WO2002090388A1 - Derives d'exendine - Google Patents

Derives d'exendine Download PDF

Info

Publication number
WO2002090388A1
WO2002090388A1 PCT/CN2002/000316 CN0200316W WO02090388A1 WO 2002090388 A1 WO2002090388 A1 WO 2002090388A1 CN 0200316 W CN0200316 W CN 0200316W WO 02090388 A1 WO02090388 A1 WO 02090388A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
lys
arg
amino acid
exendin
Prior art date
Application number
PCT/CN2002/000316
Other languages
English (en)
French (fr)
Inventor
Yukun Sun
Dengxi Wu
Zhiyong Zhu
Gang Yu
Chunjuan Shen
Shaoling Zhao
Jiaxiang Zhou
Original Assignee
Shanghai Huayi Bio Lab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Huayi Bio Lab filed Critical Shanghai Huayi Bio Lab
Priority to CA2446394A priority Critical patent/CA2446394C/en
Priority to BRPI0209685A priority patent/BRPI0209685B8/pt
Priority to EP02727167A priority patent/EP1386930A4/en
Priority to JP2002587464A priority patent/JP4287153B2/ja
Priority to KR1020037014451A priority patent/KR100902208B1/ko
Priority to AU2002257497A priority patent/AU2002257497B2/en
Publication of WO2002090388A1 publication Critical patent/WO2002090388A1/zh
Priority to US10/704,409 priority patent/US7329646B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to an active polypeptide compound for the treatment of diabetes mellitus, and particularly to a derivative of the insulinotropic peptide Exendin-4.
  • GIP gastric inhibitory peptide
  • GLP-1 glucagon-like peptide
  • GLP-1 is characterized by its ability to promote islet ⁇ -cells to synthesize proinsulin RNA and proinsulin, which in turn promotes insulin release.
  • the effect of GLP-1 on pancreatic ⁇ -cells depends on glucose concentration. When the blood glucose concentration is higher than 6mmol L, it can significantly promote insulin secretion; once the blood glucose concentration returns to normal, it will no longer continue to act, which is important for type II diabetes. The treatment is very useful. Clinical trials in recent years have also shown that GLP-1 can promote insulin secretion and reduce blood glucose levels in patients with type II diabetes.
  • World patent application WO 91/11457 discloses glucagon-like polypeptides GLP-1 (7-34), (7-35), (7-36), and (7-37) for treating type II diabetes ).
  • Exendin-4 has 53% homology with GLP-1 in the amino acid sequence, and can bind to the GLP-1 receptor. It also promotes insulin secretion, so it is also an insulin-promoting peptide.
  • U.S. Patent No. 05424286 discloses a comparison experiment between Exendin-4 and GLP-1 in promoting insulin secretion. Experiments have shown that its ability to promote insulin secretion is stronger than GLP-1, and the concentration required to produce insulin secretion is lower than GLP-1. The half-life in vivo is longer than GLP-1. Based on these characteristics, Exendin-4 may become an ideal medicine for treating type 2 diabetes.
  • Exendin-4 can be prepared by chemical synthesis methods, such as peptide synthesizers, but using this method, the cost of the product is high, resulting in a high price, which cannot make it a commercial drug. Therefore, people have tried to use genetic engineering methods. To large-scale low-cost production to achieve commercialization purposes. Object of the invention
  • the purpose of the present invention is to provide a class of derivatives of the polypeptide compound Exendin-4, which have the effect of promoting insulin secretion and thereby lowering blood sugar, and thus can be used for treating type II diabetes. They can be prepared not only by chemical synthesis methods, but also easily by recombinant DNA methods, thereby reducing production costs. This provides a summary of possible inventions
  • the present invention relates to a class of insulinotropic peptide derivatives of the following structure and salts suitable for use in medicine,
  • the amino acid sequence of the insulin-promoting peptide derivative of the present invention is as described in ⁇ 210> 1- ⁇ 210> 4 in the sequence listing.
  • the insulin-promoting peptide derivative provided by the present invention is an amphoteric compound and can react with acidic or basic substances to form a salt.
  • the acids commonly used to form acid addition salts are: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, Citric acid, benzoic acid, acetic acid, etc.
  • salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, Bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate , Malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-diacid, hexyne-1,6-di Acid salt, benzoate salt, chlorobenzoate salt, methylbenzoate salt, dinitrobenzoate salt, hydroxybenzoate salt, methoxybenzoate salt, phenylacetate salt, phenylpropionate Acid salt, phenylbutyrate, citrate, lactate, Y-
  • Basic substances can also react with insulin-promoting peptide derivatives to form salts.
  • These basic substances include ammonium, alkali metal or alkaline earth metal hydroxides, as well as carbonates, bicarbonates, and the like.
  • the preparation method of the insulin-promoting peptide derivative according to the present invention is a solid-phase chemical synthesis method, and the steps include: using a HMP resin as a solid-phase carrier, and using a 9-fluorenylmethoxycarbonyl group for an ⁇ -amino group of an amino acid; The (Fmoc) group is protected, and the peptide synthesizer is used to synthesize the amino acid sequence of the insulin-promoting peptide derivative, and then separated and purified, and lyophilized to obtain the product.
  • the method for preparing the insulin-promoting peptide derivative according to the present invention is a genetic engineering method, and the steps include:
  • the present invention also provides the application of the insulin secretin-promoting peptide derivative and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating II diabetes.
  • the present invention provides a new class of derivatives of the exendin-4 polypeptide compound, which expands the types of the exendin-4 derivatives of the polypeptide compound. They have biological activities that promote insulin secretion and hypoglycemic effects, and can be used to treat ⁇ diabetes. They can be prepared not only by chemical synthesis methods, but also easily by recombinant DNA methods, thus providing the possibility to reduce production costs.
  • the medicinal effect experiment proves that the non-obese diabetic mice (NOD mice) intraperitoneally injected with 0.1 insulin secretin-derived peptide derivative of the present invention can obviously promote insulin secretion and lower blood sugar.
  • FIG. 1 is an experimental diagram of the medicinal effect of the insulin-promoting peptide derivative of the invention.
  • FIG. 2 is a mass spectrum of the product obtained in Example 1.
  • the theoretical molecular weight of the derivative is 4300.6, and the measured value is 4316.7.
  • Figure 3 is Exendin 4 (Lys 20 , Arg 40 ) and Exendin 4 (Leu 14 , Lys 20 , Arg 40 ) in the present invention Schematic of the experimental results of hypoglycemic effect.
  • FIG. 4 is a schematic diagram of the experimental results of insulin secretion of E Xen din 4 (Lys 20 , Arg 40 ) in the present invention.
  • FIG. 5 is a schematic diagram of the experimental results of C-peptide secretion of Exendin 4 (Lys 20 , Arg 40 ) in the present invention. Summary of the invention
  • Example 1 A frog skin antibacterial insulin secretion peptide derivative of the structure of the present invention was prepared by a solid-phase chemical synthesis method, where X is Arg, Y is Lys, and Z is -OH.
  • Trt trityl
  • the reagents are: N-methylpyrrolidone, dichloromethane, hexahydropyridine, methanol, dimethylaminopyridine (I DMF) ⁇ , N-diisopropylethylamine ( ⁇ , ⁇ - diisopropylethylamine) / NMP, HBTU 100 mmole / 0.5 M HOBT in DMF, N, N- Dicyclohexylcarbodiimide / NMP
  • DMF is ⁇ , ⁇ -Dimet ylformamide
  • NMP is N-methylpyrrolidone
  • HOBT 1-Hydroxybenzotriazole
  • HBTU 2- (1Hydroxybenzotriazole) -tetramethylurea hexafluorophosphate (2- (1 (-benzotriazole-yl- 1,1 , 3, 3 -tetramethyl-Uronium hexafluorophosphate)
  • the 0.25mmol scale Take the 0.25mmol scale as an example, weigh 0.25g of HMP resin, place it in the reactor on the peptide synthesizer, weigh various protective amino acids into 1mmol bottles, and follow the amino acid sequence of the insulinotropic peptide derivative from The C-terminal to N-terminals are arranged in a synthesizer, and at room temperature of 25 ° C, the Fmoc protection, activation, and connection are automatically controlled by a computer program, and then the next cycle is performed to complete the synthesis. After the synthesis is completed, the obtained peptide resin with a side chain protecting group may be dried on a peptide synthesizer and then weighed.
  • the protected insulin-promoting peptide derivative polypeptide resin was placed in a conical Erlenmeyer flask, and the lysis reagent was added as shown in the following table:
  • the reaction was electromagnetically stirred for 6 hours.
  • the filtrate was collected, and the resin was washed with a small amount of trifluoroacetic acid.
  • the collection solution and the washing solution were combined, and ether was added to generate a precipitate.
  • the precipitate was filtered and washed with a small amount of ether. Dry in a desiccator to obtain a crude product.
  • the insulin-promoting peptide derivative of the present invention was prepared by genetic engineering, where X is Leu, Y is Lys, and Z is Arg- OH.
  • Plasmid In another test tube, the plasmid containing the lactose promoter Lac (or the temperature-controlled promoter P ⁇ , the tryptophan-lactose mixed promoter Tac) was digested with the restriction enzymes EcoR 1 and Hindlll, and then used. Phenol / chloroform extraction, centrifugation to take the water layer part, and then washing the water layer 3 times with chloroform, centrifugation, precipitation of the water layer with isopropanol, centrifugation, and drying.
  • Recipient bacteria culture The clone E. coli (M. coli) JM103 is cultured in a culture solution (LB culture solution) containing 10 g of peptone, 5 g of yeast extract, and 5 g of sodium chloride per liter at 37 ° C with shaking for 4 hours. The bacteria were collected by centrifugation, and after being treated with CaCl 2 solution, they were stored for 4 days.
  • a culture solution LB culture solution
  • the cloned plasmid was transformed into the recipient bacterium Escherichia coli JM103 cells, maintained in an ice bath for 30 minutes, warmed to 42 ° C, and maintained for 2 minutes, transferred to an agarose plate containing ampicillin, and incubated at 37 ° C overnight. Colony screening yielded clone-positive plasmids.
  • culture in a 300ml X 10 shaker induce, and collect the bacterial cells with lysate (1% NaCl, phosphate buffer pH 7.5, 20mM) and lysozyme, incubate at 30 ° C for 30 minutes, collect the pellet by centrifugation, add
  • the inclusion bodies were extracted with 6M guanidine hydrochloride (Gu ⁇ ⁇ (31), centrifuged, and the supernatant was dialyzed to remove Gu-HCl, and then a phosphate buffer composed of 1% NaCl and 0.1% Tween 80 ( 20 mM, pH 7.5) Wash the pellet three times to obtain inclusion bodies.
  • the inclusion bodies were dissolved in 8M urea solution, hydrochloric acid was added to 50 mM, cyanogen bromide was added, protected from light, stirred under nitrogen protection, and lysed for 2 hours, followed by HPLC analysis.
  • mice non-obese diabetic mice (NOD mice), weighing 17g ⁇ 2g, fasting for 2 hours
  • Control group 1 Intraperitoneal injection of insulin 2 g
  • Control group 2 Intraperitoneal injection of insulin 4 ⁇ g
  • Example 4 Hypoglycemic effect of Exendin 4 (Lys 20s Arg 40 ) and Exendin 4 (Leu 14 , Lys 20 , Arg 40 ) on NOD mice
  • NOD mice were fasted overnight (provided by Shanghai Animal Center, Chinese Academy of Sciences)
  • Exendin 4 (Lys 20 , Arg 40 ), Exendin 4 (Leu 14 , Lys 20 , Arg 40 ) blood glucose measurement kit (produced by Shanghai Biological Products Ministry of Health)
  • NOD mice were fasted overnight and divided into three groups.
  • the first group was injected intraperitoneally with 200 ⁇ l of 40% glucose and 1 ⁇ g Exendin 4 (Lys 20 , Arg 40 ), and the second group was injected intraperitoneally with 200 ⁇ l of 40% glucose and 2 ⁇ g Exendin 4 (Leu 14 , Lys 20 , Arg 40 )
  • the third group was the control group and only injected glucose.
  • a graduated capillary tube treated with heparin before injection
  • the blood glucose concentration of the three groups of serum was measured according to the method described in the kit to test the hypoglycemic effect of GLP-1 (7-36).
  • Radioimmunoassay kit (produced by Shanghai Biological Products Ministry of Health)
  • NOD mice were divided into two groups. First, 50 microliters of blood was collected from the fundus vein plexus with graduated capillary (the inner wall of the capillary was rinsed with 1 mg / mL heparin and dried), and then two groups of mice were injected intraperitoneally with 5 micrograms of Exendin 4 (Lys 2 , Arg 4Q ) and 200 microliters of physiological saline was recorded, and the time was zero. Then take blood at 5, 10, 20 and 30 minutes. Immediately after the blood was taken, each blood sample was added to a centrifuge tube containing 50 microliters of physiological saline, mixed, and the red blood cells were removed at 3,000 rpm to separate the hearts. The insulin concentration in the serum was measured according to the method of the radioimmunity kit to test Exendin 4 (Lys 20 , Arg 4Q ).
  • Radioimmunoassay kit (produced by Shanghai Biological Products, Ministry of Health, China)
  • Radioimmunoassay C-peptide assay kit produced by Shanghai Biological Products, Ministry of Health
  • Healthy C 57 / BL mice were divided into two groups. Firstly, 50 microliters of blood was taken from the fundus venous plexus with graduated capillaries (the inner wall of the capillary was rinsed with 1 mg / mL heparin and dried), and then two groups of mice were injected intraperitoneally with 5 micrograms of Exendin 4 (Lys 20 , Arg 4Q ) and 200 Slightly increase the physiological saline and record it as the zero moment. Then take blood at 5, 10, 20 and 30 minutes. Immediately after the blood was taken, each blood sample was added to a centrifuge tube containing 50 microliters of normal saline, mixed, and the blood cells were removed at 3000 rpm to separate the red blood cells. The concentration of C peptide in the serum was measured according to the method of using the radioimmunity kit to test Exendin 4 (Lys 20 , Arg 40 ) promotes C-peptide secretion.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Emergency Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Description

促胰岛素分泌肽衍牛物 技术领域
本发明涉及治疗 Π糖尿病的活性多肽化合物， 具体地说涉及促胰岛素分 泌肽化合物 Exendin-4的衍生物。 技术背景
研究发现， 同等数量的葡萄糖， 口服比注射产生更多的胰岛素， 究其原 因， 科研人员发现原来是肠道激素 （Incretins)起的作用。 肠道激素是一组 多肽化合物， 1985年人们了解了在肠道激素中有强力肠抑胃肽 GIP (gastric inhibitory peptide)和胰高血糖素类多肽 GLP-1 ( glucagon-like peptide ) 0 GLP-1 又有二种， 一种为 GLP-1 (7-36) - H2, 由 30个氨基酸残基组成， 另一种 为 GLP-1 (7-37), 由 31个氨基酸残基组成， 二者有相同的促胰岛素分泌作 用。 在对离体胰岛细胞作用时， 浓度为
Figure imgf000002_0001
的 GLP-1 即促 进了胰岛素的分泌。 DNA及氨基酸序列分析发现， 这些促胰岛素分泌肽是 由胰高血糖素原分子经肠道蛋白水解酶作用而产生， 因而称为胰高血糖素类 多肽。 GLP-1的特点是能促进胰岛 β -细胞合成胰岛素原信使 RNA和胰岛素 原， 进而促进胰岛素的释放。 GLP-1 对胰岛 β -细胞的作用依赖于葡萄糖浓 度， 当血糖浓度高于 6mmol L 时， 能显著促进胰岛素分泌； 而一旦血糖浓 度恢复至正常值则不再继续作用， 这一点对 II型糖尿病的治疗十分有用。 近 年的临床实验也证明， GLP-1对 II型糖尿病患者有促进胰岛素分泌和降低血 糖浓度的作用。
GLP-l(7-36)-NH2的化学结构如下：
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- NH2
式中英文缩写表示：
His组氨酸， Ala丙氨酸， Glu谷氨酸， Gly甘氨酸， Thr苏氨酸， Phe 苯丙氨酸， Ser丝氨酸， Asp天冬氨酸， Val缬氨酸， Tyr酪氨酸， Leu亮 氨酸， Gin谷氨酰胺， Lys赖氨酸， lie异亮氨酸， Arg精氨酸
世界专利申请 WO 91/11457号公幵了用于治疗 II型糖尿病的类胰高血糖 素类多肽 GLP-1 (7-34)、 （7-35)、 (7-36)和 (7-37) 的类似物。
1996年 Jean-Pierre Ranfinan在（Regulatory Peptides))杂志上向人们介 绍了一种从墨西哥巨蜥蜴 beaded Hzard(Heloderma horridum) 的毒液中发现分 离得到的含 39个氨基酸的多肽化合物 EXen(iin-4， 其 C末端为酰胺， 化学结 构如下：
10 20 His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-
30 39 Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 式中英文缩写表示：
Met 甲硫氨酸， Asn天冬酰胺， Pro脯氨酸
Exendin-4在氨基酸序列上与 GLP-1有 53%的同源性， 并且可与 GLP-1 的受体相结合， 亦有促进胰岛素分泌的作用， 因此也是一种促胰岛素分泌肽。
美国专利 USP 05424286公开了 Exendin-4与 GLP-1促进胰岛素分泌作 用的对比实验， 实验证明其促进胰岛素的分泌作用比 GLP-1 强， 产生促胰 岛素分泌作用所需浓度比 GLP-1低， 而且在体内的半衰期比 GLP-1 长。 鉴 于这些特点， Exendin-4可能成为一种较为理想的治疗 II型糖尿病的药物。
Exendin-4可采用化学合成方法， 例如用多肽合成仪来制备， 但用此种 方法制备， 产品成本高， 导致售价高， 无法使其成为一个商业化药品， 为此 人们试图采用基因工程方法来大规模低成本生产以达到商业化目的。 发明目的
本发明目的在于提供一类多肽化合物 Exendin-4的衍生物， 它们有促进 胰岛素分泌从而降低血糖的作用， 因此可用于治疗 II型糖尿病。 它们不仅可 以用化学合成方法制备， 还易于用重组 DNA方法制备， 从而为降低生产成 本提供了可能 发明概要
本发明的内容涉及一类如下结构的促胰岛素分泌肽衍生物和其适用于药 用的盐，
10 14 20
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X-Glu-Glu-Glu-Ala-Val-Y-
30 3940
Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ak-Pro-Pro-Pro-Ser-Z 其中： X是 Arg， Leu, lie或 Met, Y是 His, Arg或 Lys， Z是 Arg-OH， -OH, -]^2或1^-0 且当 X=Met， Y=Arg时 Z不能取值为 NH2。
本发明的促胰岛素分泌肽衍生物的氨基酸序列如序列表中<210>1- <210>4所述。
本发明提供的促胰岛素分泌肽衍生物是两性化合物， 可与酸性或碱性物 质反应形成盐。 通常采用的形成酸加成盐的酸为： 盐酸， 氢溴酸， 氢碘酸， 硫酸， 磷酸， 对 -甲苯磺酸， 甲磺酸， 草酸， 对溴苯基磺酸， 碳酸， 琥珀酸， 柠檬酸， 苯甲酸， 乙酸等。 这类盐的例子包括硫酸盐， 焦硫酸盐， 硫酸氢盐， 亚硫酸盐， 亚硫酸氢盐， 磷酸盐， 磷酸氢盐， 磷酸二氢盐， 偏磷酸盐， 焦磷 酸盐， 盐酸盐， 溴化物， 碘化物， 乙酸盐， 丙酸盐， 癸酸盐， 辛酸盐， 丙烯 酸盐， 甲酸盐， 异丁酸盐， 己酸盐， 庚酸盐， 丙炔酸盐， 草酸盐， 丙二酸盐， 丁二酸盐， 辛二酸盐， 癸二酸盐， 富马酸盐， 马来酸盐， 丁炔 -1,4-二酸盐， 己炔 -1,6-二酸盐， 苯甲酸盐， 氯苯甲酸盐， 甲基苯甲酸盐， 二硝基苯甲酸盐， 羟基苯甲酸盐， 甲氧基苯甲酸盐， 苯乙酸盐， 苯丙酸盐， 苯丁酸盐， 柠檬酸 盐， 乳酸盐， Y -羟基丁酸盐， 甘醇酸盐， 酒石酸盐， 甲磺酸盐， 丙磺酸盐， 萘 -1-磺酸盐， 萘 -2-磺酸盐， 扁桃酸盐等， 优选的酸加成盐是促胰岛素分泌 肽衍生物与无机酸如盐酸， 氢溴酸， 尤其是盐酸形成的盐。
碱性物质也可以与促胰岛素分泌肽衍生物反应形成盐， 这些碱性物质包 括铵， 碱金属或碱土金属的氢氧化物， 以及碳酸盐， 碳酸氢盐等。 典型的有 氢氧化钠， 氢氧化钾， 氢氧化铵， 碳酸钾等。
本发明所述的促胰岛素分泌肽衍生物的制备方法， 此制备方法为固相化 学合成法， 其步骤包括： 以 HMP树脂为固相载体， 氨基酸的 α -氨基用 9-芴 基甲氧羰基 (Fmoc)基团保护， 用多肽合成仪按促胰岛素分泌肽衍生物的氨基 酸序列进行合成， 再经分离纯化， 冻干即得产品。
本发明所述的促胰岛素分泌肽衍生物的制备方法， 此制备方法为基因工 程制备法， 其步骤包括：
a. 按促胰岛素分泌肽衍生物的氨基酸序列合成基因片段；
b. 基因片段经连接， 质粒构建， 受菌体的培养， 转化， 克隆得到菌株； c. 菌株经发酵， 破壁， 抽提得到包涵体；
d. 包涵体裂解、 分离得粗品， 经高效液相层析仪 HPLC纯化， 最后经 冻干得产品。
本发明中还提供了该促胰岛素分泌肽衍生物和其适用于药用的盐在制备 治疗 II糖尿病的药物中的应用。
本发明提供了一类新的多肽化合物 Exendin-4 的衍生物， 扩大了多肽化 合物 Exendin-4衍生物的种类， 它们具有促进胰岛素分泌的生物活性和降血 糖作用， 可用于治疗 Π糖尿病。 它们不仅可以用化学合成方法制备， 还易于 用重组 DNA方法制备， 从而为降低生产成本提供了可能。 药效实验证明， 在非肥胖型糖尿病小鼠 （NOD小鼠）腹腔内注射含 0.1 本发明的促胰岛 素分泌肽衍生物， 有明显的促进胰岛素分泌和降血糖作用。 附图说明
现结合附图和实施例对本发明的内容作进一步的说明。
图 1是本发明促胰岛素分泌肽衍生物的药效实验图。
图 2是实施例 1所得产品的质谱图， 该衍生物分子量理论值为 4300.6， 实测值为 4316.7。
图 3是本发明中 Exendin 4 (Lys20, Arg40)和 Exendin 4 (Leu14, Lys20, Arg40) 的降血糖作用实验结果示意图。
图 4是本发明中 EXendin 4 (Lys20, Arg40) 的促胰岛素分泌作用实验结果 示意图。
图 5是本发明中 Exendin 4 (Lys20, Arg40) 的促 C肽分泌作用实验结果 示意图。 发明内容
下面实施例是对本发明的进一步阐述， 而不是对本发明的限制。
实施例 1 固相化学合成法制备本发明结构的蛙皮抗菌促胰岛素分泌 肽衍生物， 其中 X为 Arg， Y为 Lys， Z为 -OH。
(1)所采用的氨基酸单体：
Figure imgf000006_0001
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 ) I DMF Ν,Ν-二异丙基乙胺 （ Ν,Ν- diisopropylethylamine ) / NMP, HBTU 100 mmole / 0.5 M HOBT in DMF, N，N- 二环己基碳二亚胺（N，N-Dicyclohexylcarbodiimide) /NMP
其中： DMF为 Ν,Ν-二甲基甲酰胺 (Ν,Ν-Dimet ylformamide)
NMP为 Ν-甲基吡咯垸酮 （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-(1Η- benzotriazole-yl- 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 lmmol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 25Ό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：
将带保护基的该促胰岛素分泌肽衍生物多肽树脂置于具塞三角烧瓶， 加 入裂解试剂如下表：
试剂 用量
K 0.50 ml
苯甲醚 0.50 ml
苯酚 0.75 g
巯基乙醇 0.20 ml
三氟乙酸 10.0 ml
然后在恒温 30°C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， 最后经冷冻干燥后得到 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 实施例 2 基因工程法制备本发明的促胰岛素分泌肽衍生物， 其中 X 为 Leu， Y为 Lys， Z为 Arg-OH。
A. 按此促胰岛素分泌肽衍生物的氨基酸序列， 合成基因片段：
(1) 5， AAT TCC ATG CAC GGC GAA GGC ACC TTC ACC AGC GAT CTG AGC AAA CAG CTG GAA GAA GAA GCG GTT AA
(2) 5， ACTG TTC ATC GAA TGG CTG AAA AAC GGC GGC CCG AGC AGC GGC GCG CCG CCG CCG AGC CGT TAG A
(3) 5， AGCTT CTA ACG GCT CGG CGG CGG CGC GCC GCT GCT CGG GCC GCC GTT TTT CAG CCA TTC GAT GA
(4) 5， ACAG TTT AAC CGC TTC TTC TTC CAG CTG TTT GCT CAG ATC GCT GGT GAA GGT GCC TTC GCC GTG CAT GG
B. 克隆
连接： 取二支试管， 一支加入光密度值为 0.1 (A26tan) 的片段 (1)和片段 (4), 另一支试管中加入光密度值为 （A26()nm) 的片段 (2)和片段 (3)， 再将聚核 苷酸激酶缓冲液 （Polynucleotide Kinase 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中， 于 37°C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 °C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°C左右保温。 过夜， 使基因 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 P^， 色氨酸- 乳糖混合启动子 Tac)的质粒用限制性内切酶 EcoR l和 Hindlll双酶酶切后 用酚 /氯仿抽提， 离心取水层部分， 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。 受体菌的培养： 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°C振荡培养 4小时， 离心收集菌体， 经 CaCl2溶液处理后， 4Ό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°C， 维持 2分钟后， 转移至含氨苄青霉素的琼脂糖平板， 37 °C过夜， 进行集落筛选， 得克隆阳性质粒。
C. 发酵
将筛选的含该种衍生物质粒的寄主菌菌株于 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， 300ml X 10 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°C 保温 30分钟， 离心收集沉 淀，加 6M盐酸胍（Gu ·Η(31)抽提包涵体，离心，将上清液透析，除去 Gu -HCl, 再用组成为 l %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尿素溶液中， 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 3 药效实验
实验动物： 非肥胖型糖尿病小鼠（NOD小鼠）， 体重 17g±2g， 禁食 2 小时 对照组 1 : 腹腔注射胰岛素 2 g
对照组 2: 腹腔注射胰岛素 4 y g
实验组： 腹腔注射含 0.1 μ g实例一所得促胰岛素分泌肽衍生物
不同时间采集的血样， 使用卫生部上海生物制品所出品的血糖测定试剂 盒进行测定。 实验结果如附图。 此实验表明本发明所得促胰岛素分泌肽衍生 物的降血糖作用十分明显。 实施例 4 Exendin 4(Lys20s Arg40)和 Exendin 4 (Leu14, Lys20, Arg40)对 NOD小鼠的降血糖作用
实验材料与方法：
NOD小鼠禁食过夜（中国科学院上海动物中心提供）
0.9%NaCl溶液， Exendin 4(Lys20, Arg40)， Exendin 4 (Leu14, Lys20, Arg40) 血糖测定试剂盒（中国卫生部上海生物制品所出品）
NOD小鼠禁食过夜， 分为三组。 第一组腹腔注射 200微升 40%葡萄糖 和 1微克 Exendin 4(Lys20, Arg40), 第二组腹腔注射 200微升 40%葡萄糖和 2 微克 Exendin 4(Leu14, Lys20, Arg40), 第三组为对照组， 只注射葡萄糖。 立刻 用刻度毛细管（注射前以肝素处理）从眼窦取血 30微升， 置入 300微升生 理盐水中混匀， 3000转 /分离心除去红血球， 血清用于血糖浓度测定。 分别 于 30， 60， 120分钟按同样操作取血， 分离血清。 三组血清均按照试剂盒所 述方法测定血糖浓度， 以检验 GLP-l(7-36)的降血糖作用。
结果如图 1所示。
对照组血糖浓度大幅度升高后， 逐渐回落到正常水平； 而给药组血糖浓 度未出现显著升高， 一直保持在正常水平附近。 这是由于给药后促进了胰岛 素分泌，从而避免了血糖浓度的大幅波动。由此可证明 Exendin 4(Lys2Q, Arg4。) 和 Exendin 4 (Leu14, Lys20, Arg4。)的降血糖作用。
'实施例 5 Exendin 4(Lys20, Arg4a)的促胰岛素分泌作用
实验材料与方法- NOD小鼠 （中国科学院上海动物中心提供）
40%葡萄糖溶液， 0.9%NaCl溶液, Exendin 4(Lys2。, Arg40)
放射免疫胰岛素测定试剂盒（中国卫生部上海生物制品所出品）
NOD小鼠分成两组。 首先用刻度毛细管 （先以 lmg/mL肝素润洗毛细 管内壁并晾干）从眼底静脉丛取血 50微升， 然后两组小鼠分别腹腔注射 5 微克 Exendin 4(Lys2。， Arg4Q)和 200微升生理盐水， 并记此时为零时刻。 然后 分别于 5， 10， 20， 30分钟按同样操作取血。 取血后各血样立刻加入盛有 50 微升生理盐水的离心管中， 混匀， 3000转 /分离心除去红血球， 按放射免疫 试剂盒使用方法测定血清中胰岛素浓度， 以检验 Exendin 4(Lys20, Arg4Q)的促 胰岛素分泌作用。
实验结果如图 2所示。 该结果表明， 腹腔注射 Exendin 4(Lys2Q, Arg4。)能 显著促进胰岛素的分泌。 实施例 6 Exendin 4(Lys20, Arg4Q)的促 C肽分泌作用
实验材料与方法- 健康 C57 BL小鼠（中国科学院上海动物中心提供）
40%葡萄糖溶液, 0.9%NaCl溶液, Exendin 4(Lys20, Arg40)
放射免疫胰岛素测定试剂盒（中国卫生部上海生物制品所出品） 放射免疫 C-肽测定试剂盒（中国卫生部上海生物制品所出品）
健康 C57/BL小鼠分成两组。 首先用刻度毛细管（先以 lmg/mL肝素润 洗毛细管内壁并晾干）从眼底静脉丛取血 50微升， 然后两组小鼠分别腹腔 注射 5微克 Exendin 4(Lys20, Arg4Q)和 200微升生理盐水， 并记此时为零时刻。 然后分别于 5， 10, 20， 30分钟按同样操作取血。 取血后各血样立刻加入盛 有 50微升生理盐水的离心管中， 混匀， 3000转 /分离心除去红血球， 按放射 免疫试剂盒使用方法测定血清中 C肽浓度， 以检验 Exendin 4(Lys20, Arg40)½ 促 C肽分泌作用。
实验结果如图 3所示。 该结果表明， 腹腔注射 Exendin 4(Ly , Arg4(^ 显著 C肽的分泌。

Claims

权利要求
1. 一类促胰岛素分泌肽衍生物及其适用于药用的盐， 该促胰岛素分泌 肽衍生物的氨基酸序列如序列表中 <210>1—< 210>4所述。
2. 根据权利要求 1 所述的促胰岛素分泌肽衍生物的制备方法， 所述方 法为固相化学合成法， 其步骤包括： 以 HMP树脂为固相载体， 氨基酸的 α - 氨基用 9-芴基甲氧羰基 (Fmoc)基团保护， 用多肽合成仪按促胰岛素分泌肽衍 生物的氨基酸序列进行合成， 再经分离纯化， 冻干即得产品。
3. 根据权利要求 1 所述的促胰岛素分泌肽衍生物的制备方法， 所述方 法为基因工程制备法， 其步骤包括：
a. 按促胰岛素分泌肽衍生物的氨基酸序列合成基因片段；
b . 基因片段经连接， 质粒构建， 受菌体的培养， 转化， 克隆得到菌株； c 菌株经发酵， 破壁， 抽提得到包涵体；
d. 包涵体裂解、 分离得粗品， 经高效液相层析仪 HPLC纯化， 最后经 冻干得产品。
4. 根据权利要求 1 所述的促胰岛素分泌肽衍生物及其适用于药用的盐 在制备治疗 II型糖尿病的药物中的应用。
II
PCT/CN2002/000316 2001-05-10 2002-05-08 Derives d'exendine WO2002090388A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA2446394A CA2446394C (en) 2001-05-10 2002-05-08 Insulinotropic peptide derivatives
BRPI0209685A BRPI0209685B8 (pt) 2001-05-10 2002-05-08 derivado de peptídeo insulinotrópico e método para a produção de um derivado de peptídeo insulinotrópico
EP02727167A EP1386930A4 (en) 2001-05-10 2002-05-08 EXENDINDERIVATE
JP2002587464A JP4287153B2 (ja) 2001-05-10 2002-05-08 インスリン指向性ペプチド誘導体
KR1020037014451A KR100902208B1 (ko) 2001-05-10 2002-05-08 인슐린 친화성 펩티드 유도체
AU2002257497A AU2002257497B2 (en) 2001-05-10 2002-05-08 Derivatives of exendin
US10/704,409 US7329646B2 (en) 2001-05-10 2003-11-07 Derivatives of the insulinotropic peptide exendin-4 and methods of production thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN01112856.9 2001-05-10
CNB011128569A CN1162446C (zh) 2001-05-10 2001-05-10 促胰岛素分泌肽衍生物

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/704,409 Continuation-In-Part US7329646B2 (en) 2001-05-10 2003-11-07 Derivatives of the insulinotropic peptide exendin-4 and methods of production thereof

Publications (1)

Publication Number Publication Date
WO2002090388A1 true WO2002090388A1 (fr) 2002-11-14

Family

ID=4659603

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2002/000316 WO2002090388A1 (fr) 2001-05-10 2002-05-08 Derives d'exendine

Country Status (9)

Country Link
US (1) US7329646B2 (zh)
EP (2) EP2223938B1 (zh)
JP (1) JP4287153B2 (zh)
KR (1) KR100902208B1 (zh)
CN (1) CN1162446C (zh)
AU (1) AU2002257497B2 (zh)
BR (1) BRPI0209685B8 (zh)
CA (1) CA2446394C (zh)
WO (1) WO2002090388A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2008091177A1 (fr) 2007-01-18 2008-07-31 Otkrytoe Aktsionernoe Obschestvo 'otechestvennye Lekarstva' Médicament pour le traitement du diabète à base d'exenatide et de dalargine, utilisation et traitement

Families Citing this family (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924264B1 (en) * 1999-04-30 2005-08-02 Amylin Pharmaceuticals, Inc. Modified exendins and exendin agonists
JP5107713B2 (ja) * 2004-10-07 2012-12-26 ノヴォ ノルディスク アー／エス 遅延性のエキセンディン−４化合物
US7893017B2 (en) 2004-10-07 2011-02-22 Novo Nordisk A/S Protracted GLP-1 compounds
CN100429227C (zh) * 2005-06-29 2008-10-29 常州制药厂有限公司 Exendin4多肽片段
CN100374462C (zh) * 2005-11-21 2008-03-12 大连帝恩生物工程有限公司 截短胰高血糖素样肽1(sGLP-1)、制法及其应用
AU2007267833B2 (en) * 2006-05-26 2012-07-26 Amylin Pharmaceuticals, Llc Composition and methods for treatment of congestive heart failure
CN101125207B (zh) 2006-11-14 2012-09-05 上海华谊生物技术有限公司 带有聚乙二醇基团的艾塞丁或其类似物及其制剂和用途
HUE058307T2 (hu) * 2007-01-08 2022-07-28 Univ Pennsylvania GLP-1 receptor antagonista veleszületett hiperinzulinizmus kezelésében történõ használatra
CN101040936B (zh) * 2007-04-28 2011-10-19 大连水产学院 防治鱼类寄生虫病的复方中草药制剂
CN101215324B (zh) * 2007-12-26 2010-11-24 吉林大学 艾塞那肽短肽模拟肽及其在制备糖尿病治疗药物中的应用
KR100864584B1 (ko) * 2008-02-25 2008-10-24 성균관대학교산학협력단 비오틴으로 수식된 엑센딘 유도체, 이의 제조방법 및 이의용도
CN101463078B (zh) * 2009-01-12 2011-07-27 华东师范大学 一种Exendin-4衍生物及其固相化学合成
CN101665799A (zh) * 2009-06-29 2010-03-10 华东师范大学 一种Exendin-4衍生物的重组制备方法和应用
CN102712690B (zh) * 2009-11-26 2016-04-13 吴晓琰 长效Exendin 4的类似物
CN102397558B (zh) * 2010-09-09 2013-08-14 中国人民解放军军事医学科学院毒物药物研究所 Exendin-4类似物的定位聚乙二醇化修饰物及其用途
CN102532301B (zh) * 2010-12-31 2014-09-03 上海医药工业研究院 一类新型的Exendin-4类似物及其制备方法
CN106928341B (zh) * 2011-03-30 2021-06-01 上海仁会生物制药股份有限公司 定点单取代聚乙二醇化Exendin类似物及其制备方法
UA116217C2 (uk) 2012-10-09 2018-02-26 Санофі Пептидна сполука як подвійний агоніст рецепторів glp1-1 та глюкагону
JP2016503771A (ja) 2012-12-21 2016-02-08 サノフイ エキセンジン−４誘導体
HUE034308T2 (en) 2013-03-21 2018-02-28 Sanofi Aventis Deutschland Preparation of hydantoin-containing peptide products
US10450343B2 (en) 2013-03-21 2019-10-22 Sanofi-Aventis Deutschland Gmbh Synthesis of cyclic imide containing peptide products
EP3080154B1 (en) 2013-12-13 2018-02-07 Sanofi Dual glp-1/gip receptor agonists
WO2015086730A1 (en) 2013-12-13 2015-06-18 Sanofi Non-acylated exendin-4 peptide analogues
EP3080149A1 (en) 2013-12-13 2016-10-19 Sanofi Dual glp-1/glucagon receptor agonists
EP3080150B1 (en) 2013-12-13 2018-08-01 Sanofi Exendin-4 peptide analogues as dual glp-1/gip receptor agonists
TW201625668A (zh) 2014-04-07 2016-07-16 賽諾菲公司 作為胜肽性雙重ｇｌｐ－１／昇糖素受體激動劑之艾塞那肽－４衍生物
TW201625670A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自ｅｘｅｎｄｉｎ－４之雙重ｇｌｐ－１／升糖素受體促效劑
TW201625669A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自艾塞那肽－４（Ｅｘｅｎｄｉｎ－４）之肽類雙重ＧＬＰ－１／升糖素受體促效劑
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
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

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011457A1 (en) 1990-01-24 1991-08-08 Buckley Douglas I Glp-1 analogs useful for diabetes treatment
US5424286A (en) 1993-05-24 1995-06-13 Eng; John Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising same
WO2000069911A1 (en) 1999-05-17 2000-11-23 Conjuchem, Inc. Long lasting insulinotropic peptides
WO2001004156A1 (en) * 1999-07-12 2001-01-18 Zealand Pharmaceuticals A/S Peptides that lower blood glucose levels

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK144093D0 (zh) * 1993-12-23 1993-12-23 Novo Nordisk As
US6265204B1 (en) * 1997-01-17 2001-07-24 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
JP2003522721A (ja) * 1997-11-14 2003-07-29 アミリン・ファーマシューティカルズ，インコーポレイテッド 新規なエキセンジンアゴニスト化合物
EP1032587B2 (en) * 1997-11-14 2013-03-13 Amylin Pharmaceuticals, Inc. Novel exendin agonist compounds
US6514500B1 (en) 1999-10-15 2003-02-04 Conjuchem, Inc. Long lasting synthetic glucagon like peptide {GLP-!}
US6506724B1 (en) 1999-06-01 2003-01-14 Amylin Pharmaceuticals, Inc. Use of exendins and agonists thereof for the treatment of gestational diabetes mellitus
US6528486B1 (en) 1999-07-12 2003-03-04 Zealand Pharma A/S Peptide agonists of GLP-1 activity
AU2025401A (en) * 1999-12-07 2001-06-18 Yong-Hwa Lee A bathtub for a pet dog
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
CN1363654A (zh) 2001-07-19 2002-08-14 上海华谊生物技术有限公司 生产促胰岛素分泌肽glp－1(7－36)的基因工程菌以及生产glp－1(7－36)的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011457A1 (en) 1990-01-24 1991-08-08 Buckley Douglas I Glp-1 analogs useful for diabetes treatment
US5424286A (en) 1993-05-24 1995-06-13 Eng; John Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising same
WO2000069911A1 (en) 1999-05-17 2000-11-23 Conjuchem, Inc. Long lasting insulinotropic peptides
US6329336B1 (en) * 1999-05-17 2001-12-11 Conjuchem, Inc. Long lasting insulinotropic peptides
WO2001004156A1 (en) * 1999-07-12 2001-01-18 Zealand Pharmaceuticals A/S Peptides that lower blood glucose levels

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1386930A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2008091177A1 (fr) 2007-01-18 2008-07-31 Otkrytoe Aktsionernoe Obschestvo 'otechestvennye Lekarstva' Médicament pour le traitement du diabète à base d'exenatide et de dalargine, utilisation et traitement

Also Published As

Publication number Publication date
CA2446394A1 (en) 2002-11-14
JP4287153B2 (ja) 2009-07-01
CA2446394C (en) 2016-08-16
KR100902208B1 (ko) 2009-06-11
CN1162446C (zh) 2004-08-18
AU2002257497B2 (en) 2007-10-11
EP1386930A4 (en) 2005-09-07
EP2223938A1 (en) 2010-09-01
BR0209685A (pt) 2004-07-13
EP1386930A1 (en) 2004-02-04
CN1363559A (zh) 2002-08-14
JP2005502595A (ja) 2005-01-27
US20040142866A1 (en) 2004-07-22
EP2223938B1 (en) 2013-07-10
BRPI0209685B1 (pt) 2016-03-01
BRPI0209685B8 (pt) 2021-05-25
KR20030094386A (ko) 2003-12-11
US7329646B2 (en) 2008-02-12

Similar Documents

Publication Publication Date Title
WO2002090388A1 (fr) Derives d&#39;exendine
JP4282485B2 (ja) 伸長グルカゴン様ペプチド−１アナログ
JP4716641B2 (ja) グルカゴン様ペプチド−１類似体
US5981488A (en) Glucagon-like peptide-1 analogs
EP1991574B1 (en) Oxyntomodulin derivatives
US8097586B2 (en) Modified exedins and uses thereof
CN104302772B (zh) 用于糖尿病治疗的蛋白、蛋白缀合物及其应用
WO2021160185A1 (zh) Glp-1r激活剂的分子改构及其二聚体在治疗代谢病中的应用
US8716221B2 (en) Modified exendins and uses thereof
US20060252916A1 (en) Modified glucagon-like peptide-1 analogs
WO2011020320A1 (zh) 促胰岛素分泌肽类似物同源二聚体及其制备方法与用途
AU2003200839A2 (en) Extended glucagon-like peptide-1 analogs
KR100997835B1 (ko) 엑센딘 4 폴리펩타이드 단편
CN111171135B (zh) 具有双重受体激动作用的胰高血糖素衍生肽及其用途
KR20230008846A (ko) 이중 수용체 아고니즘 작용을 갖는 폴리펩티드 유도체 및 그 용도
CN116589536B (zh) 一类长效glp-1/gip受体双重激动剂及其应用
JP2793216B2 (ja) 粘膜による吸収を高める、ソルビン及び誘導ペプチド

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2002727167

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 1020037014451

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2446394

Country of ref document: CA

Ref document number: 10704409

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2002587464

Country of ref document: JP

Ref document number: 1878/DELNP/2003

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 2002257497

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2002727167

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642