CN1230966A - 勒帕素拮抗剂在用于治疗ii型糖尿病胰岛素耐受性中的用途 - Google Patents
勒帕素拮抗剂在用于治疗ii型糖尿病胰岛素耐受性中的用途 Download PDFInfo
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- CN1230966A CN1230966A CN97198076A CN97198076A CN1230966A CN 1230966 A CN1230966 A CN 1230966A CN 97198076 A CN97198076 A CN 97198076A CN 97198076 A CN97198076 A CN 97198076A CN 1230966 A CN1230966 A CN 1230966A
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Abstract
本发明涉及含有用于治疗Ⅱ型糖尿病的勒帕素拮抗剂的药物组合物。勒帕素拮抗剂基于小鼠勒帕素片段并包含氨基酸116—167或116—166。还公开了治疗Ⅱ型糖尿病的方法。
Description
发明背景
本发明涉及勒帕素(leptin)拮抗剂在用于治疗Ⅱ型糖尿病胰岛素耐受性中的用途以及用于治疗这种耐受性的药物。
糖尿病是一种在工业化国家中最常发生的代谢疾病。在世界范围内有1.1亿糖尿病患者;虽然大约1千万为Ⅰ型糖尿病,但占优势的主要(约1亿)为Ⅱ型糖尿病。这种疾病由葡萄糖代谢的错误调节所导致。在Ⅰ型糖尿病中,胰腺中β细胞的缺乏导致不再形成胰岛素。这种胰岛素的缺乏导致血液葡萄糖的增加,并且如果不通过补充胰岛素来进行治疗,则导致病人酮酸中毒、糖尿病性昏迷和死亡。在Ⅱ型糖尿病中,其因果关系不同,特征在于胰岛素耐受性的最初产生,即细胞适当地对胰岛素产生应答的能力减弱。尤其是过重的体重以及缺乏体力活动被认为是导致诱导胰岛素耐受性的原因。由于通过增加的胰岛素分泌而得以补偿,因此后一种情况起初不为人所注意。然而,在持续多年的过程中,持续的胰岛素耐受性导致内源补偿机制的不足以及随之而发生的Ⅱ型糖尿病。虽然饮食和体力活动可延缓这些进程的后果,但它们通常不能防止这些疾病的出现。为了适当控制血液葡萄糖则需要医疗手段。
将血液葡萄糖维持在尽可能窄的生理范围内对于治疗的长期成功具有至关的重要性。目前认为在控制得不好的糖尿病患者(Ⅰ型和Ⅱ型糖尿病患者)中发现的已升高达几十年的葡萄糖水平对糖尿病后期并发症有着重要的影响。这些后期并发症尤其形成导致肾病、视力丧失和心血管疾病的血管损伤。这种所谓的后期损伤是导致糖尿病患者死亡的一个重要因素。
在1994年,描述了一种新的在脂肪细胞中形成并且在遗传肥胖小鼠(ob/ob小鼠)中缺乏的激素,勒帕素(Zhang,Y.,Proenca,R.,Maffei,M.,Barone,M.,Leopold,L.和Friedman,J.M.(1994)小鼠肥胖基因的定位克隆和其人类似物,自然(Nature)372,425-432)。人勒帕素和小鼠勒帕素在很大程度上是相同的。用重组制备的勒帕素注射ob/ob小鼠导致营养摄取的减少以及导致体重的降低(Pelleymounter,M.A.,Cullen,M.J.,Baker,M.B.,Hecht,R.,Winters,D.,Boone,T.和Collins,F.(1995)肥胖基因产物对ob/ob小鼠体重调节的影响,科学(Science)269,540-543)。至今没有迹象表明肥胖基因突变可能导致人肥胖的经常发生(在美国约30%的人为明显超重)。***的调查证实如在各种肥胖动物模型中一样,在肥胖人中勒帕素的血清水平增加(Dagogo-Jack,S.,Fanelli,C.,Paramore,D.,Brothers,J.和Landt,M.(1996)肥胖和不肥胖者中的血浆勒帕素与胰岛素的相互关系糖尿病(Diabetes)45,695-698;Considine,R.V.,Sinha,M.K.,Heiman,M.L.,Kriauciunas,A.,Stephens,T.W.,Nyce,M.R.,Ohannesian,J.P.,Marco,C.C.,McKee,L.J.,Bauer,T.L.和Caro,J.F.(1996)正常体重者和肥胖者中的血清免疫反应性勒帕素浓度,新英格兰医学杂志(N.Engl.J.Med.)334,292-295)。由于这种原因,设想勒帕素是将储藏在脂肪组织中的能量的量通知大脑的反馈信号。根据这种假设,那么大脑的功能一方面是通过抑制食欲减少食物的摄取,另一方面是刺激基础代谢。在肥胖者中,这种调节回路看来被打断了。
除此之外,推测勒帕素还直接作用于大脑之外的组织。
目前已公开了三项有关勒帕素对分离细胞的直接作用的研究:
Kroder等(Kroder,G.,Kellerer,M.和Hring,H.(1996)Exp.Crin.Endocrin.Diabetes104增刊2,66(摘要))作出这样一种设想,即勒帕素在胰岛素耐受性和肥胖之间建立一种联系,并报道在过度表达人胰岛素受体的大鼠成纤维细胞中,勒帕素降低胰岛素受体和胰岛素受体底物1(1RS-1)的胰岛素诱导的磷酸化。没有研究和讨论勒帕素对胰岛素作用的终点,例如葡萄糖转运或糖原合成的影响程度。
已证实在过度表达勒帕素的被转化的30A5前脂肪细胞中,对脂肪生成激素(***和胰岛素)的敏感性降低(Bai,Y.L.,Zhang,S.Y.,Kim,K.S.,Lee,J.K.和Kim,K.H.(1996)生物化学杂志(J.Biol.Chem.)271,13939-13942)。甚至在未刺激状态下,勒帕素的过度表达也降低脂肪酸的合成以及中性脂肪的合成。虽然在细胞被用***或胰岛素或这两种激素的混合物处理后,对照细胞的脂肪合成速度显示明显的增加,但在所有这些条件下,过度表达勒帕素的细胞很难被刺激。除此之外,还进行了关于在用***与胰岛素的混合物处理细胞后产生的磷酸甘油脱氢酶活性抑制以及乙酰CoA羧化酶表达抑制的研究。发现不可能刺激表达勒帕素的细胞。观察到的结果表明勒帕素以常规方式抑制脂肪代谢。没有提到肥胖和胰岛素耐受性之间的任何可能的联系。
在另一个模型***即C2C12小鼠肌管中,发现勒帕素显示胰岛素样作用(Berti,L.,Kellerer,M.,和Haring,H.(1996)糖尿病学(Diabetologia)39增刊1,A59(摘要))。该研究报道勒帕素刺激葡萄糖的转运和糖原的合成。这些发现与其它作者报道的那些以及本文提供的结果矛盾。它们可能涉及这种细胞类型所特有的效应。
在我们对勒帕素对分离的大鼠脂肪细胞,即脂肪组织的模型***的作用的研究中,现已出乎意料地发现脂肪细胞重要代谢途径-如对脂肪生成、葡萄糖转运以及糖原生成的刺激-的胰岛素敏感性被大大降低(实施例4),而同时基础值保持不变。异丙基肾上腺素刺激的脂肪生成也是如此。通过加入胰岛素(10nM)分离的大鼠脂肪细胞中的葡萄糖转运被刺激约14倍。通过与不同浓度的勒帕素预保温15小时,这种被刺激的能力被以剂量依赖性方式降低。勒帕素使细胞失敏,即产生胰岛素耐受性。不同勒帕素浓度下的胰岛素剂量/效应曲线(实施例5)证明对于胰岛素(0.1-0.2nM)和勒帕素(0.5-1nm)而言,在体外能够检测到效果的浓度在生理范围内(Dagogo-Jack等,1996;Considine等,1996)。在肥胖人中发现了较高的勒帕素水平(2-4nM)(Dagogo-Jack等,1996;Considine等,1996),这样在这些个体中胰岛素的作用可能被更强地损害。结论不言自明,如在肥胖个体中可见到的,长期升高的勒帕素导致胰岛素耐受性。如上面已经解释的,胰岛素耐受性是Ⅱ型胰岛素发病机理中的一个重要因素。
因此,本发明的一个目的是提供可被配制在药物组合物中的新的勒帕素拮抗剂。本发明的另一个目的是提供治疗Ⅱ型糖尿病和其它与胰岛素相关的疾病的方法。
本发明因此涉及勒帕素拮抗剂,尤其是衍生于勒帕素本身的那些在制备用于Ⅱ型糖尿病的药物中的用途。用于这种用途的勒帕素拮抗剂在下面被详细描述。
发明概述
根据本发明的第一个目的,提供含有勒帕素拮抗剂的药物组合物。根据此目的,公开了含有衍生于勒帕素的勒帕素拮抗剂的药物组合物。进一步根据此目的,公开了含有作为可溶性勒帕素受体的勒帕素拮抗剂或其衍生物的药物组合物。
根据本发明的第二个目的,提供在治疗Ⅱ型糖尿病中使用本发明药物组合物的方法。还根据此目的,提供恢复或加强胰岛素生理作用的方法。
附图的简要说明
表1:在大鼠脂肪细胞中勒帕素对胰岛素诱导的葡萄糖转运的抑制。实验详细描述在实施例8中。
表2:作为勒帕素浓度函数的大鼠脂肪细胞的2-脱氧葡萄糖的摄取。实验详细描述在实施例9中。
表3:就大鼠脂肪细胞中的胰岛素诱导的葡萄糖转运,勒帕素片段116-167对勒帕素作用的拮抗作用。实验详细描述在实施例10中。
表4:人勒帕素的氨基酸序列。两个半胱氨酸通过二硫桥连接。
表5:小鼠勒帕素的氨基酸序列。两个半胱氨酸通过二硫桥连接。
表6:小鼠勒帕素片段116-167的氨基酸序列。两个半胱氨酸通过二硫桥连接。
发明详述
本文提供的发明涉及通过抑制勒帕素的作用降低或完全消除胰岛素耐受性的组合物和方法。为此目的,可使用起勒帕素拮抗剂作用并在体外分离大鼠细胞中导致勒帕素诱导的胰岛素耐受性消除的肽。这些肽因而适宜于治疗优选肥胖病人中的胰岛素耐受性。
勒帕素拮抗剂
根据本发明的勒帕素拮抗剂具体包括肽拮抗剂。所述肽衍生于勒帕素片段并可例如通过化学或酶解完整的勒帕素(例如用赖氨酰内肽酶、胰蛋白酶、内切Arg C或溴化氰)而获得或通过在微生物中直接或以融合蛋白的形式表达来制备。在微生物中制备肽方面,当选择待表达的片段时,不必依赖于天然裂解位点的存在。人和动物勒帕素,例如大鼠、小鼠、猪或类人猿勒帕素适宜于产生这些肽。
根据Zhang等(1994)公开的序列,一个适宜的作为实例的肽为从氨基酸116至氨基酸167或从氨基酸116至氨基酸166(表6,SEQ ID NO:4)(实施例3和6)。在实施例6中,在10nM勒帕素和不同浓度的拮抗剂116-167勒帕素片段存在下,将脂肪细胞保温约15小时。接着用5nM胰岛素刺激细胞。在此实验中,发现递增量的拮抗剂导致被胰岛素刺激能力的恢复,并且在存在高浓度的勒帕素拮抗剂时,没有耐受性产生。因此,使用这些以及类似的分析方法,本领域技术人员可很容易地确定根据本发明有用的任何勒帕素拮抗剂的拮抗性质。除此之外,还可使用其中一个或多个氨基酸被置换或缺失的拮抗性勒帕素片段的类似物。优选的置换为保守氨基酸置换。这些保守置换包括带电荷-带电荷、极性-极性和疏水性-疏水性氨基酸置换。例如,一个或多个天冬氨酸残基可被谷氨酸残基置换和/或反之亦然和/或一个或多个亮氨酸残基可被异亮氨酸残基置换和/或反之亦然。根据空间和结构情况,如氨基酸大小和螺旋形成或断裂的倾向,可合理地进行其它置换和缺失。
分子生物学和生物技术方法可被用来以特定的方式改变和优化所述肽的拮抗性质。除此之外,为了增加它们的稳定性或调节它们的血浆半衰期和药物动力学,可通过例如乙酰化、氨基甲酰化、甲酰化、生物素化、酰化或用聚乙二醇或亲水性聚合物衍生化对这些肽进行化学修饰。
抗勒帕素的抗体,尤其是其勒帕素结合区也适宜作为用于所述目的的勒帕素拮抗剂。除此之外,可溶性的勒帕素受体和/或勒帕素受体片段以及其与其它蛋白(例如IgG Fc区)的融合物也是适宜的。类似于肽拮抗剂,为蛋白质的任何勒帕素拮抗剂可通过分子生物学方法改变。类似的,可通过在微生物或任何种标准表达体系中直接或以融合蛋白的形式表达来制备这些拮抗剂。
药物组合物
本发明进一步涉及包含本专利申请中描述的勒帕素拮抗剂的药物。
可例如以可经口服给药的药物制剂的形式使用该药物,例如片剂、包衣片剂、硬或软明胶胶囊剂、溶液剂、乳剂或悬浮剂。它们还可被例如以栓剂的形式经直肠给药,或以注射用溶液剂的形式经胃肠外给药。该药物还可通过鼻、口或肺粘膜的途径给药。为了制备药物制剂,这些化合物可被配制到治疗惰性、有机和无机赋形剂中。乳糖、玉米淀粉或其衍生物、滑石和硬脂酸或其盐为用于这些片剂、包衣片剂和硬明胶胶囊剂的赋形剂的实例。水、多元醇、蔗糖、转化糖和葡萄糖为用于制备溶液的适宜的赋形剂。水、醇、多元醇、甘油和植物油为用于注射用溶液的适宜赋形剂。植物油和硬化油、蜡、脂以及半液体多元醇为用于栓剂的适宜赋形剂。药物制剂还可包含防腐剂、溶剂、稳定剂、湿润剂、乳化剂、增甜剂、染料、调味剂、用于改变渗透压的盐、缓冲液、包衣剂、抗氧化剂以及适宜时其它治疗活性化合物。
优选口服给药和注射。为了注射,将新的勒帕素拮抗剂配制在液体溶液中,优选在生理可接受的缓冲液中,如Hank氏溶液或Ringer氏溶液。然而,也可将新的勒帕素拮抗剂配制成固体形式并在使用前将其溶解或悬浮。
典型的制剂含有治疗有益量的勒帕素拮抗剂。如下所述,治疗有益量可与治疗有效量相同。此外,治疗有益量可为单剂,一个或多个单剂可用于提供治疗有效量的勒帕素拮抗剂。因此,治疗有益量将尤其取决于待治疗疾病的性质。
治疗方法
本发明方法可用于治疗其中涉及勒帕素作用的任何疾病。根据目前有关勒帕素抑制一些胰岛素生理活性的报道和评述,本发明方法尤其可用于治疗涉及胰岛素活性紊乱的疾病,尤其是Ⅱ型糖尿病。这些胰岛素活性的紊乱包括脂肪合成、葡萄糖转运、糖原合成和脂解作用的改变。因此,本发明包括治疗Ⅱ型糖尿病的方法以及恢复或增强胰岛素作用的方法。
常规方法包括给予需要治疗的病人治疗有效量的勒帕素拮抗剂。当患有涉及勒帕素作用的疾病时,病人将需要治疗。当病人患有Ⅱ型糖尿病时,尤其需要治疗。当病人患有特征在于胰岛素活性的紊乱,如脂肪合成、葡萄糖转运、糖原合成和脂解作用的改变的疾病时也需要治疗。在涉及这种紊乱的疾病中,恢复或增强胰岛素生理作用的方法是有用的。
治疗有效量将取决于例如所治疗疾病的性质、给药途径、所选择的拮抗剂的具体特性以及尤其是临床医生的判断。治疗有效量通常为足以完成目标疾病的治疗或达到治疗方法的所述目的,例如恢复或增强胰岛素生理作用的量。归根到底治疗有效量将取决于临床确定的功效以及各种勒帕素拮抗剂的毒性。可常规性地作出这些判定,且其完全在普通临床技术人员的知识范围内。
与本发明相关的各种语法形式的术语“治疗”涉及预防、治疗、逆转、减弱、缓和、最小化、抑制或停止病情的有害影响、疾病的进程、疾病的病原体或其它异常情况。
优选用于全身给药的剂量为约0.01mg/kg至约50mg/kg体重/天。
下面通过表和实施例说明本发明而不限制本发明。
实施例
实施例1:克隆小鼠勒帕素
RNA的分离-从成年小鼠中取出附睾脂肪垫并在液氮中迅速冷冻。液氮下,将1g脂肪组织用研钵研碎,此后,加入15ml 5M硫氰酸胍的50mMTris(pH7.5)溶液,其中含有10mM EDTA和0.1M DTT,将全部溶液用力匀浆化以获得细分散液。在组织颗粒被完全消散后,加入10g固体CsCl,室温下,搅拌混合物。在加入10ml H2O后,在离心管中9ml 5.7M CsCl溶液上加25ml这种溶液。在SW28离心机中以25,000rpm(18℃)离心15小时后,将试管在液氮中深度冷冻,使用热解剖刀片切去试管底部的四分之一;取出冷冻内容物,从其底部末端刮下RNA沉淀。溶解RNA,接着用乙醇沉淀。
cDNA的合成-在混合物中,将1μg来自脂肪组织的总RNA和1μg特异性引物寡核苷酸5’-GAATGCAGAATAAATAAATA(SEQ ID NO:1;Zhang等,1994)溶解在10μl H2O中,接着热变性并在65℃下保温5分钟。在加入0.5μl RNase抑制剂、各为5nmol的dNTP和0.5μl AMV逆转录酶(Boehringer Mannheim)后,42℃下,将混合物保温1小时。此后,用水将cDNA配成200ul,贮存于-20℃。
PCR-在制造商推荐的反应缓冲液中(1.5mM MgCl2,200uM dNTPs的100ul溶液),用各0.5μg的两种引物5’-GAAAGAAGGATCCAGTGCCTATCCAGAAAGTCCA(SEQ ID NO:2)和5’-GGAGAGAAGCTTGAGGGAGAGAAATGAATGATGG(SEQ IND NO:3;Zhang等,1994)和2.5U Taq聚合酶(Perkin Elmer)将3μl特异性引导的cDNA扩增30次循环。每一次循环包括94℃ 1分钟,55℃ 1分钟和72℃2分钟。
连接-在每种情况下,在根据制造商说明的缓冲条件下,37℃下将来自PCR制备的特异性扩增的PCR产物(583bp)用限制酶BamHⅠ和HindⅢ(Boehringer Mannheim)裂解2小时,此后,将564bp片段电泳纯化并分离。30℃下,在20μl中将其与0.1μg被BamHⅠ和HindⅢ裂解的载体pQE31(Qiagen)以及20U T4 DNA连接酶(新英格兰实验室)一起保温2小时。
克隆-将5μl连接混合物与100μl HB101菌株的转化感受态大肠杆菌细胞一起在冰上放置30分钟,37℃下,将混合物在水浴中轻轻涡旋5分钟。在加入0.9Ⅲl含有10mM MgCl2的营养培养基后,37℃下,将混合物振荡1小时。在每种情况下,将100μl体积的该混合物平板接种到含有氨苄青霉素(100μg/ml)的琼脂平板中。
克隆的鉴定-将37℃下培养过夜的克隆接种到2ml体积含有氨苄青霉素的液体培养基中,培养至稳定期,接着离心沉淀。将细胞悬浮在0.1ml25mM Tris溶液中(pH8),其中含有50mM萄萄糖,10mM EDTA和溶菌酶(2mg/ml),在室温下保温5分钟后,加入0.2ml 0.2M NaOH,1%SDS使其裂解。通过加入150ul 3M乙酸钠/乙酸(pH5.2)沉淀染色体DNA并在4℃下离心(在Sigma 2MK中,10,000rpm)5分钟。用2.5体积的乙醇沉淀质粒DNA,离心(参见上文),在乙醇洗涤一次后,溶解在100μl H2O中;接着加入10ul RNase溶液(10mg/ml)。
根据制造商的说明用限制酶(BgⅡ,XhoⅠ+PvuⅡ;BoehringerMannheim)消化质粒DNA,在电泳以及用溴化乙啶染色后,在琼脂糖凝胶中根据标志DNA测定产生的DNA片段。用同样的方法使用限制酶(新英格兰实验室)测定具有正确片段图谱的克隆的谷氨酰胺49残基的存在。
通过DNA测序证实各种情况下的含有Gln49(pQEob3-9)和不含Gln49(pQEob3-4)的大肠杆菌克隆。在少量培养物中检测含有前序列MetArgGlySer(His)6ThrAspPro(来自载体pQE31)接着为来自小鼠勒帕素的氨基酸22-167的重组勒帕素的产生。
虽然在下面描述的实验中可采用两种重组勒帕素(含与不含Gln49),但实施例具体地涉及含有Gln49并通过表达pQEob3-9获得的勒帕素。
实施例2:勒帕素的制备
破碎-将来自10升发酵物的细菌以4800rpm离心20分钟。将沉淀在-20℃下冷冻,随后悬浮于溶菌缓冲液中(6M氯化胍,0.1M NaH2PO4,10mM Tris/HCl,pH8)(5ml溶菌缓冲液/克沉淀),此后,室温下将混合物搅拌1小时,接着以4800rpm离心30分钟。
Ni-NTA层析-将100ml Ni-NTA琼脂糖FF(Qiagen,Hilden)加入到含有约800mg勒帕素的粗提物中,4℃下,将整个混合物搅拌过夜。将悬浮液吸入通过含有玻璃料的玻璃柱(φ5cm)。将柱与包含于其中的Ni-NTA琼脂糖一起用300ml溶菌缓冲液洗涤,接着用100ml含有10mM咪唑的溶菌缓冲液洗涤(5ml/分钟)。通过使用在溶菌缓冲液中10-200mM咪唑的线性梯度进行勒帕素的分级洗脱(梯度体积:300ml,5ml/分钟)。用RP-HPLC分析级分,混合具有适当纯度和浓度的那些级分(=Ni-NTA洗脱液)。
折叠-用溶菌缓冲液将Ni-NTA洗脱液稀释至1-3mg/ml的浓度,用氢氧化钠溶液调节至pH9。加入β-巯基乙醇(4-6molβ-巯基乙醇/摩尔勒帕素)后,室温下,在密封容器中将混合物保温2小时。为了重新氧化和重新折叠,将溶液倒入9倍体积的折叠缓冲液中(0.1M Tris/HCl,pH9),16℃下,在允许有空气下,将整个溶液搅拌16-24小时。离心除去出现的任何混浊物(4,000rpm,45分钟)。
反向高效液相色谱-用HCl将折叠混合物调至pH3,并以20ml/分钟的速率泵入2.5×30cm RP柱中(PLRPS300埃,10μ,PolymerLaboratories,Amherst,USA)。随后将柱用300ml洗脱液A(0.1%TFA水溶液)洗涤。通过使用梯度为25-50%的洗脱液B(0.09%TFA的乙晴溶液),在100分钟内将勒帕素洗脱(流速:7ml/分钟)。通过分析型HPLC分析各级分。混合具有适当纯度和浓度的级分(RP库)。将该库用7mmolNa2HPO4处理,用NaOH调至pH3,在旋转蒸发仪中除去溶剂。用NaOH(pH7.4)中和勒帕素水溶液,4℃下贮存过夜。离心除去任何产生的混浊物(4,000rpm,10分钟)。
凝胶渗透层析-通过超滤将中和以及离心的勒帕素溶液浓缩至10-15mg/ml,接着通过过滤灭菌。将50至75mg上样到Superdex75柱(2.6×60cm,Pharmacia,瑞典)。PBS(154mM NaCl,10mM磷酸钠,pH7.4)被用作洗脱缓冲液,其流速为3ml/分钟。将以这种方法纯化的勒帕素再通过0.22μm膜过滤,贮存于-70℃。
实施例3:拮抗性116-167片段的制备
将1ml 1M Tris/HCl,pH8加入到40ml勒帕素溶液中(在PBS中,为1mg/ml),在加入160μg赖氨酰内肽酶后,室温下,将勒帕素消化3小时。将混合物调至pH3,通过如实施例2描述的RP-HPLC分级分离。通过电喷射质谱法(5532 D)鉴定116-167片段,如实施例2所述除去溶剂,用凝胶渗透层析浓缩并纯化。
实施例4:脂肪细胞的分离
通过用胶原酶(Rodbell,1964,生物化学杂志(J.Biol.Chem.)239,375-380)消化,通过飘浮法(800×g,1分钟,在小塑料试管中),用KRH(25mM Hepes游离酸,25mM Hepes钠盐,80mM NaCl,1mM MgSO4,2mMCaCl2,6mM KCl,1mM丙酮酸钠,0.5%BSA)洗涤两次,用补充有5.5mM葡萄糖、20mM Hepes(pH7.4)、2%胎牛血清、1%BSA、50U青霉素/ml、10mg链霉素/ml的DMEM(Dulbecco氏最小基本培养基)洗涤一次,并最后稀释为20ml DMEM/g脂肪组织湿重的体积(细胞滴度:约2.5×105细胞/ml)来从雄性Wistar大鼠(140-160g,Hoechst AG动物站,Kastengrund)的附睾脂肪组织制备脂肪细胞。
实施例5:脂肪细胞的原代培养和与勒帕素的保温
37℃,5%CO2气氛下,在存在100nM苯基异丙基腺苷以及存在或不存在勒帕素时,将脂肪细胞在补充的(参见上文)DMEM中轻轻振荡保温15-18小时(4ml DMEM被加入到1ml细胞中,细胞滴度为约5×104细胞/ml,在50ml无菌聚丙烯试管中)。随后,将脂肪细胞用冷KRH洗涤3次,通过向完全除去最后的洗涤溶液后剩余的细胞层中加入0.7ml无葡萄糖的KRH将细胞滴度调至约3×105细胞/ml。为了确定脂肪细胞被胰岛素刺激的能力以及它们对胰岛素的敏感性,在原代培养后,37℃下,在不存在或存在人胰岛素时(0.02-50nM的最终浓度),将洗涤后的脂肪细胞保温20分钟,并接着测定葡萄糖转运或脂肪生成。
实施例6:葡萄糖的转运
以不可代谢的葡萄糖类似物2-脱氧葡萄糖的特异性摄取来测定葡萄糖的转运(Muller和Wied,1993,糖尿病42,1852-1867)。25℃下,将50ul与或未与胰岛素预保温(参见上文)的脂肪细胞的KRH悬液与50μl补充有2-脱氧-D-[2,6-3H]葡萄糖(0.5μCi,0.2mM)的KRH保温5分钟。将保温混合物转移到薄的软塑料离心管中,其中每一个已含有200μl邻苯二甲酸二壬酯油,并立即离心(2,000×g,30秒)。使用特殊的剪刀,在油层(上缘的附近)和试管的上半部切断试管,连同飘浮在油层中的细胞层一起转移至闪烁瓶中。在加入10ml闪烁液(水为基质)后,测定与细胞相关的放射性。为了校正已包含在细胞间隙或以非特异性方式扩散到细胞中的2-脱氧葡萄糖,从各个保温混合物的与细胞相关的总放射性中减去与细胞松弛素B(20uM)预保温的细胞的放射性(Gliemann等,1972,生物化学与生物物理学报(Biochim.Biophys.Acta)286,1-9)。
实施例7:脂肪生成
以D-葡萄糖向甲苯可提取的脂肪中的掺入来测定脂肪生成(Moody等,1974,激素代谢研究(Horm.Metab.Res.)6,12-16)。37℃下,在闪烁瓶中将脂肪细胞的KRH悬液在680μl补充有3.5mM葡萄糖和20μl胰岛素溶液的KRH中保温20分钟。通过加入100μlD-[3-3H]葡萄糖(25uCi/mlKRH)启动脂肪生成。37℃,5%CO2气氛下轻轻振荡保温90分钟后,加入1Oml闪烁液(甲苯为基质),在剧烈振荡以及随后的相分离后,测定甲苯相中的放射活性(至少保温4小时)。将甲苯相中脂肪的放射性对含有相同量的[3H]葡萄糖但没有细胞的保温混合物的放射性进行校正。
实施例8:勒帕素对胰岛素诱导的葡萄糖转运以及胰岛素诱导的脂肪生成的抑制
在存在或不存在递增浓度的勒帕素下,在原代培养物中将分离的大鼠脂肪细胞保温15小时。接着洗涤细胞,测定在基础状态和胰岛素(10nM)刺激状态下的葡萄糖转运和脂肪生成。以胰岛素刺激的活性与基础活性之间的比例来计算胰岛素的刺激系数。每个值代表来自两个独立的脂肪细胞培养物的平均值,其中在每种情况下测定活性两或三次。
表1:胰岛素浓度:5nM
勒帕素 | 葡萄糖 | |
浓度[nM] | 转运* | 脂肪生成* |
0 | 13.4 | 3.9 |
0.3 | 13.4 | 3.9 |
1 | 11.75 | 3.2 |
3 | 7.4 | 2.15 |
10 | 3.5 | 1.5 |
30 | 2.35 | 1.15 |
100 | 1.5 | 1.05 |
*系数为刺激后的值与基础值的比值。
结果总结于表1中。
实施例9:勒帕素对胰岛素剂量/效应曲线的影响
在存在或不存在递增浓度的勒帕素下,在原代培养物中将分离大鼠脂肪细胞保温16.5小时。接着洗涤细胞,检测不同浓度的胰岛素对葡萄糖转运的刺激。葡萄糖转运活性以与细胞特异性结合的2-脱氧-[3H]葡萄糖的“dpm值”给出。每个值代表来自两个独立的脂肪细胞培养物的平均值,其中每种情况下测定活性4次。
表2:测定的参数:葡萄糖转运
勒帕素[nM] | ||||||||
胰岛素,[nM] | 0 | 0.05 | 1 | 2 | 5 | 10 | 30 | 100 |
0 | 671 | 654 | 634 | 688 | 712 | 755 | 688 | 747 |
0.02 | 784 | 735 | 678 | 704 | 734 | 773 | 704 | 766 |
0.05 | 1285 | 1025 | 824 | 755 | 798 | 802 | 745 | 780 |
0.1 | 2406 | 1674 | 1189 | 860 | 883 | 856 | 789 | 803 |
0.2 | 4762 | 3320 | 1587 | 1006 | 923 | 941 | 852 | 813 |
0.5 | 6976 | 5138 | 2180 | 1377 | 1167 | 1209 | 943 | 883 |
1 | 7981 | 6834 | 3904 | 1916 | 1583 | 1573 | 1183 | 896 |
2 | 8576 | 7942 | 5510 | 2680 | 2140 | 2061 | 1374 | 1034 |
5 | 8956 | 8794 | 6985 | 4323 | 2950 | 2476 | 1782 | 1205 |
10 | 9064 | 9134 | 8241 | 6107 | 3682 | 2710 | 1972 | 1451 |
50 | 9072 | 9189 | 8932 | 7032 | 4031 | 2967 | 2114 | 1723 |
给出的值均为以dpm(每分钟的蜕变值)测定的3H-标记的2-脱氧葡萄糖的摄取。
结果总结在表2中。
实施例10:片段116-167对勒帕素作用的拮抗作用
在存在或不存在勒帕素(10nM)以及递增浓度的勒帕素片段116-167下,在原代培养物中将分离的大鼠脂肪细胞保温17.5小时。接着洗涤细胞,测定5nM胰岛素对葡萄糖转运和脂肪生成的刺激作用。以胰岛素刺激的活性与基础活性之间的比例来计算胰岛素的刺激系数。每个值代表来自两个独立的脂肪细胞培养物的平均值,其中每种情况下测定活性三次。
表3:勒帕素:10nM
胰岛素:5nM
片段 | 葡萄糖 | |
浓度[nM] | 转运* | 脂肪生成 |
0 | 3.55 | 1.5 |
0.3 | 3.65 | 1.65 |
1 | 4.5 | 2.15 |
3 | 5.75 | 2.65 |
10 | 7.75 | 3.1 |
30 | 10.8 | 3.6 |
100 | 12.55 | 4 |
300 | 13.2 | 4 |
仅有胰岛素 | 13.4 | 3.9 |
*系数为刺激后的值与基础值的比值。
结果总结于表3中。附录:表4:SEQ ID NO:4人勒帕素1- Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro TyrLeu Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp AspThr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp IleSer His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly LeuAsp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys MetAsp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met ProSer Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu ArgAsp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu ProTrp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val LeuGlu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg LeuGln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser ProGly Cys-167表5:SEQ ID NO:5小鼠勒帕素1-Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp Ser TyrLeu Ser Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp AspThr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp IleSer His Thr Gln Ser Val Ser Ala Lys Gln Arg Val Thr Gly LeuAsp Phe Ile Pro Gly Leu His Pro Ile Leu Ser Leu Ser Lys MetAsp Gln Thr Leu Ala Val Tyr Gln Gln Val Leu Thr Ser Leu ProSer Gln Asn Val Leu Gln Ile Ala Asn Asp Leu Glu Asn Leu ArgAsp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys Ser Leu ProGln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly Val LeuGlu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg LeuGln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val Ser ProGlu Cys-167表6:SEQ ID NO:6来自小鼠勒帕素的片段116-Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser
Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val
Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln
Leu Asp Val Ser Pro Glu Cys-167存在于表4-6所示序列中的两个半胱氨酸通过一个二硫桥连接。
序列表
(1)一般信息:
(ⅰ)申请人:
(A)名称:Hoechst Aktengesellschaft
(B)街道:-
(C)城市:Frankfurt
(D)联邦州:-
(E)国家:德国
(F)邮编:65926
(G)电话:069-305-3005-
(H)传真:069-35-7175
(I)电传:-
(ⅱ)申请题目:勒帕素拮抗剂在用于治疗Ⅱ型糖尿病中的胰岛素耐受性中的用途
(ⅲ)序列数目:6
(ⅳ)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容
(C)操作***:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,版本#1.25(EPO)
(2)SEQ ID NO:l的信息
(ⅰ)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:DNA(基因组)
(ⅸ)特性:
(A)名称/关键词:外显子
(B)位置:1..20
(ⅹⅰ)序列描述:SEQ ID NO:1GAATGCAGAA TAAATAAATA
(3)SEQ ID NO:2的信息:
(ⅰ)序列特征:
(A)长度:34个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:DNA(基因组)
(ⅸ)特性:
(A)名称/关键词:外显子
(B)位置:1..34
(ⅹⅰ)序列描述:SEQ ID NO:2GAAAGAAGGA TCCAGTGCCT ATCCAGAAAG TCCA(2)SEQ ID NO:3的信息:
(ⅰ)序列特征:
(A)长度:34个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:DNA(基因组)
(ⅸ)特性:
(A)名称/关键词:外显子
(B)位置:1..34
(ⅹⅰ)序列描述:SEQ ID NO:3:GGAGAGAAGC TTGAGGGAGA GAAATGAATG ATGG(2)SEQ ID NO:4的信息:
(ⅰ)序列特征:
(A)长度:167个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:肽
(ⅸ)特性:
(A)名称/关键词:肽
(B)位置:1..167
(ⅹⅰ)序列描述:SEQ ID NO:4:Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu1 5 10 15Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys
20 25 30Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr
35 40 45Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro
50 55 60Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala65 70 75 80Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln
85 90 95Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala
100 105 110Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu
115 120 125Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
130 135 140Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln145 150 155 160Leu Asp Leu Ser Pro Gly Cys
165(2)SEQ ID NO:5的信息:
(ⅰ)序列特征:
(A)长度:167个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:肽
(ⅸ)特性:
(A)名称/关键词:肽
(C)位置:1..167
(ⅹⅰ)序列描述:SEQ ID NO:5:Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp Ser Tyr Lau1 5 10 15Ser Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys
20 25 30Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr
35 40 45Gln Ser Val Ser Ala Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro
50 55 60Gly Leu His Pro Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala65 70 75 80Val Tyr Gln Gln Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln
85 90 95Ile Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala
100 105 110Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro
115 120 125Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val
130 135 140Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln145 150 155 160Leu Asp Val Ser Pro Glu Cys
165(2)SEQ ID NO:6的信息:
(ⅰ)序列特征:
(A)长度:52个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑学:线性
(ⅱ)分子类型:肽
(ⅸ)特性:
(A)名称/关键词:肽
(B)位置:1..52
(ⅸ)序列描述:SEQ ID NO:6:Ser Cys Sar Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu1 5 10 15Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu
20 25 30Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val
35 40 45Ser Pro Glu Cys
50
Claims (21)
1.一种药物组合物,其包含Ⅱ型糖尿病的治疗有益量的勒帕素拮抗剂。
2.根据权利要求1的药物组合物,其中所述勒帕素拮抗剂衍生于人或动物的勒帕素。
3.根据权利要求2的药物组合物,其中所述勒帕素拮抗剂衍生于选自人、大鼠、小鼠、猪和类人猿勒帕素的勒帕素。
4.根据权利要求3的药物组合物,其中所述勒帕素拮抗剂衍生于小鼠勒帕素。
5.根据权利要求4的药物组合物,其中所述勒帕素拮抗剂为小鼠勒帕素内部片段或羧基末端片段。
6.根据权利要求5的药物组合物,其中所述勒帕素拮抗剂为包含表6氨基酸序列(SEQ ID NO:6)的小鼠勒帕素片段。
7.根据权利要求6的药物组合物,其中所述勒帕素拮抗剂含有一个或多个保守氨基酸置换。
8.根据权利要求7的药物组合物,其中所述保守氨基酸置换是将一个离子性氨基酸交换为另一个离子性氨基酸或将一个疏水性氨基酸交换为另一个疏水性氨基酸。
9.根据权利要求8的药物组合物,其中所述保守氨基酸置换选自天冬氨酸置换谷氨酸、谷氨酸置换天冬氨酸、亮氨酸置换异亮氨酸和异亮氨酸置换亮氨酸。
10.根据权利要求6的药物组合物,其中所述勒帕素拮抗剂通过选自乙酰化、氨基甲酰化、甲酰化、生物素化、酰化或用聚乙二醇衍生或用亲水性聚合物衍生的化学修饰进行修饰。
11.根据权利要求1的药物组合物,其中所述勒帕素拮抗剂选自可溶性勒帕素受体、勒帕素受体片段、与可溶性勒帕素受体的融合物以及与勒帕素受体片段的融合物。
12.一种Ⅱ型糖尿病的治疗方法,包括给予需要这种治疗的病人治疗有效量的根据权利要求1的组合物。
13.根据权利要求12的方法,其中所述组合物包含一种勒帕素拮抗剂,其中勒帕素拮抗剂为含有表6氨基酸序列(SEQ ID NO:6)的小鼠勒帕素片段。
14.根据权利要求13的方法,其中所述勒帕素拮抗剂含有一个或多个保守氨基酸置换。
15.根据权利要求12的方法,其中所述组合物包含选自可溶性勒帕素受体、勒帕素受体片段、与可溶性勒帕素受体的融合物以及与勒帕素受体片段的融合物的勒帕素拮抗剂。
16.一种恢复或增强胰岛素生理作用的方法,包括对需要所述恢复或增强作用的病人给予治疗有效量的根据权利要求1的组合物。
17.根据权利要求16的方法,其中所述组合物包含一种勒帕素拮抗剂,其中勒帕素拮抗剂为含有表6氨基酸序列(SEQ ID NO:6)的小鼠勒帕素片段。
18.根据权利要求17的方法,其中所述勒帕素拮抗剂含有一个或多个保守氨基酸置换。
19.根据权利要求16的方法,其中所述组合物包含选自可溶性勒帕素受体、勒帕素受体片段、与可溶性勒帕素受体的融合物以及与勒帕素受体片段的融合物的勒帕素拮抗剂。
20.包含小鼠勒帕素氨基酸116-167或116-166的小鼠勒帕素片段。
21.一种制备权利要求2-11以及20任一项的勒帕素片段的方法,其中
(a)使用适宜的载体/宿主体系表达编码所述勒帕素片段的基因;以及
(b)从所述宿主体系中分离所述勒帕素片段。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE19638487 | 1996-09-20 | ||
DE19638487.7 | 1996-09-20 |
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CN1230966A true CN1230966A (zh) | 1999-10-06 |
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CN97198076A Pending CN1230966A (zh) | 1996-09-20 | 1997-09-15 | 勒帕素拮抗剂在用于治疗ii型糖尿病胰岛素耐受性中的用途 |
Country Status (18)
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US (1) | US6399745B1 (zh) |
EP (1) | EP0956302A1 (zh) |
JP (1) | JP2001500869A (zh) |
KR (1) | KR20010029537A (zh) |
CN (1) | CN1230966A (zh) |
AR (1) | AR008444A1 (zh) |
AU (1) | AU735178B2 (zh) |
BR (1) | BR9711513A (zh) |
CA (1) | CA2266585A1 (zh) |
CZ (1) | CZ90999A3 (zh) |
HU (1) | HUP9904023A3 (zh) |
ID (1) | ID21861A (zh) |
IL (1) | IL129057A0 (zh) |
NO (1) | NO991186D0 (zh) |
PL (1) | PL332458A1 (zh) |
RU (1) | RU2201249C2 (zh) |
WO (1) | WO1998012224A1 (zh) |
ZA (1) | ZA978455B (zh) |
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WO2016176935A1 (zh) * | 2015-05-06 | 2016-11-10 | 广东省昆虫研究所 | 一条cd环和e螺旋区突变的瘦素活性肽及其编码基因和应用 |
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ATE278952T1 (de) | 1998-07-28 | 2004-10-15 | Vlaams Interuniv Inst Biotech | Leptin vermittelte geninduktion |
US6777388B1 (en) | 1998-08-21 | 2004-08-17 | Clf Medical Technology Acceleration Program, Inc. | Leptin-related peptides |
US7208572B2 (en) | 1998-08-21 | 2007-04-24 | Albany Medical College | Leptin-related peptides |
US20050272652A1 (en) | 1999-03-29 | 2005-12-08 | Gault Victor A | Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity |
CA2388417A1 (en) * | 1999-09-22 | 2001-03-29 | Genset | Methods of screening for compounds that modulate the lsr-leptin interaction and their use in the prevention and treatment of obesity-related diseases |
EP1612221A3 (en) | 2000-05-22 | 2008-07-23 | Vlaams Interuniversitair Instituut voor Biotechnologie vzw. | Receptor-based interaction trap |
US7235629B2 (en) | 2000-11-14 | 2007-06-26 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Functional fragment of the leptin receptor |
US8076288B2 (en) | 2004-02-11 | 2011-12-13 | Amylin Pharmaceuticals, Inc. | Hybrid polypeptides having glucose lowering activity |
CN1961000B (zh) | 2004-02-11 | 2011-05-04 | 安米林药品公司 | 具有可选择特性的杂合多肽 |
US7407929B2 (en) * | 2004-05-07 | 2008-08-05 | Boston Biomedical Research Institute | Leptin peptide antagonists |
US7423113B2 (en) | 2004-08-25 | 2008-09-09 | Vib Vzw | Leptin antagonist |
EP2127676A3 (en) | 2004-11-01 | 2013-09-04 | Amylin Pharmaceuticals, LLC | Treatment of obesity and related disorders |
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US7575878B2 (en) | 2004-11-18 | 2009-08-18 | Vib Vzw | Methods of inhibiting leptin-induced signaling with fibronectin III domain antibodies |
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US8263545B2 (en) | 2005-02-11 | 2012-09-11 | Amylin Pharmaceuticals, Inc. | GIP analog and hybrid polypeptides with selectable properties |
CA2597649A1 (en) | 2005-02-11 | 2006-08-17 | Amylin Pharmaceuticals, Inc. | Gip analog and hybrid polypeptides with selectable properties |
BRPI0614649A2 (pt) | 2005-08-11 | 2011-04-12 | Amylin Pharmaceuticals Inc | polipeptìdeos hìbridos com propriedades selecionáveis |
CA2617649A1 (en) | 2005-08-11 | 2007-02-22 | Amylin Pharmaceuticals, Inc. | Hybrid polypeptides with selectable properties |
EA200870365A1 (ru) * | 2006-03-23 | 2009-02-27 | Амилин Фармасьютикалз, Инк. | Эндотелин и агонисты рецепторов к эндотелину в лечении заболеваний обмена веществ |
US8497240B2 (en) | 2006-08-17 | 2013-07-30 | Amylin Pharmaceuticals, Llc | DPP-IV resistant GIP hybrid polypeptides with selectable properties |
EA201070609A1 (ru) * | 2007-11-14 | 2010-12-30 | Амилин Фармасьютикалз, Инк. | Способы лечения ожирения и ассоциированных с ожирением заболеваний и расстройств |
US8778890B2 (en) | 2009-03-31 | 2014-07-15 | Temple University—Of the Commonwealth System of Higher Education | Leptin antagonist and methods of use |
US20120071401A1 (en) | 2009-04-10 | 2012-03-22 | Amylin Pharamaceuticals, Inc. | Amylin agonist compounds for estrogen-deficient mammals |
DK3241558T3 (da) | 2010-09-28 | 2021-04-26 | Aegerion Pharmaceuticals Inc | Højopløselige leptiner |
JP6040464B2 (ja) | 2011-07-08 | 2016-12-07 | アエゲリオン・ファーマシューティカルズ・インコーポレイテッドAegerion Pharmaceuticals, Inc. | 作用持続期間が増大し、免疫原性が減少した操作されたポリペプチド |
EP2900230B1 (en) | 2012-09-27 | 2018-08-15 | The Children's Medical Center Corporation | Compounds for the treatment of obesity and methods of use thereof |
AU2014354831B2 (en) | 2013-11-26 | 2017-10-26 | The Children's Medical Center Corporation | Compounds for the treatment of obesity and methods of use thereof |
US20170209408A1 (en) | 2014-04-03 | 2017-07-27 | The Children's Medical Center Corporation | Hsp90 inhibitors for the treatment of obesity and methods of use thereof |
WO2017013270A1 (en) | 2015-07-23 | 2017-01-26 | Universite De Strasbourg | Use of leptin signaling inhibitor for protecting kidneys from patients having ciliopathy |
BR112019004715A2 (pt) | 2016-09-12 | 2019-07-16 | Aegerion Pharmaceuticals Inc | métodos para detectar anticorpos neutralizantes anti-leptina |
EP3675961A1 (en) * | 2017-08-31 | 2020-07-08 | University Of Dundee | Leptin peptides and their use for treating neurological disorders |
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US6048837A (en) * | 1994-08-17 | 2000-04-11 | The Rockefeller University | OB polypeptides as modulators of body weight |
US6309853B1 (en) * | 1994-08-17 | 2001-10-30 | The Rockfeller University | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
US6001968A (en) * | 1994-08-17 | 1999-12-14 | The Rockefeller University | OB polypeptides, modified forms and compositions |
US5525705A (en) * | 1995-01-31 | 1996-06-11 | Eli Lilly And Company | Anti-obesity proteins |
US5574133A (en) * | 1995-01-31 | 1996-11-12 | Eli Lilly And Company | Anti-obesity proteins |
US5569743A (en) * | 1995-01-31 | 1996-10-29 | Eli Lilly And Company | Anti-obesity proteins |
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US5521283A (en) * | 1995-01-31 | 1996-05-28 | Eli Lilly And Company | Anti-obesity proteins |
PL324284A1 (en) * | 1995-06-30 | 1998-05-11 | Lilly Co Eli | Methods of treating diabetes |
US7063958B1 (en) | 1996-01-16 | 2006-06-20 | The Rockefeller University | Nucleic acids db, the receptor for leptin |
US5922678A (en) * | 1996-06-28 | 1999-07-13 | Eli Lilly And Company | Methods for treating diabetes |
-
1997
- 1997-09-15 WO PCT/EP1997/005035 patent/WO1998012224A1/en not_active Application Discontinuation
- 1997-09-15 EP EP97942929A patent/EP0956302A1/en not_active Withdrawn
- 1997-09-15 IL IL12905797A patent/IL129057A0/xx unknown
- 1997-09-15 CZ CZ99909A patent/CZ90999A3/cs unknown
- 1997-09-15 PL PL97332458A patent/PL332458A1/xx unknown
- 1997-09-15 JP JP10514270A patent/JP2001500869A/ja active Pending
- 1997-09-15 KR KR1019997002389A patent/KR20010029537A/ko not_active Application Discontinuation
- 1997-09-15 CA CA002266585A patent/CA2266585A1/en not_active Abandoned
- 1997-09-15 BR BR9711513A patent/BR9711513A/pt not_active Application Discontinuation
- 1997-09-15 ID IDW990105A patent/ID21861A/id unknown
- 1997-09-15 CN CN97198076A patent/CN1230966A/zh active Pending
- 1997-09-15 US US09/147,805 patent/US6399745B1/en not_active Expired - Fee Related
- 1997-09-15 AU AU44586/97A patent/AU735178B2/en not_active Ceased
- 1997-09-15 HU HU9904023A patent/HUP9904023A3/hu unknown
- 1997-09-15 RU RU99107759/14A patent/RU2201249C2/ru not_active IP Right Cessation
- 1997-09-18 AR ARP970104293A patent/AR008444A1/es unknown
- 1997-09-19 ZA ZA9708455A patent/ZA978455B/xx unknown
-
1999
- 1999-03-11 NO NO991186A patent/NO991186D0/no not_active Application Discontinuation
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016176933A1 (zh) * | 2015-05-06 | 2016-11-10 | 广东省昆虫研究所 | 一条d螺旋区突变的瘦素活性肽及其编码基因和应用 |
WO2016176935A1 (zh) * | 2015-05-06 | 2016-11-10 | 广东省昆虫研究所 | 一条cd环和e螺旋区突变的瘦素活性肽及其编码基因和应用 |
Also Published As
Publication number | Publication date |
---|---|
CZ90999A3 (cs) | 1999-06-16 |
BR9711513A (pt) | 1999-08-24 |
AU735178B2 (en) | 2001-07-05 |
AR008444A1 (es) | 2000-01-19 |
JP2001500869A (ja) | 2001-01-23 |
ID21861A (id) | 1999-08-05 |
PL332458A1 (en) | 1999-09-13 |
RU2201249C2 (ru) | 2003-03-27 |
EP0956302A1 (en) | 1999-11-17 |
IL129057A0 (en) | 2000-02-17 |
ZA978455B (en) | 1998-03-20 |
NO991186L (no) | 1999-03-11 |
CA2266585A1 (en) | 1998-03-26 |
AU4458697A (en) | 1998-04-14 |
KR20010029537A (ko) | 2001-04-06 |
HUP9904023A3 (en) | 2002-01-28 |
HUP9904023A2 (hu) | 2000-03-28 |
WO1998012224A1 (en) | 1998-03-26 |
US6399745B1 (en) | 2002-06-04 |
NO991186D0 (no) | 1999-03-11 |
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