专利名称：一种glp-1类似物、制备方法及其应用的制作方法本发明涉及的GLP-I (glucagon-1 ikepeptide-Ι,以下简称GLP_1)是主要由小肠L细胞分泌的一种37个氨基酸组成的多肽，其活性形式为GLP-I (7-37) OH和GLP-I (7-36)NH2 (Mojsov S，J Clin Invest. 1987 Feb ；79(2) :616_9)。GLP-1 明显减低人用餐后的血糖，能刺激胰岛素的产生，同时还能起到一定减肥效应，并且不会引起低血糖症(Drucker DJ7Diabetes. 1998 Feb ；47(2) 159-69) 近期研究还表明 GLP-1 有胰腺再生作用(DruckerD J,2003Dec ； 144 (12) :5145_8)。此外，因为GLP-I为完全人源化多肽，作为临床药物在安全上具有较大优势。然而，GLP-I (7-37)的血清半衰期仅仅为3-5分钟，每天多次注射给药在临床使用中非常不方便。目前已经有不少研究采用GLP-I类似物融合蛋白技术解决GLP-I类似物在体内的存留时间(CN90101167. 3、CN200710018734. 2、CN200410054397. 9、CN01820232. 2、CN200380110152. 7、CN200510039265. 3、CN200610127237. I、CN200910009642. 7)，然而，现有的技术与临床理想的目标还有很大距离，普遍都还没有达到临床标准，最近Novo Norisk生产的利拉鲁肽为GLP-I类似物，其对GLP-I进行了棕榈酸的修饰，并于2009年于美国上市。但是利拉鲁肽也存在半衰期短的问题，其剂型仍需每日注射。因此，目前存在对解决GLP-I体内半衰期短的方法的需求。
本发明的一个目的是针对临床上GLP-I类似物在体内存留时间较短，需要每天注射给药的缺限，提供一种半衰期较长的GLP-I的类似物。本发明的另一个目的是提供一种GLP-I类似物在制备治疗糖尿病的药物中的应用，以及该GLP-I类似物在制备治疗和/或预防肥胖症的药物中的应用。本发明的又一个目的是提供一种以GLP-I类似物作为有效成分的药用组合物，其包含上述GLP-I类似物。其中，所述的药物组合物还包含一种或多种药学上可接受的辅料，并且所述药物组合物优选为注射剂，进一步优选为冻干粉针或溶液注射剂。用于实现上述目的的技术方案如下一方面，本发明提供一种GLP-I类似物，所述GLP-I类似物具有下列通式I :7HAEX10T FTSX15V SSYLE X22QAAK EFIX30W LX33KGR G37H1X1C n2X2通式I，其中，Xltl为甘氨酸或半胱氨酸，X15为天冬氨酸或半胱氨酸，X22为甘氨酸或半胱氨酸，X30为丙氨酸或半胱氨酸，X33为缬氨酸或半胱氨酸，并且X1(l、Xi5> X22> X30和X33中至少有一个为半胱氨酸！II1X1表示有η个X1,!! = 3 30，且X1为甘氨酸、丙氨酸或缬氨酸；η2Χ2表示有η个Χ2，η = 3 30，且X2为甘氨酸、丙氨酸或缬氨酸；以及通式I中含有的两个半胱氨酸均形成二硫键。优选地，所述GLP-I类似物具有下列通式II 7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37H1X1C η2Χ2通式II，ηι = I 20，η2 = O 25，并且其中X1为甘氨酸、丙氨酸或缬氨酸，X2为甘氨酸、丙氨酸或纟颜氨酸；优选地，所述Ii1 = 5 15, η2 = 3 9。进一步优选地，所述GLP-I类似物为 SEQ ID NO I =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG C GGG ；SEQ ID NO 2 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG GGGGGC GGG ；SEQ ID NO 3 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37GGGGG GGGGGGGGGGC GGG ；SEQ ID NO 4=7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG C GGGGG G ；SEQ ID NO 5 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG C GGGGG GGGG ；SEQ ID NO 6 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37GGGGG GGGGGC GGGGG G ；SEQ ID NO 7 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG GGGGGC GGGGG GGGG ；SEQ ID NO 8 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG GGGGGGGGGGC GGGGG G ；SEQ ID NO 9 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37GGGGG GGGGGGGGGGC GGGGG GGGG ；SEQ ID NO 10 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA C AAA ；SEQ ID NO 11 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAC AAA ；SEQ ID NO 12 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAM AAAAAAAAAAC AM ；SEQ ID NO 13 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA C AAAAA A ；SEQ ID NO 14 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37MAAA C AAMA AMA ；SEQ ID NO 15 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37AAAM AAAMC AAAM A ；SEQ ID NO 16 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAC AAAAA AAAA ；SEQ ID NO 17 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAAAAAAC AAAAA A ；SEQ ID NO 18 =7HABGT FTSCV SSYLE GQAAK EFIAff LVKGRS37AAAM AAAAAAAAAAC AAAAA AAM ；SEQ ID NO 19 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV C VVV ；SEQ ID NO 20 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV VVVVVC VVV ；SEQ ID NO 21 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37VWVV VVVVVVVVVVC VVV ；SEQ ID NO 22 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV C VVVVV V ；SEQ ID NO 23 :7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37WVW C VVVVV VVVV ；SEQ ID NO 24 =7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37VVVVV VVVVVC VVVVV V ；SEQ ID NO 25 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VWW VWWC VWW WW ；SEQ ID NO 26 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VWW WWWWWC VWW V ;或SEQ ID NO 27 =7HABGT FTSCV SSYLE GQAAK EFIAff LVKGRS37VWW WWWWWC VWW WW0此外，具有通式I的GLP-I类似物还可以具有下列通式III :7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37H1X1Cn2X2通式III，其中，Ii1 = I 10，n2 = O 25，X1为甘氨酸、丙氨酸或缬氨酸，X2为甘氨酸、丙氨酸或缴氨酸；优选地，Ii1 = I 5, n2 = 10 20。优选地，所述GLP-I类似物为SEQ ID NO 28 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37GC GGGGG GGGGG ；SEQ ID NO 29 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37GC GGGGG GGGGGGGGGG ；SEQ ID NO 30 =7HABGT FTSDV SSYLE GQAAK EFICW LVKGR G37GC GGGGG GGGGGGGGGGGGGGGGGGGG ；SEQ ID NO 31 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37GG C GGGGG GGGGG ；SEQ ID NO 32 :7HAEGT FTSDV SSYLE GQMK EFICff LVKGRG37GG C GGGGG GGGGGGGGGG ；SEQ ID NO 33 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGRG37GG C GGGGG GGGGGGGGGGGGGGG ；SEQ ID NO 34 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGRG37GGGGG C GGGGG GGGGG ；SEQ ID NO 35 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37GGGGG C GGGGG GGGGGGGGGG ；SEQ ID NO 36 =7HABGT FTSDV SSYLE GQAAK EFICW LVKGRS37GGGGG C GGGGG GGGGGGGGGGGGGGG ；SEQ ID NO 37 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37AC AAAAA AAAAA ；SEQ ID NO 38 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37AC AAAM AAAAAAAAAA ；SEQ ID NO 39 =7HABGT FTSDV SSYLE GQAAK EFICW LVKGR G37AC AAAM AAAAAAAAAAAAAAAAAAAA ；SEQ ID NO 40 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37AA C AAAAA AAAAA ；SEQ ID NO 41 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37M C AAAM AAAAAAAAAA ；SEQ ID NO 42 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGRG37AA C AAAAA AAAAAAAAAAAAAAA ；SEQ ID NO 43 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGRG37AAAM C AAAM AAAM ；SEQ ID NO 44 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37AAAAA C AAAAA AAAAAAAAAA ；SEQ ID NO 45 =7HABGT FTSDV SSYLE GQAAK EFICW LVKGRS37AAAM C AAAM AAAAAAAAAAAAAAA ；SEQ ID NO 46 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37VC VVVVV VVVVV ；SEQ ID NO 47 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37VC VVVVV VVVVVVVVVV ；SEQ ID NO 48 =7HABGT FTSDV SSYLE GQAAK EFICW LVKGR G37VC VWW WWWWWWWWWW ；SEQ ID NO 49 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37VV C VVVVV VVVVV ；SEQ ID NO 50 :7HAEGT FTSDV SSYLE GQMK EFICff LVKGRG37VV C WVVV VVVVVVVVVV ；SEQ ID NO 51 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGRG37W C VWW WWVWWWWW ；SEQ ID NO 52 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGRG37VVVVV C VVVVV VVVVV ；SEQ ID NO 53 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGRG37VWW C VWW WWWWW ;或SEQ ID NO 54 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37VWW C VWW WWWWVWWW。另外，所述具有通式I的GLP-I类似物还可以具有下列通式IV 7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37H1X1Cn2X2 通式IV其中，Ii1 = I 10，n2 = O 30，X1为甘氨酸、丙氨酸或缬氨酸，X2为甘氨酸、丙氨酸或缴氨酸；优选地，Ii1 = I 5, n2 = 10 20。优选地，所述GLP-I类似物为SEQ ID NO 55 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37GC GGGGG GGGGG ；SEQ ID NO 56 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37GC GGGGG GGGGGGGGGG ；SEQ ID NO 57 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGR G37GC GGGGG GGGGGGGGGGGGGGG ；
SEQ ID NO 58 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37GG C GGGGG GGGGG ；
SEQ ID NO 59 :7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37GG C GGGGG GGGGGGGGGG ；SEQ ID NO 60 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGRG37GG C GGGGG GGGGGGGGGGGGGGG ；SEQ ID NO 61 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37GGGGG C GGGGG GGGGG ；SEQ ID NO 62 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37GGGGG C GGGGG GGGGGGGGGG ；SEQ ID NO 63 =7HABGT FTSDV SSYLE GQAAK EFICW LCKGRS37GGGGG C GGGGG GGGGGGGGGGGGGGG ；SEQ ID NO 64 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37AC AAAAA AAAAA ；SEQ ID NO 65 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37AC AAAM AAAAAAAAAA ；SEQ ID NO 66 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGR G37AC AAAAA AAAAAAAAAAAAAAA ；SEQ ID NO 67 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37AA C AAAAA AAAAA ；SEQ ID NO 68 :7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37M C AAAM AAAAAAAAAA ；SEQ ID NO 69 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGRG37AA C AAAAA AAAAAAAAAAAAAAA ；SEQ ID NO 70 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37AAAM C AAAM AAAM ；SEQ ID NO 71 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37AAAAA C AAAAA AAAAAAAAAA ；SEQ ID NO 72 =7HABGT FTSDV SSYLE GQAAK EFICW LCKGRS37AAAAA C AAAM AAAAAAAAAAAAAAA ；SEQ ID NO 73 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37VC VVVVV VVVVV ；SEQ ID NO 74 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37VC VVVVV VVVVVVVVVV ；SEQ ID NO 75 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGR G37VC WWV WWVWWWWW ；SEQ ID NO 76 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37VV C VVVVV VVVVV ；SEQ ID NO 77 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37W C WVVV VVVVVVVVVV ；SEQ ID NO 78 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGRG37W C VWW WWVWWWWW ；SEQ ID NO 79 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGRG37VVVVV C VVVVV VVVVV ；SEQ ID NO 80 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGRG37VWW C VWW WWWWW ;或SEQ ID NO 81 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37WVW C VWW WWWWVWWW。再另，所述具有通式I的GLP-I类似物为SEQ ID NO 82 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGG GGGGG C GGGGG GGGGG ；SEQ ID NO 83 =7HAEGT FTSDV SSYLE CQAAK EFIAff LVKGR G37GGGGG C GGGGG GGGGG ;或SEQ ID NO 84 =7HABGT FTSDV SSYLE GQAAK EFId LVKGR G37GGG C GGGGG GGGGGGGGGGGGGGG。另一方面，本发明提供上述GLP-I类似物的制备方法，所述制备方法包括Fmoc策略的固相多肽合成。又一方面，本发明提供上述GLP-I类似物在制备治疗糖尿病，肥胖症和糖尿病、月巴胖症相关疾病以及预防肥胖症的药物中的应用。并且，本发明还提供一种药物组合物，所述药物组合物包含如权利要求1-8中任一项所述的GLP-I类似物和一种或多种药学上可接受的辅料；优选地，所述药物组合物为注射剂，进一步优选为冻干粉针或溶液注射剂。以下是本发明的详细描述(I)GLP-I 类似物本发明所述的GLP-I类似物的通式如下通式I :
7HAEX10T FTSX15V SSYLE X22QAAK EFIX30W LX33KGR G37H1X1Cn2X27HAEGT FTSDV SSYLE GQAAK EFIAff LVKGR G37 为人源 GLP-1 序列，上述通式 I 的GLP-I类似物为人工序列，分别在第10、15、22、30或33位氨基酸上进行了修饰，由半胱氨酸取代了原有的氨基酸。并且在上述的氨基酸通式中X1可为Gly，Ala，Val ;通式中的&可为Gly,Ala, Val。最后通式中的Oi1及n2)指代在多肽的C末端含有η个疏水氨基酸(Gly,Ala或Val), η = 3 30。在上述的多肽通式中,两个半胱氨酸均形成二硫键。 基于15位、30位和33位氨基酸突变为半胱氨酸的氨基酸通式可优选为通式II 7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37Ii1X1Cn2X2A1 = I 20 ;η2 = O 25。其中 X1 为 Gly, Ala 或 Val ；Χ2 为 Gly, Ala 或 Val。通式III:7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37H1X1C η2Χ2, η! = I 10 ;η2 = O 25。其中 X1 为 Gly，Ala 或 Val ;X2 为 Gly，Ala 或 Val。通式IV :7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37H1X1C n2X2, η! = I 10 ;n2 = O 30。其中 X1 为 Gly，Ala 或 Val ;X2 为 Gly，Ala 或 Val。上述的多肽通式II可进一步优化为7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37H1X1C Ii2X2A1 = 5 15;n2 = 3 9。其中 X1 为 Gly, Ala 或 Val ；X2 为 Gly, Ala 或 Val。上述的多肽通式III可进一步优化为7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37H1X1C η2Χ2, η! = I 5 ;n2 = 10 20。其中 X1 为 Gly，Ala 或 Val ;X2 为 Gly，Ala 或 Val。上述的多肽通式IV可进一步优化为7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37H1X1C n2X2, η! = I 5 ;n2 = 10 20。其中 X1 为 Gly，Ala 或 Val ;X2 为 Gly，Ala 或 Val。(2)本发明的药用组合物本发明的含有GLP-I类似物可以与一种或多种药学上可接受的辅料共同制成药用组合物，这些辅料包括水溶性填充剂、PH调节剂、稳定剂、注射用水、渗透压调节剂等
坐寸ο本发明所述的水溶性填充剂辅料为选自以下的一种或一种甘露醇、低分子右旋糖苷、山梨醇、聚乙二醇、葡萄糖、乳糖、半乳糖等。PH调节剂为选自以下的一种或一种枸橼酸、磷酸、乳酸、酒石酸、盐酸等非挥发性的酸以及氢氧化钾、氢氧化钠或氢氧化钾或氢氧化铵、碳酸钠或碳酸钾或碳酸铵盐、碳酸氢钠或碳酸氢钾或碳酸氢铵盐等生理可接受的有机或无机酸和碱及盐等。稳定剂为选自以下的一种或一种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、谷氨酸、聚乙二醇6000、聚乙二醇4000、十二烷基硫酸钠或三羟甲基氨基甲烷等。优选焦亚硫酸钠、磷酸氢二钾、精氨酸、聚乙二醇6000、三羟甲基氨基甲烷。渗透压调节剂是氯化钠、氯化钾的一种或两种的组合。
(3)注射剂的制备方法本发明的药用组合物可以通过肌肉、静脉内、皮下注射途经进行给药，优选的剂型为冻干粉或溶液注射剂。冷冻干燥注射剂的制备方法取GLP-I类似物溶液适量，加入水溶性填充剂、稳定齐 、渗透压调节剂等，加入注射用水适量，调节PH值至4-8使其溶解，加水稀释至适当浓度，加入O. 1-0. 5%活性炭，在0-10°C下搅拌10-20分钟，脱碳，采用微孔滤膜过滤除菌，滤液进行分装，采用冷冻干燥法，制得白色疏松块状物，封口即得，每个规格含有GLP-I类似物在5 μ g至Ij Img之间。注射液的制备方法取GLP-I类似物溶液或冻干粉适量，加入水溶性填充剂、稳定 齐 、渗透压调节剂等，加入注射用水适量，调节PH值至4-8使其溶解，加水稀释至适当浓度，加入O. 1-0. 5%活性炭，在0-10°C下搅拌10-20分钟，脱碳，采用微孔滤膜过滤除菌，滤液进行分装，封口即得，每个规格含有GLP-I类似物在5 μ g到Img之间。(4)药用组合物的用途本发明的GLP-I类似物可以用在制备糖尿病治疗药物方面。具体地，本发明的组合物可以静脉、肌肉或皮下注射剂形式给药。虽然剂量依治疗对象、给药方式、症状及其它因素而改变，本发明的GLP-I类似物在相当宽的剂量范围内是有效的。在成人的治疗中，剂量范围在5 μ g/人一Img/人，每日一次或每几日一次给药。实际剂量应该由医生根据有关的情况来决定，这些情况包括被治疗者的身体状态、给药途径、年龄、体重、患者对药物的个体反应，患者症状的严重程度等等，因此上述剂量范围并不是以任何方式限制本发明的范围。本发明的GLP-I类似物克服了 GLP-I半衰期短的问题，所提供的GLP-I类似物在体内的半衰期可达到15 75小时以上，较单独给药的GLP-I的半衰期(半衰期仅为3-5分钟)明显延长，极大地便利了其临床推广和应用。


以下，结合附图来详细说明本发明的实施例，其中图I为实施例2中含GLP-I类似物(SEQ ID NO 1-27)的降血糖实验；图2为实施例3中含GLP-I类似物(SEQ ID NO 28-54)的降血糖实验；图3为实施例4中含GLP-I类似物(SEQ ID NO 55-81)的降血糖实验；图4为实施例5中含GLP-I类似物(SEQ ID NO 82-84)的降血糖实验。

对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。在以下的实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。实施例I :多肽的固相合成使用Fmoc策略的固相多肽合成方法，使用CSBio公司生产的CS 336X型仪器进行本发明的多肽的合成。合成的方法按照生产商的仪器说明书进行。将制得的多肽使用HPLC C18半制备柱进行纯化，流动相为乙腈。经脱盐及冷冻干燥得到多肽冻干粉。本专利包含的多肽均含有二硫键，碳酸氢铵或其他还原剂用来形成多妝中的~■硫键。
实施例2 =GLP-I类似物(通式II)相关的降血糖功能本实施例中，采用的多肽如下SEQ ID NO I =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGC GGGSEQ ID NO 2 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGGGGGGC GGGSEQ ID NO 3 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGR G37GGGGGGGGGGGGGGGC GGGSEQ ID NO 4 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGC GGGGG GSEQ ID NO 5 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGC GGGGG GGGG SEQ ID NO 6 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGGGGGGC GGGGG GSEQ ID NO 7 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGGGGGGC GGGGG GGGGSEQ ID NO 8 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGGGGGGGGGGGC GGGGG GSEQ ID NO 9 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGGGGGGGGGGGGC GGGGG GGGGSEQ ID NO 10 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA C AAASEQ ID NO 11 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAC AAASEQ ID NO 12 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37AAAM AAAAAAAAAAC AMSEQ ID NO 13 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA C AAAAA ASEQ ID NO 14 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA C AAAAA AAAASEQ ID NO 15 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37AAAM AAAMC AAAM ASEQ ID NO 16 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAMC AAAAA AAAASEQ ID NO 17 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAAAAAAC AAAAA ASEQ ID NO 18 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37AAAAA AAAAAAAAAAC AAAAA AAAASEQ ID NO 19 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV C VVVSEQ ID NO 20 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV VVVVVC VVVSEQ ID NO 21 =7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37VVVVV VVVVVVVVVVC VVVSEQ ID NO 22 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV C VVVVV VSEQ ID NO 23 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VVVVV C VVVVV VVVVSEQ ID NO 24 :7HAEGT FTSCV SSYLE GQMK EFIAff LVKGRG37VVVVV VVVVVC VVVVV VSEQ ID NO 25 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VWW WWVC VWW WWSEQ ID NO 26 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VWW WWWWWC VWW VSEQ ID NO 27 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGRG37VWW WWWWWC VWW WW将上述多肽各Img溶于Iml生理盐水，皮下注射于小鼠(200 μ I/只，6只/组，购自中科院上海实验动物中心)，给药30分钟后将400mg葡萄糖注射于每只小鼠。分别于葡萄糖注射后2小时、24小时、48小时、72小时及96小时测定小鼠的血糖(注每次测血糖前两个小时再次给相同剂量的葡萄糖)，结果见图I，其中本实施例中的control (空白)为生理盐水，用量1ml。实施例3 =GLP-I类似物(通式III)相关的降血糖功能。本实施例中，采用的多肽如下SEQ ID NO 28 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37G CGGGGG GGGGG
SEQ ID NO 29 :7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37G CGGGGG GGGGGGGGGG
SEQ ID NO 30 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G37G OGGGGG GGGGGGGGGGGGGGGGGGGGSEQ ID NO 31 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37GG CGGGGG GGGGGSEQ ID NO 32 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37GG CGGGGG GGGGGGGGGGSEQ ID NO 33 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGR G37GG CGGGGG GGGGGGGGGGGGGGGSEQ ID NO 34 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37GGGGG C GGGGG GGGGGSEQ ID NO 35 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37GGGGG C GGGGG GGGGGGGGGGSEQ ID NO 36 =7HABGT FTSDV SSYLE GQAAK EFId LVKGRS37GGGGG C GGGGG GGGGGGGGGGGGGGGSEQ ID NO 37 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37A CAAAAA AAAAASEQ ID NO 38 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37A CAMM AAAAAAAAAASEQ ID NO 39 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G37A CAAAM AAAAAAAAAAAAAAAAAAAASEQ ID NO 40 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37AA CAAAAA AAAAASEQ ID NO 41 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37M CAAAM AAAAAAAAAASEQ ID NO 42 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGR G37AA CAAAAA AAAAAAAAAAAAAAASEQ ID NO 43 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37MAAA C AAMA AMAASEQ ID NO 44=7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37AAAAA C AAAAA AAAAAAAAAASEQ ID NO 45 =7HABGT FTSDV SSYLE GQAAK EFId LVKGRS37AAAM C AAAM AAAAAAAAAAAAAAASEQ ID NO 46 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37V CVVVVV VVVVVSEQ ID NO 47 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37V CVVVVV VVVVVVVVVV
SEQ ID NO 48 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGR G37V CVWW WWWWWWWWWWSEQ ID NO 49 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGR G37VV CVVVVV VVVVVSEQ ID NO 50 =7HAEGT FTSDV SSYLE GQMK EFICff LVKGR G37VV CVVVVV VVVVVVVVVVSEQ ID NO 51 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGR G37W CVWW WWVWWWWWSEQ ID NO 52 =7HAEGT FTSDV SSYLE GQAAK EFICff LVKGRG37VVVVV C VVVVV VVVVVSEQ ID NO 53 =7HAEGT FTSDV SSYLE GQAAK EFICW LVKGRG37VWW C VWW WWWWWSEQ ID NO 54 =7HABGT FTSDV SSYLE GQAAK EFId LVKGRS37VWW C VWW WWVWWWWW将上述多肽各Img溶于Iml生理盐水，皮下注射于小鼠(200 μ I/只，6只/组，购自中科院上海实验动物中心)，给药30分钟后将400mg葡萄糖注射于每只小鼠。分别于葡萄糖注射后2小时、24小时、48小时、72小时及96小时测定小鼠的血糖(注每次测血糖前两个小时再次给相同剂量的葡萄糖)，结果见图2，其中本实施例中的control (空白)为生理盐水，用量1ml。实施例4 =GLP-I类似物(通式IV)相关的降血糖功能本实施例中，采用的多肽如下SEQ ID NO 55 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37G CGGGGG GGGGGSEQ ID NO 56 :7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37G CGGGGG GGGGGGGGGGSEQ ID NO 57 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G37G CGGGGG GGGGGGGGGGGGGGGSEQ ID NO 58 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37GG CGGGGG GGGGGSEQ ID NO 59 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37GG CGGGGG GGGGGGGGGGSEQ ID NO 60 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G37GG CGGGGG GGGGGGGGGGGGGGG
SEQ ID NO 61 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37GGGGG C GGGGG GGGGGSEQ ID NO 62 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37GGGGG C GGGGG GGGGGGGGGGSEQ ID NO 63 =7HABGT FTSDV SSYLE GQAAK EFId LCKGRS37GGGGG C GGGGG GGGGGGGGGGGGGGGSEQ ID NO 64 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37A CAAAAA AAAAASEQ ID NO 65 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37A CAMM AAAAAAAAAASEQ ID NO 66 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G37A CAAAAA AAAAAAAAAAAAAAASEQ ID NO 67 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37AA CAAAAA AAAAASEQ ID NO 68 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37M CAAAM AAAAAAAAAA SEQ ID NO 69 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGR G37AA CAAAAA AAAAAAAAAAAAAAASEQ ID NO 70 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37MAAA C AAMA AAMASEQ ID NO 71 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37AAAAA C AAAAA AAAAAAAAAASEQ ID NO 72 =7HABGT FTSDV SSYLE GQAAK EFId LCKGRS37AAAM C AAAM AAAAAAAAAAAAAAASEQ ID NO 73 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37V CVVVVV VVVVVSEQ ID NO 74 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37V CVVVVV VVVVVVVVVVSEQ ID NO 75 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGR G37V CVWW WWVWWWWWSEQ ID NO 76 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGR G37VV CVVVVV VVVVVSEQ ID NO 77 =7HAEGT FTSDV SSYLE GQMK EFICff LCKGR G37VV CVVVVV VVVVVVVVVVSEQ ID NO 78 =7HAEGT FTSDV SSYLE GQAAK EFId LCKGR G37W CVWW WWVWWWWWSEQ ID NO 79 =7HAEGT FTSDV SSYLE GQAAK EFICff LCKGRG37VVVVV C VVVVV VVVVVSEQ ID NO 80 =7HAEGT FTSDV SSYLE GQAAK EFICW LCKGRG37VWW C VWW WWWWWSEQ ID NO 81 =7HABGT FTSDV SSYLE GQAAK EFId LCKGRS37WVW C VWW WWVWWWWW将上述多肽各Img溶于Iml生理盐水，皮下注射于小鼠(200 μ I/只，6只/组，购自中科院上海实验动物中心)，给药30分钟后将400mg葡萄糖注射于每只小鼠。分别于葡萄糖注射后2小时、24小时、48小时、72小时及96小时测定小鼠的血糖(注每次测血糖前两个小时再次给相同剂量的葡萄糖)，结果见图3，其中本实施例中的control (空白)为生理盐水，用量1ml。实施例5 :基于其他位点的氨基酸突变为半胱氨酸的GLP-I类似物本实施例中，采用的多肽如下SEQ ID NO 82 =7HAEGT FTSCV SSYLE GQAAK EFIAff LVKGR G37GGGGG GGGGG C GGGGG GGGGGSEQ ID NO 83 =7HAEGT FTSDV SSYLE CQAAK EFIAff LVKGR G37GGGGG C GGGGG GGGGGSEQ ID NO 84 =7HAEGT FTSDV SSYLE GQAAK EFId LVKGR G37GGGC GGGGG GGGGGGGGGGGGGGG将上述多肽各Img溶于Iml生理盐水，皮下注射于小鼠(200 μ I/只，6只/组，购自中科院上海实验动物中心)，给药30分钟后将400mg葡萄糖注射于每只小鼠。分别于葡萄糖注射后2小时、24小时、48小时、72小时及96小时测定小鼠的血糖，结果见图4，其中本实施例中的control (空白)为生理盐水，用量1ml。


本发明公开了所述GLP-1类似物具有下列通式I7HAEX10T FTSX15V SSYLE X22QAAK EFIX30W LX33KGR G37n1X1C n2X2通式I，其中，X10为甘氨酸或半胱氨酸，X15为天冬氨酸或半胱氨酸，X22为甘氨酸或半胱氨酸，X30为丙氨酸或半胱氨酸，X33为缬氨酸或半胱氨酸，并且X10、X15、X22、X30和X33中至少有一个为半胱氨酸；n1X1表示有n个X1，n＝3～30，且X1为甘氨酸、丙氨酸或缬氨酸；n2X2表示有n个X2，n＝3～30，且X2为甘氨酸、丙氨酸或缬氨酸；以及通式I中含有的两个半胱氨酸均形成二硫键。本GLP-1类似物能够有效增加GLP-1的血液半衰期，克服其因为半衰期短而无法在临床上使用的现状，在糖尿病、肥胖症治疗药物领域较有应用前景。本发明还公开了所述GLP-1类似物的制备方法及其应用。