尿酸氧化酶长效制剂的研制及体内外研究

发布时间：2018-05-31 20:35

  本文选题：重组尿酸氧化酶 + 聚乙二醇修饰　； 参考：《浙江大学》2014年硕士论文

【摘要】：目的：制备一种新型支链(Y型)聚乙二醇(polyethylene glycol, PEG)修饰的尿酸氧化酶,并通过大鼠体内药效学、药代动力学和免疫原性评估该修饰酶的体内外酶活性和稳定性,为其临床研究提供实验基础与理论依据。 方法：(1)通过不同pH、时间、投料比,考察对PEG耦合尿酸氧化酶的影响,采用离子交换色谱、超滤等手段对修饰产物进行分离纯化,制备PEG修饰尿酸氧化酶耦合物；(2)运用凝胶电泳、高效液相色谱技术、TNBS法、酶法研究支链PEG修饰产物的理化特征、修饰度等；(3)采用酶反应—紫外分光光度法对修饰的尿酸酶生物活性及其体外稳定性进行检测；(4)并以大鼠为模型,研究不同聚乙二醇修饰的尿酸氧化酶在体内的药代动力学和药效动力学；(5)通过ELISA方法分析比较修饰产物的免疫原性。 结果：(1)在pH8.0的磷酸盐缓冲溶液中,在室温(25℃)条件下,以1：5的摩尔比加入尿酸氧化酶和Y型SPA-mPEG20000MW (SPA-mPEG2)反应30min,可以得到修饰度和保留活性较高的PEG化蛋白药物；采用Q Sepharose Fast Flow层析柱和截留分子量为50kD的超滤膜堆进行分离纯化,可以得到纯度≥95%的PEG化尿酸氧化酶样品。(2)Y型PEG尿酸氧化酶修饰物rUOX-mPEG2的表观分子量比线性PEG修饰的产物rUOX-mPEG更大,PEG分子的修饰度更低而活性更高；(3)在相同温度、pH条件下的活性稳定性明显高于直链型PEG尿酸氧化酶修饰物；(4)Y型PEG尿酸氧化酶修饰物在体内半衰期显著延长,在体内具有更长的药效维持时间；(5)免疫原性实验结果表明,Y型PEG修饰能有效降低免疫原性。 结论：新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商业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。
[Abstract]:Aim: to prepare a novel branched poly (ethylene glycol) polyethylene-glycolene (peg) modified uric acid oxidase and evaluate its activity and stability in vitro and in vivo by pharmacodynamics, pharmacokinetics and immunogenicity in rats. To provide experimental and theoretical basis for its clinical research. Methods the effects of different pH, time and feed ratio on PEG coupled uric acid oxidase were investigated. The modified product was separated and purified by ion exchange chromatography and ultrafiltration. The coupling compound of PEG modified uric acid oxidase was prepared by gel electrophoresis. High performance liquid chromatography (HPLC) was used to study the physicochemical characteristics of branched PEG modified products and the degree of modification. The bioactivity and in vitro stability of modified uric acid enzymes were determined by enzyme reaction-UV spectrophotometry) and the rat model was used as a model. The pharmacokinetics and pharmacokinetics of uric acid oxidase modified by polyethylene glycol in vivo were studied. The immunogenicity of the modified product was analyzed by ELISA method. Results (1) in the phosphate buffer solution of pH8.0, at room temperature (25 鈩，

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