CN1195293A - 治疗糖尿病的方法 - Google Patents
治疗糖尿病的方法 Download PDFInfo
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- CN1195293A CN1195293A CN96196457A CN96196457A CN1195293A CN 1195293 A CN1195293 A CN 1195293A CN 96196457 A CN96196457 A CN 96196457A CN 96196457 A CN96196457 A CN 96196457A CN 1195293 A CN1195293 A CN 1195293A
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Abstract
本发明描述治疗肥胖性Ⅱ型糖尿病患者的糖尿病的方法。具体地说,要求保护的是与各种内源性leptin水平有关的肥胖性Ⅱ型糖尿病的治疗方法。
Description
糖尿病是一种代谢性疾病,特征在于机体组织不能以正常速率贮存碳水化合物。对胰岛素作用的抗性是II型糖尿病的最重要因素。当该抗性超过β细胞产生胰岛素的能力时,人就会患糖尿病。在70年代末期,患糖尿病的人十分得益于接受了控制量的胰岛素。
肥胖,尤其是过度机体肥胖,常与非胰岛素依赖型糖尿病(NIDDM)有关。这些称为II型糖尿病的对胰岛素没有绝对需求,因为他们的β细胞能够分泌胰岛素,尽管通常是以降低的水平分泌。此外,这类患者通常肥胖并且对胰岛素不产生反应。
对于治疗肥胖II型糖尿病所熟知的最佳方法是饮食调整和通过锻炼减肥。不幸的是,这些方案一般都不成功。减肥的失败是由于遗传因素,主要是食欲好、对高卡路里食品的喜好、体育活动少和脂肪生成代谢高。尽管他们对疾病斗争付出了努力,但继承了这些遗传因素的人易于肥胖,常成为II型糖尿病患者。ob/ob小鼠是一种肥胖和糖尿病模型,它已知携带与第六染色体的突变有关的常染色体隐性遗传特性。最近,Yiying Zhang和协作者发表了与该疾病有关的小鼠基因(ob)的位置克隆(positional cloning)。Yiying Zhang等,自然(Nature)372:425-32(1994)。该报道公开了专门在脂肪组织中表达的编码167个氨基酸蛋白质(下文leptin)的基因,该蛋白质带有21个氨基酸信号肽。
据显示肥胖者中leptin的循环水平差异很大。结果,现在人们认为使用leptin治疗肥胖性II型糖尿病患者亚群十分顺理成章。因此有生物活性的、并具有leptin相似活性的药理学物质可用于治疗肥胖性II型糖尿病,尤其是循环leptin水平异常高或低的患者。
一方面,本发明是一种治疗或预防糖尿病的方法,包括给肥胖性II型糖尿病患者服用有效量的leptin、leptin类似物或它们的可药用盐。在优选的实施方案中,本发明包括治疗低内源性leptin水平的肥胖性II型糖尿病的方法。
肥胖是指个体的身体质量指数大于27公斤/平方米的一种疾病。
leptin是指由肥胖基因经下列途经生产的蛋白质:转录和缺失内含子,翻译为蛋白并加工成带有分泌信号肽的成熟蛋白质(例如除去成熟蛋白的N末端缬氨酸-脯氨酸至C末端半胱氨酸)。Zhang等在自然(Nature)372:425-32(1994)中公开了小鼠和人的leptin蛋白序列。Murakami等在生物化学和生物物理学研究通讯(Biochemical and Biophysical Research Comm.)209(3):944-52(1995)中公开了大鼠leptin序列。在人、鼠和大鼠的leptin中与二硫化物形成有关的Cys位于96和145位。但是在使用鼠和人leptin时,观察到了desGln(28)变体。因此,与二硫键形成有关的Cys可位于95或96位和145或146位。本说明书中Leptin还可以指肥胖蛋白、OB或ob基因产物。因而,leptin包括SEQ ID Nos:1-6。
本领域专业技术人员将认识到某些氨基酸易发生重排。例如,Asp可重排为天冬酰亚胺和异天冬酰胺,正如1.Schn等在国际肽和蛋白质研究杂志(International Journal of Peptide and ProteinResearch),14:485-94(1979)中所述,该文献引入本文以供参考。这些重排衍生物也包括在本发明的范围之中。除非另有说明,氨基酸均是L构型的。
用于本权利要求方法的优选形式的Leptin是天然序列。更优选使用人leptin。用于本发明方法的最优选的leptins包括蛋白质的SEQID Nos:1-6
鼠Leptin
SEQ ID NO:1Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Ash Asp Ile Ser His Thr Xaa Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145其中:28位的Xaa是Gln或没有。
猪Leptin
SEQ ID NO:2Val Pro Ile Trp Arg Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Ser Asp Ile Ser His Met Gln Ser Val Ser Ser
20 25 30Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val
35 40 45Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
50 55 60Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys
85 90 95Pro Leu Pro Gln Ala Arg Ala Leu Glu ThrLeu Glu Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ala Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145
牛Leptin
SEQ ID NO:3Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thrl 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
20 25 30Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu
35 40 45Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
50 55 60Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys
85 90 95Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val
100 105 110Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145其中:28位的Xaa是Gln或没有。
人Leptin
SEQ ID NO:4Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Ash Asp Ile Ser His Xaa Xaa Ser Val Ser Ser
20 25 30Lvs Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 53 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:27位的Xaa是Thr或Ala;并且28位的Xaa是Gln或没有。
恒河猴Leptin
SEQ ID NO:5Val Pro Ile Gln Lys Val Gln Ser Asp Thr Lys Thr Leu Ile Lys1 5 10 15Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val
20 25 30Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu
35 40 45His Pro Val Leu Thr Leu Ser Gln Met Asp Gln Thr Leu Ala Ile
50 55 60Tyr Gln Gln Ile Leu Ile Asn Leu Pro Ser Arg Asn Val Ile Gln
65 70 75Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu
80 85 90Ala Phe Ser Lys Ser Cys His Leu Pro Leu Ala Ser Gly Leu Glu
95 100 105Thr Leu Glu Ser Leu Gly Asp Val Leu Glu Ala Ser Leu Tyr Ser
110 115 120Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp
125 130 135Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
140 145
大鼠Leptin
SEQ ID NO:6Val Pro Ile His Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ala
20 25 30Arg Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln ILe
50 55 60Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala His Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys
85 90 95Ser Leu Pro Gln Thr Arg Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly
100 105 110Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Leu Ser Pro
130 135 140Glu Cys145
除上述确定的蛋白序列外,通常认为使用一种或两种氨基酸前导序列,尤其是包含甲硫氨酸的前导序列制备这类抗糖尿病的蛋白质是高效的。两种经常使用的前导序列是Met-Arg和Met-Asp。这些蛋白质在下文可以鉴定为Met-Arg-leptin或Met-Asp-leptin,或者标记为Met-Arg-SEQ ID NO:X,其中X是1-6。
Leptin类似物和片断也可用于本发明的方法。Leptin类似物通常由下式(I)(SEQ ID NO:7)所定义:Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Xaa Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gln Ile Xaa Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:22位的Xaa是Asn,Asp或Glu;27位的Xaa是Thr或Ala;28位的Xaa是Gln,Glu或没有;54位的Xaa是Met或Ala;68位的Xaa是Met或Leu;72位的Xaa是Asn,Asp或Glu;77位的Xaa是Ser或Ala;118位的Xaa是Gly或Leu;所述蛋白质具有至少一个选自下述的取代基:97位的His被Ser或Pro替代;100位的Trp被Gln,Ala或Leu替代;101位的Ala被Thr或Val替代;102位的Ser被Arg替代;103位的Gly被Ala替代;105位的Glu被Gln替代;106位的Thr被Lys或Ser替代;107位的Leu被Pro替代;108位的Asp被Glu替代;或者111位的Gly被Asp替代。Leptin类似物优选是如下式(II)(SEQ ID NO:8)所示的化合物:Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gln Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Xaa Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:27位的Xaa是Thr或Ala;77位的Xaa是Ser或Ala;118位的Xaa是Gly或Leu;
所述蛋白质具有至少一个取代基,优选具有一至五个取代基,最优选具有一至二个取代基,所述取代基选自:97位的His被Ser替代;100位的Trp被Gln替代;101位的Ala被Thr替代;105位的Glu被Gln替代;106位的Thr被Lys替代;107位的Leu被Pro替代;108位的Asp被Glu替代;或者111位的Gly被Asp替代。
本发明优选蛋白质的实施例包括SEQ ID NO:8的蛋白质,其中27位的Xaa是Thr;77位的Xaa是Ser;118位的Xaa是Gly;并且97、100、101、105、106、107、108和111位的氨基酸残基是如下表I所述的残基。提供该天然的人序列与用于本发明方法的蛋白质进行比较。
表I
化合物 | 氨基酸位置 | |||||||
97 | 100 | 101 | 105 | 106 | 107 | 108 | 111 |
天然人1234567891011121314151617181920212223242526272829303132333435 | HisSerHisHisHisHisHisHisHisSerSerSerSerSerSerSerHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHis | TrpTrpGlnTrpTrpTrpTrpTrpTrpGlnTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrp | AlaAlaAlaThrAlaAlaAlaAlaAlaAlaThrAlaAlaAlaAlaAlaThrAlaAlaAlaAlaAlaThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAla | GluGluGluGluGlnGluGluGluGluGluGluGlnGluGluGluGluGluGlnGluGluGluGluGlnGluGluGluGluGlnGlnGlnGlnGluGluGluGluGlu | ThrThrThrThrThrLysThrThrThrThrThrThrLysThrThrThrThrThrLysThrThrThrThrLysThrThrThrLysThrThrThrLysLysLysThrThr | LeuLeuLeuLeuLeuLeuProLeuLeuLeuLeuLeuLeuProLeuLeuLeuLeuLeuProLeuLeuLeuLeuProLeuLeuLeuProLeuLeuProLeuLeuProPro | AspAspAspAspAspAspAspGluAspAspAspAspAspAspGluAspAspAspAspAspGluAspAspAspAspGluAspAspAspGluAspAspGluAspGluAsp | GlyGlyGlyGlyGlyGlyGlyGlyAspGlyGlyGlyGlyGlyGlyAspGlyGlyGlyGlyGlyAspGlyGlyGlyGlyAspGlyGlyGlyAspGlyGlyAspGlyAsp |
3637383940414243444546474849505l5253545556575859606162636465666768697071 | HisSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerHisHisHisHisHisHisHisHisHisHisHisHisHisHis | TrpGlnGlnGlnGlnGlnGlnTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln | AlaThrAlaAlaAlaAlaAlaThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAla | GluGluGlnGluGluGluGluGlnGluGluGluGluGlnGlnGlnGlnGluGluGluGluGluGluGlnGluGluGluGluGlnGlnGlnGlnGluGluGluGluGlu | ThrThrThrLysThrThrThrThrLysThrThrThrLysThrThrThrLysLysLysThrThrThrThrLysThrThrThrLysThrThrThrLysLysLysThrThr | LeuLeuLeuLeuProLeuLeuLeuLeuProLeuLeuLeuProLeuLeuProLeuLeuProProLeuLeuLeuProLeuLeuLeuProLeuLeuProLeuLeuProPro | GluAspAspAspAspGluAspAspAspAspGluAspAspAspGluAspAspGluAspGluAspGluAspAspAspGluAspAspAspGluAspAspGluAspGluAsp | AspGlyGlyGlyGlyGlyAspGlyGlyGlyGlyAspGlyGlyGlyAspGlyGlyAspGlyAspAspGlyGlyGlyGlyAspGlyGlyGlyAspGlyGlyAspGlyAsp |
72737475767778798081828384858687888990919293949596979899100101102103104105106107 | HisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisSerSerSerSerSerSerSerSerSerSerSerSerSerSerSer | GlnTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln | AlaThrThrThrThrThrThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAlaAla | GluGlnGlnGlnGlnGluGluGluGluGluGluGlnGlnGlnGlnGlnGlnGluGluGluGluGlnGluGluGluGluGlnGlnGlnGlnGluGluGluGluGluGlu | ThrLysThrThrThrLysLysLysThrThrThrLysLysLysThrThrThrLysLysLysThrThrLysThrThrThrLysThrThrThrLysLysLysThrThrThr | LeuLeuProLeuLeuProLeuLeuProProLeuProLeuLeuProProLeuProProLeuProLeuLeuProLeuLeuLeuProLeuLeuProLeuLeuProProLeu | GluAspAspGluAspAspGluAspGluAspGluAspGluAspGluAspGluGluAspGluGluAspAspAspGluAspAspAspGluAspAspGluAspGluAspGlu | AspGlyGlyGlyAspGlyGlyAspGlyAspAspGlyGlyAspGlyAspAspGlyAspAspAspGlyGlyGlyGlyAspGlyGlyGlyAspGlyGlyAspGlyAspAsp |
108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143 | SerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHis | TrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln | ThrThrThrThrThrThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaThrThrThrThrThrThrThrThrThrThrAlaAlaAlaAlaAlaAla | GlnGlnGlnGlnGluGluGluGluGluGluGlnGlnGlnGlnGlnGlnGluGluGluGluGlnGlnGlnGlnGluGluGluGluGluGluGlnGlnGlnGlnGlnGln | LysThrThrThrLysLysLysThrThrThrLysLysLysThrThrThrLysLysLysThrLysThrThrThrLysLysLysThrThrThrLysLysLysThrThrThr | LeuProLeuLeuProLeuLeuProProLeuProLeuLeuProProLeuProProLeuProLeuProLeuLeuProLeuLeuProProLeuProLeuLeuProProLeu | AspAspGluAspAspGluAspGluAspGluAspGluAspGluAspGluGluAspGluGluAspAspGluAspAspGluAspGluAspGluAspGluAspGluAspGlu | GlyGlyGlyAspGlyGlyAspGlyAspAspGlyGlyAspGlyAspAspGlyAspAspAspGlyGlyGlyAspGlyGlyAspGlyAspAspGlyGlyAspGlyAspAsp |
144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179 | HisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHisHis | GlnGlnGlnGlnTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln | AlaAlaAlaAlaThrThrThrThrThrThrThrThrThrThrAlaAlaAlaAlaAlaAlaThrThrThrThrThrAlaAlaAlaAlaAlaThrThrThrThrThrThr | GluGluGluGluGlnGlnGlnGlnGlnGlnGluGluGluGluGlnGlnGlnGlnGluGlnGluGlnGlnGlnGlnGluGlnGlnGlnGlnGluGluGluGluGlnGln | LysLysLysThrLysLysLysThrThrThrLysLysLysThrLysLysLysThrLysLysLysThrLysLysLysLysThrLysLysLysThrLysLysLysThrThr | ProProLeuProProLeuLeuProProLeuProProLeuProProProLeuProProProProProLeuProProProProLeuProProProLeuProProLeuPro | GluAspGluGluAspGluAspGluAspGluGluAspGluGluGluAspGluGluGluGluGluGluGluAspGluGluGluGluAspGluG1uGluAspGluGluAsp | GlyAspAspAgpGlyGlyAspGlyAspAspGlyAspAspAspGlyAspAspAspAspAspAspAspAspAspGlyAspAspAspAspGlyAspAspAspGlyAspAsp |
18018l18218318418518618718818919019l192193194195196197198199200201202203204205206207208209210211212213214215 | HisHisHisHisSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSer | GlnGlnGlnGlnTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln | ThrThrThrThrAlaAlaAlaAlaAlaThrThrThrThrThrThrThrThrThrThrAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaThrThrThrThrThrThrThr | GlnGlnGlnGlnGluGlnGlnGlnGlnGluGluGluGluGlnGlnGlnGlnGlnGlnGluGluGluGluGlnGlnGlnGlnGlnGlnGluGluGluGluGluGluGln | ThrLysLysLysLysThrLysLysLysThrLysLysLysThrThrThrLysLysLysThrLysLysLVsThrThrThrLysLysLysThrThrThrLysLysLysThr | ProLeuLeuProProProLeuProProProLeuProProLeuProProLeuLeuProProLeuProProLeuProProLeuLeuProLeuProProLeuLeuProLeu | GluAspGluAspGluGluGluAspGluGluGluAspGluGluAspGluAspGluAspGluGluAspGluGluAspGluAspGluAspGluAspGluAspGluAspAsp | GlyAspGlyGlyAspAspAspAspGlyAspAspAspGlyAspAspGlyAspGlyGlyAspAspAspGlyAspAspGlyAspGlyGlyAspAspGlyAspGlyGlyAsp |
216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245246247248249250251 | SerSerSerHisHisHisHisHisHisHisSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerSerHisSerSerSerSer | GlnGlnGlnTrpGlnGlnGlnGlnGlnGlnTrpTrpTrpTrpTrpTrpGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnTrpGlnGlnGln | ThrThrThrThrAlaThrThrThrThrThrAlaThrThrThrThrThrAlaAlaAlaAlaAlaThrThrThrThrThrThrThrThrThrThrThrThrAlaThrThr | GlnGlnGlnGlnGlnGluGlnGlnGlnGlnGlnGluGlnGlnGlnGlnGluGlnGlnGlnGlnGluGluGluGluGlnGlnGlnGlnGlnGlnGlnGlnGlnGluGln | ThrThrLysLysLysLysThrLysLysLysLysLysThrLysLysLysLysThrLysLysLysThrLysLysLysThrThrThrLysLysLysLysLysLysLysThr | LeuProLeuProProProProLeuProProProProProLeuProProProProLeuProProProLeuProProLeuProProLeuLeuProProProProProPro | GluAspAspGluGluGluGluGluAspGluGluGluGluGluAspGluGluGluGluAspGluGluGluAspGluGluAspGluAspGluAspGluGluGluGluGlu | GlyGlyGlyAspAspAspAspAspAspGlyAspAspAspAspAspGlyAspAspAspAspGlyAspAspAspGlyAspAspGlyAspGlyGlyAspAspAspAspAsp |
252253254255 | SerSerSerSer | GlnGlnGlnGln | ThrThrThrThr | GlnGlnGlnGln | LysLysLysLys | LeuProProPro | GluAspGluGlu | AspAspGlyAsp |
其它优选的蛋白质是那些其中27位的Xaa是Ala;77位的Xaa是Ser;118位的Xaa是Gly;并且97、100、101、105、106、107、108和111位的氨基残基如表I所述的。
本发明提供可有效治疗肥胖性II型糖尿病的生物活性蛋白质。出乎意料地是,表I的leptin蛋白质由于带有相对于人肥胖蛋白的特定取代基而具有改进特性。这些蛋白质比小鼠和人的肥胖蛋白都更稳定,因此是更卓越的治疗药物。
使用Jackson Laboratories(Bar Harbor,Maine)或Harlan(英格兰)的五至六月龄雄性近交正常ICR小鼠、近交正常(ob/+)、肥胖-糖尿病小鼠(ob/ob)和肥胖-糖尿病(db/db)小鼠进行实验。
将正常的和糖尿病小鼠都圈养在带垫的塑料笼中,每个笼中三或六只,并不限量地给予水和食物。动物室的温度保持在23±2℃,光强为0600-1800h。从尾静脉收集血样。因此,最密切相关的生物试验是通过数种给药途径中的任一种(如静脉、皮下、腹膜内或借助微型泵或导管)注射试验物质,然后监测不同时间段的食物和水的消耗、体重增加、血浆化学或激素(葡萄糖、胰岛素、ACTH、皮质酮、GH、T4)水平。适宜的实验动物包括正常小鼠(ICR等)和肥胖小鼠(ob/ob,Avy/a,KK-Ay,矮胖,肥胖)。对这些注射非特效的对照试验可以通过给相同动物使用含有或不含试验物质的类似组合物的载体监测相同参数进行或给认为缺乏所述受体的动物(db/db小鼠,fa/fa或cp/cp大鼠)施用试验物质本身进行对照试验。
血葡糖水平通过葡萄糖氧化酶方法或偶联己糖激酶方法测定。血浆胰岛素使用放射免疫试剂盒、以大鼠胰岛素为标准进行测定。血浆甘油三酯使用市售试剂盒、用甘油为标准进行测定。
上述研究证明leptin和leptin类似物调节正常ICR和遗传肥胖ob/ob小鼠的食物摄取和体重。长期给予ob/ob小鼠leptin和leptin类似物可总体改善这些动物的糖尿病症状,显示出这些抗糖尿病蛋白质作为治疗肥胖性II型糖尿病药物的潜在希望。本发明提供治疗肥胖性II型糖尿病,尤其是那些具有低leptin循环水平的糖尿病的方法。
本发明的化合物可采用本领域已知的化学方法生产,例如液相或固相肽合成法或通过常规溶液方法由蛋白片段偶联开始在溶液中的半合成法。这类方法是已知的,记述于本领域的普通教科书,例如H.Dugas和C.Penney的生物有机化学(BIOOR GANIC CHEMISTRY),(1981)54-92页。
用于本权利要求方法的蛋白质也可采用熟知的重组DNA技术制备,例如Maniatis等(1988)分子克隆:实验室手册(MolecularCloning:A Laboratory Manual),Cold Spring Harbor Press,ColdSpring Harbor Laboratory,Cold Spring Harbor,New York或者Current Protocols in Molecular Biology(1989)和增补。在已知序列DNA的预定位置上进行取代突变的技术是熟知的,例如M13引物诱变法。在编码本发明抗糖尿病蛋白质的DNA上产生的突变必须不位于阅读框架之外的序列,并优选所述突变不会产生可导致二级mRNA结构的互补区。参见DeBoer等,欧洲专利公开075444A(1983)。本发明提供一种治疗肥胖性II型糖尿病的方法。该方法包括施用有效量的leptin或leptin类似物,剂量为约1-10000μg/kg。优选剂量为约20-10000μg/kg。更优选剂量为约200-600μg/kg。在实践该方法中,抗糖尿病蛋白质可每日给药一次或多次剂量。治疗方案可要求长时间给药。每剂的给药量或给药总量可由医师决定,取决于如下因素例如患者体重、年龄和患者健康状况及对化合物的耐受量。
本发明还提供含有本发明化合物的药物制剂。蛋白质、优选是可药用盐形式的蛋白质可配制成非胃肠给药的制剂。例如可将化合物与常规药物载体和赋形剂混合。含有所要求保护的蛋白质的组合物含约0.1-90%重量的活性蛋白质、优选是可溶形式的活性蛋白质,更优选10-30%。另外,本发明的蛋白质可单独给药或与其它抗肥胖症的药物或用于治疗糖尿病的药物联合给药。
对于静脉给药,蛋白质常以常用的静脉流体并通过静脉输注给药。对于肌内制剂、无菌制剂,优选将适宜的可溶盐形式的蛋白质,例如盐酸盐,溶解在药物稀释剂中如去热源水或生理盐水中给药。适宜的不溶形式的化合物可制备成水基或可药用油基中的悬浮液,例如长链脂肪酸的酯如油酸乙酯的悬浮液给药。
在优选的实施方案中,本发明提供一种治疗低leptin水平的肥胖性II型糖尿病的方法,尽管具有高内源性leptin水平的糖尿病患者也受益于本发明所要求保护的方法。测定血清或血浆leptin水平的方法可使用标准基于抗体的方法学完成。Leptin测试试剂盒可从Linco Research,Inc.(14 Research Park Dr.,St Louis,MO 63304)购得。
优选治疗具有0-80ng/ml的leptin水平的肥胖性II型糖尿病。较优选治疗leptin水平在0-50ng/ml的肥胖性II型糖尿病。更优选治疗leptin水平在0-30ng/ml的肥胖性II型糖尿病。最优选治疗leptin水平在0-15ng/ml的肥胖性II型糖尿病。
提供下列实施例以说明如何实施和实践本发明的各种实施方案。这些实施例并不旨在限定本发明的范围。
实施例1
采用标准PCR方法,从人脂肪细胞文库(从CLONETECH购得)得到编码下列蛋白序列:
Met-Arg-SEQ ID NO:4。的DNA序列,概括地说,根据所公开的人ob基因的氨基酸序列设计简并引物。使用Model 308A DNA合成仪(PE-Applied Biosystem,Inc.,850 Lincoln center Drive,Foster City,CA 94404)以聚合物酶链反应(PCR)扩增方法制备引物。接下来,将引物OB.F1M(5-GG GG CATATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC)和OB.F2H(5-GG GG CAT ATG AGG GTA CCC ATC CAG AAG GTGCAG GAC GA)(和逆转录引物OB.R1M(5-GG GG GGATC GATAAT TTA GCA TCC AGG GCT AAG ATC CAA CTG CCA AAGCAT)和OB.R2H(5-GG GG GGATC CTA TTA GCA CCC GGG AGACAG GTC CAG CTG CCA CAA CAT)与作为模板的PCR-准备好的(PCR-ready)人脂肪细胞cDNA(Clontech Laboratories,Inc.,4030 Fabian Way,Palo Alto,CA 94303;Item #7128-1)混合。
使用2.5单位的Amplitag DNA聚合物酶(Perkin Elmer Cetus)或2单位的Vent DNA聚合物酶(New England Biolabs)在100ul的反应中进行2组PCR扩增反应。PCR反应包含1ul人脂肪细胞cDNA,10pmol每种引物(将全部四种混合)。对“TouchdownPCR”采用下列条件:2个循环:94℃×30秒,60℃×30秒,72℃×45秒;2个循环:94℃×30秒,56℃×30秒,72℃×45秒;2次循环:94℃×30秒,52℃×30秒,72℃×45秒;2个循环:94℃×30秒,48℃×30秒,72℃×45秒;2个循环:94℃×30秒,44℃×30秒,72℃×45秒;28个循环:94℃×30秒,52℃×30秒,72℃×45秒。
将所得PCR反应产物在1%琼脂糖凝胶上展开,经溴化乙啶染色可见一条约450bp大小的谱带。该谱带在两组PCR反应中均存在。切下该带并以上述条件(94℃×30秒,52℃×30秒,72℃×45秒)再扩增30次循环。将使用Vent DNA聚合物酶得到的PCR产物进行凝胶纯化并克隆到pCR-SCRIPT克隆载体(Stratagene)中。然后使用该载体转化E.coli细胞。从20个E.coli的白色茵落中分离质粒DNA并将从3个克隆得到的样品进行排序。将两个这样的菌落,即E.coliDH10B/pOJ717知E.coli DH10B/pOJ718依布达佩斯条约寄存于Northern Regional Research Laboratories(NRRL),保藏号分别为B-21408和B-21409。
实施例2
载体构建
构建含编码所需蛋白质的DNA顺序的质粒以包括NdeI和BamHI限制位点。用NdeI和BamHI限制酶消化携带克隆的PCR产物的质粒。凝胶纯化小的~450bp片段,然后连接载体pRB182,从该载体中缺失了A-C-B胰岛素原的编码序列。将连接产物转化到大肠杆菌(E.coli)DH10B(可从市场上购买的)中,分析在用10μg/ml四环素补充的胰胨-酵母平板上生长的菌落。分离质粒DNA,用NdeI和BamHI消化,琼脂糖凝胶电泳分离所得的片段。保留含期望的~450 bp NdeI-BamHI片段的质粒。用所述的第二质粒转化大肠杆菌B BL21(DE3),该质粒表达适合进行蛋白质生产的培养。
使用所述的载体转化细胞的技术是本领域已知的,常见于普通文献例如Maniatis等的分子克隆:实验室手册,Cold SpringHarbourPress,Cold Spring Hargor Laboratory,Cold Spring Harbor,New York(1988),或者CRURRENT PROTOCOLS IN MOLECULARBIOLOGY,(F.Ausabel,ed.,1989)和其增补。本发明的实践中作为举例说明使用的涉及E.coli细胞转化的技术是本领域所熟知的。培养转化E.coli细胞的具体条件取决于所用E.coli宿主细胞系和表达或克隆载体的性质。例如掺入到可热诱导启动子-操作子区域(例如c1857可热诱导λ噬茵体启动子-操作子区域)的载体需要的培养条件温度在约30℃-约40℃内变化,以引导蛋白质的合成。
在本发明的优选实施方案中,使用的宿主细胞是E.coli K12 RV308细胞,但也可使用其它细胞,例如但不限于E.coli K12 L201,L687,L693,L507,L640,L641,L695,L814(E.coli B)。然后在对应于表达质粒中存在的抗性基因的抗生素的选择压力下,将转化的宿主细胞平铺于适当的培养基中。之后将培养物在适于所用的宿主细胞系的温度下保温一段时间。
在高水平细菌表达系统中表达的蛋白质特征性地以颗粒或包合体形式聚集,它们含有高水平的过表达蛋白。参见例如Kreuger等,PROTEIN FOLDING(Gierasch和King,eds.,1990),136-142页。American Association for the Advancement of Science Publication No.89-18S,Washington,D.C.。这类蛋白质聚集物必须被溶解以进一步纯化分离所需蛋白质产物。出处同上。可采用各种技术使用强烈变性溶液例如盐酸胍和/或弱变性溶液例如二硫苏糖醇(DTT)溶解这些蛋白质。
在溶液中逐渐地除去变性试剂(通常通过透析),使变性蛋白质恢复天然构型。具体的变性和折叠条件取决于具体的蛋白质表达体系和/或所述的蛋白质。
本发明的蛋白质优选表达为Met-Arg-SEQ ID NO:X,这样所表达的蛋白质可快速地转化为要求保护的具有组织蛋白酶C的蛋白质(还称为二氨基肽酶)。蛋白质的纯化是本领域的已知技术,包括反相色谱、亲和色谱和空间排阻色谱法。
所要求的蛋白质包含两个半胱氨酸残基。因此,可形成二硫键以稳定蛋白质。本发明包括其中96位Cys与146位Cys交联的蛋白质以及那些不含该二硫键的蛋白质。
蛋白质,除本发明使用的蛋白质外,特别的当配制时,还可作为二聚物、三聚物、四聚物和其它多聚物存在。此类多聚物均包括在本发明的范围内。
Claims (19)
1.leptin或leptin类似物用于制备治疗或预防糖尿病的药物的用途。
2.权利要求1的方法,其中所述的leptin或leptin类似物选自(a)Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Qln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145其中:28位的Xaa是Gln或没有;
(SEQ ID NO:1)(b)Val pro Ile Trp Arg Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thrl 5 10 15Ile Val Thr Arg Ile Ser Asp Ile Ser His Met Gln Ser Val Ser Ser
20 25 30Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val
35 40 45Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
50 55 60Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys
85 90 95Pro Leu Pro Gln Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ala Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145(SEQ ID NO:2)(c)Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
20 25 30Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu
35 40 45Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
50 55 60Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys
85 90 95Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val
100 105 110Val Leu Glu Ala Ser Leu Iyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
130 135 140Gly Cys145其中:28位的Xaa是Gln或没有;(SEQ ID NO:3)(d)Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Xaa Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Glv Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:27位的Xaa是Thr或Ala;并且28位的Xaa是Gln或没有;(SEQ ID NO:4)(e)Val Pro Ile Gln Lys Val Gln Ser Asp Thr Lys Thr Leu Ile Lys1 5 l0 15Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val
20 25 30Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu
35 40 45His Pro Val Leu Thr Leu Ser Gln Met Asp Gln Thr Leu Ala Ile
50 55 60Tyr Gln Gln Ile Leu Ile Asn Leu Pro Ser Arg Asn Val Ile Gln
65 70 75Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu
80 85 90Ala Phe Ser Lys Ser Cys His Leu Pro Leu Ala Ser Gly Leu Glu
95 100 105Thr Leu Glu Ser Leu Gly Asp Val Leu Glu Ala Ser Leu Tyr Ser
110 115 120Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp
125 130 135Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys
140 145 ;(SEQ ID NO:5)(f)Val Pro Ile His Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ala
20 25 30Arg Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Ser Leu Ser Lys Met Asp Gln ThrLeu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala H1s Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys
85 90 95Ser Leu Pro Gln Thr Arg Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly
100 105 110Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Leu Ser Pro
130 135 140Glu Cys145,(SEQ ID NO:5)(g)Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr1 5 10 15Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Xaa Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gln Ile Xaa Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:22位的Xaa是Asn,Asp或Glu;27位的Xaa是Thr或Ala;28位的Xaa是Gln,Glu或没有;54位的Xaa是Met或Ala;68位的Xaa是Met或Leu;72位的Xaa是Asn,Asp或Glu;77位的Xaa是Ser或Ala;118位的Xaa是Gly或Leu;所述蛋白质具有至少一个选自下述的取代基:97位的His被Ser或Pro替代;100位的Trp被Gln,Ala或Leu替代;101位的Ala被Thr或Val替代;102位的Ser被Arg替代;103位的Gly被Ala替代;105位的Glu被Gln替代;106位的Thr被Lys或Ser替代;107位的Leu被Pro替代;108位的Asp被Glu替代;或者111位的Gly被Asp替代;和,(SEQ ID NO:7)(h)Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thrl 5 10 15Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gln Ser Val Ser Ser
20 25 30Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Xaa Asn Asp Leu65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140145Gly Cys其中:
27位的Xaa是Thr或Ala;
77位的Xaa是Ser或Ala;
118位的Xaa是Gly或Leu;
所述蛋白质具有至少一个取代基,优选具有一至五个取代基,和最优选具有一至二个取代基,所述取代基选自:
97位的His被Ser替代;
100位的Trp被Gln替代;
101位的Ala被Thr替代;
105位的Glu被Gln替代;
106位的Thr被Lys替代;
107位的Leu被Pro替代;
108位的Asp被Glu替代;或
111位的Gly被Asp替代;
或者它们的可药用盐或溶剂化物。
(SEQ ID NO:8)
3.权利要求1或2任一项的方法,其中的糖尿病与高内源性leptin水平有关。
4.权利要求1或3任一项的方法,其中的糖尿病与低内源性leptin水平有关。
5.权利要求1或3任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-80ng/ml。
6.权利要求1或3任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-50ng/ml。
7.权利要求1或3任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-30ng/ml。
8.权利要求1或3任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-15ng/ml。
9.一种治疗或预防糖尿病的方法,该方法包括给糖尿病患者施用leptin或leptin类似物。
10.权利要求9的方法,其中所述的leptin或leptin类似选自SEQ IDNO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ IDNO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8。
11.如权利要求9或10任一项的方法,其中糖尿病与高内源性leptin水平有关。
12.如权利要求9或10任一项的方法,其中糖尿病与低内源性leptin水平有关。
13.权利要求9或10任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-80ng/ml。
14.权利要求9或10任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-50ng/ml。
15.权利要求9或10任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-30ng/ml。
16.权利要求9或10任一项的方法,其中所述的肥胖性II型糖尿病的内源性leptin水平为0-15ng/ml。
17.一种治疗或预防糖尿病的制剂,该制剂含有作为活性成分的leptin或leptin类似物。
18.权利要求17的制剂,其中所述的leptin或leptin类似物选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8。
19.权利要求17或18任一项的方法,其中糖尿病与高或低内源性leptin水平有关。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US71895P | 1995-06-30 | 1995-06-30 | |
US60/000,718 | 1995-06-30 |
Publications (1)
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CN1195293A true CN1195293A (zh) | 1998-10-07 |
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ID=21692725
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CN96196457A Pending CN1195293A (zh) | 1995-06-30 | 1996-06-26 | 治疗糖尿病的方法 |
Country Status (14)
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US (1) | US5756461A (zh) |
EP (1) | EP0836479A2 (zh) |
JP (1) | JPH11508895A (zh) |
KR (1) | KR19990028388A (zh) |
CN (1) | CN1195293A (zh) |
AU (1) | AU695934B2 (zh) |
CA (1) | CA2225454A1 (zh) |
CZ (1) | CZ416797A3 (zh) |
EA (1) | EA199800104A1 (zh) |
HU (1) | HUP9802609A2 (zh) |
IL (1) | IL122718A0 (zh) |
NO (1) | NO976063L (zh) |
PL (1) | PL324284A1 (zh) |
WO (1) | WO1997002004A2 (zh) |
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-
1996
- 1996-06-26 AU AU64795/96A patent/AU695934B2/en not_active Withdrawn - After Issue
- 1996-06-26 HU HU9802609A patent/HUP9802609A2/hu unknown
- 1996-06-26 CZ CZ974167A patent/CZ416797A3/cs unknown
- 1996-06-26 CA CA002225454A patent/CA2225454A1/en not_active Abandoned
- 1996-06-26 JP JP9505211A patent/JPH11508895A/ja active Pending
- 1996-06-26 CN CN96196457A patent/CN1195293A/zh active Pending
- 1996-06-26 WO PCT/US1996/010983 patent/WO1997002004A2/en not_active Application Discontinuation
- 1996-06-26 EP EP96924308A patent/EP0836479A2/en not_active Withdrawn
- 1996-06-26 EA EA199800104A patent/EA199800104A1/ru unknown
- 1996-06-26 IL IL12271896A patent/IL122718A0/xx unknown
- 1996-06-26 PL PL96324284A patent/PL324284A1/xx unknown
- 1996-06-26 KR KR1019970709704A patent/KR19990028388A/ko not_active Application Discontinuation
- 1996-06-28 US US08/678,369 patent/US5756461A/en not_active Expired - Fee Related
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102552225A (zh) * | 2010-01-25 | 2012-07-11 | 四川省中医药科学院 | 四氢姜黄素的新用途 |
CN103547590A (zh) * | 2010-09-28 | 2014-01-29 | 艾米琳制药有限责任公司 | 高度可溶性瘦蛋白 |
CN103547590B (zh) * | 2010-09-28 | 2017-11-28 | 埃格里昂制药股份有限公司 | 高度可溶性瘦蛋白 |
Also Published As
Publication number | Publication date |
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HUP9802609A2 (hu) | 1999-03-29 |
WO1997002004A3 (en) | 1997-05-22 |
AU6479596A (en) | 1997-02-05 |
KR19990028388A (ko) | 1999-04-15 |
IL122718A0 (en) | 1998-08-16 |
EA199800104A1 (ru) | 1998-10-29 |
PL324284A1 (en) | 1998-05-11 |
EP0836479A2 (en) | 1998-04-22 |
WO1997002004A2 (en) | 1997-01-23 |
NO976063L (no) | 1998-02-16 |
US5756461A (en) | 1998-05-26 |
JPH11508895A (ja) | 1999-08-03 |
NO976063D0 (no) | 1997-12-23 |
MX9800133A (es) | 1998-03-29 |
CZ416797A3 (cs) | 1998-06-17 |
CA2225454A1 (en) | 1997-01-23 |
AU695934B2 (en) | 1998-08-27 |
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