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目的制备一种新型支链(Y型)聚乙二醇(Y-shape polyethylene glycol,PEG)修饰的尿酸氧化酶,并对该修饰酶的体外酶活性和稳定性,在大鼠体内药效学、药动学和免疫原性进行评估,为其临床研究给药方案的设计和优化提供依据。方法运用凝胶电泳、高效液相色谱技术研究聚乙二醇修饰产物的理化特征,采用酶反应-紫外分光光度法对酶生物活性进行检测,结合ELISA方法分析修饰酶的免疫原性。以大鼠为模型,研究不同聚乙二醇修饰的尿酸氧化酶在体内的药动学。结果 Y型聚乙二醇尿酸氧化酶修饰物rUOX-mPEG2的表观相对分子质量比线性聚乙二醇修饰的产物rUOX-mPEG更大,聚乙二醇分子的修饰度更高,在相同温度和pH条件下的活性明显高于直链型聚乙二醇尿酸氧化酶修饰物。动物实验表明,该聚乙二醇-尿酸氧化酶修饰物在体内呈现良好的动力学特征,并在体内具有更长的药效维持时间。免疫原性实验结果也表明,Y型聚乙二醇修饰能有效降低免疫原性。结论 Y型聚乙二醇修饰尿酸氧化酶的酶学性质明显优于重组酶和直链型修饰的尿酸氧化酶,药效维持时间显著高于后者,免疫原性和国外品种采用的直链修饰尿酸氧化酶相当,且明显低于重组酶,在大鼠体内呈线性动力学特征。
Objective To prepare a novel uric acid oxidase modified by Y-shape polyethylene glycol (PEG), and to study the in vitro enzymatic activity and stability of the modified enzyme. In vivo pharmacodynamics , Pharmacokinetics and immunogenicity were evaluated to provide the basis for the design and optimization of its clinical research dosing regimen. Methods The physical and chemical characteristics of polyethylene glycol modified products were studied by gel electrophoresis and high performance liquid chromatography (HPLC). The enzyme activity - UV spectrophotometry was used to detect the bioactivity of the enzyme. The immunogenicity of the modified enzyme was analyzed by ELISA. Using rat as a model, pharmacokinetics of different uric acid oxidase modified by polyethylene glycol in vivo was studied. Results The apparent relative molecular mass of rUOX-mPEG2, a modification of Y-type polyethylene glycol uricase, was larger than that of rUOX-mPEG, a product of linear polyethylene glycol. The degree of modification of polyethylene glycol was higher, and at the same temperature And pH were significantly higher than the linear polyethylene glycol uricase oxidase modification. Animal experiments show that the PEG-UA oxidase modification exhibits good kinetic characteristics in vivo and has a longer duration of efficacy in vivo. The results of the immunogenicity experiments also showed that Y-type polyethylene glycol modification can effectively reduce the immunogenicity. Conclusion The Y-type polyethylene glycol modified uricase oxidase has better enzymatic properties than that of the recombinant enzyme and the linear modified uricase, and the pharmacodynamic maintenance time is significantly higher than that of the latter. The immunogenicity and the linearity Modified uricase was equivalent, and significantly lower than the recombinase in rats showed a linear kinetic characteristics.