專利名稱：帶有聚乙二醇基團(tuán)的Exendin或其類似物及其制劑和用途的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及一種修飾的Exendin或其類似物，特別地涉及一種由單個(gè)或多個(gè)聚乙二醇修飾的Exendin或其類似物及其制劑、給藥方法以及應(yīng)用。

背景技術(shù)：
胰高血糖素樣肽-1首次于1987年被研究人員認(rèn)定為一種葡萄糖依賴的腸分泌激素肽。這種多肽通過(guò)細(xì)胞上G蛋白耦聯(lián)受體傳遞信號(hào)，從而產(chǎn)生刺激β胰島細(xì)胞分泌胰島素，抑制胰高血糖素分泌，抑制胃排空和胃酸分泌等生理作用。
Exendin是一種在美國(guó)毒蜥和珠毒蜥唾液中發(fā)現(xiàn)的一類多肽(J.Biol.Chem.1990，265，20259-20262；J.Biol.Chem.1992，267，7402-7405)，以Exendin-4為代表的這類多肽與胰高血糖素樣肽-1[7-36]具有高度同源性。研究發(fā)現(xiàn)，Exendin同樣可以與胰高血糖素樣肽-1受體結(jié)合，并在藥理上表現(xiàn)出相類似的效果，如促進(jìn)胰島素分泌，有效控制餐后血糖，降低糖化血紅蛋白，抑制胃排空等。在動(dòng)物體內(nèi)的長(zhǎng)期研究發(fā)現(xiàn)，長(zhǎng)期使用這類胰高血糖素樣肽-1受體多肽還能有效改善胰島素抵制，從而逆轉(zhuǎn)糖尿病的病程。此外，人們還發(fā)現(xiàn)，以胰高血糖素樣肽-1和Exendin-4為代表的一系列促胰島素分泌激動(dòng)劑能促進(jìn)β胰島細(xì)胞再生(Nat.Biotech.2005，23，857-861)和改善非酒精性脂肪肝(Hepatology 2006，43，173-181)。這使得該類多肽一直成為糖尿病及肥胖制藥領(lǐng)域的研究熱點(diǎn)。伍登熙和孫玉昆等人(中國(guó)專利ZL01112856)對(duì)Exendin-4進(jìn)行了多肽改構(gòu)，得到了一系列相同功能的多肽。尤其是在前不久，美國(guó)安米林(Amylin)和禮來(lái)(Eli Lilly)公司共同開(kāi)發(fā)的一天注射兩次的Exendin——Exendin-4(Byetta)在美國(guó)上市，使得該類藥物得到全世界糖尿病及肥胖治療領(lǐng)域的廣泛關(guān)注。但是經(jīng)過(guò)臨床研究發(fā)現(xiàn)，這種異源的多肽在給藥30周后會(huì)使受試病人體內(nèi)出現(xiàn)Exendin-4抗體，占總?cè)藬?shù)的41％(Diabetes Care.2004，27，2628-2635)。
由于蛋白質(zhì)/多肽類藥物普遍存在體內(nèi)半衰期較短，物理、化學(xué)穩(wěn)定性較差，易被體內(nèi)各種蛋白酶降解等特性，使得這些藥物通常需要在一天之內(nèi)多次注射，這給病人帶來(lái)了許多痛苦和諸多不便。如何延長(zhǎng)這類藥物的給藥周期一直長(zhǎng)期困擾人們，目前還沒(méi)一種普遍適用的給藥方式。
上世紀(jì)70年代出現(xiàn)的聚乙二醇化技術(shù)被證明是當(dāng)前蛋白質(zhì)/多肽類給藥領(lǐng)域一項(xiàng)較適用的技術(shù)。通常采用線性直鏈或分支狀的聚乙二醇對(duì)蛋白質(zhì)/多肽進(jìn)行修飾，這樣的修飾賦予蛋白質(zhì)/多肽具有如下特點(diǎn)1)物理和化學(xué)穩(wěn)定性得到顯著提高，2)免疫抗原性得到顯著下降，3)抗蛋白酶降解能力顯著提高，4)隨著聚乙二醇分子量的增加，能明顯降低腎清除作用對(duì)藥物的代謝，從而藥物的體內(nèi)半衰期顯著增加，5)藥物溶解度提高，以及對(duì)細(xì)胞膜的穿透力增強(qiáng)。A·楊和K·普里克特的研究證明了Exendin-4主要是通過(guò)腎清除來(lái)代謝的，因此他們將分子量范圍在500至20,000道爾頓(Dalton)的聚乙二醇對(duì)Exendin進(jìn)行了修飾(中國(guó)專利CN1372570A)，從而實(shí)現(xiàn)降低腎過(guò)濾對(duì)藥物的清除。
但是，修飾后藥物的活性會(huì)普遍下降是該技術(shù)的一個(gè)缺陷。Haim Tsubery等人使用9羥甲基7硫代芴N(yùn)羥基琥珀酰亞胺(9-hydroxymethyl-7-sulfofluorene-Nhydroxysuccinimide，F(xiàn)MS)活化聚乙二醇，在體內(nèi)聚乙二醇可以從Exendin-4上水解下來(lái)，從而使未經(jīng)修飾的Exendin-4發(fā)揮作用。這種方式雖然解決了聚乙二醇修飾后所導(dǎo)致的生物活性降低的問(wèn)題，但是由于發(fā)揮作用的還是未經(jīng)修飾的Exendin-4，因此長(zhǎng)期注射而產(chǎn)生的免疫抗原性問(wèn)題也依舊沒(méi)能解決(J.Biol.Chem.2004，279，38118-38124)。
我們使用新型的聚乙二醇修飾的Exendin或其類似物，取得了半衰期長(zhǎng)、活性高、免疫抗原性低的聚乙二醇修飾的Exendin或其類似物。


發(fā)明內(nèi)容
本發(fā)明所要解決的技術(shù)問(wèn)題是Exendin或其類似物在體內(nèi)使用單次給藥后的時(shí)間間隔較短，長(zhǎng)期注射會(huì)使體內(nèi)產(chǎn)生抗體，現(xiàn)存的聚乙二醇修飾后的Exendin或其類似物生物活性嚴(yán)重下降，使給藥劑量大大增加，而無(wú)法實(shí)際應(yīng)用。本發(fā)明提供新型的聚乙二醇修飾的Exendin或其類似物，具有所述的半衰期長(zhǎng)、活性高、免疫抗原性低的特點(diǎn)。
聚乙二醇修飾 本發(fā)明涉及新型的聚乙二醇修飾的Exendin或其類似物，其特征是Exendin或其類似物由一個(gè)或多個(gè)聚乙二醇修飾。
聚乙二醇衍生物不同的化學(xué)結(jié)構(gòu)對(duì)修飾后的多肽生物活性影響不同。本發(fā)明所述的聚乙二醇可以采用線性直鏈或分叉支鏈的結(jié)構(gòu)。本發(fā)明所述的分叉支鏈(或稱分支叉狀)結(jié)構(gòu)包括雙臂支鏈的結(jié)構(gòu)，也包括多臂支鏈的結(jié)構(gòu)。本發(fā)明所述的聚乙二醇雙臂支鏈結(jié)構(gòu)包括但不限于如下列式I、式II、式III和式IV所示結(jié)構(gòu)。
本發(fā)明所述的聚乙二醇支鏈結(jié)構(gòu)包括但不限于下列式I所示結(jié)構(gòu)
其中，X為H或保護(hù)基團(tuán)。這里所說(shuō)的保護(hù)基團(tuán)可以是任何能夠與自由羥基反應(yīng)，并防止其進(jìn)一步與其它基團(tuán)反應(yīng)的基團(tuán)，包括但不限于低碳烷基如甲基、二甲基、乙基、丙基、異丙基等。
另外，在上述結(jié)構(gòu)中，PEG為-O(CH2CH2O)q-，q為正整數(shù)；n1為0-5的整數(shù)；m為0-5的整數(shù)；Y為O，S，SO，SO2或NR1，其中R1為H或C1-8經(jīng)取代或未經(jīng)取代的烷基或經(jīng)取代或未經(jīng)取代的環(huán)烷；k為0或1；p為0-6的整數(shù)。上述的帶式I結(jié)構(gòu)的聚乙二醇可使用常規(guī)的化學(xué)方法合成，美國(guó)專利U.S.6,566,506提供了一些帶上述式I結(jié)構(gòu)的聚乙二醇的合成方法。
在本發(fā)明一優(yōu)選的實(shí)施例中，本發(fā)明使用的帶有式I所示支鏈結(jié)構(gòu)的聚乙二醇帶有如下式II所示具體結(jié)構(gòu)
其中，PEG為-O(CH2CH2O)q-，q為正整數(shù)，Me為甲基。
本發(fā)明所述的聚乙二醇支鏈結(jié)構(gòu)包括但不限于下列式III所示結(jié)構(gòu)
其中，X為H或保護(hù)基團(tuán)；PEG為-O(CH2CH2O)q-，q為正整數(shù)；n2為0-10的整數(shù)；Z為選自于以下組中的基團(tuán)(CH2)i、(CH2)iOCO、(CH2)iNHCO、(CH2)iCO，i為0-10的整數(shù)；R2為H、C1-12經(jīng)取代或未經(jīng)取代的烷基、取代芳基、芳烷基或雜烷基；j為1-12的整數(shù)。上述的帶式III結(jié)構(gòu)的聚乙二醇可使用常規(guī)的化學(xué)方法合成，中國(guó)專利ZL03801105.0提供了一些帶上述式III結(jié)構(gòu)的聚乙二醇的合成方法。
在本發(fā)明一優(yōu)選的實(shí)施例中，本發(fā)明使用的帶有式III所示支鏈結(jié)構(gòu)的聚乙二醇帶有如下式IV所示具體結(jié)構(gòu)
其中，PEG為-O(CH2CH2O)q-，q為正整數(shù)，Me為甲基。
聚乙二醇的分子量大小對(duì)修飾后的多肽生物活性也有不同影響。本發(fā)明使用的直線型和支鏈型聚乙二醇的分子量范圍優(yōu)選為200道爾頓或其以上，更優(yōu)選為5,000道爾頓或其以上，再優(yōu)選為20,000道爾頓或其以上。在一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇分子量為5,000道爾頓至50,000道爾頓之間，包括5,000道爾頓和50,000道爾頓。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇分子量為20,000道爾頓至50,000道爾頓之間，包括20,000道爾頓和50,000道爾頓。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇分子量為20,000道爾頓至45,000道爾頓之間，包括20,000道爾頓和45,000道爾頓。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇分子量為20,000道爾頓至40,000道爾頓之間，包括20,000道爾頓和40,000道爾頓。本發(fā)明所述的聚乙二醇分子量除特別指出外均指通過(guò)凝膠滲透色譜法(Gel PermeationChromatography，GPC)(董炎明，《高分子分析手冊(cè)》，北京中國(guó)石化出版社2004，416-427)測(cè)定得到的數(shù)均分子量(Mn)。帶不同分子量的聚乙二醇可以在市場(chǎng)上買到也可以使用本技術(shù)領(lǐng)域公知的技術(shù)合成。
在本發(fā)明一優(yōu)選的實(shí)施方案中，本發(fā)明所述的聚乙二醇采用分子量為20,000以上的支鏈結(jié)構(gòu)。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇為分子量在20,000道爾頓以上至50,000道爾頓之間的支鏈結(jié)構(gòu)，包括50,000道爾頓。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇為分子量在20,000道爾頓以上至45,000道爾頓之間的支鏈結(jié)構(gòu)，包括45,000道爾頓。在另一個(gè)優(yōu)選的實(shí)施例中，本發(fā)明使用的聚乙二醇為分子量在20,000道爾頓以上至40,000道爾頓之間的支鏈結(jié)構(gòu)，包括40,000道爾頓。
本發(fā)明所述的聚乙二醇修飾的Exendin或其類似物是通過(guò)活化的聚乙二醇與多肽氨基酸殘基的側(cè)鏈，N末端和C末端連接。使用帶不同活化基團(tuán)的聚乙二醇，可以實(shí)現(xiàn)對(duì)不同氨基酸側(cè)鏈、N末端和C末端的化學(xué)結(jié)合，也可以針對(duì)某一氨基酸位點(diǎn)專一性結(jié)合?？梢耘c活化的聚乙二醇化學(xué)結(jié)合的氨基酸基團(tuán)包括但不限于多肽的N末端α氨基、賴氨酸側(cè)鏈ε氨基、組氨酸側(cè)鏈咪唑基上的亞氨基、多肽的C末端羧基、天冬氨酸和谷氨酸側(cè)鏈羧基、絲氨酸和蘇氨酸側(cè)鏈羥基，半胱氨酸側(cè)鏈巰基等。通常情況下，這種化學(xué)連接通過(guò)親電親核方式進(jìn)行。例如，經(jīng)N羥基琥珀酰亞胺活化的聚乙二醇與多肽的N末端α氨基，賴氨酸側(cè)鏈ε氨基以及組氨酸側(cè)鏈咪唑基上的亞氨基等游離氨基連接。另外，還可以采用活化基團(tuán)為醛基的聚乙二醇，通過(guò)還原烷基化定點(diǎn)結(jié)合到多肽分子的N末端。本發(fā)明包括通過(guò)不同活化基團(tuán)使聚乙二醇與Exendin或其類似物結(jié)合，及其產(chǎn)生的聚乙二醇修飾的Exendin或其類似物。帶不同活化基團(tuán)的聚乙二醇可以在市場(chǎng)上買到也可以使用本技術(shù)領(lǐng)域公知的技術(shù)合成。
本發(fā)明包括Exendin或其類似物上一種或多種氨基酸與聚乙二醇結(jié)合的Exendin或其類似物。本發(fā)明所述的Exendin或其類似物上能同時(shí)連接1個(gè)或數(shù)個(gè)聚乙二醇，所結(jié)合聚乙二醇的個(gè)數(shù)視Exendin或其類似物上游離反應(yīng)基團(tuán)的數(shù)量，聚乙二醇所帶活化基團(tuán)以及聚乙二醇的分子量大小而定。一般來(lái)說(shuō)，游離反應(yīng)基團(tuán)的數(shù)量越多，就能結(jié)合更多的活化聚乙二醇；活化的聚乙二醇分子量越大，其能結(jié)合到多肽上的數(shù)量就越少。優(yōu)選地，Exendin或其類似物由一個(gè)、二個(gè)、三個(gè)或四個(gè)聚乙二醇修飾。在一個(gè)優(yōu)選的實(shí)施例中，Exendin或其類似物由一個(gè)聚乙二醇修飾。在另一個(gè)優(yōu)選的實(shí)施例中，Exendin或其類似物由二個(gè)聚乙二醇修飾。
聚乙二醇與Exendin或其類似物連接反應(yīng)后的混合物可以通過(guò)常用的離子交換、凝膠過(guò)濾或反相色譜等方式實(shí)現(xiàn)對(duì)這些產(chǎn)物的有效分離。通常使用的離子交換色譜法有陰離子交換色譜和陽(yáng)離子交換色譜兩種，選擇不同的離子交換基質(zhì)可以影響到分離純化的效果。由于不同的Exendin或其類似物在氨基酸個(gè)數(shù)和種類略有不同，而使各自的分子量略有不同，因此聚乙二醇單取代的Exendin或其類似物的最終分子量為聚乙二醇的分子量加上Exendin或其類似物的分子量。通過(guò)分離純化、超濾濃縮和緩沖液置換等方法處理后，可以得到本發(fā)明所述的聚乙二醇修飾的Exendin或其類似物，并可以制成藥用制劑。
Exendin或其類似物 本發(fā)明所述的Exendin或其類似物是指與Exendin全部或部分氨基酸結(jié)構(gòu)序列相類似的，能與胰高血糖素樣肽-1受體結(jié)合，并產(chǎn)生細(xì)胞信號(hào)級(jí)聯(lián)傳導(dǎo)的一類多肽。這些多肽可以通過(guò)化學(xué)固相合成或基因工程的宿主菌表達(dá)后，再經(jīng)過(guò)一步或多步分離純化得到。
本發(fā)明所述的Exendin或其類似物包括但不限于天然的Exendin-3或Exendin-4。天然的Exendin-3的氨基酸序列結(jié)構(gòu)如下 [His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH](SEQID NO1) 天然的Exendin-4的氨基酸序列結(jié)構(gòu)如下 [His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2](SEQID NO2) 本發(fā)明所述的Exendin或其類似物包括但不限于那些通過(guò)對(duì)天然的Exendin-3或Exendin-4的氨基酸序列進(jìn)行氨基酸置換、增加和刪減而得到的類似多肽。本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列進(jìn)行天然氨基酸置換，同時(shí)也包括用非天然氨基酸對(duì)Exendin-3或Exendin-4的氨基酸序列進(jìn)行氨基酸置換。非天然氨基酸包括但不限于氮雜環(huán)丁烷羧酸、2-氨基己二酸、3氨基己二酸、β-丙氨酸、氨基丙酸、2-氨基丁酸、4-氨基丁酸、6-氨基己酸、2-氨基庚酸、2-氨基異丁酸、3-氨基異丁酸、2氨基庚二酸、叔丁基甘氨酸、2，4-二氨基異丁酸、鎖鏈賴氨素、2，2’-二氨基庚二酸、2，3-二氨基丙酸、N乙基甘氨酸、N-乙基天冬酰胺、高脯氨酸、羥賴氨酸、別羥賴氨酸、3羥脯氨酸、4-羥脯氨酸、異鎖鏈賴氨素、別-異亮氨酸、N-甲基丙氨酸、N-甲基甘氨酸、N甲基異亮氨酸、N-甲基戊基甘氨酸、N-甲基纈氨酸、萘丙氨酸、正纈氨酸、正亮氨酸、鳥(niǎo)氨酸、戊基甘氨酸、2哌啶酸和硫代脯氨酸。優(yōu)選地，本發(fā)明所述Exendin類似物包含1個(gè)、2個(gè)、3個(gè)、4個(gè)或5個(gè)氨基酸置換。
本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列增加和刪減一個(gè)或多個(gè)氨基酸得到的類似多肽。在本發(fā)明一優(yōu)選的實(shí)施方案中，本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列增加和刪減1個(gè)至20個(gè)氨基酸得到的類似多肽。再優(yōu)選地，本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列增加和刪減1個(gè)至15個(gè)氨基酸得到的類似多肽。進(jìn)一步優(yōu)選地，本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列增加和刪減1個(gè)至10個(gè)氨基酸得到的類似多肽。在本發(fā)明具體實(shí)施方案中，本發(fā)明所述的Exendin類似物包括那些對(duì)Exendin-3或Exendin-4的氨基酸序列增加和刪減1個(gè)、2個(gè)、3個(gè)、4個(gè)、5個(gè)、6個(gè)、7個(gè)、8個(gè)、9個(gè)或10個(gè)氨基酸得到的類似多肽。
Exendin類似物包括在Exendin-3或Exendin-4的氨基酸序列的N末端氨基、C末端羧基或其側(cè)鏈基團(tuán)可逆或不可逆的化學(xué)封閉或修飾，例如C末端羧基的酰胺化。
在本發(fā)明另一優(yōu)選的實(shí)施方案中，本發(fā)明所述的Exendin或其類似物包括SEQ ID NOs1-265的氨基酸序列。優(yōu)選地，本發(fā)明所述的Exendin或其類似物包括SEQ ID NOs1-2的氨基酸序列。另一優(yōu)選地，本發(fā)明所述的Exendin或其類似物包括SEQ ID NOs3-229的氨基酸序列。另一優(yōu)選地，本發(fā)明所述的Exendin或其類似物包括SEQ ID NOs230-265的氨基酸序列。
聚乙二醇修飾的Exendin或其類似物可以根據(jù)鞏秋紅等(中華內(nèi)分泌代謝雜志2004，20，559-560)發(fā)表的實(shí)驗(yàn)方法在細(xì)胞水平上進(jìn)行活性測(cè)定。具體地，可以在INS-1細(xì)胞中分別加入不同濃度的葡萄糖和含本發(fā)明聚乙二醇修飾的Exendin及其類似物的溶液，孵育4小時(shí)，用放射免疫法測(cè)定上清液中胰島素含量，并利用RT-PCR技術(shù)對(duì)INS-1細(xì)胞的胰島素mRNA水平作半定量分析。通過(guò)這種方法可以在體外大量篩選聚乙二醇修飾的Exendin及其類似物的活性。另外，還可以通過(guò)測(cè)定小鼠(例如C57或db/db鼠)給藥后在不同時(shí)間點(diǎn)的血糖變化情況來(lái)進(jìn)行篩選。
藥用制劑及其使用方法 本發(fā)明所述的聚乙二醇修飾的Exendin或其類似物可以與各種無(wú)機(jī)和有機(jī)酸和堿形成鹽。這類鹽包括用有機(jī)酸和無(wú)機(jī)酸制備的鹽，所述有機(jī)酸和無(wú)機(jī)酸包括但不限于鹽酸、溴化氫、硫酸、磷酸、三氟乙酸、乙酸、甲酸、甲磺酸、甲苯磺酸、馬來(lái)酸、富馬酸和樟腦磺酸。用堿制備的鹽包括但不限于銨鹽、堿金屬鹽(例如鈉鹽和鉀鹽)和堿土金屬鹽(例如鈣鹽和鎂鹽)。優(yōu)選乙酸鹽、鹽酸鹽和三氟乙酸鹽。通過(guò)常規(guī)方法，例如通過(guò)在所述鹽不溶于其中的溶劑或介質(zhì)、或在隨后可真空除去或通過(guò)經(jīng)冷凍干燥除去的溶劑(例如水)中，使所述產(chǎn)物的游離酸或堿形式與一種或更多種適當(dāng)堿或酸的等同物反應(yīng)，或通過(guò)在合適離子交換樹(shù)脂上用另一種離子交換現(xiàn)有鹽的離子，可以制備所述鹽。
所述要求保護(hù)的化合物也可以配制成藥學(xué)上可接受的鹽(例如酸加成鹽)和/或它們的絡(luò)合物。這類鹽制劑，可以通過(guò)改變所述組合物的物理及化學(xué)特性而有助于藥理學(xué)應(yīng)用，但是不防礙所述組合物發(fā)揮其生理學(xué)效應(yīng)。物理特性方面改變的實(shí)例包括降低熔點(diǎn)，以易于經(jīng)粘膜給藥；以及增加溶解度，以易于給予更高濃度的所述藥物。
藥學(xué)上可接受的鹽包括酸加成鹽，例如含有硫酸鹽、鹽酸鹽、磷酸鹽、氨基磺酸鹽、乙酸鹽、檸檬酸鹽、乳酸鹽、酒石酸鹽、甲磺酸鹽、乙磺酸鹽、苯磺酸鹽、對(duì)甲苯磺酸鹽、環(huán)己氨基磺酸鹽和奎尼酸鹽的那些酸加成鹽。藥學(xué)上可接受的鹽可以得自各種無(wú)機(jī)和有機(jī)酸例如鹽酸、硫酸、磷酸、氨基磺酸、乙酸、檸檬酸、乳酸、酒石酸、丙二酸、甲磺酸、乙磺酸、苯磺酸、對(duì)甲苯磺酸、環(huán)己氨基磺酸和奎尼酸。例如通過(guò)在所述鹽不溶于其中的溶劑或介質(zhì)、或在隨后可真空除去或通過(guò)經(jīng)冷凍干燥除去的溶劑(例如水)中，使所述產(chǎn)物的游離酸或堿形式與一種或更多種適當(dāng)堿或酸的等同物反應(yīng)，或通過(guò)在合適離子交換樹(shù)脂上用另一種離子交換現(xiàn)有鹽的離子，可以制備所述鹽。
也可以使用載體或賦形劑，以易于給予所述化合物。載體和賦形劑的實(shí)例包括碳酸鈣、磷酸鈣、各種糖(例如乳糖、葡萄糖或蔗糖)或各種類型的淀粉、纖維素衍生物、明膠、植物油(例如芝麻油、花生油、橄欖油)、聚乙二醇和生理上相容的溶劑?？梢酝ㄟ^(guò)不同的途徑，包括靜脈內(nèi)、腹膜內(nèi)、皮下、和肌內(nèi)、口服、局部或經(jīng)粘膜途徑，給予所述組合物或藥用組合物。
如有需要，上述組合物的溶液可以用增稠劑(例如甲基纖維素)增稠。它們可以乳化形式(比如油包水或者水包油)制備?？梢允褂酶鞣N各樣的藥學(xué)上可接受的乳化劑中的任一種，包括例如金合歡膠粉、非離子型表面活性劑(例如吐溫)、或離子型表面活性劑(例如堿性聚醚醇硫酸酯或磺酸酯，例如Triton)。
這些組合物可以通過(guò)常規(guī)滅菌技術(shù)滅茵或者可以過(guò)濾除茵。所述組合物可以含有需要的近似生理?xiàng)l件的藥學(xué)上可接受的輔助物質(zhì)，例如pH緩沖劑。有效的緩沖液包括例如乙酸鈉/乙酸緩沖液?？梢允褂盟▌┬问胶汀熬忈屝汀迓尫胖苿┑男问剑沟弥委熡行Я康闹苿┰诮?jīng)皮注射或傳遞后許多小時(shí)或許多天內(nèi)被傳遞至血流中。
可以用氯化鈉或其它藥學(xué)上可接受的試劑，例如葡萄糖、硼酸、酒石酸鈉、丙二醇、多元醇(例如甘露醇和山梨糖醇)或其它無(wú)機(jī)溶質(zhì)或有機(jī)溶質(zhì)，達(dá)到所需的等滲性。對(duì)于含鈉離子的緩沖液特別優(yōu)選氯化鈉。
對(duì)于一名體重為70kg患者而言，所述化合物的有效劑量通常的范圍是0.01mg/天或0.03mg/天至約5mg/天，優(yōu)選約0.01或0.5至2mg/天，更優(yōu)選約0.01或0.1至1mg/天，以單劑量或分次劑量給予。將給予的精確劑量由主治醫(yī)師確定，并且依賴于所述特定化合物是否在上述提供的范圍內(nèi)以及取決于個(gè)體的年齡、體重和病癥。給藥應(yīng)在糖尿病剛出現(xiàn)癥狀時(shí)開(kāi)始，或在診斷糖尿病后立即開(kāi)始。
雖然通常將所述化合物用于治療人類患者，但也可以用它們治療其它脊椎動(dòng)物例如其它靈長(zhǎng)類、家畜(例如豬、牛和家禽)以及運(yùn)動(dòng)用動(dòng)物和寵物(例如馬、狗和貓)中的相同或相似疾病。



附圖1是帶式I所示支鏈聚乙二醇結(jié)構(gòu)。
附圖2是帶式III所示支鏈聚乙二醇結(jié)構(gòu)。
附圖3是分子量為40,000道爾頓的聚乙二醇(式IV)取代的Exendin-4類似物的分離純化色譜圖。其中標(biāo)記1為上樣時(shí)的吸收峰，標(biāo)記2為聚乙二醇多取代Exendin-4類似物的洗脫吸收峰，標(biāo)記3為聚乙二醇單取代Exendin-4類似物的洗脫吸收峰，標(biāo)記4為未結(jié)合聚乙二醇Exendin-4類似物的洗脫吸收峰。
附圖4是分子量為40,000道爾頓的聚乙二醇(式IV)單取代Exendin-4類似物電泳圖。其中標(biāo)記1道顯示聚乙二醇單取代的Exendin-4類似物的條帶。
附圖5是經(jīng)分子量為5,000道爾頓和21,000道爾頓直線型聚乙二醇單取代Exendin-4類似物24小時(shí)動(dòng)物藥效試驗(yàn)結(jié)果。
附圖6是經(jīng)分子量為21,000道爾頓直線型和分子量為40,000道爾頓聚乙二醇(式IV)單取代Exendin-4類似物72小時(shí)動(dòng)物藥效試驗(yàn)結(jié)果。
附圖7是經(jīng)分子量分別為21,000道爾頓(U21K)、30,000道爾頓(U30K)和40,000道爾頓(U40K)聚乙二醇(式II)單取代的Exendin-4類似物72小時(shí)動(dòng)物藥效試驗(yàn)結(jié)果。
附圖8是經(jīng)分子量分別為21,000道爾頓(Y21K)、30,000道爾頓(Y30K)和40,000道爾頓(Y40K)聚乙二醇(式IV)單取代Exendin 4類似物72小時(shí)動(dòng)物藥效試驗(yàn)結(jié)果。
附圖9是經(jīng)分子量分別為21,000道爾頓(L21K)和30,000道爾頓(L30K)直線型聚乙二醇單取代Exendin-4類似物的72小時(shí)動(dòng)物藥效試驗(yàn)結(jié)果。
附圖10是分別經(jīng)分子量為30,000道爾頓的聚乙二醇(式II)單取代和經(jīng)分子量為40,000道爾頓的聚乙二醇(式IV)單取代的Exendin-4類似物72小時(shí)動(dòng)物藥代(血漿半衰期)試驗(yàn)結(jié)果。

具體實(shí)施例方式 本實(shí)驗(yàn)所用的聚乙二醇衍生物中，分子量為5,000道爾頓的直線型聚乙二醇購(gòu)自西格瑪奧德里奇公司(Sigma-Aldrich)，其它聚乙二醇衍生物均購(gòu)自北京鍵凱科技有限公司。
實(shí)驗(yàn)中的離子交換法所用A、B雙泵AKTA purifier 100分離純化設(shè)備，其它預(yù)裝柱及填料等均購(gòu)自美國(guó)通用電器公司(General Electric)，其它相關(guān)化學(xué)試劑購(gòu)自西格瑪奧德里奇公司。實(shí)施例三到實(shí)施例九中所用的Exendin-4及其類似物購(gòu)自成都圣諾科技發(fā)展有限公司。
實(shí)施例一Exendin-4類似物的固相化學(xué)合成 本實(shí)施例介紹用固相化學(xué)的方法合成具有下列結(jié)構(gòu)的Exendin-4類似物 [His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Arg-Glu-Glu-Glu-Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH](SEQID NO232)。
(1)所采用的氨基酸單體 上式中縮寫表示 Fmoc9-芴基甲氧羰基 BOC叔丁氧羰基(tert-butyloxycarbonyl) Trt三苯甲基(trityl) OtBu叔丁基酯 tBu叔丁基(tert-butyl) (2)合成所采用的儀器設(shè)備及試劑 儀器為Applied Biosystem多肽合成儀，433A型，美國(guó) 試劑有N-甲基吡咯烷酮，二氯甲烷，六氫吡啶，甲醇，二甲氨基吡啶(Dimethylaminopyridine)/DMF，N，N-二異丙基乙胺(N，N-diisopropylethylamine)/NMP，HBTU 100mmole/0.5M HOBT in DMF，N，N-二環(huán)乙基碳二亞胺(N，N-Dicyclohexylcarbodiimide)/NMP。
其中DMF為N，N-二甲基甲酰胺(N，N-Dimethylformamide)， NMP為N-甲基吡咯烷酮(N-methylpyrrolidone)， HOBT為1-羥基苯并三唑(1-Hydroxybenzotriazole)， HBTU為2-(1氫-苯并三唑基)-四甲基脲六氟磷酸鹽 (2-1H-benzotriazole-y1-1，1，3，3-tetramethyl-Uronium hexafluorophosphate)。
(3)操作 a.合成 以0.25mmol規(guī)模為例，稱取HMP樹(shù)脂0.25g，置入多肽合成儀上的反應(yīng)器中，將各種帶保護(hù)基氨基酸稱取1mmol裝瓶，按該促胰島素分泌肽衍生物的氨基酸序列從C-端向N-端排列在合成儀中，于25℃室溫條件下，由計(jì)算機(jī)程序控制自動(dòng)進(jìn)行脫Fmoc保護(hù)、活化、連結(jié)，然后再進(jìn)行下一輪循環(huán)，如此完成合成，合成結(jié)束后，將得到的帶側(cè)鏈保護(hù)基的多肽樹(shù)脂在多肽合成儀上吹干后稱重即可。
b.脫保護(hù)基及切斷樹(shù)脂 將帶保護(hù)基的多肽樹(shù)脂置于具塞三角燒瓶，加入裂解試劑如下表 然后在恒溫30℃條件下，電磁攪拌反應(yīng)6小時(shí)；過(guò)濾、收集濾液，樹(shù)脂用少量三氟乙酸洗滌；合并收集液與洗滌液，加入乙醚產(chǎn)生沉淀，過(guò)濾，沉淀用少量乙醚洗滌后放入干燥器中干燥，得到粗品。
c.HPLC分離純化、凍干 將得到的粗品用制備型HPLC進(jìn)行分離、提純，最后經(jīng)冷凍干燥得到產(chǎn)品，經(jīng)色質(zhì)聯(lián)用檢測(cè)，該衍生物分子量理論值為4300.6，實(shí)測(cè)值為4316.7。
同樣地，本領(lǐng)域技術(shù)人員可以采用相同的方法合成其它Exendin或其類似物。
實(shí)施例二Exendin-4類似物的基因工程法制備 本實(shí)施例介紹用基因工程的方法合成具有下列結(jié)構(gòu)的Exendin-4類似物 [His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Leu-Glu-Glu-Glu-Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Arg-OH] (SEQ ID NO251)。
A.按此Exendin-4類似物的氨基酸序列，合成基因片段 (1)5’AAT TCC ATG CAC GGC GAA ACC TTC ACC AGC GAT CTG AGC AAA CAG CTG GAA GAA GAAGCG GTT AA(SEQ ID NO266) (2)5’ACTG TTC ATC GAA TGG CTG AAA AAC GGC GGC CCG AGC AGC GGC CCG CCG CCG CCG AGCCGT TAG A(SEQ ID NO267) (3)5’AGCTT CTA ACG GCT CGG CGG CGG CGC GCT GCT CGG GCC GCC GTT TTT CAG CCA TTC GATGA(SEQ ID NO268) (4)5’ACAG TTT AAC CGC TTC TTC TTC CAG CTG TTT GCT CAG ATC GCT GGT GAA GGT GCC TTCGCC GTG CAT GG(SEQ ID NO269) B、克隆 連接取二支試管，一支加入光密度值為0.1(A260nm)的片段(1)和片段(4)，另一支試管中加入光密度值為(A260nm)的片段(2)和片段(3)，再將聚核苷酸激酶緩沖液(PolynucleotideKinase Buffer)，聚核苷酸激酶(Polynucleotide Kinase)及三磷酸腺苷(ATP)分別加入該二支試管，于37℃保溫60分鐘，使基因片段5’端磷酸化。然后，將二支試管轉(zhuǎn)移至95℃水浴中保溫10分鐘，停止加溫，自然冷卻退火降至室溫。再將T4連接酶緩沖液和T4連接酶分別加入該二支試管中，16℃左右保溫。過(guò)夜，使基團(tuán)片段連接。
質(zhì)粒另取一支試管將含有乳糖啟動(dòng)子Lac(或溫控啟動(dòng)子PL，色氨酸-乳糖混合啟動(dòng)子Tac)的質(zhì)粒用限制性內(nèi)切酶EcoR I和Hind III雙酶酶切后用酚/氯仿抽提，離心取水層部分，再用氯仿抽提，離心抽取水層部分，再用氯仿將水層洗滌3次，離心，將水層用異丙醇沉淀，離心，干燥。
將上述酶切質(zhì)粒與連接后的基因片段混合，加T4連接酶緩沖液和T4連接酶，室溫連接3-4小時(shí)。
受菌體的培養(yǎng)克隆菌大腸桿菌(E.Coli)JM103于每升含蛋白胨10g、酵母抽提粉5g、氯化鈉5g的培養(yǎng)液(LB培養(yǎng)液)中37℃振蕩培養(yǎng)4小時(shí)，離心收集菌體，經(jīng)CaCl2溶液處理后，4℃保存?zhèn)溆谩?br> 轉(zhuǎn)化將克隆的質(zhì)粒轉(zhuǎn)化至受體菌大腸桿菌JM103細(xì)胞中，冰浴維持30分鐘，升溫至42℃，維持2分鐘后，轉(zhuǎn)移至含氨芐青霉素的瓊脂糖平板，37℃過(guò)夜，進(jìn)行菌落篩選，得克隆陽(yáng)性質(zhì)粒。
C、發(fā)酵 將篩選得含該種衍生物質(zhì)粒的寄主菌株于LB培養(yǎng)液中振蕩培養(yǎng)，加0.5mM異丙基硫代半乳糖苷(IPTG)誘導(dǎo)，過(guò)夜后，離心收集菌體，以十二烷基磺酸鈉＝12％的丙烯酰胺凝膠(PAGE)電泳，鑒定表達(dá)的蛋白。
D、包涵體 按上述條件，300ml×10瓶，搖床培養(yǎng)，誘導(dǎo)，收集的菌體加裂解液(1％NaCl，磷酸緩沖液pH7.5，20mM)及溶菌酶，30℃保溫30分鐘，離心收集沉淀，加6M鹽酸胍(Gu.HCl)抽提包涵體，離心，將上清液透析，除去Gu.IICL，再用組成為1％NaCl，0.1％吐溫80(Tween 80)的磷酸緩沖液(20mM，PH7.5)洗滌沉淀三次，即得包涵體。
E、裂解 將包涵體溶于8M尿素溶液，加鹽酸至50mM，加溴化氰，避光，氮?dú)獗Ｗo(hù)條件下攪拌，裂解2小時(shí)，以HPLC跟蹤分析。
F、純化 裂解完成，通過(guò)瓊脂糖凝膠分子篩G-25分離得粗品，再經(jīng)HPLC純化，得產(chǎn)品，與化學(xué)合成品相同，質(zhì)譜分析結(jié)果表明所得衍生物分子量與理論值相吻合。
實(shí)施例三直線型單甲氧基聚乙二醇(分子量＝5,000道爾頓)修飾Exendin-4 每個(gè)試管中稱取1.0mg Exendin-4，共三個(gè)試管。分別使用不同pH值的磷酸緩沖液溶解，再在每個(gè)試管中加入N羥基琥珀酰亞胺活化的單甲氧基聚乙二醇(分子量＝5,000道爾頓)5.8mg，置于搖床，室溫反應(yīng)1小時(shí)。之后使用安捷倫1100系列色譜儀(Agilent 1100)分析樣品(分析條件為采用0.1％磷酸流動(dòng)相A和0.1％磷酸+80％乙腈流動(dòng)相B，梯度為35-70％流動(dòng)相B/25分鐘)，檢測(cè)沒(méi)有被單甲氧基聚乙二醇修飾的Exendin-4的量。結(jié)果如下表 表不同pH對(duì)Exendin-4聚乙二醇化的影響 使用旋轉(zhuǎn)蒸發(fā)器對(duì)反相分離得到的收集液進(jìn)行去除乙腈處理后，可以獲得pH為3-4，溶解有單甲氧基聚乙二醇修飾的Exendin-4的溶液。
實(shí)施例四直線型單甲氧基聚乙二醇(分子量＝21,000道爾頓)修飾Exendin-4類似物 使用梅特勒(AG285，Mettler Toledo)天平先準(zhǔn)確稱量1.0mg Exendin-4類似物(SEQ INNO251)，加入2.0ml溶解有氰硼氫化鈉(NaBH3CN)磷酸緩沖液(pH4.5)，再準(zhǔn)確稱取經(jīng)由醛基活化的分子量為21,000道爾頓的單甲氧基聚乙二醇衍生物5mg。將衍生物加入Exendin-4類似物，置于搖床，室溫反應(yīng)過(guò)夜。使用配有Zorbax SB-300半制備柱的安捷倫1100色譜儀對(duì)反應(yīng)液進(jìn)行純化(分離條件為采用0.1％磷酸流動(dòng)相A和0.1％磷酸+80％乙腈流動(dòng)相B，梯度為35-70％流動(dòng)相B/25分鐘)，獲得聚乙二醇N末端修飾的Exendin-4類似物。
使用旋轉(zhuǎn)蒸發(fā)器對(duì)反相分離得到的收集液進(jìn)行去除乙腈處理后，使用脫鹽柱對(duì)樣品進(jìn)行緩沖液置換，將得到的聚乙二醇修飾的Exendin-4類似物溶解于pH為7.0-8.0的磷酸鹽緩沖液，并加入0.001-1.0％(w/v)間甲酚。
實(shí)施例五帶式IV結(jié)構(gòu)單甲氧基聚乙二醇(分子量＝40,000道爾頓)修飾Exendin-4類似物 使用梅特勒(AG285，Mettler Toledo)天平先準(zhǔn)確稱量2.0mg Exendin-4類似物(SEQ INNO251)，加入4.0ml pH7.0磷酸緩沖液，再準(zhǔn)確稱取分子量為40,000道爾頓的N羥基琥珀酰亞胺活化的帶式IV結(jié)構(gòu)單甲氧基聚乙二醇衍生物280mg。將衍生物加入Exendin-4類似物，置于搖床，室溫反應(yīng)1小時(shí)。使用200mlBistris緩沖液(pH＝7.0)稀釋反應(yīng)溶液于250ml燒杯中，待用。
使用Bistris緩沖液(pH＝7.0)平衡DEAE FF或ANX FF陰離子交換預(yù)裝柱，將稀釋后的反應(yīng)液上樣，使目標(biāo)物質(zhì)吸附到離子交換填料上。上樣完成后采用線性梯度洗脫的方式，使用不同濃度的NaCl將多取代和單取代Exendin-4類似物分別洗脫下來(lái)，同時(shí)使用餾分收集器收集，最后使用1M NaCl將未修飾的Exendin-4多肽洗脫下來(lái)。整個(gè)分離純化過(guò)程如圖3所示，圖上標(biāo)記2、3和4分別顯示多取代Exendin-4類似物、單取代Exendin-4類似物和未修飾的Exendin-4類似物的洗脫峰。將收集得到的聚乙二醇單取代Exendin-4類似物(理論分子量為44,300道爾頓)做SDS-PAGE凝膠電泳得到的實(shí)際分子量略大于43,000道爾頓的標(biāo)準(zhǔn)分子量(如圖4所示)，說(shuō)明分離獲得的產(chǎn)品確實(shí)是聚乙二醇(分子量＝40,000道爾頓)單取代Exendin-4類似物。
使用超濾裝置(超濾離心管或超濾設(shè)備)對(duì)收集到的聚乙二醇單取代Exendin-4類似物洗脫液進(jìn)行濃縮，之后再將濃縮液過(guò)脫鹽柱進(jìn)行乙酸緩沖液置換，最后在溶解有樣品的乙酸緩沖液中加入等滲調(diào)節(jié)劑(如甘露醇和0.9％NaCl)和0.001-1.0％(w/v)抑菌劑(如間甲酚)等。
實(shí)施例六經(jīng)5,000道爾頓和21,000道爾頓直線型單甲氧基聚乙二醇單取代Exendin-4類似物24小時(shí)動(dòng)物藥效試驗(yàn) 實(shí)驗(yàn)動(dòng)物C57小鼠，體重20g±2g，每組8只動(dòng)物，共4組。
對(duì)照組1每只皮下注射注射用水。
對(duì)照組2每只皮下注射Exendin-4類似物0.025μg。
給藥組1每只皮下注射單甲氧基聚乙二醇(分子量＝5,000道爾頓)單取代Exendin-4類似物0.625μg。
給藥組2每只皮下注射單甲氧基聚乙二醇(分子量＝21,000道爾頓)單取代Exendin-4類似物0.625μg。
每組小鼠分別在給藥后4、8、12、16和24小時(shí)取血，并在采血前30min給所有受試小鼠腹腔注射200μl的20％葡萄糖溶液。使用葡萄糖試劑盒(上海申索試劑有限公司)對(duì)血糖進(jìn)行測(cè)定。結(jié)果如圖5所示，經(jīng)單甲氧基聚乙二醇(分子量＝21,000道爾頓)修飾的Exendin-4類似物單次給藥后在16小時(shí)能保持藥效；單甲氧基聚乙二醇(分子量＝5,000道爾頓)修飾的Exendin-4類似物單次給藥后能保持藥效的時(shí)間范圍在12-16小時(shí)；而未經(jīng)修飾的Exendin-4類似物的作用時(shí)間不超過(guò)12小時(shí)。實(shí)驗(yàn)結(jié)果顯示，分子量為21,000道爾頓的直線型聚乙二醇修飾的Exendin-4類似物的降糖作用保持時(shí)間比分子量為5,000道爾頓的直線型聚乙二醇修飾的Exendin-4類似物長(zhǎng)。
實(shí)施例七經(jīng)21,000道爾頓(直線型)和40,000道爾頓(式IV所示支鏈結(jié)構(gòu))單甲氧基聚乙二醇單取代Exendin-4類似物72小時(shí)動(dòng)物藥效試驗(yàn) 實(shí)驗(yàn)動(dòng)物C57小鼠，體重20g±2g，每組8只動(dòng)物，共5組。
對(duì)照組1每只皮下注射注射用水。
對(duì)照組2每只皮下注射Exendin-4多肽0.025μg。
對(duì)照組3每只皮下注射Exendin-4類似物0.025μg。
給藥組1每只皮下注射單甲氧基聚乙二醇(分子量＝21,000道爾頓)取代Exendin-4類似物0.625μg。
給藥組2每只皮下注射單甲氧基聚乙二醇(分子量＝40,000道爾頓)取代Exendin-4類似物0.625μg。
每組小鼠分別在給藥后4、8、24、48和72小時(shí)取血，并在采血前30min給所有受試小鼠腹腔注射200μl的20％葡萄糖溶液。使用葡萄糖試劑盒對(duì)血糖進(jìn)行測(cè)定。結(jié)果如圖6所示，經(jīng)支鏈型聚乙二醇(分子量＝40,000道爾頓)修飾的Exendin-4類似物單次給藥后能保持至少72小時(shí)的藥效；直線型聚乙二醇(分子量＝21,000道爾頓)修飾的Exendin-4類似物單次給藥后能保持大約24小時(shí)的藥效。
實(shí)施例八單甲氧基聚乙二醇單取代Exendin-4類似物藥效試驗(yàn) 試驗(yàn)樣品總共8個(gè)樣品，分別為Exendin-4類似物(SEQ ID NO251)上單取代有分子量為21,000道爾頓，30,000道爾頓和40,000道爾頓的單甲氧基聚乙二醇(式II)；Exendin-4類似物上單取代有分子量為21,000道爾頓，30,000道爾頓和40,000道爾頓的單甲氧基聚乙二醇(式IV)；Exendin-4類似物上單取代有分子量為21,000道爾頓和30,000道爾頓的直線型單甲氧基聚乙二醇。
實(shí)驗(yàn)動(dòng)物C57小鼠，體重20g±2g，共72只，分為3個(gè)采血組，每組24只。
由于動(dòng)物不耐受多次采血，對(duì)三組動(dòng)物分不同時(shí)間段采血，給藥后對(duì)應(yīng)的采血時(shí)間點(diǎn)如下 每個(gè)采血組再平均分為8個(gè)樣品小組，每小組3只老鼠。每小組老鼠在試驗(yàn)開(kāi)始時(shí)皮下注射含0.625μg Exendin-4類似物分子的不同類型單甲氧基聚乙二醇單取代Exendin-4類似物，研發(fā)代號(hào)為L(zhǎng)21K、L30K(分子量分別為21,000、30,000道爾頓的直線型單甲氧基聚乙二醇單取代)，Y21K、Y30K、Y40K(分子量分別為21,000、30,000、40,000道爾頓的式IV結(jié)構(gòu)單甲氧基聚乙二醇單取代)，U21K、U30K和U40K(分子量分別為21,000、30,000、40,000道爾頓的式II結(jié)構(gòu)單甲氧基聚乙二醇單取代)。給藥同時(shí)給所有受試小鼠腹腔注射200μl 20％葡萄糖溶液，并在給藥之后0.5小時(shí)對(duì)所有受試?yán)鲜蟛裳?。然后再按上述分組分別在給藥后2、4、8、16、24、36、48、60和72小時(shí)采血。每次采血前0.5小時(shí)對(duì)所有采血小鼠腹腔注射200μl 20％葡萄糖溶液。結(jié)果如圖7、圖8和圖9所示，顯示聚乙二醇分子結(jié)構(gòu)和分子量對(duì)聚乙二醇單取代Exendin-4類似物的降糖生物活性的影響很大。式II結(jié)構(gòu)單甲氧基聚乙二醇單取代Exendin-4類似物在給藥后的72小時(shí)中，降糖活性的穩(wěn)定性要好于直線型及式IV結(jié)構(gòu)單甲氧基聚乙二醇單取代Exendin-4類似物；而同樣結(jié)構(gòu)的支鏈型單甲氧基聚乙二醇單取代Exendin-4類似物中，分子量越大，其在第72小時(shí)降糖活性越好。直線型單甲氧基聚乙二醇單取代Exendin-4類似物中L30K在給藥后的72小時(shí)中均無(wú)明顯降糖活性，說(shuō)明直線型聚乙二醇分子量太大對(duì)其所修飾的Exendin-4類似物的降糖活性有負(fù)面影響。
實(shí)施例九單甲氧基聚乙二醇單取代的Exendin-4類似物藥代(血漿半衰期)試驗(yàn)實(shí)驗(yàn)動(dòng)物SD大鼠，雄性，體重250g-300g，每組4只，共兩組。
給藥組1每只皮下注射含4.375μg Exendin-4類似物分子的單甲氧基聚乙二醇(式II，分子量＝30,000道爾頓)單取代Exendin-4類似物(SEQ ID N0251)，研發(fā)代號(hào)U30K。
給藥組2每只皮下注射含4.375μg Exendin-4類似物分子的單甲氧基聚乙二醇(式IV，分子量＝40,000道爾頓)單取代Exendin-4類似物(SEQ ID N0251)，研發(fā)代號(hào)Y40K。
每組小鼠分別在給藥后0、2、4、8、12、16、24、36、48、60和72小時(shí)采血。血藥濃度檢測(cè)使用美國(guó)康肽公司(Phoenix Pharmaceuticals，Inc.，California，USA)Enzyme Immunoassaykit(EIA)試劑盒檢測(cè)Exendin-4濃度。結(jié)果如圖10所示，經(jīng)使用上海宏能軟件有限公司的計(jì)算機(jī)軟件Pharmaceutical Kinetics Software(版本1.0.2)計(jì)算，U30K的吸收半衰期為10.78小時(shí)，消除半衰期為21.44小時(shí)，Y40K的吸收半衰期為5.53小時(shí)，消除半衰期為40.77小時(shí)，兩個(gè)樣品的達(dá)峰時(shí)間均為給藥后第12小時(shí)左右，最大藥峰濃度均為20ng/mL左右。
序列表
<110>上海華誼生物技術(shù)有限公司
<120>聚乙二醇修飾的Exendin或其類似物及其制劑
<130>GHY001
<160>269
<170>PatentIn version 3.3
<210>1
<211>39
<212>PRT
<213>Exendin-3
<400>1
His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>2
<211>39
<212>PRT
<213>Exendin-4
<400>2
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>3
<211>30
<212>PRT
<213>Exendin類似物1
<400>3
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210>4
<211>30
<212>PRT
<213>Exendin類似物2
<400>4
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210>5
<211>28
<212>PRT
<213>Exendin類似物3
<400>5
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>6
<211>39
<212>PRT
<213>Exendin類似物4
<400>6
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu PheIle Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>7
<211>28
<212>PRT
<213>Exendin類似物5
<400>7
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>8
<211>28
<212>PRT
<213>Exendin類似物6
<400>8
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Ala Ile Glu Phe Leu Lys Asn
20 25
<210>9
<211>18
<212>PRT
<213>Exendin類似物7
<220>
<221>misc_feature
<222>(9)..(9)
<223>Xaa stands for Pgly
<400>9
His Gly Glu Phe Thr Ser Asp Leu Xaa Phe Ile Glu Phe Pro Pro Pro
1 5 10 15
Pro Ser
<210>10
<211>18
<212>PRT
<213>Exendin類似物8
<220>
<221>misc_feature
<222>(10)..(10)
<223>Xaa stands for naph
<400>10
His Gly Glu Phe Thr Ser Asp Leu Met Xaa Ile Glu Trp Pro Pro Pro
1 5 10 15
Pro Ser
<210>11
<211>20
<212>PRT
<213>Exendin類似物9
<400>11
His Gly Glu Phe Thr Ser Asp Leu Met Phe Val Ala Leu Glu Trp Pro
1 5 10 15
Pro Pro Pro Ser
20
<210>12
<211>20
<212>PRT
<213>Exendin類似物10
<400>12
His Gly Glu Phe Thr Ser Asp Leu Leu Phe Val Ala Leu Glu Phe Pro
1 5 10 15
Pro Pro Pro Ser
20
<210>13
<211>18
<212>PRT
<213>Exendin類似物11
<220>
<221>misc_feature
<222>(11)..(11)
<223>Xaa stands for tBug
<400>13
His Gly Glu Phe Thr Ser Asp Leu Met Phe Xaa Glu Trp Pro Pro Pro
1 5 10 15
Pro Ser
<210>14
<211>18
<212>PRT
<213>Exendin類似物12
<220>
<221>misc_feature
<222>(11)..(11)
<223>Xaa stands for tBug
<400>14
His Gly Glu Phe Thr Ser Asp Leu Leu Phe Xaa Glu Phe Pro Pro Pro
1 5 10 15
Pro Ser
<210>15
<211>18
<212>PRT
<213>Exendin類似物13
<400>15
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Asp Trp Pro Pro Pro
1 5 10 15
Pro Ser
<210>16
<211>18
<212>PRT
<213>Exendin類似物14
<400>16
His Ala Glu Phe Thr Ser Asp Leu Leu Phe Ile Glu Phe Pro Pro Pro
1 5 10 15
Pro Ser
<210>17
<211>18
<212>PRT
<213>Exendin類似物15
<220>
<221>misc_feature
<222>(14)..(17)
<223>Xaa stands for hPro
<400>17
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Xaa Xaa Xaa
1 5 10 15
Xaa Ser
<210>18
<211>18
<212>PRT
<213>Exendin類似物16
<220>
<221>misc_feature
<222>(15)..(17)
<223>Xaa stands for hPro
<400>18
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Pro Xaa Xaa
1 5 10 15
Xaa Ser
<210>19
<211>17
<212>PRT
<213>Exendin類似物17
<220>
<221>misc_feature
<222>(14)..(16)
<223>Xaa stands for hPro
<400>19
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Xaa Xaa Xaa
1 5 10 15
Ser
<210>20
<211>18
<212>PRT
<213>Exendin類似物18
<220>
<221>misc_feature
<222>(15)..(17)
<223>Xaa stands for hPro
<400>20
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Pro Xaa Xaa
1 5 10 15
Xaa Ser
<210>21
<211>18
<212>PRT
<213>Exendin類似物19
<220>
<221>misc_feature
<222>(14)..(17)
<223>Xaa stands for hPro
<400>21
His Gly Glu Phe Thr Ser Asp Leu Leu Phe Ile Glu Phe Xaa Xaa Xaa
1 5 10 15
Xaa Ser
<210>22
<211>18
<212>PRT
<213>Exendin類似物20
<220>
<221>misc_feature
<222>(14)..(17)
<223>Xaa stands for hPro
<400>22
His Gly Glu Phe Thr Ser Asp Leu Leu Phe Ile Glu Phe Xaa Xaa Xaa
1 5 10 15
Xaa Ser
<210>23
<211>18
<212>PRT
<213>Exendin類似物21
<220>
<221>misc_feature
<222>(14)..(17)
<223>Xaa stands for MeAla
<400>23
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Xaa Xaa Xaa
1 5 10 15
Xaa Ser
<210>24
<211>18
<212>PRT
<213>Exendin類似物22
<220>
<221>misc_feature
<222>(15)..(17)
<223>Xaa stands for MeAla
<400>24
His Gly Glu Phe Thr Ser Asp Leu Met Phe Ile Glu Trp Pro Xaa Xaa
1 5 10 15
Xaa Ser
<210>25
<211>18
<212>PRT
<213>Exendin類似物23
<220>
<221>misc_feature
<222>(14)..(17)
<223>Xaa stands for MeAla
<400>25
His Gly Glu Phe Thr Ser Asp Leu Leu Phe Ile Glu Phe Xaa Xaa Xaa
1 5 10 15
Xaa Ser
<210>26
<211>30
<212>PRT
<213>Exendin類似物24
<400>26
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210>27
<211>28
<212>PRT
<213>Exendin類似物25
<400>27
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>28
<211>28
<212>PRT
<213>Exendin類似物26
<400>28
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>29
<211>28
<212>PRT
<213>Exendin類似物27
<400>29
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>30
<211>28
<212>PRT
<213>Exendin類似物28
<400>30
His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>31
<211>28
<212>PRT
<213>Exendin類似物29
<400>31
His Gly Glu Gly Thr Ala Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>32
<211>28
<212>PRT
<213>Exendin類似物30
<400>32
His Gly Glu Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>33
<211>28
<212>PRT
<213>Exendin類似物31
<400>33
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>34
<211>28
<212>PRT
<213>Exendin類似物32
<400>34
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>35
<211>28
<212>PRT
<213>Exendin類似物33
<400>35
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>36
<211>28
<212>PRT
<213>Exendin類似物34
<400>36
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>37
<211>28
<212>PRT
<213>Exendin類似物35
<400>37
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>38
<211>28
<212>PRT
<213>Exendin類似物36
<400>38
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Ala Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>39
<211>28
<212>PRT
<213>Exendin類似物37
<400>39
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Ala
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>40
<211>28
<212>PRT
<213>Exendin類似物38
<400>40
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>41
<211>28
<212>PRT
<213>Exendin類似物39
<400>41
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>42
<211>28
<212>PRT
<213>Exendin類似物40
<400>42
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>43
<211>28
<212>PRT
<213>Exendin類似物41
<400>43
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys Asn
20 25
<210>44
<211>28
<212>PRT
<213>Exendin類似物42
<400>44
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Ala Phe Leu Lys Asn
20 25
<210>45
<211>28
<212>PRT
<213>Exendin類似物43
<400>45
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn
20 25
<210>46
<211>28
<212>PRT
<213>Exendin類似物44
<400>46
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 510 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Ala Lys Asn
20 25
<210>47
<211>28
<212>PRT
<213>Exendin類似物45
<400>47
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala Asn
20 25
<210>48
<211>28
<212>PRT
<213>Exendin類似物46
<400>48
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Ala
20 25
<210>49
<211>38
<212>PRT
<213>Exendin類似物47
<400>49
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro
35
<210>50
<211>38
<212>PRT
<213>Exendin類似物48
<400>50
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro
35
<210>51
<211>37
<212>PRT
<213>Exendin類似物49
<400>51
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro
35
<210>52
<211>37
<212>PRT
<213>Exendin類似物50
<400>52
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro
35
<210>53
<211>36
<212>PRT
<213>Exendin類似物51
<400>53
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro
35
<210>54
<211>36
<212>PRT
<213>Exendin類似物52
<400>54
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro
35
<210>55
<211>35
<212>PRT
<213>Exendin類似物53
<400>55
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>56
<211>35
<212>PRT
<213>Exendin類似物54
<400>56
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>57
<211>34
<212>PRT
<213>Exendin類似物55
<400>57
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly
<210>58
<211>34
<212>PRT
<213>Exendin類似物56
<400>58
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly
<210>59
<211>33
<212>PRT
<213>Exendin類似物57
<400>59
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser
<210>60
<211>33
<212>PRT
<213>Exendin類似物58
<400>60
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser
<210>61
<211>32
<212>PRT
<213>Exendin類似物59
<400>61
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
<210>62
<211>32
<212>PRT
<213>Exendin類似物60
<400>62
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
<210>63
<211>31
<212>PRT
<213>Exendin類似物61
<400>63
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
20 25 30
<210>64
<211>31
<212>PRT
<213>Exendin類似物62
<400>64
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
20 25 30
<210>65
<211>30
<212>PRT
<213>Exendin類似物63
<400>65
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly
20 25 30
<210>66
<211>29
<212>PRT
<213>Exendin類似物64
<400>66
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly
20 25
<210>67
<211>29
<212>PRT
<213>Exendin類似物65
<400>67
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly
20 25
<210>68
<211>38
<212>PRT
<213>Exendin類似物66
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for hPro
<220>
<221>misc_feature
<222>(36)..(38)
<223>Xaa stands for hPro
<400>68
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa Xaa
35
<210>69
<211>38
<212>PRT
<213>Exendin類似物67
<220>
<221>misc_feature
<222>(36)..(38)
<223>Xaa stands for hPro
<400>69
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Xaa Xaa Xaa
35
<210>70
<211>37
<212>PRT
<213>Exendin類似物68
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for Nme
<400>70
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Pro Pro
35
<210>71
<211>37
<212>PRT
<213>Exendin類似物69
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for Nme
<220>
<221>misc_feature
<222>(36)..(37)
<223>Xaa stands for Nme
<400>71
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa
35
<210>72
<211>37
<212>PRT
<213>Exendin類似物70
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for hPro
<220>
<221>misc_feature
<222>(36)..(37)
<223>Xaa stands for hPro
<400>72
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa
35
<210>73
<211>36
<212>PRT
<213>Exendin類似物71
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for hPro
<220>
<221>misc_feature
<222>(36>..(36)
<223>Xaa stands for hPro
<400>73
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25v30
Ser Gly Ala Xaa
35
<210>74
<211>35
<212>PRT
<213>Exendin類似物72
<400>74
Arg Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>75
<211>30
<212>PRT
<213>Exendin類似物73
<400>75
His Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210>76
<211>28
<212>PRT
<213>Exendin類似物74
<220>
<221>misc_feature
<222>(6)..(6)
<223>Xaa stands for naph
<400>76
His Gly Glu Gly Thr Xaa Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>77
<211>28
<212>PRT
<213>Exendin類似物75
<400>77
His Gly Glu Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>78
<211>28
<212>PRT
<213>Exendin類似物76
<400>78
His Gly Glu Gly Thr Phe Ser Thr Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>79
<211>28
<212>PRT
<213>Exendin類似物77
<400>79
His Gly Glu Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Ala Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>80
<211>28
<212>PRT
<213>Exendin類似物78
<220>
<221>misc_feature
<222>(10)..(10)
<223>Xaa stands for Pgly
<400>80
His Gly Glu Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>81
<211>28
<212>PRT
<213>Exendin類似物79
<220>
<221>misc_feature
<222>(22)..(22)
<223>Xaa stands for naph
<400>81
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Xaa Ile Glu Phe Leu Lys Asn
20 25
<210>82
<211>28
<212>PRT
<213>Exendin類似物80
<220>
<221>misc_feature
<222>(23)..(23)
<223>Xaa stands for tBug
<400>82
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Xaa Glu Trp Leu Lys Asn
20 25
<210>83
<211>28
<212>PRT
<213>Exendin類似物81
<400>83
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Asp Phe Leu Lys Asn
20 25
<210>84
<211>33
<212>PRT
<213>Exendin類似物82
<400>84
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser
<210>85
<211>29
<212>PRT
<213>Exendin類似物83
<400>85
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly
20 25
<210>86
<211>37
<212>PRT
<213>Exendin類似物84
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for Pgly
<220>
<221>misc_feature
<222>(36)..(37)
<223>Xaa stands for Pgly
<400>86
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa
35
<210>87
<211>27
<212>PRT
<213>Exendin類似物85
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-Imidazolyl Ppionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>87
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn
20 25
<210>88
<211>27
<212>PRT
<213>Exendin類似物86
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>88
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn
20 25
<210>89
<211>29
<212>PRT
<213>Exendin類似物87
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>89
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly
20 25
<210>90
<211>29
<212>PRT
<213>Exendin類似物88
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>90
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn Gly Gly
20 25
<210>91
<211>27
<212>PRT
<213>Exendin類似物89
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for 4-ImidazolylPpionyl G
<400>91
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ilc Glu Trp Leu Asn Xaa
20 25
<210>92
<211>27
<212>PRT
<213>Exendin類似物90
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>92
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa
20 25
<210>93
<211>29
<212>PRT
<213>Exendin類似物91
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>93
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa Gly Gly
20 25
<210>94
<211>29
<212>PRT
<213>Exendin類似物92
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<400>94
Xaa Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa Gly Gly
20 25
<210>95
<211>28
<212>PRT
<213>Exendin類似物93
<400>95
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>96
<211>28
<212>PRT
<213>Exendin類似物94
<400>96
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>97
<211>28
<212>PRT
<213>Exendin類似物95
<400>97
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>98
<211>28
<212>PRT
<213>Exendin類似物96
<400>98
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>99
<211>28
<212>PRT
<213>Exendin類似物97
<400>99
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>100
<211>28
<212>PRT
<213>Exendin類似物98
<400>100
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>101
<211>28
<212>PRT
<213>Exendin類似物99
<400>101
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>102
<211>28
<212>PRT
<213>Exendin類似物100
<400>102
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>103
<211>28
<212>PRT
<213>Exendin類似物101
<400>103
His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>104
<211>28
<212>PRT
<213>Exendin類似物102
<400>104
Ala Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>105
<211>28
<212>PRT
<213>Exendin類似物103
<400>105
Ala Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>106
<211>28
<212>PRT
<213>Exendin類似物104
<400>106
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>107
<211>28
<212>PRT
<213>Exendin類似物105
<400>107
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>108
<211>28
<212>PRT
<213>Exendin類似物106
<400>108
Ala Gly Asp Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>109
<211>28
<212>PRT
<213>Exendin類似物107
<400>109
Ala Gly Asp Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>110
<211>28
<212>PRT
<213>Exendin類似物108
<220>
<221>misc_feature
<222>(6)..(6)
<223>Xaa stands for Nala
<400>110
Ala Gly Asp Gly Thr Xaa Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>111
<211>28
<212>PRT
<213>Exendin類似物109
<220>
<221>misc_feature
<222>(6)..(6)
<223>Xaa stands for Nala
<400>111
Ala Gly Asp Gly Thr Xaa Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>112
<211>28
<212>PRT
<213>Exendin類似物110
<400>112
Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>113
<211>28
<212>PRT
<213>Exendin類似物111
<400>113
Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>114
<211>28
<212>PRT
<213>Exendin類似物112
<400>114
Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>115
<211>28
<212>PRT
<213>Exendin類似物113
<400>115
Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>116
<211>28
<212>PRT
<213>Exendin類似物114
<400>116
Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>117
<211>28
<212>PRT
<213>Exendin類似物115
<400>117
Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>118
<211>28
<212>PRT
<213>Exendin類似物116
<400>118
Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>119
<211>28
<212>PRT
<213>Exendin類似物117
<400>119
Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Leu Glu Glu
15 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>120
<211>28
<212>PRT
<213>Exendin類似物118
<400>120
Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn
20 25
<210>121
<211>28
<212>PRT
<213>Exendin類似物119
<400>121
Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>122
<211>28
<212>PRT
<213>Exendin類似物120
<220>
<221>misc_feature
<222>(10)..(10)
<223>Xaa stands for Pgly
<400>122
Ala Gly Asp Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>123
<211>28
<212>PRT
<213>Exendin類似物121
<220>
<221>misc_feature
<222>(10)..(10)
<223>Xaa stands for Pgly
<400>123
Ala Gly Asp Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>124
<211>28
<212>PRT
<213>Exendin類似物122
<400>124
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>125
<211>28
<212>PRT
<213>Exendin類似物123
<400>125
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>126
<211>28
<212>PRT
<213>Exendin類似物124
<400>126
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>127
<211>28
<212>PRT
<213>Exendin類似物125
<400>127
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>128
<211>28
<212>PRT
<213>Exendin類似物126
<400>128
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>129
<211>28
<212>PRT
<213>Exendin類似物127
<400>129
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>130
<211>28
<212>PRT
<213>Exendin類似物128
<400>130
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>131
<211>28
<212>PRT
<213>Exendin類似物129
<400>131
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>132
<211>28
<212>PRT
<213>Exendin類似物130
<220>
<221>misc_feature
<222>(14)..(14)
<223>Xaa stands for Pgly
<400>132
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>133
<211>28
<212>PRT
<213>Exendin類似物131
<220>
<221>misc_feature
<222>(14)..(14)
<223>Xaa stands for Pgly
<400>133
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>134
<211>28
<212>PRT
<213>Exendin類似物132
<400>134
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Ala Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>135
<211>28
<212>PRT
<213>Exendin類似物133
<400>135
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Ala Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>136
<211>28
<212>PRT
<213>Exendin類似物134
<400>136
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Ala
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>137
<211>28
<212>PRT
<213>Exendin類似物135
<400>137
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Ala
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>138
<211>28
<212>PRT
<213>Exendin類似物136
<400>138
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Ala Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>139
<211>28
<212>PRT
<213>Exendin類似物137
<400>139
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>140
<211>28
<212>PRT
<2l3>Exendin類似物138
<400>140
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Ala Arg Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>141
<211>28
<212>PRT
<213>Exendin類似物139
<400>141
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>142
<211>28
<212>PRT
<213>Exendin類似物140
<400>142
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Ala Leu Phe Ile Glu Trp Leu Lys Asn
20 25
<210>143
<211>28
<212>PRT
<213>Exendin類似物141
<400>143
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys Asn
20 25
<210>144
<211>28
<212>PRT
<213>Exendin類似物142
<400>144
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Ala Phe Ile Glu Trp Leu Lys Asn
20 25
<210>145
<211>28
<212>PRT
<213>Exendin類似物143
<400>145
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys Asn
20 25
<210>146
<211>28
<212>PRT
<213>Exendin類似物144
<220>
<221>misc_feature
<222>(22)..(22)
<223>Xaa stands for Nala
<400>146
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Xaa Ile Glu Trp Leu Lys Asn
20 25
<210>147
<211>28
<212>PRT
<213>Exendin類似物145
<220>
<221>misc_feature
<222>(22)..(22)
<223>Xaa stands for Nala
<400>147
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Xaa Tle Glu Phe Leu Lys Asn
20 25
<210>148
<211>28
<212>PRT
<213>Exendin類似物146
<400>148
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Val Glu Trp Leu Lys Asn
20 25
<210>149
<211>28
<212>PRT
<213>Exendin類似物147
<400>149
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Val Glu Phe Leu Lys Asn
20 25
<210>150
<211>28
<212>PRT
<213>Exendin類似物148
<220>
<221>misc_feature
<222>(23)..(23)
<223>Xaa stands for Tgly
<400>150
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Xaa Glu Trp Leu Lys Asn
20 25
<210>151
<211>28
<212>PRT
<213>Exendin類似物149
<220>
<221>misc_feature
<222><23)..<23)
<223>Xaa stands for Tgly
<400>151
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Xaa Glu Phe Leu Lys Asn
20 25
<210>152
<211>28
<212>PRT
<213>Exendin類似物150
<400>152
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Asp Trp Leu Lys Ash
20 25
<210>153
<211>28
<212>PRT
<213>Exendin類似物151
<400>153
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Asp Phe Leu Lys Asn
20 25
<210>154
<211>28
<212>PRT
<213>Exendin類似物152
<400>154
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn
20 25
<210>155
<211>28
<212>PRT
<213>Exendin類似物153
<400>155
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn
20 25
<210>156
<211>28
<212>PRT
<213>Exendin類似物154
<400>156
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Ala Lys Asn
20 25
<210>157
<211>28
<212>PRT
<213>Exendin類似物155
<400>157
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Ala Lys Asn
20 25
<210>158
<211>28
<212>PRT
<213>Exendin類似物156
<400>158
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe I1e Glu Trp Leu Ala Asn
20 25
<210>159
<211>28
<212>PRT
<213>Exendin類似物157
<400>159
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala Asn
20 25
<210>160
<211>28
<212>PRT
<213>Exendin類似物158
<400>160
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Ala
20 25
<210>161
<211>28
<212>PRT
<213>Exendin類似物159
<400>161
Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Ala
20 25
<210>162
<211>38
<212>PRT
<213>Exendin類似物160
<400>162
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro
35
<210>163
<211>38
<212>PRT
<213>Exendin類似物161
<400>163
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro
35
<210>164
<211>37
<212>PRT
<213>Exendin類似物162
<400>164
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro
35
<210>165
<211>36
<212>PRT
<213>Exendin類似物163
<400>165
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro
35
<210>166
<211>36
<212>PRT
<213>Exendin類似物164
<400>166
Ala Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro
35
<210>167
<211>35
<212>PRT
<213>Exendin類似物165
<400>167
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>168
<211>35
<212>PRT
<213>Exendin類似物166
<400>168
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>169
<211>34
<212>PRT
<213>Exendin類似物167
<400>169
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly
<210>170
<211>32
<212>PRT
<213>Exendin類似物168
<400>170
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
<210>171
<211>32
<212>PRT
<213>Exendin類似物169
<400>171
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
<210>172
<211>32
<212>PRT
<213>Exendin類似物170
<400>172
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
<210>173
<211>31
<212>PRT
<213>Exendin類似物171
<400>173
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
20 25 30
<210>174
<211>30
<212>PRT
<213>Exendin類似物172
<400>174
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly
20 25 30
<210>175
<211>29
<212>PRT
<213>Exendin類似物173
<400>175
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly
20 25
<210>176
<211>38
<212>PRT
<213>Exendin類似物174
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for tPro
<220>
<221>misc_feature
<222>(36)..(38)
<223>Xaa stands for tPro
<400>176
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa Xaa
35
<210>177
<211>38
<212>PRT
<213>Exendin類似物175
<220>
<221>misc_feature
<222>(36)..(38)
<223>Xaa stands for tPro
<400>177
His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Xaa Xaa Xaa
35
<210>178
<211>37
<212>PRT
<213>Exendin類似物176
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for Nme
<220>
<221>misc_feature
<222>(36)..(37)
<223>Xaa stands for Nme
<400>178
His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa Xaa
35
<210>179
<211>36
<212>PRT
<213>Exendin類似物177
<220>
<221>misc_feature
<222>(31)..(31)
<223>Xaa stands for tPro
<220>
<221>misc_feature
<222>(36)..(36)
<223>Xaa stands for tPro
<400>179
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30
Ser Gly Ala Xaa
35
<210>180
<211>35
<212>PRT
<213>Exendin類似物178
<400>180
His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala
35
<210>181
<211>30
<212>PRT
<213>Exendin類似物179
<400>181
His Gly Asp Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
<210>182
<211>39
<212>PRT
<213>Exendin類似物180
<400>182
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>183
<211>39
<212>PRT
<213>Exendin類似物181
<400>183
Ala Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>184
<211>27
<212>PRT
<213>Exendin類似物182
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>184
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn
20 25
<210>185
<211>27
<212>PRT
<213>Exendin類似物183
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>185
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn
20 25
<210>186
<211>29
<212>PRT
<213>Exendin類似物184
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>186
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly
20 25
<210>187
<211>29
<212>PRT
<213>Exendin類似物185
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(26)..(26)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>187
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn Gly Gly
20 25
<210>188
<211>27
<212>PRT
<213>Exendin類似物186
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>188
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa
20 25
<210>189
<211>27
<212>PRT
<213>Exendin類似物187
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>189
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa
20 25
<210>190
<211>29
<212>PRT
<213>Exendin類似物188
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>190
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa Gly Gly
20 25
<210>191
<211>29
<212>PRT
<213>Exendin類似物189
<220>
<221>misc_feature
<222>(1)..(1)
<223>Xaa stands for 4-ImidazolylPpionyl-G
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>191
Xaa Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu
1 5 10 15
Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa Gly Gly
20 25
<210>192
<211>28
<212>PRT
<213>Exendin類似物190
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>192
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn
20 25
<210>193
<211>28
<212>PRT
<213>Exendin類似物191
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>193
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn
20 25
<210>194
<211>30
<212>PRT
<213>Exendin類似物192
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>194
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly
20 25 30
<210>195
<211>30
<212>PRT
<213>Exendin類似物193
<220>
<221>misc_feature
<222>(27)..(27)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>195
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn Gly Gly
20 25 30
<210>196
<211>28
<212>PRT
<213>Exendin類似物194
<220>
<221>misc_feature
<222>(28)..(28)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>196
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa
20 25
<210>197
<211>28
<212>PRT
<213>Exendin類似物195
<220>
<221>misc_feature
<222>(28)..(28)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>197
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa
20 25
<210>198
<211>30
<212>PRT
<213>Exendin類似物196
<220>
<221>misc_feature
<222>(28)..(28)
<223>Xaa stands for Lys-NH(epsilon)octaneyl
<400>198
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa Gly Gly
20 25 30
<210>199
<211>30
<212>PRT
<213>Exendin類似物197
<220>
<221>misc_feature
<222>(28)..(28)
<223>Xaa stands for Lys-NH(epsilon)octanoyl
<400>199
Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa Gly Gly
20 25 30
<210>200
<211>39
<212>PRT
<213>Exendin類似物198
<220>
<221>misc_feature
<222>(12)..(12)
<223>Lys is Pegylated
<400>200
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>201
<211>39
<212>PRT
<213>Exendin類似物199
<220>
<221>misc_feature
<222>(27)..(27)
<223>Lys is Pegylated
<400>201
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>202
<211>39
<212>PRT
<213>Exendin類似物200
<220>
<221>misc_feature
<222>(2)..(2)
<223>Lys is Pegylated
<400>202
His Lys Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>203
<211>39
<212>PRT
<213>Exendin類似物201
<220>
<221>misc_feature
<222>(5)..(5)
<223>Lys is Pegylated
<400>203
His Gly Glu Gly Lys Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>204
<211>39
<212>PRT
<213>Exendin類似物202
<220>
<221>misc_feature
<222>(8)..(8)
<223>Lys is Pegylated
<400>204
His Gly Glu Gly Thr Phe Thr Lys Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>205
<211>39
<212>PRT
<213>Exendin類似物203
<220>
<221>misc_feature
<222>(10)..(10)
<223>Lys is Pegylated
<400>205
His Gly Glu Gly Thr Phe Thr Ser Asp Lys Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>206
<211>38
<212>PRT
<213>Exendin類似物204
<220>
<221>misc_feature
<222>(11)..(11)
<223>Lys is Pegylated
<400>206
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Lys Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gly Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser
35
<210>207
<211>39
<212>PRT
<213>Exendin類似物205
<220>
<221>misc_feature
<222>(13)..(13)
<223>Lys is Pegylated
<400>207
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Lys Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>208
<211>39
<212>PRT
<213>Exendin類似物206
<220>
<221>misc_feature
<222>(16)..(16)
<223>Lys is Pegylated
<400>208
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Lys
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>209
<211>39
<212>PRT
<213>Exendin類似物207
<220>
<221>misc_feature
<222>(17)..(17)
<223>Lys is Pegylated
<400>209
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Lys Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>210
<211>39
<212>PRT
<213>Exendin類似物208
<220>
<221>misc_feature
<222>(19)..(19)
<223>Lys is Pegylated
<400>210
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Lys Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>211
<211>39
<212>PRT
<213>Exendin類似物209
<220>
<221>misc_feature
<222>(21)..(21)
<223>Lys is Pegylated
<400>211
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Ser Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>212
<211>39
<212>PRT
<213>Exendin類似物210
<220>
<221>misc_feature
<222>(24)..(24)
<223>Lys is Pegylated
<400>212
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Lys Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>213
<211>39
<212>PRT
<213>Exendin類似物211
<220>
<221>misc_feature
<222>(25)..(25)
<223>Lys is Pegylated
<400>213
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Lys Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>214
<211>39
<212>PRT
<213>Exendin類似物212
<220>
<221>misc_feature
<222>(28)..(28)
<223>Lys is Pegylated
<400>214
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>215
<211>39
<212>PRT
<213>Exendin類似物213
<220>
<221>misc_feature
<222>(29)..(29)
<223>Lys is Pegylated
<400>215
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Lys Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>216
<211>39
<212>PRT
<213>Exendin類似物214
<220>
<221>misc_feature
<222>(30)..(30)
<223>Lys is Pegylated
<400>216
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Lys Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>217
<211>39
<212>PRT
<213>Exendin類似物215
<220>
<221>misc_feature
<222>(1)..(1)
<223>His is Acylated
<400>217
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>218
<211>39
<212>PRT
<213>Exendin類似物216
<220>
<221>misc_feature
<222>(1)..(1)
<223>His is Alkylated
<400>218
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>219
<211>39
<212>PRT
<213>Exendin類似物217
<220>
<221>misc_feature
<222>(27)..(27)
<223>Lys is Pegylated
<400>219
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>220
<211>39
<212>PRT
<213>Exendin類似物218
<220>
<221>misc_feature
<222>(12)..(12)
<223>Lys is Pegylated
<400>220
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>221
<211>39
<212>PRT
<213>Exendin類似物219
<400>221
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>222
<211>39
<212>PRT
<213>Exendin類似物220
<220>
<221>misc_feature
<222>(29)..(29)
<223>Lys is Pegylated
<400>222
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>223
<211>39
<212>PRT
<213>Exendin類似物221
<220>
<221>misc_feature
<222>(30)..(30)
<223>Lys is Pegylated
<400>223
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Lys Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>224
<211>39
<212>PRT
<213>Exendin類似物222
<220>
<221>misc_feature
<222>(27)..(27)
<223>Lys is Pegylated
<400>224
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>225
<211>39
<212>PRT
<213>Exendin類似物223
<220>
<221>misc_feature
<222>(12)..(12)
<223>Lys is Pegylated
<400>225
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>226
<211>39
<212>PRT
<213>Exendin類似物224
<220>
<221>misc_feature
<222>(1)..(1)
<223>His is Pegylated
<400>226
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>227
<211>39
<212>PRT
<213>Exendin類似物225
<220>
<221>misc_feature
<222>(1)..(1)
<223>His is Pegylated
<400>227
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>228
<211>39
<212>PRT
<213>Exendin類似物226
<220>
<221>misc_feature
<222>(39)..(39)
<223>Lys is Pegylated
<400>228
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>229
<211>39
<212>PRT
<213>Exendin類似物227
<220>
<221>misc_feature
<222>(30)..(30)
<223>Lys is Pegylated
<400>229
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Arg Asn Gly Lys Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>230
<211>39
<212>PRT
<213>Exendin類似物228
<400>230
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>231
<211>39
<212>PRT
<213>Exendin類似物229
<400>231
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>232
<211>39
<212>PRT
<213>Exendin類似物230
<400>232
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>233
<211>40
<212>PRT
<213>Exendin類似物231
<400>233
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>234
<211>39
<212>PRT
<213>Exendin類似物232
<400>234
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>235
<211>40
<212>PRT
<213>Exendin類似物233
<400>235
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>236
<211>40
<212>PRT
<213>Exendin類似物234
<400>236
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>237
<211>39
<212>PRT
<213>Exendin類似物235
<400>237
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>238
<211>40
<212>PRT
<213>Exendin類似物236
<400>238
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val His Leu PheIle Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>239
<211>40
<212>PRT
<213>Exendin類似物237
<400>239
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>240
<211>39
<212>PRT
<213>Exendin類似物238
<400>240
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>241
<211>40
<212>PRT
<213>Exendin類似物239
<400>241
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Arg Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>242
<211>39
<212>PRT
<213>Exendin類似物240
<400>242
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>243
<211>39
<212>PRT
<213>Exendin類似物241
<400>243
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>244
<211>39
<212>PRT
<213>Exendin類似物242
<400>244
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>245
<211>40
<212>PRT
<213>Exendin類似物243
<400>245
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>246
<211>39
<212>PRT
<213>Exendin類似物244
<400>246
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>247
<211>40
<212>PRT
<213>Exendin類似物245
<400>247
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>248
<211>40
<212>PRT
<213>Exendin類似物246
<400>248
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>249
<211>39
<212>PRT
<213>Exendin類似物247
<400>249
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>250
<211>40
<212>PRT
<213>Exendin類似物248
<400>250
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>251
<211>40
<212>PRT
<213>Exendin類似物249
<400>251
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Lys Leu PheIle Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>252
<211>39
<212>PRT
<213>Exendin類似物250
<400>252
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>253
<211>40
<212>PRT
<213>Exendin類似物251
<400>253
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>254
<211>39
<212>PRT
<213>Exendin類似物252
<400>254
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>255
<211>39
<212>PRT
<213>Exendin類似物253
<400>255
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>256
<211>39
<212>PRT
<213>Exendin類似物254
<400>256
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>257
<211>40
<212>PRT
<213>Exendin類似物255
<400>257
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>258
<211>39
<212>PRT
<213>Exendin類似物256
<400>258
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>259
<211>40
<212>PRT
<213>Exendin類似物257
<400>259
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>260
<211>40
<212>PRT
<213>Exendin類似物258
<400>260
His GIy Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>261
<211>39
<212>PRT
<213>Exendin類似物259
<400>261
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>262
<211>40
<212>PRT
<213>Exendin類似物260
<400>262
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val His Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>263
<211>40
<212>PRT
<213>Exendin類似物261
<400>263
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Arg
35 40
<210>264
<211>39
<212>PRT
<213>Exendin類似物262
<400>264
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>265
<211>40
<212>PRT
<213>Exendin類似物263
<400>265
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu
1 5 10 15
Glu Ala Val Lys Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys
35 40
<210>266
<211>65
<212>DNA
<213>Artificial Sequence
<220>
<223>Chemically Synthesized
<400>266
aattccatgc acggcgaaac cttcaccagc gatctgagca aacagctgga agaagaagcg60
gttaa65
<210>267
<211>65
<212>DNA
<213>Artificial Sequence
<220>
<223>Chemically Synthesized
<400>267
actgttcatc gaatggctga aaaacggcgg cccgagcagc ggcccgccgc cgccgagccg60
ttaga65
<210>268
<211>61
<212>DNA
<213>Artificial Sequence
<220>
<223>Chemically Synthesized
<400>268
agcttctaac ggctcggcgg cggcgcgctg ctcgggccgc cgtttttcag ccattcgatg60
a61
<210>269
<211>69
<212>DNA
<213>Artificial Sequence
<220>
<223>Chemically Synthesized
<400>269
acagtttaac cgcttcttct tccagctgtt tgctcagatc gctggtgaag gtgccttcgc60
cgtgcatgg69
權(quán)利要求
1.一種帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)的分子量在20,000道爾頓以上、為支鏈結(jié)構(gòu)，其中，所述的聚乙二醇基團(tuán)是通過(guò)將一個(gè)或一個(gè)以上的聚乙二醇與所述的Exendin或其類似物中的一個(gè)或一個(gè)以上的氨基酸相連接而連接到所述的Exendin或其類似物上。
2.一種帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)帶如下式I所示支鏈結(jié)構(gòu)
其中，X為H或保護(hù)基團(tuán)；PEG為-O(CH2CH2O)q-，q為正整數(shù)；n1為0-5的整數(shù)；m為0-5的整數(shù)；Y為O，S，SO，SO2或NR1，其中R1為H或C1-8經(jīng)取代或未經(jīng)取代的烷基或經(jīng)取代或未經(jīng)取代的環(huán)烷；k為0或1；p為0-6的整數(shù)；
其中，所述的聚乙二醇基團(tuán)是通過(guò)將一個(gè)或一個(gè)以上的聚乙二醇與所述的Exendin或其類似物中的一個(gè)或一個(gè)以上的氨基酸相連接而連接到所述的Exendin或其類似物上。
3.如權(quán)利要求2所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇具有如下式II所示的支鏈結(jié)構(gòu)
其中，PEG為-O(CH2CH2O)q-，q為正整數(shù)，Me為甲基。
4.一種帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)帶如下式III所示的支鏈結(jié)構(gòu)
其中，X為H或保護(hù)基團(tuán)；PEG為-O(CH2CH2O)q-，q為正整數(shù)；n2為0-10的整數(shù)；Z為選自于以下組中的基團(tuán)(CH2)i、(CH2)iOCO、(CH2)iNHCO、(CH2)iCO，i為0-10的整數(shù)；R2為H、C1-12經(jīng)取代或未經(jīng)取代的烷基、取代芳基、芳烷基或雜烷基；j為1-12的整數(shù)；
其中，所述的聚乙二醇基團(tuán)是通過(guò)將一個(gè)或一個(gè)以上的聚乙二醇與所述的Exendin或其類似物中的一個(gè)或一個(gè)以上的氨基酸相連接而連接到所述的Exendin或其類似物上。
5.如權(quán)利要求4所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇具有如下式IV所示的支鏈結(jié)構(gòu)
其中，PEG為-O(CH2CH2O)q-，q為正整數(shù)，Me為甲基。
6.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的Exendin或其類似物包含SEQ ID NO1所列的氨基酸序列。
7.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的Exendin或其類似物包含SEQ ID NO2所列的氨基酸序列。
8.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的Exendin或其類似物包含的氨基酸序列選自SEQ ID NO3-SEQ ID NO229所列氨基酸序列之一。
9.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的Exendin或其類似物包含的氨基酸序列選自SEQ ID NO230-SEQ ID NO265所列氨基酸序列之一。
10.如權(quán)利要求1所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)的分子量為20,000道爾頓以上至50,000道爾頓之間。
11.如權(quán)利要求2-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)的分子量為5,000道爾頓至50,000道爾頓之間。
12.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的聚乙二醇基團(tuán)是經(jīng)N羥基琥珀酰亞胺活化而連接到所述的Exendin或其類似物上。
13.如權(quán)利要求1-5之一所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物中包含一個(gè)、兩個(gè)、三個(gè)或四個(gè)帶支鏈結(jié)構(gòu)的聚乙二醇基團(tuán)。
14.如權(quán)利要求13所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物，其特征在于，所述的帶有聚乙二醇基團(tuán)的Exendin或其類似物中包含一個(gè)帶支鏈結(jié)構(gòu)的聚乙二醇基團(tuán)。
15.一種由帶有聚乙二醇基團(tuán)的Exendin或其類似物形成的制劑，其特征在于，該制劑包括如權(quán)利要求1-5所述的一種或多種帶有聚乙二醇基團(tuán)的Exendin或其類似物。
16.如權(quán)利要求1-5所述的一種或多種帶有聚乙二醇基團(tuán)的Exendin或其類似物在制備治療糖尿病藥物方面的用途。
全文摘要
一種帶有聚乙二醇基團(tuán)的Exendin或其類似物及其制劑，涉及一種由單個(gè)或多個(gè)聚乙二醇修飾的Exendin或其類似物及其制劑技術(shù)領(lǐng)域。本發(fā)明所要解決的技術(shù)問(wèn)題是單次給藥后的時(shí)間間隔較短，經(jīng)聚乙二醇修飾后生物活性嚴(yán)重下降，使給藥劑量大大增加，而無(wú)法實(shí)際應(yīng)用。本發(fā)明使用新型的聚乙二醇修飾Exendin或其類似物，修飾后的Exendin或其類似物具有半衰期長(zhǎng)、活性高、免疫抗原性低的特點(diǎn)。
文檔編號(hào)A61P3/10GK101125207SQ200710138718
公開(kāi)日2008年2月20日 申請(qǐng)日期2007年7月23日 優(yōu)先權(quán)日2006年11月14日
發(fā)明者包文超, 徐宏景, 剛 余, 左亞軍 申請(qǐng)人:上海華誼生物技術(shù)有限公司