新型Y型聚乙二醇-尿酸氧化酶修饰物的制备及免疫原性和生物活性评价

Construction and Assessment for Immunogenicity and Bioactivity of Novel Recombinant Urate Oxidase Modified with PEG

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 construct and evaluate the stability, and in vitro bioactivity for new Y-shape PEG-labeled recombi- nant mammalian urate oxidase rUOX-mPEG2, as well as pharmacokinetics and immunogenicity compared with the line-shape rUOX- mPEG in rats. METHODS Physicochemical characteristics and modification degree of PEG were detected by gel electrophoresis and HPLC,bio-activity by enzyme-spectrophotometric method, and immunogenicity though ELISA. Pharmacokinetic and tolerability proper- ty were assessed in vivo. RESULTS The novel Y-shape PEG modified urate oxidase rUOX-mPEG2 presented higher molecular weight, modification degree of PEG compared to line-shape rUOX-mPEG. And pharmacokinetic assay showed that rUOX-mPEG2 more excellent curative effects in vivo. 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-mPEG2 were 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 lineari- ty kinetic characteristic of rUOX-mPEG2, implying its potential excellent clinical supplication.