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Polypeptide complex, pharmaceutical composition, as well as preparation method and application of polypeptide complex

A polypeptide composition and composition technology, applied in the direction of drug combination, pharmaceutical formula, chemical instrument and method, etc., can solve the problems of short half-life of liraglutide, inconvenient clinical use, failure to meet clinical standards, etc., and achieve convenience in clinical practice The effect of promotion and application

Active Publication Date: 2014-07-23
TIANJIN INSTITUTE OF PHARMA RESEARCH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the serum half-life of GLP-1(7-37) is only 3-5 minutes, and multiple injections per day are very inconvenient in clinical use
[0003] At present, many studies have adopted GLP-1 analog fusion protein technology to solve the retention time of GLP-1 analog in vivo (CN90101167.3, CN200710018734.2, CN200410054397.9, CN01820232.2, CN200380110152.7, CN200510039265.3 , CN200610127237.1, CN200910009642.7), however, there is still a great distance between the existing technology and the ideal clinical goal, and generally have not reached the clinical standard. Recently, the liraglutide produced by Novo Norisk is a GLP-1 analog , which modified GLP-1 with palmitic acid, and was launched in the United States in 2009
However, liraglutide also has the problem of short half-life, and its dosage form still needs daily injections
[0004]Therefore, there is currently a need for methods that address the short in vivo half-life of GLP-1

Method used

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  • Polypeptide complex, pharmaceutical composition, as well as preparation method and application of polypeptide complex
  • Polypeptide complex, pharmaceutical composition, as well as preparation method and application of polypeptide complex
  • Polypeptide complex, pharmaceutical composition, as well as preparation method and application of polypeptide complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Preparation of carrier polypeptide A

[0056] According to the preferred codon (but not limited to the preferred codon) of Escherichia coli, the base sequence containing polypeptide A is synthesized by the method of total gene synthesis, and the DNA sequence is as follows:

[0057] SEQ ID NO 3:

[0058] 5 TATG ATTGAAGGCCGT GGCCTGTGGTGGAAAGCGTGGTGGAAAGCGTGGTGGAAATCCCTGTGGTGGCGTAAACGTAAACGTAAAGCGTAATAAG 3

[0059] SEQ ID NO 4:

[0060] 5 GATCCTTATTACGCTTTACGTTTACGTTTACGCCACCACAGGGATTTCCACCACGCTTTCCACCACGCTTTCCACCACAGGCC ACGGCCTTCAAT 3

[0061] To bind DNA sequence 3 and sequence 4, add equal molar amounts of SEQ ID NO3 and SEQ ID NO 4 respectively, incubate in a water bath at 95°C for 5 minutes, and then slowly cool down to room temperature. This double-stranded DNA can be connected with the Escherichia coli expression vector pET15b after being digested by NdeI / BamHI. As shown below, the double-stranded DNA synthesized by the above method conta...

Embodiment 2

[0065] Embodiment 2: Preparation of carrier polypeptide B

[0066] According to the preferred codon (but not limited to the preferred codon) of Escherichia coli, the DNA sequence of Polypeptide B synthesized by the method of total gene synthesis is as follows:

[0067] SEQ ID NO 5:

[0068] 5 TATG ATTGAAGGCCGT GGCCTGTGGTGGAAAGTGTGGTGGAAACTGTGGTGGAAAAGCCTGTGGTGGCGCAAACGCCTGCGCAAAGCGTAATAAG 3

[0069] SEQ ID NO 6:

[0070] 5 GATCCTTATTACGCTTTGCGCAGGCGTTTGCGCCACCACAGGCTTTTCCACCACAGTTTCCACCACACTTTTCCACCACAGGCC ACGGCCTTCAAT 3

[0071] To bond DNA sequence 5 and sequence 6, add equal molar amounts of SEQ ID NO3 and SEQ ID NO 4 respectively, place in a water bath at 95°C for 5 minutes, and then slowly cool to room temperature. This double-stranded DNA can be connected with the Escherichia coli expression vector pET15b after being digested by NdeI / BamHI. As shown below, the double-stranded DNA synthesized by the above method contains the sharp ends of 5'NdeI and 3'BamHI,...

Embodiment 3

[0074] Embodiment 3: Preparation and purification technology of recombinant GLP-1

[0075] GLP-1 is a natural product without patent restrictions. In the present invention, biological methods are used to prepare GLP-1, and this patent is also applicable to the use of other polypeptide preparation methods, such as polypeptide chemical synthesis.

[0076] SEQ ID NO 9: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0077] The coding DNA was synthesized and prepared according to the amino acid sequence of GLP-1 (SEQ ID NO 9). Two complementary DNA single strands were synthesized by Shanghai Sangong, see SEQ ID NO: 7, SEQ ID NO: 8. And referring to the description in Example 1, a double-stranded DNA encoding GLP-1 was prepared, and the double-stranded DNA had an enzyme cutting site (NdeI / BamHI) connected to the vector (pET15b). Referring to the content of Example 1, the expression plasmid of GLP-1 was constructed.

[0078] Recombinant GLP-1 was prepared according to the protein expression, ...

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PUM

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Abstract

The invention discloses a polypeptide complex, which comprises GLP-1 (glucagon-like peptide-1) and a carrier polypeptide, wherein the carrier polypeptide can effectively prolong blood half life of GLP-1, thus the existing condition that GLP-1 cannot be clinically used due to short half life is overcome, and GLP-1 has better application prospects in the field of medicaments for treating diabetes and obesity. The invention also discloses a preparation method and an application of the polypeptide complex. In addition, the invention further discloses a pharmaceutical composition comprising the polypeptide complex.

Description

technical field [0001] The present invention relates to the field of medicines related to diabetes, in particular, the present invention relates to a polypeptide complex, which has prolonged half-life of glucagon-like peptides in vivo. The invention also relates to the preparation method and application of the polypeptide complex. Background technique [0002] GLP-1 (glucagon-likepeptide-1, hereinafter referred to as: GLP-1) involved in the present invention is a polypeptide composed of 37 amino acids mainly secreted by small intestinal L cells, and its active form is GLP-1(7-37) OH and GLP-1(7-36)NH2 (Mojsov S, J Clin Invest. 1987 Feb;79(2):616-9). GLP-1 significantly reduces blood sugar after meals, can stimulate the production of insulin, and at the same time has a certain weight loss effect, and will not cause hypoglycemia (Drucker D J, Diabetes.1998Feb; 47(2): 159-69) . Recent studies have also shown that GLP-1 has a role in pancreatic regeneration (Drucker D J, 2003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/02A61K38/17A61P3/10A61P3/04
Inventor 龚珉任晓文王玉丽徐为人汤立达付刚郑学敏李心
Owner TIANJIN INSTITUTE OF PHARMA RESEARCH
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