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mPEG-SC5K modified matrix metalloproteinase-9 inhibitor polypeptide P2 and application thereof

A matrix metal and protease technology, which is applied in the direction of protease inhibitors, peptide/protein components, peptide preparation methods, etc., can solve the problems of short half-life of polypeptide P2 and high cost of modification, and achieve the effect of prolonging the half-life

Inactive Publication Date: 2017-05-31
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the short half-life of existing matrix metalloproteinase-9 inhibitor polypeptide P2, the present invention provides an mPEG-SC 5K Modified matrix metalloproteinase-9 inhibitor polypeptide P2 and its application, mPEG-SC was carried out on polypeptide P2 5K modify
Meanwhile, in order to solve mPEG-SC 5K Costly modification problem for mPEG-SC 5K The reaction conditions of the modified polypeptide P2 were explored many times to screen out the reaction conditions with high yields of the modified products; and the mPEG-SC was detected 5K -The in vivo half-life of P2, as well as a variety of in vivo and in vitro drug efficacy tests on the modified product, proved that mPEG-SC 5K Modifying P2 can improve or enhance its biological activity, which can be better applied to the prevention and treatment of acute inflammation and autoimmune diseases

Method used

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  • mPEG-SC5K modified matrix metalloproteinase-9 inhibitor polypeptide P2 and application thereof
  • mPEG-SC5K modified matrix metalloproteinase-9 inhibitor polypeptide P2 and application thereof
  • mPEG-SC5K modified matrix metalloproteinase-9 inhibitor polypeptide P2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] mPEG-SC 5K Steps to modify P2

[0041] 1. PEGSC 5K Preliminary exploration of modification conditions

[0042] Weigh 32mg mPEG-SC respectively 5K And 10mg P2 (molar ratio is 1.5: 1) when the concentration is 0.05mol / L, 0.1mol / L, 0.15mol / L, 0.2mol / L, 0.25mol / L, prepare the Na of pH7.0 2 HPO 4 -NaH 2 PO 4 buffer, and reacted at 4°C for 3 hours.

[0043] 2. mPEG-SC 5K Optimization of modification conditions

[0044] Weigh 23mg mPEG-SC respectively 5K And 10mg P2 (molar ratio is 1.1:1), 27mg mPEG-SC 5K And 10mg P2 (molar ratio is 1.3:1), 32mg mPEG-SC 5K And 10mg P2 (molar ratio is 1.5:1), 36mg mPEG-SC 5K And 10mg P2 (molar ratio is 1.7:1), 42mg mPEG-SC 5K and 10 mg of P2 (molar ratio: 2.0:1) were placed in 40 mL of prepared PBS buffer solution with pH 8.0-8.5, and reacted at 4° C. for 3-12 h.

[0045] 3. Re-optimization of various PEG modification conditions

[0046] Weigh 128mg mPEG-SC respectively 5K and 40 mg of P2 (molar ratio: 1.5:1) were placed in 40 m...

Embodiment 2

[0063] mPEG-SC 5K Separation and purification steps of modified P2

[0064] 1. Separation

[0065] The sample after the reaction was purified by semi-preparative high-performance liquid phase (HPLC, Shimadzu), and the purification conditions were:

[0066] Semi-preparative chromatographic column: YMC, 250mm×10mm (5μm filler);

[0067] Mobile phase: Phase A is water, phase B is acetonitrile;

[0068] Sample volume: 1mL;

[0069] Flow rate: 2mL / min;

[0070] Detection wavelength: 220nm;

[0071] Elution gradient: see Table 3.

[0072] Table 3 Elution Gradient 2

[0073]

[0074] Collect the product in a centrifuge tube during the eluting process of the target peak.

[0075] 2. Purification

[0076] The product collected by semi-preparative HPLC was pre-frozen overnight in a -80°C low-temperature refrigerator after rotary evaporation, and then lyophilized in a pre-cooled freeze dryer until it was completely white powder by visual inspection (about 48h). Harvest the f...

Embodiment 3

[0090] Enzyme inhibition activity test

[0091] 1. Preparation of buffer solution: Accurately weigh 2.42g Tris, then accurately weigh 1.16g NaCl, add these two samples into 200mL double-distilled water and dissolve them completely, adjust the pH to 7.4 with HCl, and finally accurately Weigh 1.11gCaCl 2 , was added to the solution to dissolve it completely, and the solution was filtered through a 0.22 μm microporous membrane.

[0092] 2. Detection of mPEG-SC by cleavage of fluorescent substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) by matrix metalloproteinase-9 5K -P2 enzyme inhibitory activity, when MMP-9 is cleaved, the fluorescence value of the reaction system shows an increasing trend (set the excitation wavelength to 328nm and the emission wavelength to 392nm), the analysis process should be carried out in a 100μL reaction system at 37°C, This system includes MMP-9 solution, substrate solution, buffer and inhibitor). All the solutions in the reaction system should be ad...

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Abstract

The invention discloses an mPEG-SC5K modified matrix metalloproteinase-9 inhibitor polypeptide P2 and application thereof, and belongs to the technical field of polypeptide medicines. The polypeptide disclosed by the invention has a sequence of mPEG-SC5K-Pro-(D-Pyr)-(D-Cys)-Bip-Arg-Gly-Glu-Gly-Gly-Gly-Gly-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro. Due to mPEG-SC5K modification, the polypeptide has an effect of inhibiting the activity of matrix metalloproteinase-9, has pharmacokinetic properties which are greatly improved compared with those of P2, and can be used for treatment of acute inflammations and autoimmune diseases taking overexpression and over-activity of the matrix metalloproteinase-9 as characteristics. The mPEG-SC5K modified polypeptide P2 designed in the invention is prepared by a synthetic method. The mPEG-SC5K modified polypeptide P2 achieves the effects of increasing the survival rate of endotoxic shock mice in acute inflammation tests and improving the paralysis degree of model mice in multiple sclerosis animal tests, and has potential new drug development value.

Description

technical field [0001] The invention belongs to the field of polypeptide medicine, more specifically, relates to a mPEG-SC 5K Modified matrix metalloproteinase-9 inhibitor polypeptide P2 and applications thereof. Background technique [0002] Matrix metalloproteinases (MMPs) are a class of calcium- and zinc-dependent endopeptidases that mediate degradation of the extracellular matrix (ECM) and tissue remodeling, and regulate host defense responses and normal cellular functions. MMP inhibitors (MMPIs) can be used to prepare medicines for treating various acute inflammatory diseases and autoimmune diseases characterized by the overexpression and overactivation of matrix metalloproteinases. [0003] Endotoxic shock is a frequent cause of death in hospitals, and multiorgan failure is the result of an acute, exaggerated inflammatory response of the body to bacterial products such as endotoxins during the development of shock symptoms. During endotoxic shock, excessive activatio...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/113A61K38/55A61P37/02A61P29/00A61P25/00A61P31/04
CPCC07K14/8146A61K38/00
Inventor 胡加亮龚成欣徐寒梅
Owner CHINA PHARM UNIV
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