The Preparation And In Vitro/in Vivo Evaluation Of Uric Acid Oxidase Long-acting Complex

Objective:To construct and evaluate the stability, and in vitro bioactivity for new Y-shape PEG-labeled recombinant mammalian urate oxidase rUOX-mPEG2. Through pharmacodynamics, pharmacokinetics, and immunogenicity assessments of the modified enzymes in vivo and in vitro, providing experimental foundation and theoretical basis for its clinical research.Methods:(1)Through different pH, time, weight ratio of the PEG coupling effect of uric acid oxidase,and use the means such as ion exchange chromatography, ultrafiltration of modified product purification, preparation of PEG modification uric acid oxidase coupling;(2)Using gel electrophoresis, high performance liquid chromatography (HPLC) technique, TNBS method, the enzymatic branched chain of PEG modification product physical and chemical characteristics, modification, etc.(3)Using enzyme reaction-ultraviolet spectrophotometry to modify uricase biological activity and its stability test in vitro.(4)And in rats model, the different polyethylene glycol modified uric acid oxidase in vivo pharmacokinetics and pharmacodynamics.(5)By ELISA method to compare the immunogenicity of modified products.Results:(1)In phosphate buffer solution pH8.0, under the condition of room temperature (25℃), with the molar ratio of1:5to join Y type SPA-mPEG20000MW and uric acid oxidase (SPA-mPEG2) reaction30min, protein drugs can be modified and keep high activity.Adopted the Q Sepharose Fast Flow chromatography column and intercept molecular weight is50kd ultrafiltration membrane reactor for separation and purification, can get a purity of95%or more samples of PEG-UOX.(2)Y-shape PEG uric acid oxidase membranes apparent molecular weight than linear PEG modified product, PEG molecular modification degree of lower and higher activity.(3) At the same temperature, the pH activity stability under the condition of PEG uric acid oxidase membranes.it was significantly higher than linear type.(4) And pharmacokinetic assay showed that rUOX-mPEG2significantly prolong half-life and more excellent curative effects in vivo.(5)Plus, the results of experimental on immunogenicity indicated that the Y-shape PEG modified urate oxidase could reduce immunogenicity effectively.Conclusion:The bioavailability, efficacy and stability of rUOX-mPEG2were greater than those of line-shape rUOX-mPEG and recombinant mammalian control, bioavailability, efficacy, and tolerability were also superior to that of line-shape and PEG-free urate oxidase. The determination of oxidase pharmacokinetic parameters revealed the linearity kinetic characteristic of rUOX-mPEG2, implying its potential excellent clinical supplication.