CN102827284B - 带有聚乙二醇基团的Exendin或其类似物及其制剂和用途 - Google Patents
带有聚乙二醇基团的Exendin或其类似物及其制剂和用途 Download PDFInfo
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- CN102827284B CN102827284B CN201210248202.9A CN201210248202A CN102827284B CN 102827284 B CN102827284 B CN 102827284B CN 201210248202 A CN201210248202 A CN 201210248202A CN 102827284 B CN102827284 B CN 102827284B
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Classifications
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Landscapes
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Abstract
一种带有聚乙二醇基团的Exendin或其类似物及其制剂，涉及一种由单个或多个聚乙二醇修饰的Exendin或其类似物及其制剂技术领域。本发明所要解决的技术问题是单次给药后的时间间隔较短，经聚乙二醇修饰后生物活性严重下降，使给药剂量大大增加，而无法实际应用。本发明使用新型的聚乙二醇修饰Exendin或其类似物，修饰后的Exendin或其类似物具有半衰期长、活性高、免疫抗原性低的特点。
Description
分案声明
本申请是2007年7月23日提交的申请号为200710138718.7、发明名称为“带有聚乙二醇基团的艾塞丁或其类似物及其制剂和用途”的中国专利申请的分案申请；并要求中国专利申请200610118326.X作为优先权，该申请于2006年11月14日提交。
技术领域
本发明涉及一种修饰的Exendin或其类似物，特别地涉及一种由单个或多个聚乙二醇修饰的Exendin或其类似物及其制剂、给药方法以及应用。
背景技术
胰高血糖素样肽-1首次于1987年被研究人员认定为一种葡萄糖依赖的肠分泌激素肽。这种多肽通过细胞上G蛋白耦联受体传递信号，从而产生刺激β胰岛细胞分泌胰岛素，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是一种在美国毒蜥和珠毒蜥唾液中发现的一类多肽（J.Biol.Chem.1990,265,20259-20262;J.Biol.Chem.1992,267,7402-7405），以Exendin-4为代表的这类多肽与胰高血糖素样肽-1[7-36]具有高度同源性。研究发现，Exendin同样可以与胰高血糖素样肽-1受体结合，并在药理上表现出相类似的效果，如：促进胰岛素分泌，有效控制餐后血糖，降低糖化血红蛋白，抑制胃排空等。在动物体内的长期研究发现，长期使用这类胰高血糖素样肽-1受体多肽还能有效改善胰岛素抵制，从而逆转糖尿病的病程。此外，人们还发现，以胰高血糖素样肽-1和Exendin-4为代表的一系列促胰岛素分泌激动剂能促进β胰岛细胞再生（Nat.Biotech.2005,23,857-861）和改善非酒精性脂肪肝（Hepatology2006，43，173-181）。这使得该类多肽一直成为糖尿病及肥胖制药领域的研究热点。伍登熙和孙玉昆等人（中国专利：ZL01112856）对Exendin-4进行了多肽改构，得到了一系列相同功能的多肽。尤其是在前不久，美国安米林(Amylin)和礼来(Eli Lilly)公司共同开发的一天注射两次的Exendin——Exendin-4在美国上市，使得该类药物得到全世界糖尿病及肥胖治疗领域的广泛关注。但是经过临床研究发现，这种异源的多肽在给药30周后会使受试病人体内出现Exendin-4抗体，占总人数的41％（Diabetes Care.2004,27,2628-2635）。
由于蛋白质/多肽类药物普遍存在体内半衰期较短，物理、化学稳定性较差，易被体内各种蛋白酶降解等特性，使得这些药物通常需要在一天之内多次注射，这给病人带来了许多痛苦和诸多不便。如何延长这类药物的给药周期一直长期困扰人们，目前还没一种普遍适用的给药方式。
上世纪70年代出现的聚乙二醇化技术被证明是当前蛋白质/多肽类给药领域一项较适用的技术。通常采用线性直链或分支状的聚乙二醇对蛋白质/多肽进行修饰，这样的修饰赋予蛋白质/多肽具有如下特点：1）物理和化学稳定性得到显著提高，2）免疫抗原性得到显著下降，3）抗蛋白酶降解能力显著提高，4）随着聚乙二醇分子量的增加，能明显降低肾清除作用对药物的代谢，从而药物的体内半衰期显著增加，5）药物溶解度提高，以及对细胞膜的穿透力增强。A·杨和K·普里克特的研究证明了Exendin-4主要是通过肾清除来代谢的，因此他们将分子量范围在500至20,000道尔顿（Dalton）的聚乙二醇对Exendin进行了修饰（中国专利：CN1372570A），从而实现降低肾过滤对药物的清除。
但是，修饰后药物的活性会普遍下降是该技术的一个缺陷。Haim Tsubery等人使用9羟甲基7硫代芴N羟基琥珀酰亚胺（9-hydroxymethyl-7-sulfofluorene-Nhydroxysuccinimide，FMS）活化聚乙二醇，在体内聚乙二醇可以从Exendin-4上水解下来，从而使未经修饰的Exendin-4发挥作用。这种方式虽然解决了聚乙二醇修饰后所导致的生物活性降低的问题，但是由于发挥作用的还是未经修饰的Exendin-4，因此长期注射而产生的免疫抗原性问题也依旧没能解决（J.Biol.Chem.2004,279,38118-38124）。
我们使用新型的聚乙二醇修饰的Exendin或其类似物，取得了半衰期长、活性高、免疫抗原性低的聚乙二醇修饰的Exendin或其类似物。
发明内容
本发明所要解决的技术问题是Exendin或其类似物在体内使用单次给药后的时间间隔较短，长期注射会使体内产生抗体，现存的聚乙二醇修饰后的Exendin或其类似物生物活性严重下降，使给药剂量大大增加，而无法实际应用。本发明提供新型的聚乙二醇修饰的Exendin或其类似物，具有所述的半衰期长、活性高、免疫抗原性低的特点。
聚乙二醇修饰
本发明涉及新型的聚乙二醇修饰的Exendin或其类似物，其特征是Exendin或其类似物由一个或多个聚乙二醇修饰。
聚乙二醇衍生物不同的化学结构对修饰后的多肽生物活性影响不同。本发明所述的聚乙二醇可以采用线性直链或分叉支链的结构。本发明所述的分叉支链（或称分支叉状）结构包括双臂支链的结构，也包括多臂支链的结构。本发明所述的聚乙二醇双臂支链结构包括但不限于如下列式I、式II、式III和式IV所示结构。
本发明所述的聚乙二醇支链结构包括但不限于下列式I所示结构：
其中，X为H或保护基团。这里所说的保护基团可以是任何能够与自由羟基反应，并防止其进一步与其它基团反应的基团，包括但不限于低碳烷基如甲基、二甲基、乙基、丙基、异丙基等。
另外，在上述结构中，PEG为-O(CH2CH2O)q-，q为正整数；n1为0-5的整数；m为0-5的整数；Y为O,S,SO,SO2或NR1,其中R1为H或C1-8经取代或未经取代的烷基或经取代或未经取代的环烷；k为0或1；p为0-6的整数。上述的带式I结构的聚乙二醇可使用常规的化学方法合成，美国专利U.S.6,566,506提供了一些带上述式I结构的聚乙二醇的合成方法。
在本发明一优选的实施例中，本发明使用的带有式I所示支链结构的聚乙二醇带有如下式II所示具体结构：
其中，PEG为-O(CH2CH2O)q-，q为正整数，Me为甲基。
本发明所述的聚乙二醇支链结构包括但不限于下列式III所示结构：
其中，X为H或保护基团；PEG为-O(CH2CH2O)q-，q为正整数；n2为0-10的整数；Z为选自于以下组中的基团：(CH2)i、(CH2)iOCO、(CH2)iNHCO、(CH2)iCO,i为0-10的整数；R2为H、C1-12经取代或未经取代的烷基、取代芳基、芳烷基或杂烷基；j为1-12的整数。上述的带式III结构的聚乙二醇可使用常规的化学方法合成，中国专利ZL03801105.0提供了一些带上述式III结构的聚乙二醇的合成方法。
在本发明一优选的实施例中，本发明使用的带有式III所示支链结构的聚乙二醇带有如下式IV所示具体结构：
其中，PEG为-O(CH2CH2O)q-，q为正整数，Me为甲基。
聚乙二醇的分子量大小对修饰后的多肽生物活性也有不同影响。本发明使用的直线型和支链型聚乙二醇的分子量范围优选为200道尔顿或其以上，更优选为5,000道尔顿或其以上，再优选为20,000道尔顿或其以上。在一个优选的实施例中，本发明使用的聚乙二醇分子量为5,000道尔顿至50,000道尔顿之间，包括5,000道尔顿和50,000道尔顿。在另一个优选的实施例中，本发明使用的聚乙二醇分子量为20,000道尔顿至50,000道尔顿之间，包括20,000道尔顿和50,000道尔顿。在另一个优选的实施例中，本发明使用的聚乙二醇分子量为20,000道尔顿至45,000道尔顿之间，包括20,000道尔顿和45,000道尔顿。在另一个优选的实施例中，本发明使用的聚乙二醇分子量为20,000道尔顿至40,000道尔顿之间，包括20,000道尔顿和40,000道尔顿。本发明所述的聚乙二醇分子量除特别指出外均指通过凝胶渗透色谱法（Gel PermeationChromatography，GPC）（董炎明，《高分子分析手册》，北京：中国石化出版社2004，416-427）测定得到的数均分子量（Mn）。带不同分子量的聚乙二醇可以在市场上买到也可以使用本技术领域公知的技术合成。
在本发明一优选的实施方案中，本发明所述的聚乙二醇采用分子量为20,000以上的支链结构。在另一个优选的实施例中，本发明使用的聚乙二醇为分子量在20,000道尔顿以上至50,000道尔顿之间的支链结构，包括50,000道尔顿。在另一个优选的实施例中，本发明使用的聚乙二醇为分子量在20,000道尔顿以上至45,000道尔顿之间的支链结构，包括45,000道尔顿。在另一个优选的实施例中，本发明使用的聚乙二醇为分子量在20,000道尔顿以上至40,000道尔顿之间的支链结构，包括40,000道尔顿。
本发明所述的聚乙二醇修饰的Exendin或其类似物是通过活化的聚乙二醇与多肽氨基酸残基的侧链，N末端和C末端连接。使用带不同活化基团的聚乙二醇，可以实现对不同氨基酸侧链、N末端和C末端的化学结合，也可以针对某一氨基酸位点专一性结合。可以与活化的聚乙二醇化学结合的氨基酸基团包括但不限于多肽的N末端α氨基、赖氨酸侧链ε氨基、组氨酸侧链咪唑基上的亚氨基、多肽的C末端羧基、天冬氨酸和谷氨酸侧链羧基、丝氨酸和苏氨酸侧链羟基，半胱氨酸侧链巯基等。通常情况下，这种化学连接通过亲电亲核方式进行。例如，经N羟基琥珀酰亚胺活化的聚乙二醇与多肽的N末端α氨基，赖氨酸侧链ε氨基以及组氨酸侧链咪唑基上的亚氨基等游离氨基连接。另外，还可以采用活化基团为醛基的聚乙二醇，通过还原烷基化定点结合到多肽分子的N末端。本发明包括通过不同活化基团使聚乙二醇与Exendin或其类似物结合，及其产生的聚乙二醇修饰的Exendin或其类似物。带不同活化基团的聚乙二醇可以在市场上买到也可以使用本技术领域公知的技术合成。
本发明包括Exendin或其类似物上一种或多种氨基酸与聚乙二醇结合的Exendin或其类似物。本发明所述的Exendin或其类似物上能同时连接1个或数个聚乙二醇，所结合聚乙二醇的个数视Exendin或其类似物上游离反应基团的数量，聚乙二醇所带活化基团以及聚乙二醇的分子量大小而定。一般来说，游离反应基团的数量越多，就能结合更多的活化聚乙二醇；活化的聚乙二醇分子量越大，其能结合到多肽上的数量就越少。优选地，Exendin或其类似物由一个、二个、三个或四个聚乙二醇修饰。在一个优选的实施例中，Exendin或其类似物由一个聚乙二醇修饰。在另一个优选的实施例中，Exendin或其类似物由二个聚乙二醇修饰。
聚乙二醇与Exendin或其类似物连接反应后的混合物可以通过常用的离子交换、凝胶过滤或反相色谱等方式实现对这些产物的有效分离。通常使用的离子交换色谱法有阴离子交换色谱和阳离子交换色谱两种，选择不同的离子交换基质可以影响到分离纯化的效果。由于不同的Exendin或其类似物在氨基酸个数和种类略有不同，而使各自的分子量略有不同，因此聚乙二醇单取代的Exendin或其类似物的最终分子量为聚乙二醇的分子量加上Exendin或其类似物的分子量。通过分离纯化、超滤浓缩和缓冲液置换等方法处理后，可以得到本发明所述的聚乙二醇修饰的Exendin或其类似物，并可以制成药用制剂。
Exendin或其类似物
本发明所述的Exendin或其类似物是指与Exendin全部或部分氨基酸结构序列相类似的，能与胰高血糖素样肽-1受体结合，并产生细胞信号级联传导的一类多肽。这些多肽可以通过化学固相合成或基因工程的宿主菌表达后，再经过一步或多步分离纯化得到。
本发明所述的Exendin或其类似物包括但不限于天然的Exendin-3或Exendin-4。天然的Exendin-3的氨基酸序列结构如下：
[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 NO:1)
天然的Exendin-4的氨基酸序列结构如下：
[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 NO:2)
本发明所述的Exendin或其类似物包括但不限于那些通过对天然的Exendin-3或Exendin-4的氨基酸序列进行氨基酸置换、增加和删减而得到的类似多肽。本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列进行天然氨基酸置换，同时也包括用非天然氨基酸对Exendin-3或Exendin-4的氨基酸序列进行氨基酸置换。非天然氨基酸包括但不限于氮杂环丁烷羧酸、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-氨基异丁酸、3-氨基异丁酸、2-氨基庚二酸、叔丁基甘氨酸、2，4-二氨基异丁酸、锁链赖氨素、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、羟赖氨酸、别-羟赖氨酸、3-羟脯氨酸、4-羟脯氨酸、异锁链赖氨素、别-异亮氨酸、N-甲基丙氨酸、N-甲基甘氨酸、N-甲基异亮氨酸、N-甲基戊基甘氨酸、N-甲基缬氨酸、萘丙氨酸、正缬氨酸、正亮氨酸、鸟氨酸、戊基甘氨酸、2-哌啶酸和硫代脯氨酸。优选地，本发明所述Exendin类似物包含1个、2个、3个、4个或5个氨基酸置换。
本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列增加和删减一个或多个氨基酸得到的类似多肽。在本发明一优选的实施方案中，本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列增加和删减1个至20个氨基酸得到的类似多肽。再优选地，本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列增加和删减1个至15个氨基酸得到的类似多肽。进一步优选地，本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列增加和删减1个至10个氨基酸得到的类似多肽。在本发明具体实施方案中，本发明所述的Exendin类似物包括那些对Exendin-3或Exendin-4的氨基酸序列增加和删减1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸得到的类似多肽。
Exendin类似物包括在Exendin-3或Exendin-4的氨基酸序列的N末端氨基、C末端羧基或其侧链基团可逆或不可逆的化学封闭或修饰，例如C末端羧基的酰胺化。
在本发明另一优选的实施方案中，本发明所述的Exendin或其类似物包括SEQ ID NOs:1-265的氨基酸序列。优选地，本发明所述的Exendin或其类似物包括SEQ ID NOs:1-2的氨基酸序列。另一优选地，本发明所述的Exendin或其类似物包括SEQ ID NOs:3-229的氨基酸序列。另一优选地，本发明所述的Exendin或其类似物包括SEQ ID NOs:230-265的氨基酸序列。
聚乙二醇修饰的Exendin或其类似物可以根据巩秋红等（中华内分泌代谢杂志2004,20,559-560）发表的实验方法在细胞水平上进行活性测定。具体地，可以在INS-1细胞中分别加入不同浓度的葡萄糖和含本发明聚乙二醇修饰的Exendin及其类似物的溶液，孵育4小时，用放射免疫法测定上清液中胰岛素含量，并利用RT-PCR技术对INS-1细胞的胰岛素mRNA水平作半定量分析。通过这种方法可以在体外大量筛选聚乙二醇修饰的Exendin及其类似物的活性。另外，还可以通过测定小鼠（例如C57或db/db鼠）给药后在不同时间点的血糖变化情况来进行筛选。
药用制剂及其使用方法
本发明所述的聚乙二醇修饰的Exendin或其类似物可以与各种无机和有机酸和碱形成盐。这类盐包括用有机酸和无机酸制备的盐，所述有机酸和无机酸包括但不限于盐酸、溴化氢、硫酸、磷酸、三氟乙酸、乙酸、甲酸、甲磺酸、甲苯磺酸、马来酸、富马酸和樟脑磺酸。用碱制备的盐包括但不限于铵盐、碱金属盐(例如钠盐和钾盐)和碱土金属盐(例如钙盐和镁盐)。优选乙酸盐、盐酸盐和三氟乙酸盐。通过常规方法，例如通过在所述盐不溶于其中的溶剂或介质、或在随后可真空除去或通过经冷冻干燥除去的溶剂(例如水)中，使所述产物的游离酸或碱形式与一种或更多种适当碱或酸的等同物反应，或通过在合适离子交换树脂上用另一种离子交换现有盐的离子，可以制备所述盐。
所述要求保护的化合物也可以配制成药学上可接受的盐(例如酸加成盐)和／或它们的络合物。这类盐制剂,可以通过改变所述组合物的物理及化学特性而有助于药理学应用，但是不防碍所述组合物发挥其生理学效应。物理特性方面改变的实例包括降低熔点，以易于经粘膜给药；以及增加溶解度，以易于给予更高浓度的所述药物。
药学上可接受的盐包括酸加成盐，例如含有硫酸盐、盐酸盐、磷酸盐、氨基磺酸盐、乙酸盐、柠檬酸盐、乳酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐、环已氨基磺酸盐和奎尼酸盐的那些酸加成盐。药学上可接受的盐可以得自各种无机和有机酸例如盐酸、硫酸、磷酸、氨基磺酸、乙酸、柠檬酸、乳酸、酒石酸、丙二酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、环已氨基磺酸和奎尼酸。例如通过在所述盐不溶于其中的溶剂或介质、或在随后可真空除去或通过经冷冻干燥除去的溶剂(例如水)中，使所述产物的游离酸或碱形式与一种或更多种适当碱或酸的等同物反应，或通过在合适离子交换树脂上用另一种离子交换现有盐的离子，可以制备所述盐。
也可以使用载体或赋形剂，以易于给予所述化合物。载体和赋形剂的实例包括碳酸钙、磷酸钙、各种糖(例如乳糖、葡萄糖或蔗糖)或各种类型的淀粉、纤维素衍生物、明胶、植物油(例如芝麻油、花生油、橄榄油)、聚乙二醇和生理上相容的溶剂。可以通过不同的途径，包括静脉内、腹膜内、皮下、和肌内、口服、局部或经粘膜途径，给予所述组合物或药用组合物。
如有需要，上述组合物的溶液可以用增稠剂(例如甲基纤维素)增稠。它们可以乳化形式(比如油包水或者水包油)制备。可以使用各种各样的药学上可接受的乳化剂中的任一种，包括例如金合欢胶粉、非离子型表面活性剂(例如吐温)、或离子型表面活性剂(例如碱性聚醚醇硫酸酯或磺酸酯，例如Triton)。
这些组合物可以通过常规灭菌技术灭茵或者可以过滤除茵。所述组合物可以含有需要的近似生理条件的药学上可接受的辅助物质，例如pH缓冲剂。有效的缓冲液包括例如乙酸钠／乙酸缓冲液。可以使用栓剂形式和“缓释型"慢释放制剂的形式，使得治疗有效量的制剂在经皮注射或传递后许多小时或许多天内被传递至血流中。
可以用氯化钠或其它药学上可接受的试剂，例如葡萄糖、硼酸、酒石酸钠、丙二醇、多元醇(例如甘露醇和山梨糖醇)或其它无机溶质或有机溶质，达到所需的等渗性。对于含钠离子的缓冲液特别优选氯化钠。
对于一名体重为70kg患者而言，所述化合物的有效剂量通常的范围是0.01mg/天或0.03mg/天至约5mg/天，优选约0.01或0.5至2mg/天，更优选约0.01或0.1至1mg/天，以单剂量或分次剂量给予。将给予的精确剂量由主治医师确定，并且依赖于所述特定化合物是否在上述提供的范围内以及取决于个体的年龄、体重和病症。给药应在糖尿病刚出现症状时开始，或在诊断糖尿病后立即开始。
虽然通常将所述化合物用于治疗人类患者，但也可以用它们治疗其它脊椎动物例如其它灵长类、家畜(例如猪、牛和家禽)以及运动用动物和宠物(例如马、狗和猫)中的相同或相似疾病。
附图说明
附图1是带式I所示支链聚乙二醇结构。
附图2是带式III所示支链聚乙二醇结构。
附图3是分子量为40,000道尔顿的聚乙二醇（式IV）取代的Exendin-4类似物的分离纯化色谱图。其中标记1为上样时的吸收峰，标记2为聚乙二醇多取代Exendin-4类似物的洗脱吸收峰，标记3为聚乙二醇单取代Exendin-4类似物的洗脱吸收峰，标记4为未结合聚乙二醇Exendin-4类似物的洗脱吸收峰。
附图4是分子量为40,000道尔顿的聚乙二醇（式IV）单取代Exendin-4类似物电泳图。其中标记1道显示聚乙二醇单取代的Exendin-4类似物的条带。
附图5是经分子量为5,000道尔顿和21,000道尔顿直线型聚乙二醇单取代Exendin-4类似物24小时动物药效试验结果。
附图6是经分子量为21,000道尔顿直线型和分子量为40,000道尔顿聚乙二醇（式IV）单取代Exendin-4类似物72小时动物药效试验结果。
附图7是经分子量分别为21,000道尔顿（U21K）、30,000道尔顿（U30K）和40,000道尔顿（U40K）聚乙二醇（式II）单取代的Exendin-4类似物72小时动物药效试验结果。
附图8是经分子量分别为21,000道尔顿（Y21K）、30,000道尔顿（Y30K）和40,000道尔顿（Y40K）聚乙二醇（式IV）单取代Exendin-4类似物72小时动物药效试验结果。
附图9是经分子量分别为21,000道尔顿（L21K）和30,000道尔顿（L30K）直线型聚乙二醇单取代Exendin-4类似物的72小时动物药效试验结果。
附图10是分别经分子量为30,000道尔顿的聚乙二醇（式II）单取代和经分子量为40,000道尔顿的聚乙二醇（式IV）单取代的Exendin-4类似物72小时动物药代（血浆半衰期）试验结果。
具体实施方式
本实验所用的聚乙二醇衍生物中，分子量为5,000道尔顿的直线型聚乙二醇购自西格玛奥德里奇公司（Sigma－Aldrich），其它聚乙二醇衍生物均购自北京键凯科技有限公司。
实验中的离子交换法所用A、B双泵AKTA purifier 100分离纯化设备，其它预装柱及填料等均购自美国通用电器公司（General Electric），其它相关化学试剂购自西格玛奥德里奇公司。实施例三到实施例九中所用的Exendin-4及其类似物购自成都圣诺科技发展有限公司。
实施例一 Exendin-4类似物的固相化学合成
本实施例介绍用固相化学的方法合成具有下列结构的Exendin-4类似物：
[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 NO:232）。
（1）所采用的氨基酸单体
| Fmoc-L-Ala-OH | Fmoc-L-Lys(Boc)-OH |
| Fmoc-L-Asn(Trt)-OH | Fmoc-L-Met-OH |
| Fmoc-L-Asp(OtBu)-OH | Fmoc-L-Phe-OH |
| Fmoc-L-Gln(Trt)-OH | Fmoc-L-Pro-OH |
| Fmoc-L-Glu(OtBu)-OH | Fmoc-L-Ser(tBu)-OH |
| Fmoc-L-Gly-OH | Fmoc-L-Thr(tBu)-OH |
| Fmoc-L-His(Trt)-OH | Fmoc-L-Trp-OH |
| Fmoc-L-Ile-OH | Fmoc-L-Tyr(tBu)-OH |
| Fmoc-L-Leu-OH | Fmoc-L-Val-OH |
上式中缩写表示：
Fmoc：9－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）合成所采用的仪器设备及试剂：
仪器为：Applied Biosystem多肽合成仪，433A型，美国
试剂有：N-甲基吡咯烷酮，二氯甲烷，六氢吡啶，甲醇，二甲氨基吡啶（Dimethylaminopyridine）/DMF,N,N－二异丙基乙胺（N,N-diisopropylethylamine）/NMP，HBTU 100mmole/0.5M HOBT in DMF，N,N-二环乙基碳二亚胺（N,N-Dicyclohexylcarbodiimide）/NMP。
其中：DMF为N,N-二甲基甲酰胺（N,N-Dimethylformamide），
NMP为N-甲基吡咯烷酮(N-methylpyrrolidone)，
HOBT为1-羟基苯并三唑（1-Hydroxybenzotriazole），
HBTU为2-（1氢-苯并三唑基）－四甲基脲六氟磷酸盐
（2-1H-benzotriazole-y1-1,1,3,3-tetramethyl-Uronium hexafluorophosphate）。
(3)操作：
a．合成
以0.25mmol规模为例，称取HMP树脂0.25g，置入多肽合成仪上的反应器中，将各种带保护基氨基酸称取1mmol装瓶，按该促胰岛素分泌肽衍生物的氨基酸序列从C-端向N-端排列在合成仪中，于25℃室温条件下，由计算机程序控制自动进行脱Fmoc保护、活化、连结，然后再进行下一轮循环，如此完成合成，合成结束后，将得到的带侧链保护基的多肽树脂在多肽合成仪上吹干后称重即可。
b.脱保护基及切断树脂：
将带保护基的多肽树脂置于具塞三角烧瓶，加入裂解试剂如下表：
然后在恒温30℃条件下，电磁搅拌反应6小时；过滤、收集滤液，树脂用少量三氟乙酸洗涤；合并收集液与洗涤液，加入乙醚产生沉淀，过滤，沉淀用少量乙醚洗涤后放入干燥器中干燥，得到粗品。
c.HPLC分离纯化、冻干
将得到的粗品用制备型HPLC进行分离、提纯，最后经冷冻干燥得到产品，经色质联用检测，该衍生物分子量理论值为4300.6，实测值为4316.7。
同样地，本领域技术人员可以采用相同的方法合成其它Exendin或其类似物。
实施例二 Exendin-4类似物的基因工程法制备
本实施例介绍用基因工程的方法合成具有下列结构的Exendin-4类似物：
[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-GlyAla-Pro-Pro-Pro-Ser-Arg-OH]（SEQ ID NO:251）。
A.按此Exendin-4类似物的氨基酸序列，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 NO:266）
（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 NO:267）
（3）5’AGCTT CTA ACG GCT CGG CGG CGG CGC GCT GCT CGG GCC GCC GTT TTT CAG CCA TTC GATGA（SEQ ID NO:268）
（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 NO:269）
B、克隆
连接：取二支试管，一支加入光密度值为0.1（A260nm）的片段（1）和片段（4），另一支试管中加入光密度值为（A260nm）的片段（2）和片段（3），再将聚核苷酸激酶缓冲液（PolynucleotideKinase Buffer），聚核苷酸激酶（Polynucleotide Kinase）及三磷酸腺苷（ATP）分别加入该二支试管，于37℃保温60分钟，使基因片段5’端磷酸化。然后，将二支试管转移至95℃水浴中保温10分钟，停止加温，自然冷却退火降至室温。再将T4连接酶缓冲液和T4连接酶分别加入该二支试管中，16℃左右保温。过夜，使基团片段连接。
质粒：另取一支试管将含有乳糖启动子Lac（或温控启动子PL，色氨酸-乳糖混合启动子Tac）的质粒用限制性内切酶EcoRⅠ和Hind Ⅲ双酶酶切后用酚/氯仿抽提，离心取水层部分，再用氯仿抽提，离心抽取水层部分，再用氯仿将水层洗涤3次，离心，将水层用异丙醇沉淀，离心，干燥。
将上述酶切质粒与连接后的基因片段混合，加T4连接酶缓冲液和T4连接酶，室温连接3－4小时。
受菌体的培养：克隆菌大肠杆菌（E.Coli）JM103于每升含蛋白胨10g、酵母抽提粉5g、氯化钠5g的培养液（LB培养液）中37℃振荡培养4小时，离心收集菌体，经CaCl2溶液处理后，4℃保存备用。
转化：将克隆的质粒转化至受体菌大肠杆菌JM103细胞中，冰浴维持30分钟，升温至42℃，维持2分钟后，转移至含氨苄青霉素的琼脂糖平板，37℃过夜，进行菌落筛选，得克隆阳性质粒。
C、发酵
将筛选得含该种衍生物质粒的寄主菌株于LB培养液中振荡培养，加0.5mM异丙基硫代半乳糖苷（IPTG）诱导，过夜后，离心收集菌体，以十二烷基磺酸钠＝12％的丙烯酰胺凝胶（PAGE）电泳，鉴定表达的蛋白。
D、包涵体
按上述条件，300mlx10瓶，摇床培养，诱导，收集的菌体加裂解液（1％NaCl，磷酸缓冲液pH7.5，20mM）及溶菌酶，30℃保温30分钟，离心收集沉淀，加6M盐酸胍（Gu.HCl）抽提包涵体，离心，将上清液透析，除去Gu.HCL，再用组成为1％NaCl，0.1％吐温80（Tween 80）的磷酸缓冲液（20mM，PH7.5）洗涤沉淀三次，即得包涵体。
E、裂解
将包涵体溶于8M尿素溶液，加盐酸至50mM,加溴化氰，避光，氮气保护条件下搅拌，裂解2小时，以HPLC跟踪分析。
F、纯化
裂解完成，通过琼脂糖凝胶分子筛G-25分离得粗品，再经HPLC纯化，得产品，与化学合成品相同，质谱分析结果表明所得衍生物分子量与理论值相吻合。
实施例三 直线型单甲氧基聚乙二醇（分子量＝5,000道尔顿）修饰Exendin-4
每个试管中称取1.0mg Exendin-4，共三个试管。分别使用不同pH值的磷酸缓冲液溶解，再在每个试管中加入N羟基琥珀酰亚胺活化的单甲氧基聚乙二醇（分子量＝5,000道尔顿）5.8mg，置于摇床，室温反应1小时。之后使用安捷伦1100系列色谱仪（Agilent 1100）分析样品（分析条件为：采用0.1％磷酸流动相A和0.1％磷酸＋80％乙腈流动相B，梯度为：35－70％流动相B/25分钟），检测没有被单甲氧基聚乙二醇修饰的Exendin-4的量。结果如下表：
表：不同pH对Exendin-4聚乙二醇化的影响
使用旋转蒸发器对反相分离得到的收集液进行去除乙腈处理后，可以获得pH为3-4，溶解有单甲氧基聚乙二醇修饰的Exendin-4的溶液。
实施例四 直线型单甲氧基聚乙二醇（分子量＝21,000道尔顿）修饰Exendin-4类似物
使用梅特勒（AG285,Mettler Toledo）天平先准确称量1.0mg Exendin－4类似物（SEQ INNO:251），加入2.0ml溶解有氰硼氢化钠（NaBH3CN）磷酸缓冲液（pH4.5），再准确称取经由醛基活化的分子量为21,000道尔顿的单甲氧基聚乙二醇衍生物5mg。将衍生物加入Exendin－4类似物，置于摇床，室温反应过夜。使用配有Zorbax SB－300半制备柱的安捷伦1100色谱仪对反应液进行纯化（分离条件为：采用0.1％磷酸流动相A和0.1％磷酸＋80％乙腈流动相B，梯度为：35－70％流动相B/25分钟），获得聚乙二醇N末端修饰的Exendin－4类似物。
使用旋转蒸发器对反相分离得到的收集液进行去除乙腈处理后，使用脱盐柱对样品进行缓冲液置换，将得到的聚乙二醇修饰的Exendin-4类似物溶解于pH为7.0-8.0的磷酸盐缓冲液，并加入0.001-1.0％（w/v）间甲酚。
实施例五 带式IV结构单甲氧基聚乙二醇（分子量＝40,000道尔顿）修饰Exendin-4类似物
使用梅特勒（AG285,Mettler Toledo）天平先准确称量2.0mg Exendin－4类似物（SEQ INNO:251），加入4.0ml pH7.0磷酸缓冲液，再准确称取分子量为40,000道尔顿的N羟基琥珀酰亚胺活化的带式IV结构单甲氧基聚乙二醇衍生物280mg。将衍生物加入Exendin－4类似物，置于摇床，室温反应1小时。使用200mlBistris缓冲液(pH=7.0)稀释反应溶液于250ml烧杯中，待用。
使用Bistris缓冲液(pH=7.0)平衡DEAE FF或ANX FF阴离子交换预装柱，将稀释后的反应液上样，使目标物质吸附到离子交换填料上。上样完成后采用线性梯度洗脱的方式，使用不同浓度的NaCl将多取代和单取代Exendin-4类似物分别洗脱下来，同时使用馏分收集器收集,最后使用1M NaCl将未修饰的Exendin-4多肽洗脱下来。整个分离纯化过程如图3所示，图上标记2、3和4分别显示多取代Exendin-4类似物、单取代Exendin-4类似物和未修饰的Exendin-4类似物的洗脱峰。将收集得到的聚乙二醇单取代Exendin-4类似物（理论分子量为44,300道尔顿）做SDS-PAGE凝胶电泳得到的实际分子量略大于43,000道尔顿的标准分子量（如图4所示），说明分离获得的产品确实是聚乙二醇（分子量=40,000道尔顿）单取代Exendin-4类似物。
使用超滤装置（超滤离心管或超滤设备）对收集到的聚乙二醇单取代Exendin－4类似物洗脱液进行浓缩，之后再将浓缩液过脱盐柱进行乙酸缓冲液置换，最后在溶解有样品的乙酸缓冲液中加入等渗调节剂（如：甘露醇和0.9％NaCl）和0.001-1.0％（w/v）抑菌剂（如：间甲酚）等。
实施例六 经5,000道尔顿和21,000道尔顿直线型单甲氧基聚乙二醇单取代Exendin-4类似物24小时动物药效试验
实验动物：C57小鼠，体重20g±2g，每组8只动物，共4组。
对照组1：每只皮下注射注射用水。
对照组2：每只皮下注射Exendin-4类似物0.025μg。
给药组1：每只皮下注射单甲氧基聚乙二醇（分子量＝5,000道尔顿）单取代Exendin-4类似物0.625μg。
给药组2：每只皮下注射单甲氧基聚乙二醇（分子量＝21,000道尔顿）单取代Exendin-4类似物0.625μg。
每组小鼠分别在给药后4、8、12、16和24小时取血，并在采血前30min给所有受试小鼠腹腔注射200μl的20％葡萄糖溶液。使用葡萄糖试剂盒（上海申索试剂有限公司）对血糖进行测定。结果如图5所示，经单甲氧基聚乙二醇（分子量＝21,000道尔顿）修饰的Exendin－4类似物单次给药后在16小时能保持药效；单甲氧基聚乙二醇（分子量＝5,000道尔顿）修饰的Exendin－4类似物单次给药后能保持药效的时间范围在12-16小时；而未经修饰的Exendin－4类似物的作用时间不超过12小时。实验结果显示，分子量为21,000道尔顿的直线型聚乙二醇修饰的Exendin-4类似物的降糖作用保持时间比分子量为5,000道尔顿的直线型聚乙二醇修饰的Exendin-4类似物长。
实施例七 经21,000道尔顿（直线型）和40,000道尔顿（式IV所示支链结构）单甲氧基聚乙二醇单取代Exendin-4类似物72小时动物药效试验
实验动物：C57小鼠，体重20g±2g，每组8只动物，共5组。
对照组1：每只皮下注射注射用水。
对照组2：每只皮下注射Exendin-4多肽0.025μg。
对照组3：每只皮下注射Exendin-4类似物0.025μg。
给药组1：每只皮下注射单甲氧基聚乙二醇（分子量＝21,000道尔顿）取代Exendin－4类似物0.625μg。
给药组2：每只皮下注射单甲氧基聚乙二醇（分子量＝40,000道尔顿）取代Exendin－4类似物0.625μg。
每组小鼠分别在给药后4、8、24、48和72小时取血，并在采血前30min给所有受试小鼠腹腔注射200μl的20％葡萄糖溶液。使用葡萄糖试剂盒对血糖进行测定。结果如图6所示，经支链型聚乙二醇（分子量＝40,000道尔顿）修饰的Exendin－4类似物单次给药后能保持至少72小时的药效；直线型聚乙二醇（分子量＝21,000道尔顿）修饰的Exendin－4类似物单次给药后能保持大约24小时的药效。
实施例八 单甲氧基聚乙二醇单取代Exendin-4类似物药效试验
试验样品：总共8个样品，分别为：Exendin-4类似物（SEQ ID NO:251）上单取代有分子量为21，000道尔顿，30，000道尔顿和40，000道尔顿的单甲氧基聚乙二醇（式II）；Exendin-4类似物上单取代有分子量为21，000道尔顿，30，000道尔顿和40，000道尔顿的单甲氧基聚乙二醇（式IV）；Exendin-4类似物上单取代有分子量为21,000道尔顿和30,000道尔顿的直线型单甲氧基聚乙二醇。
实验动物：C57小鼠，体重20g±2g，共72只，分为3个采血组，每组24只。
由于动物不耐受多次采血，对三组动物分不同时间段采血，给药后对应的采血时间点如下：
| 第一组 | 第二组 | 第三组 |
| 2、8、72小时 | 4、24、48小时 | 16、36、60小时 |
每个采血组再平均分为8个样品小组，每小组3只老鼠。每小组老鼠在试验开始时皮下注射含0.625μg Exendin-4类似物分子的不同类型单甲氧基聚乙二醇单取代Exendin-4类似物，研发代号为L21K、L30K(分子量分别为21,000、30,000道尔顿的直线型单甲氧基聚乙二醇单取代)，Y21K、Y30K、Y40K(分子量分别为21,000、30,000、40,000道尔顿的式IV结构单甲氧基聚乙二醇单取代)，U21K、U30K和U40K(分子量分别为21,000、30,000、40,000道尔顿的式II结构单甲氧基聚乙二醇单取代)。给药同时给所有受试小鼠腹腔注射200μl20%葡萄糖溶液，并在给药之后0.5小时对所有受试老鼠采血。然后再按上述分组分别在给药后2、4、8、16、24、36、48、60和72小时采血。每次采血前0.5小时对所有采血小鼠腹腔注射200μl20%葡萄糖溶液。结果如图7、图8和图9所示，显示聚乙二醇分子结构和分子量对聚乙二醇单取代Exendin-4类似物的降糖生物活性的影响很大。式II结构单甲氧基聚乙二醇单取代Exendin-4类似物在给药后的72小时中，降糖活性的稳定性要好于直线型及式IV结构单甲氧基聚乙二醇单取代Exendin-4类似物；而同样结构的支链型单甲氧基聚乙二醇单取代Exendin-4类似物中，分子量越大，其在第72小时降糖活性越好。直线型单甲氧基聚乙二醇单取代Exendin-4类似物中L30K在给药后的72小时中均无明显降糖活性，说明直线型聚乙二醇分子量太大对其所修饰的Exendin-4类似物的降糖活性有负面影响。
实施例九 单甲氧基聚乙二醇单取代的Exendin-4类似物药代（血浆半衰期）试验
实验动物：SD大鼠，雄性，体重250g-300g，每组4只，共两组。
给药组1：每只皮下注射含4.375μg Exendin-4类似物分子的单甲氧基聚乙二醇（式II，分子量=30,000道尔顿）单取代Exendin-4类似物（SEQ ID NO:251），研发代号U30K。
给药组2：每只皮下注射含4.375μg Exendin-4类似物分子的单甲氧基聚乙二醇（式IV，分子量=40,000道尔顿）单取代Exendin-4类似物（SEQ ID NO:251），研发代号Y40K。
每组小鼠分别在给药后0、2、4、8、12、16、24、36、48、60和72小时采血。血药浓度检测使用美国康肽公司（Phoenix Pharmaceuticals,Inc.,California,USA）Enzyme Immunoassaykit（EIA）试剂盒检测Exendin-4浓度。结果如图10所示，经使用上海宏能软件有限公司的计算机软件Pharmaceutical Kinetics Software（版本1.0.2）计算，U30K的吸收半衰期为10.78小时，消除半衰期为21.44小时，Y40K的吸收半衰期为5.53小时，消除半衰期为40.77小时，两个样品的达峰时间均为给药后第12小时左右，最大药峰浓度均为20ng/mL左右。
Claims (9)
1.一种带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的聚乙二醇基团带如下式II所示支链结构：
（II）
其中，PEG为-O(CH2CH2O)q-，q为正整数，Me为甲基；所述聚乙二醇基团的分子量在20000-50000道尔顿之间；
其中，所述的聚乙二醇基团是通过将一个或一个以上的聚乙二醇与所述的Exendin或其类似物中的一个或一个以上的氨基酸相连接而连接到所述的Exendin或其类似物上；
所述的Exendin或其类似物是指与Exendin全部或部分氨基酸结构序列相类似的、能与胰高血糖素样肽-1受体结合、并产生细胞信号级联传导的一类多肽。
2.一种带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的聚乙二醇基团带如下式IV所示的支链结构：
（IV）
其中，PEG为-O(CH2CH2O)q-，q为正整数，Me为甲基；所述聚乙二醇基团的分子量在20000-50000道尔顿之间；
其中，所述的聚乙二醇基团是通过将一个或一个以上的聚乙二醇与所述的Exendin或其类似物中的一个或一个以上的氨基酸相连接而连接到所述的Exendin或其类似物上；
所述的Exendin或其类似物是指与Exendin全部或部分氨基酸结构序列相类似的、能与胰高血糖素样肽-1受体结合、并产生细胞信号级联传导的一类多肽。
3.如权利要求1或2所述的带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的Exendin或其类似物为SEQ ID NO:2所列的氨基酸序列。
4.如权利要求1或2所述的带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的Exendin或其类似物为SEQ ID NO:251所列的氨基酸序列。
5.如权利要求1或2所述的带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的聚乙二醇基团是经N羟基琥珀酰亚胺活化而连接到所述的Exendin或其类似物上。
6.如权利要求1或2所述的带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的带有聚乙二醇基团的Exendin或其类似物中包含一个、两个、三个或四个带支链结构的聚乙二醇基团。
7.如权利要求6所述的带有聚乙二醇基团的Exendin或其类似物，其特征在于，所述的带有聚乙二醇基团的Exendin或其类似物中包含一个带支链结构的聚乙二醇基团。
8.一种由带有聚乙二醇基团的Exendin或其类似物形成的制剂，其特征在于，该制剂包括如权利要求1-7所述的一种或多种带有聚乙二醇基团的Exendin或其类似物。
9.如权利要求1-7之一所述的一种或多种带有聚乙二醇基团的Exendin或其类似物在制备治疗糖尿病药物方面的用途。
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| AR105319A1 (es) | 2015-06-05 | 2017-09-27 | Sanofi Sa | Profármacos que comprenden un conjugado agonista dual de glp-1 / glucagón conector ácido hialurónico |
| AR105284A1 (es) | 2015-07-10 | 2017-09-20 | Sanofi Sa | Derivados de exendina-4 como agonistas peptídicos duales específicos de los receptores de glp-1 / glucagón |
| CN106554403B (zh) | 2015-09-25 | 2021-08-31 | 博瑞生物医药（苏州）股份有限公司 | 艾塞那肽修饰物及其用途 |
| EP3426678B1 (en) * | 2016-03-10 | 2025-07-02 | Shenzhen Hightide Biopharmaceutical, Ltd. | Conjugates of islet neogenesis peptides and analogs, and methods thereof |
| US20200164083A1 (en) * | 2016-03-16 | 2020-05-28 | Prolynx Llc | Extended release conjugates of exenatide analogs |
| CN107952064B (zh) * | 2016-10-14 | 2023-10-20 | 江苏豪森药业集团有限公司 | 含有聚乙二醇洛塞那肽的药物制剂及其制备方法 |
| CN109400695B (zh) | 2018-10-31 | 2020-06-30 | 中南大学湘雅医院 | 一种多肽的修饰方法及应用 |
| AU2020253560B2 (en) | 2019-04-05 | 2026-01-15 | Prolynx Llc | Improved conjugation linkers |
| CA3184544A1 (en) * | 2020-07-02 | 2022-01-06 | Sanofi | Glp-1r agonistic peptides with reduced activity |
| CN114075275A (zh) * | 2020-08-17 | 2022-02-22 | 成都奥达生物科技有限公司 | 一种长效胰岛素类似物 |
| CN114478694A (zh) * | 2020-11-13 | 2022-05-13 | 成都奥达生物科技有限公司 | 一种长效mc4r激动剂 |
| CN114478744A (zh) * | 2020-11-13 | 2022-05-13 | 成都奥达生物科技有限公司 | 一种长效glp-1拮抗剂 |
| WO2023055081A1 (ko) * | 2021-09-28 | 2023-04-06 | 한양대학교 산학협력단 | 암 치료용 글리시리진-분지형 폴리에틸렌글리콜 컨쥬게이트 |
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| WO2000066629A1 (en) * | 1999-04-30 | 2000-11-09 | Amylin Pharmaceuticals, Inc. | Modified exendins and exendin agonists |
| CN1162446C (zh) | 2001-05-10 | 2004-08-18 | 上海华谊生物技术有限公司 | 促胰岛素分泌肽衍生物 |
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| HRP20050683A2 (en) | 2003-03-19 | 2006-07-31 | Eli Lilly And Company | Polyethylene glycol linked glp-1 compounds |
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| BRPI0417717A (pt) | 2003-12-18 | 2007-04-03 | Novo Nordisk As | composto, composição farmacêutica, e, uso de um composto |
| US7745516B2 (en) * | 2005-10-12 | 2010-06-29 | E. I. Du Pont De Nemours And Company | Composition of polyimide and sterically-hindered hydrophobic epoxy |
| WO2007047834A2 (en) * | 2005-10-18 | 2007-04-26 | Biocon Limited | Oral peptide conjugates for metabolic diseases |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004022004A2 (en) * | 2002-09-06 | 2004-03-18 | Bayer Pharmaceuticals Corporation | Modified glp-1 receptor agonists and their pharmacological methods of use |
| CN1676163A (zh) * | 2004-09-30 | 2005-10-05 | 华东师范大学 | 一种人胰高血糖素样肽－1的复合物及其制备方法 |
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| JP2014088384A (ja) | 2014-05-15 |
| AU2007321649A1 (en) | 2008-05-22 |
| CA2666182A1 (en) | 2008-05-22 |
| CN102827284A (zh) | 2012-12-19 |
| RU2498814C2 (ru) | 2013-11-20 |
| CN101125207B (zh) | 2012-09-05 |
| WO2008058461A1 (en) | 2008-05-22 |
| CA2933795C (en) | 2019-01-08 |
| AU2007321649B2 (en) | 2013-01-10 |
| JP2010509248A (ja) | 2010-03-25 |
| KR20090089316A (ko) | 2009-08-21 |
| EP2081957B1 (en) | 2019-08-14 |
| CA2933795A1 (en) | 2008-05-22 |
| CN101125207A (zh) | 2008-02-20 |
| RU2009122582A (ru) | 2010-12-20 |
| BRPI0717183B8 (pt) | 2021-05-25 |
| EP2081957A1 (en) | 2009-07-29 |
| BRPI0717183A2 (pt) | 2015-06-16 |
| US9175060B2 (en) | 2015-11-03 |
| BRPI0717183B1 (pt) | 2019-05-21 |
| US20100240586A1 (en) | 2010-09-23 |
| EP2081957A4 (en) | 2013-05-08 |
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