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Urate oxidase modified by polyethylene glycol

A uric acid oxidase and polyethylene glycol technology, applied in the field of biomedicine, can solve problems such as unavailable long-term treatment, unresolved immunogenicity, and weakened efficacy of uricase

Pending Publication Date: 2020-11-10
CHONGQING PEG BIO BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In September 2010, the FDA approved the PEG-modified recombinant porcine uricase (Pegloticase) produced by Savient Company of the United States for the treatment of intractable gout, but because it did not solve the problem of immunogenicity, it has limited clinical application. 50% of patients are ineffective
However, the uricase derived from Aspergillus flavus is less than 40% homologous to the inferred human uricase (Lee C C, Wu X, Gibbs R A, Cook R G, Muzny D M, Caskey C T. Science. 1988.239: 1288-1291.) , the human body is prone to produce anti-uricase antibodies, the efficacy of Aspergillus flavus uricase rapidly weakens, and at the same time cause severe allergic reactions, so it cannot be used for long-term treatment

Method used

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  • Urate oxidase modified by polyethylene glycol
  • Urate oxidase modified by polyethylene glycol
  • Urate oxidase modified by polyethylene glycol

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preparation example Construction

[0109] In the preparation method of the polyethylene glycol-modified urate oxidase, various purification methods are used to obtain the high-purity polyethylene glycol-modified urate oxidase.

[0110] In another preferred embodiment, the purification of the modified sample includes but not limited to molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination. More preferred are molecular sieve chromatography and tangential flow ultrafiltration.

[0111] In another aspect of the present invention, the above polyethylene glycol-modified urate oxidase and its application are provided. The conjugate can achieve long-acting effect in vivo and significantly reduce the blood uric acid level, and can be used for the treatment of hyperuricemia and ventilation.

[0112] The polyethylene glycol urate oxidase is more suitable as a medicine for treating chronic hyperuricemia or ventilation and its composition....

Embodiment 1

[0131] The preparation of embodiment 1 recombinant uric acid oxidase

[0132] 1.1 Construction of genes and expression plasmids for uricase expression

[0133] According to the codon usage bias data of E.coli, combined with factors such as codon bias and GC content, the cDNA sequence of uricase protein (code name: PHC) (SEQ ID NO: 1) was designed, the whole gene was synthesized, and named as pUC-57-PHC plasmid. Nde I and BamH I were used as the insertion site of the target gene, and the pET-30a plasmid was used as the expression vector (pET-30a-PHC).

[0134] 1.2 Transformation of expression plasmids into bacterial host cells

[0135] Using CaCl 2 The expression vector pET-30a-PHC was introduced into Escherichia coli BL21(DE3) by the method, and Kanamycin was screened for resistance, and high-expression clones were screened out, and the original seed bank strain (E3B) was preserved. These steps are in accordance with the molecular biology field Common methods are implement...

Embodiment 2

[0140] Embodiment 2 Preparation of pegylated oxidative uricase

[0141] Dissolve 5KD molecular weight N-succinimide propionate PEG (5K-PEG-SPA) with 2mmol / L HCl into a 200mmol / L PEG solution. Enzyme: 5K-PEG-SPA), wherein the molar ratio of urate oxidase to 5K-PEG-SPA is calculated according to the monomer form, that is, the molar ratio of 1:55 to 1:95 refers to the monomer form of urate oxidase and 5K -The molar ratio of PEG-SPA is added to the carbonate buffer solution with a carbonate concentration of 0.1-0.3 mol / L and a pH of 10.0 dissolved in urate oxidase, so that the coupling reaction between PEG and urate oxidase occurs, and the coupling reaction Stir the reaction at 5-30°C for more than 60 minutes until the degree of PEG coupling no longer changes with time. After the reaction is complete, unmodified PEG and by-products are removed from the reaction by ultrafiltration and / or chromatography. A suitable molecular sieve chromatography medium can be selected to separate ...

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Abstract

The invention provides urate oxidase modified by polyethylene glycol. At least 11 of the following amino acid sites in the urate oxidase have PEG modifications: T1, K3, K4, K30, K35, K76, K79, K97, K112, K116, K120, K152, K179, K222, K231, K266, K272, K285, K291 and K293. Compared with similar drugs appearing on the market, at least 11 sites of the amino acid sites in the polyethylene glycol modified urate oxidase have PEG modifications, so that the in-vivo stability of the urate oxidase can be greatly improved and the immunogenicity is reduced under the premise of ensuring the enzyme activityto the maximum extent. The in-vivo drug effect after intramuscular injection can reach the equivalent in-vivo drug effect after intravenous injection of commercially available drugs.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to polyethylene glycol-modified urate oxidase, more specifically, the present invention relates to polyethylene glycol-modified urate oxidase, drug combination, polyethylene glycol-modified uric acid Pharmaceutical uses of oxidase and methods of reducing the immunogenicity of urate oxidase. Background technique [0002] Gout is a disease caused by purine metabolism disorder, its clinical feature is hyperuricemia, and tophi is formed due to the deposition of urate in the subcutaneous, joints and kidneys. Purine in the human body undergoes a series of changes, and the final product is uric acid. When the blood uric acid concentration exceeds 70mg / L, hyperuricemia can be caused. Among them, 5% to 12% of patients with hyperuricemia can develop gout. In blood or bursa fluid, when the concentration of sodium urate reaches saturation, microcrystals of sodi...

Claims

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Application Information

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IPC IPC(8): C12N9/06A61K38/44A61P19/06A61P13/12A61P19/02A61P29/00A61P9/12A61P3/10A61P3/06A61P3/00A61P9/10A61P35/00
CPCC12N9/0048A61P19/06A61P13/12A61P19/02A61P29/00A61P9/12A61P3/10A61P3/06A61P3/00A61P9/10A61P35/00C12Y107/03003A61K38/00A61K38/44A61K47/60A61P13/00A61P13/02
Inventor 范开王志明刘日勇王彧何云凤闫天文付志成苏国威胡春兰丁旭朋谭长城王宏英杨辉丁琼文海燕
Owner CHONGQING PEG BIO BIOTECH CO LTD
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