PEG (polyethylene glycol) modified recombinant arginine deiminase (ADI) as well as preparation method and application thereof

A technology of arginine deiminase and ADI-SC-PEG20, which is applied in the field of medical bioengineering, can solve problems such as short half-life, achieve good in vitro stability, and improve plasma elimination half-life

Inactive Publication Date: 2014-07-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mainly aiming at the short half-life of arginine deiminase in vivo, a new arginine deiminase modifying enzyme ADI-PEG is provided

Method used

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  • PEG (polyethylene glycol) modified recombinant arginine deiminase (ADI) as well as preparation method and application thereof
  • PEG (polyethylene glycol) modified recombinant arginine deiminase (ADI) as well as preparation method and application thereof
  • PEG (polyethylene glycol) modified recombinant arginine deiminase (ADI) as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The purification of embodiment 1 ADI recombinant protein

[0028](1) Preparation of recombinant ADI crude enzyme solution: take the Escherichia coli fermentation broth expressing recombinant arginine deiminase and centrifuge to collect the bacteria, wash with 10-500mmol / L, pH 6-8 sodium phosphate buffer for 2 Second, according to the wet cell weight: buffer weight ratio of 1: 2-20, add buffer to resuspend, carry out ultrasonic crushing under ice bath conditions for 15 minutes, centrifuge the crushed liquid, and the obtained supernatant is the recombinant ADI crude enzyme liquid ; The ultrasonic crushing process is 400W, ultrasonic for 1s, stop for 3s;

[0029] (2) Purification of recombinant ADI:

[0030] Solution A: 20 mmol / L, pH 7.0 sodium phosphate buffer;

[0031] Solution B: 20 ​​mmol / L, pH 7.0 sodium phosphate buffer, containing 1 mol / L NaCl;

[0032] Solution C: 20 mmol / L, pH 7.0 sodium phosphate buffer, containing 0.15 mol / L NaCl.

[0033] a.HiTrap TM DEAE...

Embodiment 2

[0043] Embodiment 2 PEG modification reaction

[0044] Adjust the concentration of ADI pure enzyme solution to 0.5 mg / mL and pH 8.0 with 20 mmol / L PBS buffer. According to the molar ratio of 1:120, three PEG modifiers (mPEG-SS 20 kDa , mPEG-SC 20 kDa , mPEG-SPA 20 kDa ), and stirred at room temperature for 2 h. SDS-PAGE analysis of modified products see figure 1 .

[0045] The modified product was tested for enzyme activity, modification rate, and molecular weight (SDS-PAGE). The results are shown in Table 1.

[0046] Table 1 Enzyme activity and modification rate of different ADI-PEG

[0047]

Embodiment 3

[0048] Example 3 ADI-SPA-PEG 20 PD / PK studies

[0049] ADI-SPA-PEG 20 Determination of pharmacological parameters:

[0050] (1) Experimental group settings:

[0051] ADI: intravenous injection group, intramuscular injection group, subcutaneous injection group;

[0052] ADI-SPA-PEG 20 : intravenous injection group; intramuscular injection group; subcutaneous injection group.

[0053] Three mice in each group were used as parallel experiments, and three kinds of drug doses were injected, namely 5 U / mouse, 1 U / mouse, and 0.2 U / mouse. The sampling time was 1 hour after injection, and then sampling was performed at a fixed time point every day for 7 consecutive days.

[0054] Blood collection site: tail vein.

[0055] (2) Determination of Pharmacodynamics parameters

[0056] Serum sample treatment: Add 10% sulfosalicylic acid solution to the serum sample at a volume ratio of 1:1, stir and mix well, let stand at 4°C for 6 h, centrifuge at 10,000 r / min for 10 min, and take t...

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Abstract

The invention discloses a PEG (polyethylene glycol) modified recombinant arginine deiminase (ADI) as well as a preparation method and application thereof, belonging to the technical field of biomedical engineering. The stability of ADI-PEG obtained by modifying ADI with PEG in vitro and in mouse plasma is remarkably improved. A pharmacokinetics / pharmacodynamics study shows that compared with free ADI before modification, the half-life period of ADI-PEG in a mouse is prolonged by more than 11 times; the concentration of arginine in the plasma can be reduced and maintained for more than 5 days in the limiting level through single injection of 5U ADI-PEG and can be maintained for 1 day only through injection of unmodified ADI; an H22 liver cancer model mouse is treated for 15 days, and the cancer cell inhibition rate of ADI-PEG (15U) is 95.02% close to 98.34% (the inhibition rate) of a positive control group treated with 5-fluorouracil. PEG modified recombinant arginine deiminase is relatively long in in-vivo half-life period and relatively low in immunogenicity, and serves as an excellent tumor treating or inhibiting drug.

Description

technical field [0001] The invention relates to a PEG-modified recombinant arginine deiminase, including the PEGylation modification method of recombinant protein arginine deiminase (ADI) and the research on the in vivo and in vitro activities of the modified product, and also includes PEGylation The optimization of the modification method, the research and application of the in vivo and in vitro stability of the modified enzyme and the treatment of tumors belong to the technical field of medical bioengineering. Background technique [0002] Arginine deiminase (arginine deiminase, EC 3.5.3.6, referred to as ADI) is a potential new anticancer drug for the treatment of arginine-deficient tumors, including hepatoma, which currently lacks effective drugs (hepatocellular carcinomas, referred to as HCCs) and melanoma (melanomas). Arginine required by normal cells can be produced through the urea cycle in the body through the joint action of argininosuccinate synthetase (ASS) and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80A61P35/00
CPCC12N9/78C12Y305/03006
Inventor 倪晔张龙刘梦晗司海明
Owner JIANGNAN UNIV
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