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    • 专利摘要:
    The present invention relates to insulin affinity peptide exendin-4 derivatives and pharmaceutically acceptable salts thereof, which are useful for treating type II diabetes. Derivatives of exendin-4 have the following chemical structure: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X-Glu-Glu-Glu-Ala-Val-Y-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Z. Wherein X is Arg, Leu or Ile; Y is His, Arg or Lys; Z is Arg-OH, -OH, -NH 2 or Lys-OH. Derivatives of the present invention can be prepared by synthetic chemical means, more easily by recombinant techniques, which allows for commercial production.
    公开号:KR20030094386A
    申请号:KR10-2003-7014451
    申请日:2002-05-08
    公开日:2003-12-11
    发明作者:유쿤 선；뎅시 우；지용 주；강 유；춘주안 센；샤오링 자오；지악시앙 조우
    申请人:상하이 후아이 바이오 랩；
    IPC主号:
    专利说明:

    Exendin derivatives {Derivatives of exendin}
    [2] It has been known that oral administration of glucose at the same amount promotes more insulin secretion than injection. After much research to find out why, incretin proved to play an important role in this phenomenon.
    [3] Incretin is a group of peptide compounds, among which gastric inhibitory peptides (GIP) and glucagon-like peptides (GLP-1) with strong inhibitory activity were disclosed in 1985. There are two kinds of GLP-1, one of which is GLP-1 (7-36) amide, which consists of 30 amino acid residues and the other is GLP-1 (7-37), which is 31 amino acid residues. It consists of. The two have similar activity in stimulating insulin secretion. When acting on pancreatic islet cells ex vivo, GLP-1 can stimulate insulin secretion at concentrations as low as 1 × 10 −10 to 1 × 10 −11 mol / L. Analysis of DNA and amino acid sequences revealed that these insulin affinity peptides are derived from the proteolysis of the glucagon precursor "proglucagon" under the action of intestinal proteolytic enzymes. Thus, they are called glucagon like peptides.
    [4] One feature of GLP-1 is that it can stimulate pancreatic beta cells to synthesize proinsulin mRNA and proinsulin and release insulin. The effect of GLP-1 on pancreatic beta cells depends on glucose concentration. GLP-1 significantly stimulates insulin secretion when blood glucose levels are higher than 6 mmol / L. GLP-1 stimulation activity is no longer observed when blood glucose levels return to normal levels. Such a feature makes it very useful for treating type II diabetes. Clinical trials in patients with type II diabetes in recent years have shown that GLP-1 stimulates insulin secretion and lowers glucose levels.
    [5] The chemical structure of GLP-1 (7-36) amide is as follows:
    [6] 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-NH 2
    [7] In the above, His is histidine, Ala is alanine, Glu is glutamic acid, Gly is glycine, Thr is threonine, Phe is phenylalanine, Ser is serine, Asp is aspartic acid, Val is valine, Tyr For tyrosine, Leu for leucine, Gln for glutamine, Lys for lysine, Ile for isoleucine, and Arg for arginine.
    [8] International Publication WO 91/11467 discloses analogs of GLP-1 peptides 7-34, 7-35, 7-36 and 7-37, which are useful for treating Type II diabetes.
    [9] Peptide compound exendin-4 isolated from venom from beaded lizard (Heloderma horridum) was disclosed to Regulatory Peptides in 1996 by Jean-Pierre Ranfman. Exendin-4 consists of 39 amino acid residues with amidated carboxy termini.
    [10] The chemical composition of exendin-4 is as follows:
    [11] 10
    [12] His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-
    [13] 20 30
    [14] Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-
    [15] 39
    [16] Ala-Pro-Pro-Pro-Ser-NH 2
    [17] In the above, Met means methionine, Asn means asparagine, and Pro means proline.
    [18] Exendin-4 exhibits 53% homology with GLP-1 in its amino acid sequence. Exendin-4 also has the ability to be combined with the GLP-1 receptor to promote insulin secretion. Thus, it is considered an insulin affinity peptide.
    [19] US patent 05424286 discloses the results of several comparative experiments performed between exendin-4 and GLP-1 on the stimulatory effect on insulin secretion. As a result, exendin-4 had a stronger ability to stimulate insulin secretion compared to GLP-1 and needed a lower concentration to show such stimulatory activity. Exendin-4 also had a longer lifespan in vivo than GLP-1. In view of this feature, exendin-4 may be a more desirable treatment for type II diabetes.
    [20] Exendin-4 can be prepared by chemical synthesis methods such as solid phase synthesis on peptide synthesizers, but such methods are expensive and present an obstacle to the commercialization of exendin-4. Therefore, bio-engineering techniques have been tried to reduce production costs for commercialization purposes.
    [1] The present invention relates to active peptide compounds useful for treating type II diabetes. In particular, the present invention relates to derivatives of the insulin affinity peptide exendin-4.
    [44] The invention will be explained in more detail with reference to the following figures and examples.
    [45] 1 shows the results of pharmacokinetic studies of the insulin affinity peptide derivative of the present invention.
    [46] 2 shows the mass spectrum analysis of the product prepared in Example 1 (theoretical molecular weight 4300.6, measured molecular weight 4316.7).
    [47] Figure 3 schematically shows the experimental results of the glycemic effect of exendin 4 (Lys 20 , Arg 40 ) and exendin 4 (Leu 14 , Lys 20 , Arg 40 ).
    [48] 4 schematically shows the experimental results of the stimulatory effect of exendin 4 (Lys 20 , Arg 40 ) on insulin secretion.
    [49] 5 schematically shows the experimental results of the C-peptide-secretory stimulating effect of exendin 4 (Lys 20 , Arg 40 ).
    [21] Purpose of the Invention
    [22] The present invention is directed to providing several derivatives of the insulin affinity peptide exendin-4. Such derivatives help lower insulin levels by promoting insulin secretion. Thus, they may be useful for treating Type II diabetes. Such derivatives can be prepared by synthetic chemical means, and more easily by recombinant techniques, which can reduce production costs.
    [23] Summary of the Invention
    [24] The present invention provides insulin affinity peptide derivatives and pharmaceutically acceptable salts thereof. The insulin affinity peptide has the following amino acid sequence.
    [25] 10 14
    [26] His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X-Glu-Glu-Glu-
    [27] 20 30
    [28] Ala-Val-Y-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-
    [29] 39 40
    [30] Ala-Pro-Pro-Pro-Ser-Z
    [31] Wherein X is Arg, Leu, Ile or Met; Y is His, Arg or Lys; Z is Arg-OH, -OH, -NH 2 or Lys-OH; ego,
    [32] Z cannot be NH 2 when X is Met and Y is Arg.
    [33] Insulin affinity peptide derivatives of the invention have the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 4 (<210> 1 to <210> 4).
    [34] Insulin affinity peptide derivatives of the present invention are positive compounds that are sufficiently acidic or sufficiently basic to react with any of a number of inorganic bases, inorganic acids and organic acids to form salts. Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, Organic acids such as citric acid, benzoic acid and acetic acid. Examples of such salts are sulfates, pyrosulfates, bisulfates, sulfites, bisulfates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromide, iodides, acetates, Propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suverate, sebacate, fumarate , Maleate, butyne-1,4-dioate, hexyn-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, Sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lac Tate, gamma-hydroxybutyrate, glycolate, tartarate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. Preferred acid addition salts are salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid, in particular hydrochloric acid.
    [35] Alkali may also be used to react with the derivatives of the present invention to form salts. Representative examples of such alkalis include ammonium, alkali metals, alkali metal hydroxides, carbonates and bicarbonates. Typically such alkalis can be sodium hydroxide, potassium hydroxide, ammonium hydroxide and potassium carbonate.
    [36] The present invention also provides a method for producing an insulin- affinity peptide derivative by solid phase synthesis, which uses HMP resin as a solid phase carrier and converts the alpha-amine of the amino acid to 9-fluorenyl methoxycarbonyl (Fmoc). , And synthesizing the residue on a peptide synthesizer according to the amino acid sequence of the insulin affinity peptide derivative, and obtaining the product through separation, purification and lyophilization steps.
    [37] The present invention also provides a method for producing insulin affinity peptide derivative by recombinant technique, the method comprising:
    [38] a. Synthesizing gene fragments according to the amino acid sequence of the insulin affinity peptide derivative,
    [39] b. Obtaining cloned bacteria through ligation of the gene fragments, production of recombinant plasmids, culture of bacterial cells and transformation of the recombinant plasmids,
    [40] c. Extracting cell contents through fermentation of the bacterial strains and destruction of cell walls,
    [41] d. Lysing the cell contents, separating the crude product, purifying by HPLC, and lyophilizing to obtain the final product.
    [42] On the other hand, the present invention also relates to the use of said insulin affinity peptide derivative and pharmaceutically acceptable salts thereof in the treatment of type II diabetes.
    [43] The present invention provides novel derivatives of the peptide compound exendin-4, which broaden the range of exendin-4 derivatives. The derivatives of the present invention have a biological activity of promoting insulin secretion and lowering blood sugar levels. They can be used to treat type II diabetes. Such derivatives can be prepared by chemical synthesis, more readily by recombinant techniques, thereby reducing the production cost. In addition, the biological activity of such derivatives has been demonstrated through pharmacokinetic studies using non-obese diabetic (NOD) mice. When 0.1 μg of the derivative of the present invention is injected into the abdomen of the mouse, the insulin affinity peptide derivative secretes insulin. And in reducing blood sugar levels.
    [50] Detailed description of the invention
    [51] The following examples are illustrative and are not intended to limit the scope of the invention in any way.
    [52] Example 1
    [53] Preparation by solid phase synthesis of insulin affinity peptide derivatives wherein X is Arg, Y is Lys and Z is -OH
    [54] (1) amino acid monomer:
    [55]
    [56] In the above, Fmoc is 9-fluoroenyl methoxycarbonyl,
    [57] BOC is tert- butyloxycarbonyl,
    [58] Trt is trityl,
    [59] OtBu is tertiary butyl ester,
    [60] tBu stands for tert-butyl.
    [61] (2) apparatus and reagents
    [62] Device: Model 433A Peptide Synthesizer (Applied Biosystem, US)
    [63] Reagents: N-methyl ketopyrrolidine, methylene chloride, hexahydropyridine, methanol, dimethylaminopyridine / DMF, N, N-diisopropylethylamine / NMP, 0.5M HOBT in 100mmole HBTU / DMF, N, N- Dicyclohexylcarbodiimide / NMP
    [64] In the above,
    [65] DMF is N, N-dimethylformamide
    [66] NMP is N-methylpyrrolidone
    [67] HOBT is 1-hydroxybenzotriazole, and
    [68] HBTU stands for 2- (1H-benzotriazol-yl-1,1,3,3-tetramethyl-uronium hexafluorophosphate).
    [69] (3) procedure
    [70] a. synthesis
    [71] Taking the synthesis scale 0.25, for example, the synthesis process was as follows. 0.25 g of HMP resin was weighed and placed in the reactor vessel of the synthesizer. Various residues of 1 mmol, each of which was bound to a protecting group, were measured and arranged in the synthesizer according to the amino acid sequence of the insulin affinity peptide derivative from the carboxy terminus to the amino terminus. Removal of Fmoc protection, activation of residues and attachment of activated residues to HMP resin were performed automatically under the control of a computer program at room temperature of 25 ° C. Such reaction was repeated until the entire peptide was synthesized. After completion of the synthesis, the residue-attached resin with each residue bound to the side chain protecting group was weighed after air drying on a peptide synthesizer.
    [72] b. Remove protector and attach resin
    [73] The residue-adhesive resin having each residue of the insulin affinity peptide derivative bound to the protecting group was placed in a plugged Erlenmeyer flask and the cleavage reagents shown below were added.
    [74] reagentVolume water0.50ml Methyl phenate0.50ml phenol0.75 g Mercaptoethanol0.20ml Trifluoroacetic acid10.0ml
    [75] The electromagnetic stirring reaction was carried out for 6 hours at a constant temperature of 30 ° C. The aqueous filtrate was collected after the filtration step. The resin was washed with a small amount of trifluoroacetic acid. The collected aqueous filtrate and washing solution were then mixed together and precipitated by addition of ether. The mixture was filtered and the resulting precipitate was washed with a small amount of ether. After evaporation in the dehumidifier, the crude product was obtained.
    [76] c. Purification and Lyophilization by HPLC
    [77] Preparative HPLC was used to perform separation and purification of the crude product. The final product was obtained after the freeze drying step. The molecular weight of the product was determined using chromatogram-mass spectrogram joint analysis. The peptide derivative had a molecular weight of 4316.7 and a theoretical molecular weight of 4300.6.
    [78] Example 2
    [79] Preparation by Insulin Affinity Peptide Derivatives Where X is Leu, Y is Lys and Z is Arg-OH
    [80] A. Synthesis of Gene Fragments by Amino Acid Sequences of Insulin Affinity Peptide Derivatives
    [81]
    [82] B. Cloning
    [83] Ligation:
    [84] Take two tubes and put the fragment 1 and fragment 4 with OD 260 nm (optical density at 260 nm) = 0.1 into one tube, and the fragment 2 and fragment 3 with the same optical density into the other tube. Put in. Polynucleotide kinase buffer, polynucleotide kinase and ATP were then added to the two tubes, respectively. The reaction mixture was incubated at 37 ° C. for 60 minutes to produce the 5 ′ end of the phosphorylated gene fragment. The two tubes were then placed in a water bath at 95 ° C. and incubated for 10 minutes. The water bath was stopped and naturally cooled to room temperature in a warm state, during which an annealing reaction was carried out. T4 and T4 ligase buffers were added to each of these two tubes and the mixture was incubated overnight at 16 ° C. for ligation of gene fragments.
    [85] Plasmid
    [86] Promoter containing plasmids such as Lac, P L or Tac were placed in one tube and digested with restriction enzyme endonucleases EcoRI and HindIII. The digested plasmid was extracted with hydroxybenzene: chloroform solvent and collected by centrifugation. The aqueous phase was left behind and washed three times with chloroform solvent. Centrifugation was continued and the resulting aqueous phase was precipitated with isopropanol solvent, then centrifuged and air-dried.
    [87] The digested plasmid and the ligated fragments were mixed together. T4 ligase and ligase buffer were added to the mixture. Ligation reactions were carried out at room temperature for 3-4 hours.
    [88] Culture of Host Bacteria Cells:
    [89] The bacterial cells of E. coli JM103 were incubated with stirring at 37 ° C. for 4 hours in LB liquid medium (1000 ml of LB liquid medium containing 10 g of peptone, 5 g of yeast extract and 5 g of sodium chloride). The bacterial cultures were centrifuged and the harvested bacterial cells were treated with calcium chloride solution and kept at -4 ° C for further use.
    [90] Transformation
    [91] Cloned plasmids were transformed with this E. coli JM103 host cell. The transformed bacterial cells were incubated for 30 minutes in an ice bath. It was then incubated at 42 ° C. for 2 minutes. The bacterial cells were spread on agar plates containing ampicillin antibiotics and incubated overnight at 37 ° C. Colony screening was then performed and colonies containing the recombinant plasmids were made as positive colonies.
    [92] C. Fermentation:
    [93] Screened colonies with recombinant plasmids carrying the derivative genes were incubated with stirring in LB liquid medium. 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG) was added for protein induction purposes. The bacterial cells were incubated overnight and harvested by centrifugation. The expressed protein was confirmed by polyacrylamide gel electrophoresis (PAGE) containing 12% sodium dodecanesulfonate.
    [94] D. Cell Contents
    [95] Ten bottles, each containing 300 ml of bacterial culture, were incubated with stirring under the conditions mentioned above. After the protein induction step, lysate solution (20 mM phosphate buffer with 1% sodium chloride, pH 7.5) and lysozyme were added. The bacterial cultures were incubated at 30 ° C. for 30 minutes and centrifuged. The collected precipitate was treated with 6M guanidine hydrochloride (Gu.HCl) to extract cell contents. Centrifugation was continued to dialysis the resulting supernatant to remove guanidine hydrochloride. The dialysate was washed three times with 20 mM phosphate buffer (pH 7.5, containing 1% sodium chloride and 0.1% Tween 80), after which cell contents were obtained.
    [96] E. Lysis
    [97] Cell contents were lysed in 8M carbamide solution. Hydrochloric acid was added to make a concentration of 50 mM. After adding cyanogen bromide, the solution was stirred under conditions that block light and protect nitrogen. The lysis reaction was carried out for 2 hours and then analyzed by HPLC.
    [98] F. Tablet
    [99] After completion of the lysis reaction, the crude product was obtained by partition chromatography on Sephadex G-25 and the final product was obtained by HPLC purification. Similar to the solid phase synthesis results, mass spectrometry showed that the molecular weight of the peptide derivative was consistent with the theoretical value.
    [100] Example 3
    [101] Pharmacokinetic Research
    [102] The experiment was performed with non-obese diabetic mice weighing 17 ± 2 g and fasted for 2 hours. Mice of control group 1 were injected abdominally with 2 μg of insulin, mice of control group 2 were injected with 4 μg of insulin abdominally, and experimental groups were injected with 0.1 μg of the insulin-affinity peptide derivative obtained as described in Example 1. It was.
    [103] Blood samples were collected at different time points. Plasma glucose concentration was determined using the Plasma Glucose Testing Kit (Shanghai Institute of Biological Products Ministry of Health). The results are shown in the figure. The effect of reducing the insulin affinity peptide derivative on blood glucose is very clearly observed.
    [104] Example 4
    [105] Reduction Effect of Exendin 4 (Lys 20 , Arg 40 ) and Exendin 4 (Leu 14 , Lys 20 , Arg 40 ) on Blood Glucose in NOD Mice
    [106] Material and method:
    [107] NOD mice were provided through the Shanghai Laboratory Animal Center of Chinese Academy of Sciences. 0.9% sodium chloride solution, Exendin 4 (Lys 20 , Arg 40 ) and Exendin 4 (Leu 14 , Lys 20 , Arg 40 ) peptides were used for the analysis. Plasma glucose testing kit was purchased from Shanghai Institute of Biological Products Ministry of Health.
    [108] The overnight fasted NOD was divided into three groups. The first group of mice were injected abdominally with 200 μl solution containing 40% glucose and 1 μg exendin 4 (Lys 20 , Arg 40 ). The second group of mice was injected abdominally with 200 μl solution containing 40% glucose and 2 μg exendin 4 (Leu 14 , Lys 20 , Arg 40 ). On the other hand, mice in the third group, the control group, were injected with a glucose solution abdominally.
    [109] 30 μl of blood samples were immediately taken from retro-orbital zeinous sinus using graduated capillaries treated with alfl with heparin. The sample was placed in 300 μl of normal saline and mixed with saline. Red blood cells were removed by centrifugation at 3000 rpm and serum was obtained for glucose determination. Other blood samples were taken as described above at 30, 60 and 120 minutes, respectively. Then serum was separated. Glucose concentrations of the three plasma samples were measured by a testing kit according to the method described above and the effect of reducing GLP-1 (7-36) on blood glucose concentrations was detected.
    [110] As shown in FIG. 1, after a significant increase, the blood glucose concentration of the control group gradually returned to normal level, whereas the glucose concentration of the experimental group showed no noticeable increase and remained normal at all times. The reason is that administration of exendin 4 derivatives stimulated insulin secretion and thus avoided significant fluctuations in blood glucose. Therefore, the reducing effect of exendin 4 (Lys 20 , Arg 40 ) and exendin 4 (Leu 14 , Lys 20 , Arg 40 ) on blood glucose is supported by the experimental results.
    [111] Example 5
    [112] Stimulating Effect of Exendin 4 (Lys 20 , Arg 40 ) on Insulin Secretion
    [113] Material and method:
    [114] NOD mice were provided through the Shanghai Laboratory Animal Center of Chinese Academy of Sciences. 40% glucose solution, 0.9% sodium chloride solution and exendin 4 (Lys 20 , Arg 40 ) were used in this experiment. Insulin radioimmunoassay kits were purchased from Shanghai Institute of Biological Products Ministry of Health.
    [115] NOD mice were divided into 2 groups. 50 μl of blood samples were taken from the venous plexus of the eye, using graduated capillaries that rinsed the inner wall with 1 mg / mL heparin and were previously air dried. Each group of mice was intraperitoneally injected with 200 μl of normal saline containing 5 μg exendin 4 (Lys 20 , Arg 40 ) and this time point was recorded as 0 min. Other blood samples were taken at 5, 10, 20 and 30 minutes respectively as described above. After sampling, each blood sample was immediately placed into a centrifuge tube containing 50 μl of normal saline and mixed with saline. Erythrocytes were removed by centrifugation at 3000 rpm. Insulin concentrations of the other samples were measured according to the method described by the radioimmunoassay kit and the stimulatory effect of exendin 4 (Lys 20 , Arg 40 ) on insulin secretion was detected.
    [116] As shown in FIG. 2, abdominal injection of exendin 4 (Lys 20 , Arg 40 ) can significantly stimulate insulin secretion.
    [117] Example 6
    [118] Stimulating Effect of Exendin 4 (Lys 20 , Arg 40 ) on C-peptide Secretion
    [119] Material and method:
    [120] Healthy C 57 / BL mice were provided through the Shanghai Laboratory Animal Center of Chinese Academy of Sciences. 40% glucose solution, 0.9% sodium chloride solution and exendin 4 (Lys 20 , Arg 40 ) were used in this experiment. Insulin radioimmunoassay kits and C-peptide radioimmunoassay kits were purchased from Shanghai Institute of Biological Products Ministry of Health.
    [121] Healthy C 57 / BL mice were divided into 2 groups. 50 μl of blood samples were taken from the venous plexus of the eye, using graduated capillaries that rinsed the inner wall with 1 mg / mL heparin and were previously air dried. Each group of mice was intraperitoneally injected with 200 μl of normal saline containing 5 μg exendin 4 (Lys 20 , Arg 40 ) and this time point was recorded as 0 min. Other blood samples were taken at 5, 10, 20 and 30 minutes respectively as described above. After sampling, each blood sample was immediately placed into a centrifuge tube containing 50 μl of normal saline and mixed with saline. Erythrocytes were removed by centrifugation at 3000 rpm. The C-peptide concentrations of the other samples were measured according to the method described by the radioimmunoassay kit and the stimulatory effect of exendin 4 (Lys 20 , Arg 40 ) on C-peptide secretion was detected.
    [122] As shown in FIG. 3, abdominal injection of exendin 4 (Lys 20 , Arg 40 ) can significantly stimulate the secretion of C-peptide.
    权利要求:
    Claims (4)
    [1" claim-type="Currently amended] Insulin affinity peptide derivative having the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4 (<210> 1-<210> 4), and a pharmaceutically acceptable salt thereof.
    [2" claim-type="Currently amended] A method for producing the insulin affinity peptide derivative according to claim 1 by solid phase synthesis,
    Using HMP resin as the solid carrier,
    Protecting the alpha-amine of the amino acid with 9-fluoroenyl methoxycarbonyl (Fmoc),
    Synthesizing the residues in a peptide synthesizer according to the amino acid sequence of the insulin affinity peptide derivative,
    A method for producing an insulin affinity peptide derivative, comprising obtaining the product through separation, purification and lyophilization steps.
    [3" claim-type="Currently amended] A method for producing the insulin affinity peptide derivative according to claim 1 by recombinant techniques,
    a. Synthesizing gene fragments according to the amino acid sequence of the insulin affinity peptide derivative;
    b. Obtaining cloned bacterial strains through ligation of the gene fragments, production of recombinant plasmids, culture of bacterial cells and transformation of the recombinant plasmids;
    c. Extracting the cell contents through fermentation of the bacterial strain and destruction of the cell wall; And
    d. A method for producing an insulin affinity peptide derivative, comprising the step of obtaining the final product through lysis of cell contents, separation of crude product, purification by HPLC, and lyophilization.
    [4" claim-type="Currently amended] Use of the insulin affinity peptide derivative according to claim 1 and a pharmaceutically acceptable salt thereof in the treatment of type II diabetes.
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    同族专利:
    公开号 | 公开日
    JP4287153B2|2009-07-01|
    JP2005502595A|2005-01-27|
    EP2223938A1|2010-09-01|
    BRPI0209685B1|2016-03-01|
    EP2223938B1|2013-07-10|
    CA2446394A1|2002-11-14|
    AU2002257497B2|2007-10-11|
    CA2446394C|2016-08-16|
    WO2002090388A1|2002-11-14|
    EP1386930A1|2004-02-04|
    US7329646B2|2008-02-12|
    KR100902208B1|2009-06-11|
    CN1363559A|2002-08-14|
    CN1162446C|2004-08-18|
    US20040142866A1|2004-07-22|
    EP1386930A4|2005-09-07|
    BR0209685A|2004-07-13|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-05-10|Priority to CN01112856.9
    2001-05-10|Priority to CNB011128569A
    2002-05-08|Application filed by 상하이 후아이 바이오 랩
    2002-05-08|Priority to PCT/CN2002/000316
    2003-12-11|Publication of KR20030094386A
    2009-06-11|Application granted
    2009-06-11|Publication of KR100902208B1
    优先权:
    申请号 | 申请日 | 专利标题
    CN01112856.9|2001-05-10|
    CNB011128569A|CN1162446C|2001-05-10|2001-05-10|Insulinotropic hormone secretion peptide derivative|
    PCT/CN2002/000316|WO2002090388A1|2001-05-10|2002-05-08|Derivatives of exendin|
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