CN117186189A - 一种兼具降糖和减重作用的glp-1/cck-1受体双重激动多肽及其应用 - Google Patents
一种兼具降糖和减重作用的glp-1/cck-1受体双重激动多肽及其应用 Download PDFInfo
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- CN117186189A CN117186189A CN202311397222.7A CN202311397222A CN117186189A CN 117186189 A CN117186189 A CN 117186189A CN 202311397222 A CN202311397222 A CN 202311397222A CN 117186189 A CN117186189 A CN 117186189A
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Abstract
本发明提供了一种兼具降糖和减重作用的GLP‑1/CCK‑1受体双重激动多肽及其应用,所述多肽的结构如以下通式所示:His‑Xaa1‑Asp‑Gly‑Thr‑Phe‑Thr‑Ser‑Asp‑Met‑Ser‑Ser‑Tyr‑Leu‑Glu‑Glu‑Xaa2‑Ala‑Ala‑Xaa3‑Glu‑Phe‑Val‑Asp‑Trp‑Leu‑Ile‑Lys‑Gly‑Arg‑Pro‑Al a‑AEEA‑AEEA‑Asp‑Phe(4sm)‑Nle‑Gly‑Trp‑Nle‑DMeAsp‑MePhe‑NH2。该多肽能够选择性激动胰高血糖素样肽‑1(GLP‑1)受体和胆囊收缩素‑1(CCK‑1)受体,在更为有效的降低血糖的同时具有显著的抑制进食和减重作用,并且不会导致恶心和呕吐副作用。并且化学性质稳定,适合作为治疗代谢性疾病,如糖尿病、肥胖、非酒精性脂肪肝病、非酒精性脂肪肝炎、血脂障碍等药物的活性成分。
Description
技术领域
本发明涉及一种兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽及其应用,属于多肽技术领域。
背景技术
肥胖及其相关代谢综合征已成为全球性的公众健康问题,许多代谢综合征如2型糖尿病(T2DM)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪肝炎(NASH)、血脂代谢异常的发病率与病程发展都与肥胖密切相关。GLP-1是由小肠L细胞所分泌的一种葡萄糖依赖性降血糖多肽激素,而其中最主要的功能就是促进胰岛素的分泌。GLP-1可以抑制食欲、延缓胃排空实现降低体重的作用。虽然GLP-1具有优异的降糖作用和一定的减重效果,但是如果需要实现较好的体重减轻作用,一般需要加大给药剂量,而大剂量给予GLP-1类药物容易产生胃肠道副作用,耐受性差而导致治疗窗较窄。因此,仍然需要更为安全耐受的,可有效减轻体重和控制血糖的治疗剂。
胆囊收缩素(cholecystokinin,CCK)是肠道I细胞分泌的一种胃肠道激素,CCK最主要的生理作用是通过增加饱腹感来调节能量代谢。此外,CCK也具有抑制胃排空、增加胰岛β细胞面积和增强胰岛素分泌的作用。CCK的生理作用是通过激动CCK受体来实现的,CCK受体属于G蛋白偶联受体家族,有CCK-1受体和CCK-2受体两种亚型。CCK-1受体为涉及食物摄入及饱腹感的主要受体,而CCK-2受体介导其他效果,包括中枢神经系统效果和增加胰岛β细胞面积等。CCK-8是一种CCK的8肽类似物,其对CCK-1受体和CCK-2受体都有激动活性。选择性的激动CCK-1受体更有利于抑制食物摄入,专利TW201716432A公开了一类具有CCK-1受体选择激动活性的CCK-8长效类似物(NN9056)。但是,CCK-1受体激动较为容易导致急性胰腺炎的发生,这是CCK-1受体作为减重药物靶向受体所面临的主要障碍,目前尚无作用于CCK-1受体的减重药物上市。激动CCK-1受体抑制摄食和促进饱腹感的作用,可以提高GLP-1类药物的减重作用。J.L.Trevaskis等人描述了同时联用CCK-8类似物和GLP-1受体激动剂毒蜥外泌肽-4(exendin-4),相较于exendin-4单独使用显著提高了减重作用(DiabetesObes.Metab.,2015,17,61–73)。但是,选择性的同时激动GLP-1受体和CCK-1受体,如何避免产生急性胰腺炎这种严重的副作用,是此类药物设计所要考虑的重要问题。
发明内容
发明目的:为了解决上述技术问题,本发明提供了一种兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽。所述GLP-1/CCK-1受体双重激动多肽是基于牛蛙GLP-1序列设计的变体,保留GLP-1类似物对糖尿病的治疗作用同时具有CCK-1对食欲抑制的有益作用,并且能够避免CCK导致急性胰腺炎的副作用,具有极佳的安全性,从而对糖、脂、能量代谢产生协同影响,比单一受体激动剂在制备用于治疗代谢综合征,诸如糖尿病、肥胖症、非酒精性脂肪肝病、非酒精性脂肪肝炎、血脂障碍等疾病的药物方面更具潜力。
技术方案:为了实现上述目的,本发明采用以下技术方案:
第一方面,本发明提供了一种兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,所述多肽的结构(氨基酸序列)如以下通式所示:His-Xaa1-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa2-Ala-Ala-Xaa3-Glu-Phe-Val-Asp-Trp-Leu-Ile-Lys-Gly-Arg-Pro-Ala-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
其中:
Xaa1选自Ala或Aib;
Xaa2选自Glu或侧链经过化学修饰的Lys-R1;
Xaa3选自Lys或侧链经过化学修饰的Lys-R1;
所述Lys-R1的化学结构为:
优选的,所述兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽选自如下:(1)多肽1
(2)多肽2
优选的,所述盐为GLP-1/CCK-1受体双重激动多肽与下述化合物中的一种所形成的盐:甲酸、乙酸、丙酮酸、丁酸、己酸、苯磺酸、双羟萘酸、苯甲酸、水杨酸、月桂酸、肉桂酸、丙酸、十二烷基硫酸、柠檬酸、抗坏血酸、酒硬脂酸、石酸、草酸、乳酸、琥珀酸、丙二酸、马来酸、富马酸、天冬氨酸、磺基水杨酸。
第二方面,本发明提供了所述的兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽的合成方法,包括以下步骤:
首先溶胀树脂,脱除Fmoc保护基,然后合成Fmoc-MePhe-Rink amide-MBHA树脂,而后进行肽链的延长,Lys侧链修饰,最后将树脂上多肽进行裂解,纯化,即得。
第三方面,本发明提供了一种药物组合物,包含治疗有效量的至少一种所述的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,以及药学上可接受的载体和/或辅料。
所述药物组合物可以是任何一种药剂学上所述片剂、胶囊、糖浆、酊剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、乳剂、栓剂或者复方制剂。
第四方面,本发明提供了所述的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐、以及所述的药物组合物在制备治疗代谢性疾病药物中的应用。作为具体实施方式,所述代谢性疾病为糖尿病、肥胖症、非酒精性脂肪肝病、非酒精性脂肪肝炎和/或血脂障碍。进一步的,所述糖尿病为T1DM、T2DM或妊娠糖尿病。
本发明所制备的GLP-1/CCK-1受体双重激动多肽,对GLP-1受体和CCK-1受体具有强激动活性和激动选择性,实现了更好的降糖、减重、调脂作用,具有更低的胃肠道副作用,并且不会导致急性胰腺炎的发生,为制备此类高效、低毒的多重激动剂提供了新的思路。
有益效果:与现有技术相比,本发明具有以下优势:
(1)本发明的多肽在更为有效的降低血糖的同时具有显著的减重和防止增重作用,更好的调节脂质代谢;
(2)本发明的多肽左侧GLP-1部分使用的是牛蛙GLP-1类似物序列,右侧CCK部分使用的是对CCK-1受体具有高激动选择性的CCK类似物,中间连接臂选择了两个重复的AEEA,使得本发明的多肽对GLP-1受体和CCK-1受体具有强激动活性和激动选择性,带来了显著提高的减重和调节脂质代谢作用,并且没有胃肠道副作用,也不会导致急性胰腺炎,具有意想不到的有益效果;
(3)与已报道的GLP-1/CCK-1受体双重激动剂相比,本发明的多肽具有明显更强的GLP-1受体和CCK-1受体激动活性,以及更好的GLP-1受体和CCK-1受体激动选择性,减重、调节脂质代谢和降糖活性显著提高,胃肠道副作用显著降低,并且没有导致急性胰腺炎的副作用,在治疗代谢性疾病方面更有潜力,为此类多重激动剂的药物研发提供了新的思路;
(4)本发明提供的多肽化学性质稳定,具有支持至少每周一次给药的药代动力学特征;本发明提供的多肽对T2DM、肥胖、血脂障碍等代谢性疾病的治疗作用优于现有上市药物。因此,本发明提供的多肽,适合作为治疗代谢性疾病,如糖尿病、肥胖症、非酒精性脂肪肝病、非酒精性脂肪肝炎、血脂障碍等药物的活性成分。
附图说明
图1显示的是各受试物单次给药在C57BL/6J小鼠上的摄食抑制作用;
图2显示的是各受试物在DIO小鼠长期给药21天的体重变化百分比;
具体实施方式
在下文中,将更详细描述本发明。
除非本文另有定义,否则本申请书中所使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。通常,本文所述的与化学、生物学、药理学关联使用的术语和方法是本领域中公知和常用的。
另外,本发明体积的氨基酸根据IUPAC-IUB的命名规则缩写如下:
丙氨酸(Ala,A);精氨酸(Arg,R);天冬酰胺(Asn,N);天冬氨酸(Asp,D);半胱氨酸(Cys,C);谷氨酸(Glu,E);谷氨酰胺(Gln,Q);甘氨酸(Gly,G);组氨酸(His,H);异亮氨酸(Ile,I);亮氨酸(Leu,L);赖氨酸(Lys,K);甲硫氨酸(Met,M);苯丙氨酸(Phe,F);脯氨酸(Pro,P);丝氨酸(Ser,S);苏氨酸(Thr,T);色氨酸(Trp,W);酪氨酸(Tyr,Y);缬氨酸(Val,V)。
另外,除非明确标明,本发明的多肽化合物中的所有氨基酸残基优选为L构型。
另外,所述序列的C端上的“-NH2”部分表明C端上的酰胺基(-CONH2)。
另外,本发明序列中除了天然氨基酸以外,还使用了非天然氨基酸α-氨基异丁酸(Aib)、8-氨基-3,6-二氧杂辛酸(AEEA)、2-氨基-3-(4-(磺甲基)苯基)丙酸(Phe(4sm))、2-氨基己酸(Nle)、N-甲基-D-天冬氨酸(DMeAsp)、N-甲基苯丙氨酸(MePhe)。
本发明是通过下列实施例来进行说明的,但这些实施例不做任何限制本发明权利的解释。
实施例1
多肽化合物1的合成
(1)树脂的溶胀
称取担载量为0.36mmol/g的Rink Amide MBHA树脂0.278g(0.1mmol当量),放入25mL的反应器中,用7mL的DCM和甲醇交替清洗树脂1次,7mL的DCM清洗树脂2次,然后用7mL的DCM溶胀树脂1h,最后用7mL DMF清洗树脂3次。
(2)树脂Fmoc保护基的脱除
将溶胀后的树脂转入PSI-200多肽合成仪,加入7mL 20%哌啶/DMF(v/v)室温反应5min,滤去脱保护溶液,7mL DMF清洗树脂一次,再加入7mL 20%哌啶/DMF(v/v)脱保护溶剂与树脂反应15min,最后7mL DMF清洗树脂4次,每次2min,得到脱除Fmoc保护基的Rink树脂。
(3)Fmoc-MePhe-Rink amide-MBHA Resin的合成
称Fmoc-MePhe-OH(0.4mmol),用3mL 10%DMF/DMSO(v/v)溶解,加入2mL DIC/HOBt(0.4mmol/0.44mmol)缩合剂,预活化30min后,将活化好的氨基酸加入反应器中,室温震荡反应2h,滤去反应液后用7mL DMF清洗树脂4次,使用Kaiser试剂检测反应耦合是否完全,如不完全则2次耦合。
(4)肽链的延长
按照肽链的序列,重复上述脱保护和耦合的步骤依次连接上相应的氨基酸,直至肽链合成完毕。其中17位Lys可以采用Fmoc-Lys(Alloc)-OH、Fmoc-Lys(Dde)-OH、Fmoc-Lys(Mtt)-OH或Fmoc-Lys(ivDde)-OH等。本实例中采用Fmoc-Lys(Dde)-OH保护策略,同时N末端的His使用的是Boc-His(Boc)-OH。此外,在Phe(4sm)处选择的是侧链TCE保护的Fmoc-Phe(4sm-TCE)-OH。
(5)Lys侧链的修饰
肽链合成完毕后,加入7mL 2%水合肼/DMF(v/v)选择性脱除17位Lys的Dde保护基,Dde保护基脱除后加入0.4mmol的Fmoc-AEEA-OH,0.4mmol的HBTU及0.44mmol的HOBt,震荡缩合反应2h。脱除Fmoc保护基后,再次加入0.4mmol的Fmoc-AEEA-OH,0.4mmol的HBTU及0.44mmol的HOBt,震荡缩合反应2h。脱除Fmoc保护基后,加入0.4mmol的Fmoc-Glu-OtBu,0.4mmol的HBTU及0.44mmol的HOBt,震荡缩合反应2h。脱除Fmoc保护基后,加入0.4mmol的十八烷二酸单叔丁酯,0.4mmol的HBTU及0.44mmol的HOBt缩合反应2h,反应完全后用7mL DMF清洗树脂4次。
(6)多肽的裂解
将上述得到的连有多肽的树脂转移至圆底瓶中,使用切割剂(三异丙基硅烷/水/TFA,2.5:2.5:95,V/V)5mL切割树脂,在油浴中恒温30℃反应2h,切割液倾入40mL冰乙醚中,冷冻离心后粗品用15mL冰乙醚洗涤3次,最后用氮气吹干,得到粗肽。接下来,为了脱除Phe(4sm-TCE)-OH的TCE保护基,粗肽溶解在最小体积的乙酸中。在另一个试管中,锌粉(1.5mmol)在磁力搅拌器中与1M HCl搅拌5分钟。液相被移走以得到活化的锌粉。上述制备的粗肽溶液加入,然后加入0.2mmol的醋酸铵溶于水中,并反应1小时。然后用水和乙腈稀释混合物,并加入26%的氨水以将pH调整到7.6,然后进行纯化处理。
(7)多肽的纯化
用0.25μm微孔滤膜过滤上述反应液后进岛津制备型反相HPLC系统纯化。色谱条件为C18反相制备柱(250mm×4.6mm,5μm);流动相A:0.1% TFA/水(V/V),流动相B:甲醇(V/V);流速为0.8mL/min;检测波长为214nm。采用线性梯度(50%B~90% B/15min)洗脱,收集目标峰,除去甲醇后冻干得纯品0.11g,纯度大于99%,通过MS确认目标多肽的分子量。理论相对分子质量为5736.4。ESI-MS m/z:计算值[M+4H]4+1435.1,[M+5H]5+1148.3;观察值[M+4H]4+1435.0,[M+5H]5+1148.4。
实施例2
多肽化合物2的合成
合成方法同实施例1,收集目标峰冻干得纯品0.10g,纯度大于99%,通过MS确认目标多肽的分子量。理论相对分子质量为5737.4。ESI-MS m/z:计算值[M+4H]4+1435.4,[M+5H]5+1148.5;观察值[M+4H]4+1435.2,[M+5H]5+1148.4。
实施例3
多肽化合物对人GLP-1受体、GIP受体、glucagon受体、CCK-1受体、CCK-2受体的激动活性测定
通过功能测定法来确定多肽化合物对受体的激动作用,所述测定法测量稳定表达人GLP-1受体、glucagon受体或GIP受体的HEK-293细胞系的cAMP响应。分别将稳定表达上述三种受体的细胞分入T175培养瓶并在培养基中过夜生长至接近汇合状态,然后除去培养基,并用无钙和镁的PBS洗涤细胞,然后用Accutase酶进行蛋白酶处理。洗涤脱离的细胞并将其重悬于测定缓冲液(20mM HEPES,0.1%BSA,2mM IBMX,1×HBSS)中,并确定细胞密度,并将25μL的等分试样分装至96孔板的孔中。为了测量,将25μL的测试多肽化合物在测定缓冲液中的溶液添加到孔中,然后室温温育30分钟。用Cisbio的试剂盒,基于均相时间分辨荧光(HTRF)来确定细胞的cAMP含量。添加稀释于裂解缓冲液(试剂盒组分)中的HTRF试剂后,将平板温育1小时,然后测量665/620nm处的荧光比。通过检测引起最大响应的50%激活的浓度(EC50)来对激动剂的体外效力进行量化。
稳定表达CCK-1受体或CCK-2受体的1321-N1细胞用DMEM-31966(含有10%FBS,1%丙酮酸钠,1%青霉素,1%链霉素)培养。试验前一天,将细胞转移至384孔板中,化合物溶解在IP-One缓冲溶液中(含有10mmol/L HEPES,1mmol/L CaCl2,4.2mmol/LKCl,146mmol/LNaCl,5.5mmol/L葡萄糖,50mmol/L LiCl)并稀释,加入384孔板中。在37℃孵育1小时后,使用IP-One HTRF Assay kit测定细胞内的1-磷酸肌醇浓度,通过检测引起最大响应的50%激活的浓度(EC50)来对激动剂的体外效力进行量化。
将本专利申请实施例中的检测数据(nM)显示于下表1中,虽然用一定数量的有效数字来陈述检测数据,但不应该认为表示数据已确定精确为有效数字的数。
表1:多肽化合物对人GLP-1受体、glucagon受体及GIP受体的EC50值(以nM表示)
表2:多肽化合物对人CCK-1受体及CCK-2受体的EC50值(以nM表示)
如表1和表2所示,所有多肽化合物的GLP-1受体激动活性都优于天然GLP-1和文献报道的GLP-1/CCK-1受体双重激动剂C2816(Appetite 127(2018)334–340),同时所有多肽化合物都没有GIP受体和glucagon受体激动活性,展现出了非常好的受体激动选择性。GIP受体的激动活性降低是有益的,有文献报道糖尿病患者体内高水平的GIP有时会导致频繁的低血糖症发作(J.Clin.Endocrinol.Metab.,2010,95,1851-1855)。Glucagon受体的激动活性降低是有益的,可以避免激动glucagon受体产生的高血糖作用。此外,多肽2对CCK-1受体的激动活性也高于CCK-8、NN9056(文献报道的CCK-1受体选择性激动剂,J.Med.Chem.2019,62,1407-1419)和C2816,同时所有多肽化合物都表现出了CCK-1受体激动的高选择性,并且对CCK-1受体的激动选择性高于NN9056和C2816,而CCK-8没有CCK-1受体的激动选择性。
实施例4
多肽化合物在大鼠体内的药代动力学性质
SD大鼠给予50nmol/kg的皮下(s.c.)注射给药,在给药后0.25h、0.5h、1h、2h、4h、8h、16h、24h、36h和48h收集血样。使用乙腈沉淀蛋白质后,用LC-MS分析血浆样品。用WinonLin 5.2.1(非房室模型)计算药代参数和半衰期(表3)。
表3:多肽化合物在大鼠体内的药代动力学概貌
样品 | T1/2(h) | Cmax(ng/mL) |
Semaglutide | 9.1 | 466 |
多肽2 | 13.8 | 524 |
如表3结果显示,本发明的多肽化合物的体内半衰期显著延长,多肽2的体内半衰期明显优于已上市的一周一次给药的semaglutide,说明本发明的多肽化合物具有支持至少每周一次给药的药代动力学特征。
实施例5
多肽化合物对C57BL/6J小鼠进食的影响
雄性C57BL/6J小鼠,随机分组,每组6只。实验前小鼠禁食12h,空白组皮下注射给予生理盐水(10mg/kg),给药组小鼠分别皮下单次注射10nmol/kg的semaglutide、C2816、多肽2。然后立即给予小鼠预先称重的鼠饲料,并在2h,4h,12h,24h和48h再次称饲料重量,计算小鼠在不同时间点的摄食量。
如图1结果所示,在C57BL/6J小鼠体内的摄食实验结果表明,多肽2具有显著优于C2816和semaglutide的抑制进食的作用,说明本专利的多肽化合物具有优异的抑制进食效果。
实施例6
多肽化合物对db/db小鼠糖化血红蛋白(HbA1c)和血糖的影响
雄性db/db小鼠,随机分组,每组6只。分别为生理盐水组(空白对照组)、阳性对照组(semaglutide)和受试样品组(多肽2)。适应性饲养一周后,尾部取血测量治疗开始前初始HbA1c数值和空腹血糖数值。各组小鼠每两天一次皮下注射生理盐水(10mg/kg),semaglutide(10nmol/kg),多肽2(10nmol/kg),给药周期35天。治疗结束后小鼠禁食过夜后测量空腹血糖数值,同时取血测量HbA1c(%)数值。
表4:db/db小鼠在35天给药周期内的HbA1c(%)变化
样品(剂量) | HbA1c%(治疗前) | HbA1c%(治疗后) |
空白对照(生理盐水组) | 6.6±0.3 | 8.2±0.4 |
Semaglutide(10nmol/kg) | 6.5±0.2 | 6.3±0.2*** |
多肽2(10nmol/kg) | 6.9±0.4 | 5.2±0.3***,### |
***:与空白对照组相比P<0.001;###:与semaglutide比P<0.001(One-Way ANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如表4结果显示,本发明的多肽化合物在db/db小鼠体内连续给药35天,可以显著降低小鼠的HbA1c数值,并且在治疗后本发明的多肽化合物组小鼠的HbA1c数值显著低于阳性对照药semaglutide,说明本发明的多肽化合物具有很好的血糖控制作用。
表5:db/db小鼠在35天给药周期内的空腹血糖变化
样品(剂量) | 空腹血糖(%) |
空白对照(生理盐水组) | +5.2±0.3% |
Semaglutide(10nmol/kg) | -6.3±0.5%*** |
多肽2(10nmol/kg) | -11.9±0.6%***,### |
***:与空白对照组相比P<0.001;###:与semaglutide比P<0.001(One-Way ANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如表5结果显示,本发明实施例制备的多肽化合物在db/db小鼠体内连续给药35天,可以显著降低db/db小鼠的空腹血糖,说明本发明的多肽化合物具有优异的血糖控制作用,并且本发明的多肽化合物的血糖控制作用显著强于阳性对照药semaglutide。
实施例7
多肽化合物对饮食诱导肥胖(DIO)小鼠血脂和体重的影响
雄性C57BL/6J小鼠,体重22g左右,用Research Diets公司的D12492高脂饲料饲养18周造DIO小鼠模型。在给药开始前,各组DIO小鼠按照体重随机分组,每组6只,分别为生理盐水组(空白对照组)、阳性对照组(semaglutide和C2816)和受试样品组(多肽2)。各组小鼠每两天一次皮下注射生理盐水(10mg/kg),semaglutide(10nmol/kg),多肽2(10nmol/kg),或每天两次皮下注射C2816(50nmol/kg),给药周期21天。每天记录小鼠体重变化,实验开始前和结束时使用核磁共振(NMR)来测量体脂量。在实验结束后,各组小鼠处死,取肝脏组织测量肝脏甘油三酯(TG)和总胆固醇(TC)含量。同时取血制血清,并测量血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和总胆固醇(TC)含量。
表6:DIO小鼠在3周给药周期内的体重和体脂变化
样品(剂量) | 整体体重变化(%) | 体脂变化(%) |
空白对照(生理盐水组) | 1.8±1.3 | 2.3±0.4 |
Semaglutide(10nmol/kg) | -10.6±1.5*** | -15.9±1.5*** |
C2816(50nmol/kg) | -14.3±2.1*** | -19.9±2.3*** |
多肽2(10nmol/kg) | -29.1±1.8***,### | -47.1±4.0***,### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如图2和表6结果显示,本发明的多肽化合物多肽2在DIO小鼠体内连续给药3周,可以显著降低小鼠的体重和体脂含量,并且本发明的多肽化合物的减重和降低体脂的作用显著强于阳性对照药semaglutide和C2816。
表7:DIO小鼠治疗3周后的肝脏总胆固醇(TC)和甘油三酯(TG)含量
样品(剂量) | 总胆固醇(mg/g) | 甘油三酯(mg/g) |
空白对照(生理盐水组) | 9.5±0.6 | 97.6±7.2 |
Semaglutide(10nmol/kg) | 7.9±0.3*** | 82.6±4.6*** |
C2816(50nmol/kg) | 8.9±0.7 | 91.4±6.5 |
多肽2(10nmol/kg) | 4.2±0.2***,### | 39.7±2.9***,### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
表8:DIO小鼠治疗3周后的血清谷丙转氨酶(ALT)和谷草转氨酶(AST)含量
*:与空白对照组相比P<0.05;***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-Way ANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如表7和表8所示,本发明实施例制备的多肽化合物在DIO小鼠体内连续给药3周,可以显著降低小鼠的肝脏甘油三酯和总胆固醇含量,并且可以显著降低小鼠血清谷丙转氨酶和谷草转氨酶含量,并且本发明的多肽化合物的作用显著强于阳性对照药semaglutide和C2816,说明本发明的多肽化合物具有很好的治疗非酒精性脂肪肝病和非酒精性脂肪肝炎的前景。值得注意的是,C2816并没有降低肝脏甘油三酯和总胆固醇含量的作用,而本发明的多肽化合物显示出了极佳的肝脏甘油三酯和总胆固醇降低作用,说明本发明的多肽化合物具有意想不到的活性。
表9:DIO小鼠治疗3周后的血清总胆固醇(TC)和甘油三酯(TG)含量
样品(剂量) | 总胆固醇(mmol/L) | 甘油三酯(mmol/L) |
空白对照(生理盐水组) | 9.4±1.0 | 2.0±0.2 |
Semaglutide(10nmol/kg) | 7.5±0.3*** | 1.4±0.1*** |
C2816(50nmol/kg) | 9.2±0.4 | 1.8±0.3 |
多肽2(10nmol/kg) | 3.6±0.2***,### | 0.7±0.1***,### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如表9结果显示,本发明的多肽化合物在DIO小鼠体内连续给药3周,可以显著降低小鼠的血清甘油三酯和总胆固醇含量,并且本发明的多肽化合物的降低血清脂质(甘油三酯和胆固醇)含量的作用显著强于阳性对照药semaglutide和C2816。
实施例8
多肽化合物的胃肠道副作用影响
雄性SD大鼠(200–250g)随机分组,单笼饲养,实验前4天,各组大鼠在给予普通饲料的基础上,额外给予高岭土饲料(Research Diets),高岭土饲料放置在食物漏斗的单独隔间里,让大鼠习惯笼子里有高岭土饲料的存在。实验前大鼠禁食12h,在0h各组大鼠腹腔注射10%的DMSO/水(空白)、3mg/kg的顺铂(判断模型是否成功的对照组),以及10nmol/kg,50nmol/kg和100nmol/kg的semaglutide、C2816、多肽2。然后迅速给予各组大鼠预先称好重量的普通饲料和高岭土饲料,记录各组大鼠在24h普通饲料和高岭土饲料的进食量,根据普通饲料和高岭土饲料的消耗量,判断化合物所导致副作用的强度。
表10:SD大鼠在24小时的普通饲料和高岭土进食量
样品 | 普通饲料进食量(g) | 高岭土进食量(g) |
空白对照 | 25.4±1.3g | 0.4±0.1g |
顺铂 | 13.9±0.7g*** | 3.1±0.4g*** |
Semaglutide(10nmol/kg) | 19.2±1.2g*** | 0.5±0.1g |
C2816(10nmol/kg) | 20.4±1.9g*** | 1.2±0.2g*** |
多肽2(10nmol/kg) | 13.2±1.1g***,### | 0.05±0.01g### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只大鼠平均值±SD。
表11:SD大鼠在24小时的普通饲料和高岭土进食量
样品 | 普通饲料进食量(g) | 高岭土进食量(g) |
空白对照 | 25.4±1.3g | 0.4±0.1g |
顺铂 | 13.9±0.7g*** | 3.1±0.4g*** |
Semaglutide(50nmol/kg) | 18.1±1.9g*** | 1.0±0.2g*** |
C2816(50nmol/kg) | 19.1±1.5g*** | 1.7±0.4g*** |
多肽2(50nmol/kg) | 12.2±0.5g***,### | 0.11±0.03g### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只大鼠平均值±SD。
表12:SD大鼠在24小时的普通饲料和高岭土进食量
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只大鼠平均值±SD。
如表10-12结果显示,本发明实施例制备的多肽化合物在25nmol/kg、50nmol/kg、100nmol/kg剂量下都具有很好的抑制大鼠进食效果,显著优于阳性对照semaglutide和C2816。但是,本发明实施例制备的多肽化合物在25nmol/kg、50nmol/kg、100nmol/kg剂量下都没有导致大鼠产生高岭土进食现象,本发明实施例制备的多肽化合物组的高岭土进食量低于空白组,并且明显低于阳性对照semaglutide和C2816组大鼠的高岭土进食量。这说明本发明实施例制备的多肽化合物没有导致大鼠产生胃肠道副作用,胃肠道副作用明显低于阳性对照semaglutide和C2816。
实施例9
多肽化合物的急性胰腺炎副作用评价
7周龄雄性C57BL/6J小鼠分为8组,每组6只,适应性饲养一周后,每组分别皮下注射生理盐水,500nmol/kg的semaglutide,C2816,多肽2。给药前和48小时给药后,取血样测定血清淀粉酶(amylase)和脂肪酶(lipase)的数值。
表13:C57BL/6J小鼠给药前后血清淀粉酶和脂肪酶的数值(以U/L表示)
样品 | Amylase(给药前) | Amylase(48h) | Lipase(给药前) | Lipase(48h) |
生理盐水 | 2289±201 | 2197±305 | 6.8±0.3 | 6.7±0.4 |
Semaglutide | 2199±187 | 3269±269*** | 6.8±0.2 | 10.6±0.8*** |
C2816 | 2148±281 | 4128±319*** | 6.9±0.4 | 19.7±0.4*** |
多肽2 | 2248±145 | 2216±231### | 6.7±0.2 | 6.3±0.2### |
***:与空白对照组相比P<0.001;###:与semaglutide和C2816组比P<0.001(One-WayANOVA,Tukey post hoc test),结果表示为每组6只小鼠平均值±SD。
如表12结果显示,本发明的多肽化合物的在C57BL/6J小鼠体内大剂量给药,不会导致血清淀粉酶和脂肪酶数值的增高,说明不会导致急性胰腺炎的发生。而C2816和semaglutide给药后血清淀粉酶和脂肪酶的数值相较空白组都显著升高,具有导致急性胰腺炎的副作用。
最后所应说明的是,以上具体实施方式仅用以说明本发明的技术方案而非限制,尽管参照实例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (9)
1.一种兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,所述多肽的结构如以下通式所示:
His-Xaa1-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Ser-Tyr-Leu-Glu-Glu-Xaa2-Ala-Ala-Xaa3-Glu-Phe-Val-Asp-Trp-Leu-Ile-Lys-Gly-Arg-Pro-Ala-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
其中:
Xaa1选自Ala或Aib;
Xaa2选自Glu或侧链经过化学修饰的Lys-R1;
Xaa3选自Lys或侧链经过化学修饰的Lys-R1;
所述Lys-R1的化学结构为:
2.根据权利要求1所述的兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,其特征在于,所述兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽选自如下:
(1)多肽1
(2)多肽2
3.根据权利要求1所述的兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,其特征在于,所述盐为GLP-1/CCK-1受体双重激动多肽与下述化合物中的一种所形成的盐:甲酸、乙酸、丙酮酸、丁酸、己酸、苯磺酸、双羟萘酸、苯甲酸、水杨酸、月桂酸、肉桂酸、丙酸、十二烷基硫酸、柠檬酸、抗坏血酸、酒硬脂酸、石酸、草酸、乳酸、琥珀酸、丙二酸、马来酸、富马酸、天冬氨酸、磺基水杨酸。
4.权利要求1所述的兼具降糖和减重作用的GLP-1/CCK-1受体双重激动多肽的合成方法,其特征在于,包括以下步骤:
首先溶胀树脂,脱除Fmoc保护基,然后合成Fmoc-MePhe-Rink amide-MBHA树脂,而后进行肽链的延长,Lys侧链修饰,最后将树脂上多肽进行裂解,纯化,即得。
5.一种药物组合物,包含治疗有效量的至少一种权利要求1-3任一项所述的GLP-1/CCK-1受体双重激动多肽或其药学上可接受的盐,以及药学上可接受的载体和/或辅料。
6.根据权利要求5所述的药物组合物,其特征在于,所述药物组合物为片剂、胶囊、糖浆、酊剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、乳剂、栓剂或者复方制剂。
7.权利要求1-3任一项所述的GLP-1/CCK-1受体双重激动多肽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代谢性疾病药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述代谢性疾病为糖尿病、肥胖症、非酒精性脂肪肝病、非酒精性脂肪肝炎和/或血脂障碍。
9.根据权利要求8所述的应用,其特征在于,所述糖尿病为T1DM、T2DM或妊娠糖尿病。
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