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Application of gelatinase A inhibitory polypeptide modifier

An inhibitory, gelatinase technology, applied in drug combinations, peptide/protein components, medical preparations with inactive ingredients, etc., can solve the problems of easy degradation and unstable peptide drugs, and achieve high activity and good anti-tumor effect. Good effect and stability

Inactive Publication Date: 2013-06-05
NANJING SENMU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at the same time, there are also problems that peptide drugs are unstable and easy to degrade in vivo, and it is impossible for the drug to exert its unique biological activity in vivo

Method used

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  • Application of gelatinase A inhibitory polypeptide modifier
  • Application of gelatinase A inhibitory polypeptide modifier
  • Application of gelatinase A inhibitory polypeptide modifier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of single modified M205C4-PEG

[0031] Precisely weigh 1 mg and 15 mg of M205C4 and mPEG-SC dry powder with an analytical balance (the molar ratio is about 1:3), and dissolve them in 5 ml of PBS buffer (pH7.4). The working concentration of M205C4 is 100 uM at 4 °C The reaction was carried out for 3 hours, and the whole reaction was completed on a silent mixer.

[0032] Preparative liquid phase separation and purification M205C4-PEG: model: Waters 2545-2767-UV2489; column: SunfireTM C18 OBDTM preparative chromatography column (30*150 mm, 5 μm); mobile phase: methanol-water-trifluoroacetic acid (60: 40:0.1); Flow rate: 1.0ml / min; Column temperature: 25°C; Detector: Waters 2489 (dual wavelength, 220nm and 330nm);

[0033] The recovered solution was concentrated and distributed into 2ml EP tubes, and stored in liquid nitrogen overnight after mass spectrometry detection.

[0034] Preparation of M205C4-PEG lyophilized powder: The prepared sample stored overnight...

Embodiment 2

[0040] Matrix Metalloproteinase II Enzyme Activity Detection Experiment

[0041] Kit: MMP-2 Fluorimetric Drug Discovery Kit (Biomol)

[0042] Instrument: fluorescent microplate reader (Molecular Devices, Gemini EM)

[0043] The operation method refers to the kit instructions, and the results are as follows: figure 2 Shown is the result of the matrix metalloproteinase II enzyme activity detection experiment. From this, it can be concluded that the half maximal inhibitory concentration (IC50) of the modified product to matrix metalloproteinase II (MMP2) is 101.4uM, which is 4.1 times that of the unmodified polypeptide. That is, the biological activity of the modified product decreased to 24.3%.

Embodiment 3

[0045] Pharmacokinetic behavior of M205C4-PEG in animals

[0046] 1. Pharmacokinetic behavior of M205C4-PEG in ICR mice

[0047] (1) Collection of blood drug samples:

[0048] ICR mice: female, 6 weeks old, body weight 22-24g / mouse.

[0049] Grouping: A total of 60 rats were randomly divided into 10 groups (10 time points), 6 rats in each group.

[0050] Administration and blood sample collection: Inject into tail vein, 20mg / kg / monkey (M205C4 / M205C4-PEG), take blood from eye canthus, place in dry 2ml EP tube anticoagulated with sodium heparin, 3000 r??min -1 Centrifuge for 5 minutes, take 200ul of serum from each mouse, use 600ul of acetonitrile to precipitate protein, centrifuge at 12000r? Phase 200ul was concentrated to freeze-dried powder for content determination.

[0051] Time points of blood collection: blank blood samples were collected before administration, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 16h, 24h after administration.

[0052] (2) Determination of blood drug...

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Abstract

The invention discloses an application of a gelatinase A inhibitory polypeptide modifier. The gelatinase A inhibitory peptide modifier is a single-point modification product of mPEG-sc to a polypeptide M205C4 histidine amino terminal, the molecular weight of the gelatinase A inhibitory polypeptide modifier is 11000-13000, and the gelatinase A inhibitory polypeptide modifier can be applied in the preparation of medicaments for treating tumors. The gelatinase A inhibitory polypeptide modifier can be used for overcoming the defects of poor stability and fast in vivo metabolism of an M205C4 active peptide, has the advantages of high activity, good stability, long half-life period and the like, and can play a good anti-tumor role in a pancreatic cancer animal model and provide important basis and guarantee for treatment application of the gelatinase A inhibitory polypeptide modifier in a human body.

Description

technical field [0001] The invention relates to a gelatinase A inhibitory polypeptide (M205C4), in particular to the application of a modification of the gelatinase A inhibitory polypeptide (M205C4). The modified gelatinase A inhibitory polypeptide is referred to as M205C4-PEG for short. Background technique [0002] Local invasion and distant metastasis are the main biological characteristics of malignant tumors, and they are also the main reasons for the high mortality of cancer patients. Current studies suggest that the destruction of extracellular matrix (ECM) is a key link in the process of tumor invasion and metastasis. Among the enzymes involved in destroying the extracellular matrix, matrix metalloproteinases (MMPs) are the most important proteolytic enzymes, especially MMP2 and MMP9 can not only enzymatically degrade intercellular matrix components but also It can enzymolyze type IV collagen, the main component of basement membrane. Therefore, it is particularly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/10A61K47/48A61P35/00A61K47/60
Inventor 韩晓高璐沙敏吴越孙玉洁朱云霞郑马庆
Owner NANJING SENMU BIOTECH CO LTD