WO2011063549A1 - Long-acting exendin 4 analogues - Google Patents
Long-acting exendin 4 analogues Download PDFInfo
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- WO2011063549A1 WO2011063549A1 PCT/CN2009/001321 CN2009001321W WO2011063549A1 WO 2011063549 A1 WO2011063549 A1 WO 2011063549A1 CN 2009001321 W CN2009001321 W CN 2009001321W WO 2011063549 A1 WO2011063549 A1 WO 2011063549A1
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- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C07K14/575—Hormones
- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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- the present invention relates to a polypeptide of an Exendin 4 analog which has a hypoglycemic effect and is useful for the treatment of type 2 diabetes.
- the invention also provides methods of producing these polypeptides by chemical synthesis and recombinant DNA production processes.
- type 1 diabetes insulin-dependent diabetes
- type 2 diabetes non-insulin-dependent diabetes
- Type 2 diabetic patients exhibit many characteristics, such as decreased ⁇ -cells, insufficient insulin secretion after meals, delayed secretion of insulin, increased fasting and postprandial blood glucose, elevated glycosylated hemoglobin HbAlc, and insulin resistance.
- Exendin 4 and Exendin 3 are two peptides secreted by the saliva of Gila monster, Heloderme Suspectum, which consists of 39 amino acid residues, chemically synthesized as an anti-type 2 diabetes drug. Approved by the US FDA in 2005, the trade name is Byetta, and it was listed in China in 2009.
- Exendin 4 can promote the synthesis of proinsulin, promote insulin secretion, reduce fasting, postprandial blood glucose, and no longer continue to function when blood sugar is normal, so it is not easy to produce hypoglycemia, coma, shock, and high safety. It can reduce HbAl c , increase the number and volume of pancreatic ⁇ cells, increase the sensitivity of insulin receptors in type 2 diabetes patients, and inhibit the secretion of glucagon. Inject twice a day, 5-10ug each time.
- Exendin 4 is derived from the giant lizard, and GLP-1 is a human endogenous substance.
- Exendin 4 is a chemically synthesized product that is very expensive.
- the object of the present invention is to address the current market demand for Exendin 4, which is very expensive, and the present specification provides a 32 amino acid polypeptide derivative of Exendin 4. Its peptide chain length is similar to that of human GLP-1, reducing its immunogenicity.
- the structure contains only one Lys20 residue, which allows the fatty acid acylation to specifically bind to Lys20 ⁇ (epsi lon ) - ⁇ 2 to form a fatty acid acylated derivative.
- the present invention utilizes genetic engineering technology to mass-produce the polypeptide, which can reduce the cost and provide a new therapeutic drug for a large number of type 2 diabetic patients.
- One aspect of the present invention provides a structural change, and still has hypoglycemic activity Exendin 4 Analog,
- the Exendin 4 analog of the present invention has the following structural formula:
- X2 Met, Leu or He
- X3 Arg, OH or NH2.
- XI is Tyr or Phe
- X2 is Met or Leu
- X3 is Arg
- compositions comprising the above Exendin 4 analogue, the composition comprising one or more compounds having the above formula and a pharmaceutically acceptable diluent or excipient, preferably
- the composition is in unit dosage form, such as tablets, pills, capsules (including sustained release or delayed release forms), powders, granules, elixirs, elixirs, syrups and emulsions, sterile injectable solutions or suspensions, aerosols Or liquid sprays, drops, injections, automatic injection devices or suppositories.
- the above active pharmaceutical ingredient may be combined with an oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
- an oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
- the present invention provides the use of the above compound and a pharmaceutical combination for the preparation of a therapeutic disease, preferably the disease is type 2 diabetes.
- a further aspect of the invention provides a genetic engineering method for the preparation of the above Exendi n 4 analogs and derivatives.
- the above compound in which X3 is Arg may be synthesized by a genetic engineering method or may be synthesized by a chemical method, but is preferably synthesized by a genetic engineering method.
- the above compounds in which X3 is 0H or NH2 can also be synthesized by a chemical synthesis method using a polypeptide synthesizer.
- the invention changes the chemical structure of Exend in 4, shortens the length of the peptide chain from the C-terminus and changes the amino acid structure, and maintains its hypoglycemic activity, so that the peptide chain length of Exendi n 4 is similar to that of human GLP-1, and the immunogenicity is lowered.
- the peptide chain contains only one Lys20, which facilitates the site-direct acylation of fatty acids to form long-acting derivatives.
- the need to adapt to the production of genetic engineering methods is suitable for mass production of genetic engineering, reducing costs, adapting to the needs of patients with type 2 diabetes, and providing a new therapeutic drug for patients with type 2 diabetes.
- Figure 1 is a tandem map of Exendi n 4 analog gene fragments
- Figure 2 is a flow chart of the production of Exendi n 4 analog by genetic engineering
- Figure 3 is a measure of the hypoglycemic effect of EW1 and Exend i n4.
- Embodiment 1 is a diagrammatic representation of Embodiment 1:
- the EW1 gene was synthesized by the conventional PCR method, and then digested with EcoR I /Sal I and cloned into the Lac promoter-containing plasmid.
- the DNA sequence was identical to the design.
- 487f 5' ccaatgAATTCCAGATCTAACGGCCGTCACGGCGAAG 3' 487r : 5' aattGTCGACTAGGATCCACGCGGGCCGCCCTGAACCAGCCAT 3'
- Ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, 37 ° C, shaken overnight
- the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 g/ml, aeration of 20L/ m in, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water. When the concentration of the bacteria reached the neutral line of the log curve, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.
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Abstract
Provided is an Exendin 4 analogue peptide. Compared with wild-type Exendin 4, the peptide of the present invention is 7 amino acid residues shorter at the C-terminal end. The length of the peptide chain is similar to human GLP-1, and there is only one Lys20 amino acid residue in the structure of the peptide, such that acylation of fatty acid occurs specifically at theε-NH2 of the Lys20 to form a fatty acid acylated derivative. Also provided are pharmaceutical compositions comprising such peptide and methods for producing the peptide by chemical synthesis or recombinant DNA technologies.
Description
说 明 书 一 Description
长效 Exendin 4的类似物 Long-acting Exendin 4 analogue
技术领域 Technical field
本发明涉及 Exendin 4类似物的多肽, 该多肽具有降低血糖作用, 可用于治疗 2型糖尿病。 本发明还提供了通过化学合成和重组 DNA生产工艺生产这些多肽的方 法。 The present invention relates to a polypeptide of an Exendin 4 analog which has a hypoglycemic effect and is useful for the treatment of type 2 diabetes. The invention also provides methods of producing these polypeptides by chemical synthesis and recombinant DNA production processes.
背景技术 Background technique
世界各国糖尿病患者人数逐年增多, 现在中国有 5000万, 印度 6000万, 美国 2000万, 日本 600万, 全球已达 2. 2亿。 糖尿病患者分为两种, 一是胰岛素依赖型 糖尿病 (1型糖尿病) 和非胰岛素依赖型糖尿病 (2型糖尿病) 。 其中, 2型糖尿病 占糖尿病患者的 90%以上。 The number of people with diabetes in the world has increased year by year. Now there are 50 million in China, 60 million in India, 20 million in the United States, and 6 million in Japan. The world has reached 220 million. There are two types of diabetes, one is insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes). Among them, type 2 diabetes accounts for more than 90% of diabetic patients.
2型糖尿病患者表现了许多特征, 如 β -细胞减少, 餐后胰岛素分泌量不足, 胰 岛素分泌时间滞后, 空腹及餐后血糖增高, 糖化血红蛋白 HbAlc升高, 胰岛素抵抗 等。 Type 2 diabetic patients exhibit many characteristics, such as decreased β-cells, insufficient insulin secretion after meals, delayed secretion of insulin, increased fasting and postprandial blood glucose, elevated glycosylated hemoglobin HbAlc, and insulin resistance.
根据美国 UKPDS对 2型糖尿病患者的多年跟踪研究报告指出目前 6类药物对 2 型糖尿病患者皆不能逆转糖尿病进程, 不能遏制胰脏 β细胞的不断进行性的凋亡, 也不能阻止糖尿病的并发症如冠心病、 肾功能衰竭的发生和发展, 改变糖尿病患者 预后。 因此需要研究新的 2型糖尿病治疗药物。 According to the US UKPDS multi-year follow-up study on type 2 diabetes, it is pointed out that none of the current 6 drugs can reverse the progression of diabetes in patients with type 2 diabetes, can not inhibit the progressive apoptosis of pancreatic β cells, and can not prevent the complications of diabetes. For example, the occurrence and development of coronary heart disease and renal failure change the prognosis of diabetic patients. Therefore, it is necessary to study new therapeutic drugs for type 2 diabetes.
Exendin 4禾口 Exendin 3是南美产巨晰赐(Gi la monster, Heloderme Suspectum) 的唾液中分泌的两种多肽, 其中 Exendin 4由 39个氨基酸残基组成, 化学合成品 作为抗 2型糖尿病药物, 2005年获美国 FDA批准, 商品名为 Byetta, 2009年中国 上市。 Exendin 4 and Exendin 3 are two peptides secreted by the saliva of Gila monster, Heloderme Suspectum, which consists of 39 amino acid residues, chemically synthesized as an anti-type 2 diabetes drug. Approved by the US FDA in 2005, the trade name is Byetta, and it was listed in China in 2009.
Exendin 4能促进胰岛素原的合成, 促胰岛素分泌, 降低空腹、 餐后血糖, 当 血糖正常之后不再继续作用, 因而不易产生低血糖昏迷、 休克, 安全性高。 它能降 低 HbAl c , 增加胰腺 β细胞数量及体积,增加 2型糖尿病患者胰岛素受体的敏感性, 抑制胰高血糖素分泌等作用。 每日注射 2次, 每次 5-10ug。 但也存在不足之处, Exendin 4来源于巨蜥蜴, 而 GLP- 1是人的内源物质, 临床试验报告 Exendin 4有 52%的患者产生抗体 ( Diabetes (1997) 46, 433-439; Diabetes Care
(2002) 25, 330-336; JAMA (2002) 287, 373-379; Diabetes (1995) 44. 1249-1258; Diatetes (2002) 51 2796-2803; Diabetes Care (2000) 23 64-69; N. Engl. J. Med (2002) 346, 393-463; Lancet (1998) 352, 837-853)。 另外, Exendin 4是化学合成 品, 价格十分昂贵。 Exendin 4 can promote the synthesis of proinsulin, promote insulin secretion, reduce fasting, postprandial blood glucose, and no longer continue to function when blood sugar is normal, so it is not easy to produce hypoglycemia, coma, shock, and high safety. It can reduce HbAl c , increase the number and volume of pancreatic β cells, increase the sensitivity of insulin receptors in type 2 diabetes patients, and inhibit the secretion of glucagon. Inject twice a day, 5-10ug each time. However, there are also shortcomings. Exendin 4 is derived from the giant lizard, and GLP-1 is a human endogenous substance. Clinical trials have reported that 52% of patients in Exendin 4 produce antibodies ( Diabetes (1997) 46, 433-439; Diabetes Care (2002) 25, 330-336; JAMA (2002) 287, 373-379; Diabetes (1995) 44. 1249-1258; Diatetes (2002) 51 2796-2803; Diabetes Care (2000) 23 64-69; Engl. J. Med (2002) 346, 393-463; Lancet (1998) 352, 837-853). In addition, Exendin 4 is a chemically synthesized product that is very expensive.
发明内容 Summary of the invention
本发明的目的是针对目前市场对 Exendin 4有需要,而价格又十分昂贵的问题, 本说明提供一个 Exendin 4的 32个氨基酸多肽衍生物。使其肽链长度与人源 GLP-1 相近, 降低其免疫源性。 结构中只含一个 Lys20残基, 使脂肪酸酰化能专一的与 Lys20的 ε ( epsi lon ) - ΝΗ2结合形成脂肪酸酰化衍生物。 另外本发明利用基因工 程技术, 大量生产该多肽的工艺, 能降低成本, 为广大 2型糖尿病患者提供一个新 的治疗药物本发明的一个方面提供了结构发生改变, 而仍具有降血糖活性 Exendin 4类似物, The object of the present invention is to address the current market demand for Exendin 4, which is very expensive, and the present specification provides a 32 amino acid polypeptide derivative of Exendin 4. Its peptide chain length is similar to that of human GLP-1, reducing its immunogenicity. The structure contains only one Lys20 residue, which allows the fatty acid acylation to specifically bind to Lys20 ε (epsi lon ) - ΝΗ 2 to form a fatty acid acylated derivative. In addition, the present invention utilizes genetic engineering technology to mass-produce the polypeptide, which can reduce the cost and provide a new therapeutic drug for a large number of type 2 diabetic patients. One aspect of the present invention provides a structural change, and still has hypoglycemic activity Exendin 4 Analog,
本发明的 Exendin 4类似物其具有如下的结构式: The Exendin 4 analog of the present invention has the following structural formula:
EW1 ( 1-32 ) EW1 ( 1-32 )
His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu- His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-
Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3 Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3
EW2 (1-32) EW2 (1-32)
His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu- His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-
Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3 Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3
其中: Xl=Phe或 Tyr Where: Xl=Phe or Tyr
X2=Met、 Leu或 He ; X2=Met, Leu or He;
X3=Arg, OH或 NH2。 X3 = Arg, OH or NH2.
优选地, XI为 Tyr或 Phe, X2为 Met或 Leu, 更优选地, X3为 Arg。 Preferably, XI is Tyr or Phe, X2 is Met or Leu, and more preferably, X3 is Arg.
本发明的另一方面提供了含有上述 Exendin 4类似物的药物组合物, 该组合物 含有一种或多种具有上述结构式的化合物和一种药学上可接受的稀释剂或赋形剂, 优选的该组合物为单位剂量形式, 如片剂、 丸剂、 胶囊 (包括持续释放或延迟释放 形式) 、 粉剂、 颗粒、 酏剂、 酊剂、 糖浆和乳液, 消毒的注射用溶液或悬液, 气雾 剂或液体喷剂, 滴剂、 针剂、 自动注射装置或栓剂。 例如, 以片剂或胶囊的形式口 服给药, 上述活性药物组分可以与一种口服的无毒的药学可接受的惰性载体, 如乙 醇、 甘油、 水及其类似物结合。 另外, 本发明还提供了上述化合物及药物组合的在 制备治疗疾病中的用途, 优选的该疾病是 2型糖尿病。
本发明的又一个方面提供了制备上述 Exendi n 4类似物及衍生物的基因工程方 法。 对于 X3为 Arg的上述化合物, 可以用基因工程方法来合成, 还可以通过化学 方法来合成, 但优选用基因工程方法来合成。 对于 X3为 0H或 NH2的上述化合物, 也可以通过化学方法以多肽合成仪进行合成。 Another aspect of the invention provides a pharmaceutical composition comprising the above Exendin 4 analogue, the composition comprising one or more compounds having the above formula and a pharmaceutically acceptable diluent or excipient, preferably The composition is in unit dosage form, such as tablets, pills, capsules (including sustained release or delayed release forms), powders, granules, elixirs, elixirs, syrups and emulsions, sterile injectable solutions or suspensions, aerosols Or liquid sprays, drops, injections, automatic injection devices or suppositories. For example, orally administered in the form of a tablet or capsule, the above active pharmaceutical ingredient may be combined with an oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Further, the present invention provides the use of the above compound and a pharmaceutical combination for the preparation of a therapeutic disease, preferably the disease is type 2 diabetes. A further aspect of the invention provides a genetic engineering method for the preparation of the above Exendi n 4 analogs and derivatives. The above compound in which X3 is Arg may be synthesized by a genetic engineering method or may be synthesized by a chemical method, but is preferably synthesized by a genetic engineering method. The above compounds in which X3 is 0H or NH2 can also be synthesized by a chemical synthesis method using a polypeptide synthesizer.
本发明改变 Exend i n 4的化学结构,从 C端缩短其肽链长度并改变氨基酸结构, 保持其降血糖活性, 从而使 Exendi n 4的肽链长度与人源 GLP- 1相近, 降低免疫源 性, 肽链只含一个 Lys20, 有利于脂肪酸的定点酰化形成长效衍生物。 同时改构满 足基因工程方法生产的需要, 适合基因工程大量生产, 降低成本, 适应广大 2型糖 尿病患者的需要, 为广大 2型糖尿病患者提供一个新的治疗药物 The invention changes the chemical structure of Exend in 4, shortens the length of the peptide chain from the C-terminus and changes the amino acid structure, and maintains its hypoglycemic activity, so that the peptide chain length of Exendi n 4 is similar to that of human GLP-1, and the immunogenicity is lowered. The peptide chain contains only one Lys20, which facilitates the site-direct acylation of fatty acids to form long-acting derivatives. At the same time, the need to adapt to the production of genetic engineering methods is suitable for mass production of genetic engineering, reducing costs, adapting to the needs of patients with type 2 diabetes, and providing a new therapeutic drug for patients with type 2 diabetes.
附图说明 DRAWINGS
图 1是 Exendi n 4类似物基因片断的串联图; Figure 1 is a tandem map of Exendi n 4 analog gene fragments;
图 2是基因工程法生产 Exendi n 4类似物的流程图; Figure 2 is a flow chart of the production of Exendi n 4 analog by genetic engineering;
图 3是 EW1与 Exend i n4降血糖作用的测定。 Figure 3 is a measure of the hypoglycemic effect of EW1 and Exend i n4.
具体实施方式 detailed description
实施例一: Embodiment 1:
用基因工程方法生产 EW 1 Production of EW 1 by genetic engineering
材料 Material
EW 1 EW 1
EcoRI Bgl II Arg Hi s Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu EcoRI Bgl II Arg Hi s Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu
^ _ ^ CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Asn Gly Gly Pro Arg ^ _ ^ CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Asn Gly Gly Pro Arg
GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT AAC GGC GGC CCG CGT GGATCC TAG ^ ~ GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT AAC GGC GGC CCG CGT GGATCC TAG ^ ~
BamH I Sal I BamH I Sal I
( 1 ) 根据 EW1氨基酸序列及相应 DNA (正链) 序列, 合成 DNA片段 (1) synthesizing DNA fragments based on the EW1 amino acid sequence and the corresponding DNA (plus strand) sequence
5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG 5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG
AAGAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATC CTAGTCGAC 3' AAGAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATC CTAGTCGAC 3'
合成 DNA片段 Synthetic DNA fragment
1 ) 5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG 3'
4 ) 5' CTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3' 1) 5' AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG 3' 4) 5' CTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3'
5 ) 5' GTGCCTTCGCCGTGACGAGATCTGG 3' 5) 5' GTGCCTTCGCCGTGACGAGATCTGG 3'
6 ) 5' tcgaCTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3' 6) 5' tcgaCTAGGATCCACGCGGGCCGCCGTTAACCAGCC 3'
(2) 按 PCR常规方法, 合成 EWl基因, 经 EcoR I /Sal I双酶切后, 克隆到含 Lac启 动子质粒中, 测定 DNA序列与设计完全一致。 (2) The EW1 gene was synthesized by the conventional PCR method, and then digested with EcoR I /Sal I and cloned into the Lac promoter-containing plasmid. The DNA sequence was identical to the design.
TCGAC TCGAC
( 3 )参见图 1, 按图示方案将 EW1基因进行串联, 得到含 8个拷贝基因的质粒。 定 DNA全序列, 符合设计, 转化 E. col i JM109。 实施例二: (3) Referring to Figure 1, the EW1 gene was ligated in tandem to obtain a plasmid containing 8 copies of the gene. The complete DNA sequence, in accordance with the design, was transformed into E. col i JM109. Embodiment 2:
EW2 EW2
EcoR I Bgl II Asn Gly Arg His Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu ^ _ ^ ^ _ ^ AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Gin Gly Gly Pro Arg EcoR I Bgl II Asn Gly Arg His Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Ser Gin Met Glu ^ _ ^ ^ _ ^ AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA Glu Glu Ala Val Lys Leu Phe l ie Glu Trp Leu Val Gin Gly Gly Pro Arg
GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGATCC TAG ^ _ , GAA GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGATCC TAG ^ _ ,
BamH I Sa i l 以 £ 1的 1) 为模板, 合成二个 DNA片段 BamH I Sa il synthesizes two DNA fragments using 1 1 of £ 1 as a template
487f: 5' ccaatgAATTCCAGATCTAACGGCCGTCACGGCGAAG 3'
487r : 5' aattGTCGACTAGGATCCACGCGGGCCGCCCTGAACCAGCCAT 3' 487f: 5' ccaatgAATTCCAGATCTAACGGCCGTCACGGCGAAG 3' 487r : 5' aattGTCGACTAGGATCCACGCGGGCCGCCCTGAACCAGCCAT 3'
进行 PCR扩增, 测定 DNA序列, 构建 EW2基因工程菌。 PCR amplification was carried out, DNA sequences were determined, and EW2 genetically engineered bacteria were constructed.
gAAt TCC AGA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GA'gAAt TCC AGA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GA'
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC cAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC cAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_ GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT G_
GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA GA TCT AAC GGC CGT CAC GGC GAA GGC ACC TAC ACC AGC GAT CTG AGC AGC CAG ATG GAA GAA
GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGA TCC TAGtcgac 实施例三: GAA GCG GTT AAA CTG TTC ATC GAA TGG CTG GTT CAG GGC GGC CCG CGT GGA TCC TAGtcgac Example 3:
EW1、 EW2的制备 Preparation of EW1 and EW2
( A ) 基因工程菌的发酵: Ewl基因工程菌接种于 300ml X 2瓶 LB培养基中, 加 (A) Fermentation of genetically engineered bacteria: Ewl genetically engineered bacteria are inoculated into 300 ml X 2 bottles of LB medium, plus
氨苄西林 (Ampici ll in ) 使其最后浓度为 50 y g/ml, 37'C, 摇床培养过夜 Ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, shaken overnight
( 150rpm) 。 第二日转至 20L New Brunswik 发酵罐培养, 培养液组成为 M9 培养液, 加葡萄糖浓度 1%, 氨苄西林 (Ampici ll in ) 浓度为 50 g/ml, 通 气量为 20L/min, 溶氧维持在 20%以上, 防泡剂为国产泡敌, pH维持在 7〜8, 用氨水调节。 当菌体浓度达 log曲线中线, 加 IPTG, 浓度为 0. 5mM, 继续发 酵 4〜6小时, 离心收集菌体为湿重 30〜50g/L。 (150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 g/ml, aeration of 20L/ m in, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reached the neutral line of the log curve, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.
( B) 细胞破碎: 收集的菌体用 pH 7. 0 Tris 缓冲液 20mM匀浆, 之后加溶菌酶 (B) Cell disruption: The collected cells were homogenized with pH 7.0 0 Tris buffer 20 mM, followed by lysozyme
lg/L , 37°C保温 1小时, 冷冻 一 20°C 过夜, 超声破碎 3次, 每次 1分钟 左右, 离心收集沉淀, 2M Urea 打碎洗涤 1次, 再打碎水洗 1次。 Ig/L, incubate at 37 °C for 1 hour, freeze at 20 °C overnight, sonicate 3 times, each time for 1 minute, collect the precipitate by centrifugation, 2M Urea, crush and wash once, then wash with water and wash once.
( C) 分离、 纯化包涵体: 沉淀溶于 8M 尿素, 分子筛 Sephadex G-100 A2S0nm
检测, 透析过夜, DEAE pH8.0-9.0梯度洗脱, 蛋白电泳确定目标蛋白。(C) Separation and purification of inclusion bodies: Precipitate dissolved in 8M urea, molecular sieve Sephadex G-100 A2S0nm Detection, overnight dialysis, gradient elution with DEAE pH 8.0-9.0, protein electrophoresis to determine the target protein.
(D) 融合蛋白的裂解: 二羧酸溶解, pH7.5条件下加入 1〜2%。 高纯度猪胰蛋 白酶 37Ό 搅拌 2〜3小时 , HPLC追踪。 完成酶解反应后以蠕动泵 30000 分子量超滤除去胰蛋白酶。 (D) Cleavage of the fusion protein: The dicarboxylic acid is dissolved, and 1 to 2% is added under pH 7.5. High-purity porcine trypsin 37Ό Stir 2~3 hours, HPLC trace. After the enzymatic hydrolysis reaction was completed, trypsin was removed by a peristaltic pump 30000 molecular weight ultrafiltration.
(E) 制备型 HPLC纯化。 (E) Preparative HPLC purification.
(F) 冷冻干燥获得产品。 实施例四: (F) Freeze drying to obtain the product. Embodiment 4:
肉豆蔻酸酰化的 EW2衍生物制备 Preparation of myristic acid acylated EW2 derivative
(A) 取 EW2融合蛋白 10g溶于 100ml水中, 加 50ml 2M的羟胺并调节 pH 9.0, 加 盐酸胍使其最终浓度为 5M, 45°C保温过夜。 (A) Take 10 g of EW2 fusion protein in 100 ml of water, add 50 ml of 2M hydroxylamine and adjust pH 9.0, add guanidine hydrochloride to a final concentration of 5 M, and incubate at 45 ° C overnight.
称取肉豆蔻酸 (Maristic acid) 1.14g, N_羟基琥珀酸亚胺 Weighing Marstic acid 1.14g, N-hydroxysuccinimide
(N-hydroxysuccinimide) 0.6g, Ν, Ν' -二环己基碳酰亚胺 (DCCI) 1.03g溶于 50ml 二氧六环(dioxane) 中, 室温搅拌, 4小时后, 过滤除去脲素衍生物, 取清液 20ml 加入 (A) 中, 加 ΙΟΟμ Ι四甲基胍 (Tetrameltyl- guanidine) , 室温激烈搅拌下 反应 4小时后用 2N HC1调至 pH 3-4, 8000rpm离心, 将沉淀用乙醇洗涤 2次, 将 脂肪酸酰化的 Ew2溶于水中, 以碳酸氢钠粉末调至 pH7〜8 , 按重量的 1〜2%。 加 高纯度猪胰蛋白酶 37°C 搅拌反应 2〜3小时 , HPLC追踪。 制备型 HPLC纯化。 冷冻干燥获得产品。 实施例五: (N-hydroxysuccinimide) 0.6g, Ν, Ν'-dicyclohexylcarbimide (DCCI) 1.03g dissolved in 50ml dioxane, stirred at room temperature, 4 hours later, filtered to remove urea derivatives 20 ml of the clear solution was added to (A), and Tetrameltyl-guanidine was added thereto. The reaction was carried out under vigorous stirring at room temperature for 4 hours, then adjusted to pH 3-4 with 2N HCl, centrifuged at 8000 rpm, and the precipitate was washed with ethanol. Two times, the fatty acid acylated Ew2 was dissolved in water, and the sodium hydrogencarbonate powder was adjusted to pH 7 to 8, 1 to 2% by weight. High purity porcine trypsin was stirred at 37 ° C for 2 to 3 hours and traced by HPLC. Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 5:
EWi、 EW2的降血糖作用的测定 Determination of hypoglycemic effect of EWi and EW2
C57BL/6的 8周龄小鼠 , 禁食过夜 (只给饮水) C57BL/6 8-week-old mice, fasted overnight (only for drinking water)
对照组、 EWi组、 Exendin 4组 Control group, EWi group, Exendin 4 group
每组 4只, 腹腔注射药物一次, 然后注射 30%葡萄糖溶液 300 μ ΐ,每 2小 时注射一次葡萄糖, 20分钟后睚旁取静脉血 5 μ 1,以葡萄糖氧化酶试纸测定 快速血糖 , 参见图 2, 结果表明 EWi的降血糖作用与 Exendin 4相当。
4 rats in each group were injected intraperitoneally once, then 30 μg of 30% glucose solution was injected, glucose was injected every 2 hours, and venous blood was taken 5 μl after 20 minutes. Glucose oxidase test paper was used to measure fast blood glucose. 2, the results show that the hypoglycemic effect of EWi is comparable to Exendin 4.
Claims
1. 长效 Exendin 4类似物, 其结构式为: 1. Long-acting Exendin 4 analogue, its structural formula is:
EW1 ( 1 -32 ) EW1 ( 1 -32 )
His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu- His-Gly-Glu-Gly-Thr-Xi-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-
Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3 Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-X3
其中: Xl=Phe或 Tyr; Where: Xl=Phe or Tyr;
X2=Met或 Leu或 lie; X2=Met or Leu or lie;
X3=Arg或 OH或 NH2。 X3 = Arg or OH or NH2.
2. 长效 Exendin 4类似物, 其结构式为: 2. Long-acting Exendin 4 analogue, its structural formula is:
EW2 ( 1 -32) EW2 ( 1 -32)
His-Gly-Glu-Gly-Thr-Xl-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu- His-Gly-Glu-Gly-Thr-Xl-Thr-Ser-Asp-Leu-Ser-Ser-Gln-X2-Glu-Glu-Glu-
Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3 Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Val-Gln-Gly-Gly-Pro-X3
其中: Xl=Phe或 Tyr; Where: Xl=Phe or Tyr;
X2=Met或 Leu或 l ie; X2=Met or Leu or l ie;
X3=Arg或 OH或 NH2。 X3 = Arg or OH or NH2.
3. 如权利要求 1或 2所述的 Exendin 4类似物, 其中, 肽链长度为 32个氨基 酸。 The Exendin 4 analog according to claim 1 or 2, wherein the peptide chain has a length of 32 amino acids.
4. 如权利要求 1或 2所述的 Exendi n 4类似物, 其中, 所述的 X3是 0H或- NH2。 The Exendi n 4 analog according to claim 1 or 2, wherein the X3 is 0H or -NH2.
5. 如权利要求 1或 2所述的 Exendin 4类似物,其中,所述的 Lys2Q的 ε ( epsi lon 的缩写) -NH2是肉豆蔻酸或棕榈酸酰化物。 The Exendin 4 analogue according to claim 1 or 2, wherein the ε (abbreviation of epsi lon)-NH2 of Lys 2 Q is myristic acid or palmitic acid acylate.
6. 一种含有如权利要求 1或 2所述的 Exendin 4类似物的药物组合物, 其进一 步含有药学上可接受的稀释剂、 赋形剂或载体。 A pharmaceutical composition comprising the Exendin 4 analogue according to claim 1 or 2, which further comprises a pharmaceutically acceptable diluent, excipient or carrier.
7. 如权利要求 6所述的药物组合物, 其中, 所述的载体选自于如下一组物质: 乙醇、 甘油和水。 The pharmaceutical composition according to claim 6, wherein the carrier is selected from the group consisting of ethanol, glycerin and water.
8. 如权利要求 1或 2所述的 Exendin 4类似物, 其用途为: 用于制备治疗糖尿 病的药物, 其中, 所述的糖尿病是 2型糖尿病。 The Exendin 4 analog according to claim 1 or 2, which is used for the preparation of a medicament for treating diabetes, wherein the diabetes is type 2 diabetes.
9. 如权利要求 6或 7所述的药物组合物, 其用途为: 用于制备治疗糖尿病的药 物, 其中, 所述的糖尿病是 2型糖尿病。 The pharmaceutical composition according to claim 6 or 7, which is used for the preparation of a medicament for treating diabetes, wherein the diabetes is type 2 diabetes.
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CN1746185A (en) * | 2004-09-06 | 2006-03-15 | 上海华谊生物技术有限公司 | Analog of Exendin 4 |
US7329646B2 (en) * | 2001-05-10 | 2008-02-12 | Shanghai Huayi Bio-Lab Co., Ltd. | Derivatives of the insulinotropic peptide exendin-4 and methods of production thereof |
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US7329646B2 (en) * | 2001-05-10 | 2008-02-12 | Shanghai Huayi Bio-Lab Co., Ltd. | Derivatives of the insulinotropic peptide exendin-4 and methods of production thereof |
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JP2017510636A (en) * | 2014-03-21 | 2017-04-13 | エニジェン カンパニー.,リミテッド. | Novel exenatide analogues and uses thereof |
CN106661096A (en) * | 2014-03-21 | 2017-05-10 | 安尼根有限公司 | Novel exenatide analogue and use thereof |
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US11103557B2 (en) | 2014-03-21 | 2021-08-31 | Anygen Co., Ltd. | Exenatide analogue and use thereof |
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