悬,计数。用含 10%FBS的DMEM/F12培养基稀释细胞至1×105/ml,96孔板中每孔铺100μl,即1×104/孔,贴壁后换成含0.2%FBS的DMEM/F12培养基培养。铺在96孔板的细胞弃去上清后,将纯化的重组蛋白用含0.1%FBS的DMEM/F12培养基稀释至一系列指定浓度,加入到细胞培养孔中,100μl/孔,刺激6h后检测。根据lucifersae reporter kit(Ray Biotech,Cat:68-LuciR-S200)说明书进行检测。图9A、9B为GLP-1R激动活性检测结果。
(二)GCGR激动活性检测方法:
GCGR激动活性检测同样也采用荧光素酶报告基因检测法。将人源GCGR基因克隆至哺乳动物细胞表达质粒pcDNA3.1中,构建成重组表达质粒pCDNA3.1-GCGR,转染HEK 293T及稳转细胞株的筛选构建同上。图9C、9D为GCGR激动活性检测结果。
表5
(三)GIPR激动活性检测方法:
pcDNA3.1-GIPR质粒转染CHO细胞并筛选阳性稳转细胞株。在96孔细胞培养板中接种约200,000个细胞/孔,培养过夜,用Hanks平衡盐溶液洗涤后,待测蛋白稀释至一系列指定浓度与200μM的3-异丁基-1-甲基黄嘌呤(IBMX)共同加入到细胞中,37℃培养20min后,弃去培养上清,加入裂解液裂解细胞后用cAMP Parameter assay kit参照说明书测定cAMP含量 (美国R&D公司,货号:SKGE002B)。结果如图9E-H所示。
实施例5:葡萄糖刺激的胰岛素分泌实验
本实施例参照Aisling M.Lynch等人的方法(A novel DPP IV-resistant C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1receptor activation,Aisling M.Lynch等, Diabetologia,57:1927–1936,2014)采用大鼠BRIN-BD11细胞用于测定活性蛋白刺激引起的胰岛素释放,但稍作修改,即在24孔板(Orange Scientific,Brainel’Alleud,Belgium)中每孔加入 1.0×106个细胞,37℃培养过夜后离心去上清,再每孔加入1.0ml KRB(115mM NaCl、4.7mM KCl、1.28mM CaCl2、1.2mMMgSO4、1.2mM KH2PO4、25mM HEPES、10mM NaHCO3, NaOH调节pH至7.4)、0.1%(wt/vol.)BSA和1.1mM葡萄糖。细胞置于37℃培养40分钟后,离心去上清并替换成1.0ml新鲜的KRB溶液与梯度浓度的活性蛋白。37℃培养20分钟后,离心去除缓冲液并于-20℃储存过夜,再作免疫放射检测胰岛素含量。结果如图10所示。
实施例6:小鼠免疫原性实验
7周龄Balb/c小鼠,每组6只,给药前各鼠尾静脉采血获得50ul血清作为空白对照。每日注射对应胰高血糖素类似物(30nmol/kg,在PBS缓冲液中),连续注射28天。到第45 天眼眶采血,凝固分离血清。采用直接ELISA法测定其抗体滴度。对应多肽包被酶标板,将小鼠血清按1:50;1:200,1:1000,1:5000梯度稀释加入酶标板,羊抗鼠二抗为检测抗体。各小鼠给药前血清为阴性对照,在相同稀释度前提下,试验样品OD450值平均值大于阴性对照血清OD450值平均值2.1倍的结果判定为阳性(+),反之判定为阴性(-),结果为阳性的最高稀释度即为抗体滴度。
表6
实施例7:正常ICR小鼠的葡萄糖耐量试验(IPGTT)
正常ICR小鼠分为27组,每组6只。被禁食过夜,尾部采血(记为t=0分钟血糖样),皮下注射溶媒体对照(醋酸盐缓冲液,20mM醋酸,250mM甘露糖醇,pH5.0)和本发明表1 中的胰高血糖素类似物(30nmol/kg,PBS缓冲液中)及溶媒对照体,利拉鲁肽(商品名 40nmol/kg,稀释到PBS缓冲液中)。15分钟后腹腔注射葡萄糖(2克/千克体重),并在 t=30分钟、t=45分钟、t=60分钟、t=120分钟测量血液葡萄糖水平。在实验期间动物仍禁食以防止食物摄取的干扰。具体结果参见图11A-C。
实施例8:饮食诱导肥胖(DIO)小鼠中的减重实验
DIO鼠模型的制备:约7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60%kcal fromfat) 继续饲养约16周(共23周),到体重约为45g时进行试验。DIO小鼠随机分为组,每组6只,基础体重无差异,每组小鼠每天分别皮下注射各胰高血糖素类似物(30nmol/kg,PBS中)或PBS等体积,对照组利拉鲁肽(商品名30nmol/kg)、每天给药2次,每天称重至30天。
图12A-C为各胰高血糖素类似物给药后DIO小鼠体重的每日变化,最终的体重减少百分比见图12D。
实施例9:饮食诱导肥胖(DIO)小鼠中的减重实验
DIO小鼠随机分为组,每组6只,基础体重无差异,每组小鼠每天分别皮下注射各GCG 类似物(30nmol/kg,PBS中)或PBS,对照组利拉鲁肽(商品名30nmol/kg,稀释到PBS中)每天给药2次,脂肪酸化的GCG类似物(30nmol/kg,PBS中)每天给药1 次,每天称重至30天。
图13为各胰高血糖素类似物给药后DIO小鼠最终的体重减少百分比。图中所示GCG类似物C381、C464及C493与氨基酸序列相同且经脂肪酸修饰的相应GCG类似物呈现相似的减重效果,而C225、C163未经脂肪酸化则无明显减重作用。
综上所述,本发明有效克服了现有技术中的种种缺点而获得了一组有潜在临床应该价值的三效激动剂。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种胰高血糖素类似物,所述胰高血糖素类似物的结构中含有如式I或式II所示的结构,式I所示结构为:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz,
式II所示结构为:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2,
其中,X10选自Y,K或L之任一,X16选自S、E或A之任一,X17选自Q、E、A或R之任一,X20选自Q或R之任一;X21选自D或E之任一;X23选自V或I之任一;X29为T或缺失,Xz选自GGPSSGAPPPS、GGPSSGAPPS、GPSSGAPPPS、GPSSGAPPS、PSSGAPPPS、PSSGAPPS、SSGAPPPS或SSGAPPS之任一;
所述胰高血糖素类似物序列如SEQ ID NO:6-11、SEQ ID NO:14-35、SEQ ID NO:44-53、SEQ ID NO:60-64之任一所示。
2.一种分离的多核苷酸,所述分离的多核苷酸编码如权利要求1所述胰高血糖素类似物。
3.一种重组表达载体,包含如权利要求2所述分离的多核苷酸。
4.一种宿主细胞,所述细胞含有如权利要求3所述重组表达载体或基因组中整合有外源的如权利要求2所述分离的多核苷酸。
5.如权利要求1所述胰高血糖素类似物的制备方法,其特征在于,选自以下之任一:
(1)利用化学合成方法合成所述胰高血糖素类似物;
(2)在合适的条件下培养如权利要求4所述宿主细胞,使之表达所述胰高血糖素类似物,而后分离及纯化获得所述胰高血糖素类似物。
6.一种组合物,含有如权利要求1所述胰高血糖素类似物或如权利要求4所述宿主细胞的培养物,以及药学上可接受的载体。
7.一种融合蛋白,其结构中含有如权利要求1所述胰高血糖素类似物。
8.根据权利要求7所述的融合蛋白,其特征在于,所述融合蛋白的结构中还含有长效单元,优选地,所述长效单元选自白蛋白,转铁蛋白和免疫球蛋白及片段。
9.如权利要求1、7或8之任一所述胰高血糖素类似物或融合蛋白在制备治疗代谢相关疾病的药物中的用途。
10.一种被修饰的多肽,其结构中含有如权利要求1所述胰高血糖素类似物,所述的胰高血糖素类似物被脂肪酸、聚乙二醇、白蛋白,转铁蛋白或免疫球蛋白及片段所修饰。
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