Method for sustained-releasing polypeptide with biological activity and application thereof

A bioactive peptide, sustained release technology, applied in the field of biomedicine, can solve the problems of loss of activity, peptide or protein drugs cannot be released, etc., to achieve the effect of ensuring activity, strong pharmacokinetic properties, and prolonging half-life

Active Publication Date: 2008-10-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Peptide or protein drugs can enhance their stability in vivo and increase their half-life through various methods, such as covalent linkage with human serum albumin or human serum album

Method used

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  • Method for sustained-releasing polypeptide with biological activity and application thereof
  • Method for sustained-releasing polypeptide with biological activity and application thereof
  • Method for sustained-releasing polypeptide with biological activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0048] Example 1 Phage display method for screening human serum albumin-binding peptides

[0049] In this example, the Ph.D.-12 phage library of New England Biolabs was used to screen human serum albumin-binding peptides.

[0050] The Ph.D.-12 phage library of New England Biolabs is designed based on the M13 phage and contains a random combination of linear sequences of 12 peptides. There are at least 10 peptide species in this library 9 , and their titer is 10 12 pfu / ml.

[0051] Phage display library screening for human serum albumin-binding peptides underwent a total of four rounds of screening:

[0052] Human serum albumin (Sigma-Aldrich, St-Louis, USA) was dissolved in 0.1mol / L NaHCO 3 Buffer solution (pH 8.6) to prepare 100 μg / ml human serum albumin solution (pH 8.6).

[0053] First round of screening:

[0054] 1. Take 1.5ml of human serum albumin solution (100μg / ml, pH 8.6) and add it to a sterile polystyrene petri dish (60×15mm), then place the plate in a humid con...

Example Embodiment

[0068] Embodiment 2 Enzyme-linked immunosorbent assay (ELISA) method measures the affinity of screened phage and human serum albumin

[0069] The phage plaques respectively containing the 9 amino acid sequences shown in Table 1 obtained in Example 1 were subjected to monoclonal screening and pure culture respectively to obtain phage monoclonals containing the above-mentioned 9 peptide chains respectively, which were used in the test of this embodiment .

[0070] With 0.1mol / L NaHCO 3 Dilute the human serum albumin solution to 100 μg / ml, cover a row of ELISA multiwell plate wells with the solution, add 200 μL of the solution to each well, and place it in a sealed humid environment at 4°C overnight. Another multi-well plate was used for serial dilution of phage. Both plates were treated with 1% casein solution (dissolved in 0.1mol / L NaHCO 3 )cover. Each group of phage monoclonals was serially diluted four-fold, so that the first well contained about 10 12 phage particles, t...

Example Embodiment

[0078] Example 3 Preparation of anti-type 2 diabetes ABP-LK-GLP fusion peptide using recombinant DNA technology

[0079] In this example, human glucagon-like peptide 1 (GLP-1) is taken as an example to design a new therapeutic peptide for treating type 2 diabetes.

[0080] About 90% of diabetic patients suffer from type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM). People with type 2 diabetes are usually able to produce insulin, but the insulin produced cannot be used effectively by the cells of the body. This is mainly because the insulin produced in response to elevated levels of blood sugar is not sufficient for the cells to absorb glucose effectively, thereby failing to achieve the purpose of lowering blood sugar levels.

[0081] Since human glucagon-like peptide 1 was discovered in 1984, it has been proven clinically effective in lowering blood sugar concentrations in all stages of diabetes. More importantly, little risk of hypoglycemia was ...

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Abstract

The invention discloses a method for the slow release of a bioactive polypeptide and the application thereof. The method comprises the steps that tie molecules sensitive to a plasma environment are used for connecting the bioactive polypeptide with a seralbumin binding polypeptide to form a fusion polypeptide and transfer the fusion polypeptide into a host; the plasma proteinase of the host or the subalkaline pH catalytic pyrolysis tie molecules of the blood release the bioactive polypeptide contained therein, thus serving the purpose of prolonging the cyclic half-life of the bioactive polypeptide inside the human body. The method for the slow release of the bioactive polypeptide can be used for preparing polypeptide medicaments that resist human 2-type diabetes, human osteoporosis and cancer. The method for the slow release of the bioactive polypeptide not only prolongs the cyclic half-life of the bioactive polypeptide inside the human body, but also keeps the biological activity of the bioactive polypeptide. In addition, the adoption of the fusion polypeptide leads to stronger pharmacodynamic property than the individual adoption of the bioactive polypeptide.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method and application for slow-releasing biologically active polypeptides based on plasma protease or slightly alkaline pH of blood. Background technique [0002] Peptides have no specific three-dimensional structure in aqueous solution. Peptides with a molecular weight of less than 6 kilodaltons can exert unique biological effects by specifically binding to protein receptors or aptamers in cell signaling pathways. Therefore, before obtaining effective small-molecule drugs, for those diseases that urgently need to be treated, such as diabetes, cancer, AIDS, etc., peptide or polypeptide analogs can be used to target those off-track protein-protein signaling. It is an effective means of treating these diseases. However, peptide drugs are easily hydrolyzed by plasma proteases and cleared out of the systemic circulation, which shortens their half-life and limits their clini...

Claims

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Application Information

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IPC IPC(8): C12P21/06A61K38/26A61K38/23A61K38/17A61P3/10A61P19/10A61P35/00
Inventor 李弘剑苏正定周天鸿
Owner JINAN UNIVERSITY
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