重组尿酸氧化酶药代动力学与PEG化重组尿酸氧化酶免疫原性和生物活性研究
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摘要
目的：
研究重组尿酸氧化酶(rUOX)在动物体内的药代动力学(pharmacokinetics，PK)，为rUOX的药代动力学特点提供客观准确的评价，为其临床研究给药方案的设计和优化提供有力的理论依据。
研究聚乙二醇化尿酸氧化酶(PEG-rUOX)的免疫原性及体外生物活性，为其进一步的临床前研究提供可行性。
方法：
建立免疫放射方法(Immunometric Assay,IRMA)用于研究rUOX在猕猴体内的药代动力学。
采用ELISA方法研究PEG-rUOX的免疫原性。
采用紫外分光光度法研究PEG-rUOX的体外生物活性。
结果：
方法学确证表明本法测定血浆加入重组尿酸氧化酶的测量范围为1-100ng·mL~(-1)时，校正曲线线性良好，用四参数Logistic函数可获得稳定和重现性良好的校正曲线。按照制备校正曲线的方法制成10 ng·ml~(-1)浓度的重组尿酸氧化酶的样品，并使其中含有50％(V／V)的人血浆、比格犬血浆及SD大鼠血浆，结果表明测定的准确度和精密度均不受影响。在受试品浓度1-100 ng·mL~(-1)范围内，测定的日内重现性良好，日内精密度、日间精密度分别为3．88％-16．2％和8．75％-17．4％。在正常猕猴血浆内加入1、10、100 ng·mL~(-1)(低、中和高)三个浓度受试品重组尿酸氧化酶条件下，回收率分别为114．7％±15．9％、95．4％±3．7％和109．2％±10．3％。在保证上述精密度和准确度条件下，本法的定量下限(LOQ)为0．5 ng·mL~(-1)。方法学确证结果表明IRMA分析法满足rUOX动物药代动力学研究要求。
猕猴静脉注射0．15，0．6和2．4 mg·kg~(-1)rUOX后，血浆药物浓度随注射剂量的加大而增高，低中，中高或低高剂量组之间部分时间点的血浆药物浓度差别有统计学意义。实验结果表明AUC_(0-inf)在0．15，0．6和2．4 mg·kg~(-1)范围内随给药剂量的增加成正比例地增高(低、中、高三组给药剂量之比为1：4：16，AUC_(0-inf)之比为1：2．7：10．6)，具有剂量依赖性(R=0．99972，P<0．05)。曲线下面积AUC_(0-inf)分别为5742±2296，15531±5077和61125±22637 ng·h·mL~(-1)，消除半衰期T_(1/2)分别为2．1±0．4，1．9±0．2和2．2±0．2 h，清除率CL分别为30．0±14．7，41．5±13．3，42．5±13．1 mL·kg-1·h~(-1)，随着剂量的改变CL,T_(1/2)和V基本不发生改变，而且尿酸氧化酶进入到猕猴体内后具有较低的清除率及表观分布容积。从受试品rUOX与国外标准品FASTURREC浓度-时间曲线对比图中可以看出受试品rUOX与国外标准品FASTURREC在血浆中代谢趋势基本一致。t检验结果表明国外标准品FASTURREC与受试品的猕猴血浆药物浓度及代谢动力学参数无显著性差异(P>0．05)。连续给药第7天给药前后0～24 h内相应的11个时间点的血浆抗原浓度变化及代谢动力学参数同首次给药相比在统计学上无明显差异，积蓄因子为1．01，无药物蓄积。
大鼠静脉注射PEG-rUOX后体内产生的抗体水平明显低于静脉注射rUOX后体内产生的抗体水平，并且连续静脉注射rUOX后，大鼠体内抗体水平呈不断升高趋势，而连续静脉注射PEG-rUOX后，大鼠体内的抗体水平未见明显升高。
rUOX体外生物活性为0．20 IU／mg，PEG-rUOX体外生物活性为0．14 IU／mg，与国外Rasburicase的体外生物活性27．0IU／mg相比较低。
结论：
尿酸氧化酶在猕猴体内呈线性动力学特征。
重组人尿酸氧化酶经PEG化后免疫原性有所降低，与未PEG化的重组尿酸氧化酶比较，具有更优的临床应用价值。
尿酸氧化酶的体外生物活性较低，有待于进一步研究
讨论：
所谓放射免疫分析(IRMA)，就是生物化学中的免疫分析，加上放射性“标记”，创立的一种新的生物化学分析方法。新方法不仅保留了免疫反应的高度精密的专一性，而且又引入了放射性分析的高度灵敏性，为生物活性物质的分析带来一场革命。其特点是在一个复杂的混合体系生物样品中，不做任何处理和分离，直接检出特定物质极稀含量的准确值。其灵敏度可达10-9-10-12，这是以往任一种分析方法所不能达到的。IRMA通常用双抗体夹心法。实验中用的一对抗体分别针对细胞因子上的不同抗原决定簇，两个抗原决定簇相距一定的距离才能保证实验的高度敏感性，抗体可以用多克隆抗体，但一对单克隆抗体通常是最佳组合。采用微孔板进行的IRMA方法与其他经典方法比较具有可以同时处理大量样本，可自动化，耗费原料少，简单易于操作等优点。
方法学确证过程中，我们采取了一定的措施，比如用血浆稀释尿酸氧化酶制备校正曲线，把血浆样本稀释一定倍数等，来提高实验的精密度和准确度。方法学确证表明，这些改良方法可有效地降低血浆本底的干扰，提高了方法的精密度及准确度，IRMA检测尿酸氧化酶方法的特异性，灵敏度，精密度及准确度均满足药代动力学要求。
动物实验结果表明尿酸氧化酶在给药剂量范围内在猕猴体内呈线性动力学特征。并且尿酸氧化酶进入到猕猴体内后具有较低的清除率及表观分布容积，与D．Dussossoy等研究的结果相符。本研究所建立的用于检测尿酸氧化酶的IRMA法将来可能适用于尿酸氧化酶临床药代动力学研究，而且我们进行的rUOX猕猴体内药代动力学研究结果为其临床研究提供了宝贵的数据参考。我们将生物工程研究所提供的PEG化rUOX进行了其免疫原性研究，结果表明rUOX经PEG化后与未经PEG相比免疫原性大大降低，PEG化rUOX更适用于临床应用，具有很高的临床利用价值。生物活性实验结果得出PEG化的与未PEG化的尿酸氧化酶体外生物活性相对于国外Rasburicase都比较低。此结果并不能表明其在体内的生物活性也会比较低，毕竟在体外模拟体内环境并不十分准确。而且此结果也不能说明尿酸氧化酶经PEG化后生物活性降低，我们需要将未PEG化之前的尿酸氧化酶重新制备，然后在进一步考察其体外生物活性。
AIM:
To develop an immunoradiometric assay for the pharmacokinetic study of recombinant urate oxidase after intravenous administration in rhesus monkeys.
To study the pharmacokinetics property following single dosing and multiple dosing of rUOX via the route of intravenous administration in rhesus monkeys.
To study the immunogenicity and bioactivity of PEG-rUOX.
METHODS:
The plasma drug concentration was determined by immunoradiometric assay (IRMA). The pharmacokinetic parameters were calculated by noncompartment model.
An indirect ELISA method was utilized to determine the level of rUOX and PEG-rUOX antibody.
An UV to study the in vitro bioactivity of PEG-rUOX.
RESULTS:
The limit of quantification was found to be 0.5ng.ml~(-1) and the detection range was from 0.5 to 400 ng·ml~(-1). The intra- and inter-day precision (CV) of analysis were <15%, and accuracy were within±0.53%. The recovery of urate oxidase in different animal plasma were all >80%.
After intravenous administration of urate oxidase at dose of 0.15, 0.6, and 2.4mg·kg~(-1) and intravenous administration of Fasturtec at dose of 0.15 mg·kg~(-1), the plasma level of urate oxidase decreased with time and was down to the base at 24h after intravenous administration. The mean AUC_(0-t) values were 5741±2297, 15528±5076, 61099±22635 ng·h·mL~(-1), respectively. T_(1/2) values for urate oxidase at dose of 0.15, 0.6, 2.4 mg·kg~(-1) were 2.1±0.4h, 1.9±0.2h, 2.2±0.2h . The total body clearance (CL) were 30.0±14.7, 41.5±13.3, 42.5±13.1mL·kg~(-1)·h~(-1), and the volume of distribution (Vd) were 41.9±16.2, 81.0±34.3, 66.7±12.6mL·kg~(-1) at three doses respectively. The AUC increased with the increasing doses for intravenous administration, and had good linearity(r>0.99972, P<0.05). Whatever the dose, the CL,
T_(1/2) and V were roughly invariant. There was no significantly different between low dose of urate oxidase and low dose of Fasturtec on the trend of kinetics and on the plasma concentrations by T test. Moreover, there was no accumulation of urate oxidase in rhesus monkeys.
The levels of PEG-rUOX antibody were significantly lower than that of rUOX in SD rats.
The bioactivity of PEG-rUOX and rUOX were all lower than that of Rasburicase.
CONCLUSION:
The IRMA assay was sensitive, and specific for the pharmacokinetics study of urate oxidase and successfully to determine urate oxidase pharmacokinetic parameters after intravenous administration in rhesus monkeys. The kinetic characteristic of urate oxidase in rhesus monkeys was linearity kinetics.
The PEG- rUOX was more excellent than rUOX in clinical application.We have to carry further study of the bioactivity of PEG- rUOX.
DISCUSSION:
To evaluate urate oxidase pharmacokinetic parameters, a precise, sensitive, and specific method allowing the measurement of plasma concentrations of the enzyme was required. Here we describe the selection of monoclonal antibodies (m- Abs) directed against the recombinant urate oxidase to develop a sensitive and specific immunometric assay in plasma. The general advantages of such a IRMA performed in microtiter plates compared to other classical methods are the easy handling of large amount of samples at the same time, the possibility of automation, the need of less material, simple and convenient to operate.
For rhesus monkey, endogenous urate oxidase had no effect on pharmacokinetic experiment for absence of urate oxidase because of making some measures, such as diluting the plasma sample by blank plasma.
As a demonstration of the potential utility of the radiommunometric assay, pharmacokinetic studies of urate oxidase in rhesus monkeys were performed after intravenous administration. Whatever the dose, the CL, T~(1/2) and V were roughly invariant, demonstrating the linearity of the pharmacokinetics within the dose in rhesus monkeys. It can be seen that the small values of CL and V after urate oxidase was intravenously administered to thesus monkeys at the dose of 0.15, 0.6, and 2.4 mg·kg~(-1). These results suggested urate oxidase had a low clearance and a low volume of distribution, similar to Dussossoy D et al reported that the small values of CL and V after Fasturtec was intravenously administered to baboons at the dose of 0.15,0.45,1.5mg·kg~(-1). V was approximately equal to plasma volume by assessing. This suggests that the tissue distribution of urate oxidase is very restricted. It was important that the pharmacokinetic parameter of testing rUOX is similar to that of Fasturtec. These studies have shown the linear pharmacokinetics characteristic of urate oxidase and can provide reference for clinical administraton. The PEG- rUOX was more excellent than rUOX in clinical application by assessing the immunogenicity verus that of non-PEG-rUOX. Although the results showed the bioactivity of PEG- rUOX and rUOX, we could not deny the clinical application feasibility. We have to carry further study of the bioactivity of PEG- rUOX.
研究重组尿酸氧化酶(rUOX)在动物体内的药代动力学(pharmacokinetics，PK)，为rUOX的药代动力学特点提供客观准确的评价，为其临床研究给药方案的设计和优化提供有力的理论依据。
研究聚乙二醇化尿酸氧化酶(PEG-rUOX)的免疫原性及体外生物活性，为其进一步的临床前研究提供可行性。
方法：
建立免疫放射方法(Immunometric Assay,IRMA)用于研究rUOX在猕猴体内的药代动力学。
采用ELISA方法研究PEG-rUOX的免疫原性。
采用紫外分光光度法研究PEG-rUOX的体外生物活性。
结果：
方法学确证表明本法测定血浆加入重组尿酸氧化酶的测量范围为1-100ng·mL~(-1)时，校正曲线线性良好，用四参数Logistic函数可获得稳定和重现性良好的校正曲线。按照制备校正曲线的方法制成10 ng·ml~(-1)浓度的重组尿酸氧化酶的样品，并使其中含有50％(V／V)的人血浆、比格犬血浆及SD大鼠血浆，结果表明测定的准确度和精密度均不受影响。在受试品浓度1-100 ng·mL~(-1)范围内，测定的日内重现性良好，日内精密度、日间精密度分别为3．88％-16．2％和8．75％-17．4％。在正常猕猴血浆内加入1、10、100 ng·mL~(-1)(低、中和高)三个浓度受试品重组尿酸氧化酶条件下，回收率分别为114．7％±15．9％、95．4％±3．7％和109．2％±10．3％。在保证上述精密度和准确度条件下，本法的定量下限(LOQ)为0．5 ng·mL~(-1)。方法学确证结果表明IRMA分析法满足rUOX动物药代动力学研究要求。
猕猴静脉注射0．15，0．6和2．4 mg·kg~(-1)rUOX后，血浆药物浓度随注射剂量的加大而增高，低中，中高或低高剂量组之间部分时间点的血浆药物浓度差别有统计学意义。实验结果表明AUC_(0-inf)在0．15，0．6和2．4 mg·kg~(-1)范围内随给药剂量的增加成正比例地增高(低、中、高三组给药剂量之比为1：4：16，AUC_(0-inf)之比为1：2．7：10．6)，具有剂量依赖性(R=0．99972，P<0．05)。曲线下面积AUC_(0-inf)分别为5742±2296，15531±5077和61125±22637 ng·h·mL~(-1)，消除半衰期T_(1/2)分别为2．1±0．4，1．9±0．2和2．2±0．2 h，清除率CL分别为30．0±14．7，41．5±13．3，42．5±13．1 mL·kg-1·h~(-1)，随着剂量的改变CL,T_(1/2)和V基本不发生改变，而且尿酸氧化酶进入到猕猴体内后具有较低的清除率及表观分布容积。从受试品rUOX与国外标准品FASTURREC浓度-时间曲线对比图中可以看出受试品rUOX与国外标准品FASTURREC在血浆中代谢趋势基本一致。t检验结果表明国外标准品FASTURREC与受试品的猕猴血浆药物浓度及代谢动力学参数无显著性差异(P>0．05)。连续给药第7天给药前后0～24 h内相应的11个时间点的血浆抗原浓度变化及代谢动力学参数同首次给药相比在统计学上无明显差异，积蓄因子为1．01，无药物蓄积。
大鼠静脉注射PEG-rUOX后体内产生的抗体水平明显低于静脉注射rUOX后体内产生的抗体水平，并且连续静脉注射rUOX后，大鼠体内抗体水平呈不断升高趋势，而连续静脉注射PEG-rUOX后，大鼠体内的抗体水平未见明显升高。
rUOX体外生物活性为0．20 IU／mg，PEG-rUOX体外生物活性为0．14 IU／mg，与国外Rasburicase的体外生物活性27．0IU／mg相比较低。
结论：
尿酸氧化酶在猕猴体内呈线性动力学特征。
重组人尿酸氧化酶经PEG化后免疫原性有所降低，与未PEG化的重组尿酸氧化酶比较，具有更优的临床应用价值。
尿酸氧化酶的体外生物活性较低，有待于进一步研究
讨论：
所谓放射免疫分析(IRMA)，就是生物化学中的免疫分析，加上放射性“标记”，创立的一种新的生物化学分析方法。新方法不仅保留了免疫反应的高度精密的专一性，而且又引入了放射性分析的高度灵敏性，为生物活性物质的分析带来一场革命。其特点是在一个复杂的混合体系生物样品中，不做任何处理和分离，直接检出特定物质极稀含量的准确值。其灵敏度可达10-9-10-12，这是以往任一种分析方法所不能达到的。IRMA通常用双抗体夹心法。实验中用的一对抗体分别针对细胞因子上的不同抗原决定簇，两个抗原决定簇相距一定的距离才能保证实验的高度敏感性，抗体可以用多克隆抗体，但一对单克隆抗体通常是最佳组合。采用微孔板进行的IRMA方法与其他经典方法比较具有可以同时处理大量样本，可自动化，耗费原料少，简单易于操作等优点。
方法学确证过程中，我们采取了一定的措施，比如用血浆稀释尿酸氧化酶制备校正曲线，把血浆样本稀释一定倍数等，来提高实验的精密度和准确度。方法学确证表明，这些改良方法可有效地降低血浆本底的干扰，提高了方法的精密度及准确度，IRMA检测尿酸氧化酶方法的特异性，灵敏度，精密度及准确度均满足药代动力学要求。
动物实验结果表明尿酸氧化酶在给药剂量范围内在猕猴体内呈线性动力学特征。并且尿酸氧化酶进入到猕猴体内后具有较低的清除率及表观分布容积，与D．Dussossoy等研究的结果相符。本研究所建立的用于检测尿酸氧化酶的IRMA法将来可能适用于尿酸氧化酶临床药代动力学研究，而且我们进行的rUOX猕猴体内药代动力学研究结果为其临床研究提供了宝贵的数据参考。我们将生物工程研究所提供的PEG化rUOX进行了其免疫原性研究，结果表明rUOX经PEG化后与未经PEG相比免疫原性大大降低，PEG化rUOX更适用于临床应用，具有很高的临床利用价值。生物活性实验结果得出PEG化的与未PEG化的尿酸氧化酶体外生物活性相对于国外Rasburicase都比较低。此结果并不能表明其在体内的生物活性也会比较低，毕竟在体外模拟体内环境并不十分准确。而且此结果也不能说明尿酸氧化酶经PEG化后生物活性降低，我们需要将未PEG化之前的尿酸氧化酶重新制备，然后在进一步考察其体外生物活性。
AIM:
To develop an immunoradiometric assay for the pharmacokinetic study of recombinant urate oxidase after intravenous administration in rhesus monkeys.
To study the pharmacokinetics property following single dosing and multiple dosing of rUOX via the route of intravenous administration in rhesus monkeys.
To study the immunogenicity and bioactivity of PEG-rUOX.
METHODS:
The plasma drug concentration was determined by immunoradiometric assay (IRMA). The pharmacokinetic parameters were calculated by noncompartment model.
An indirect ELISA method was utilized to determine the level of rUOX and PEG-rUOX antibody.
An UV to study the in vitro bioactivity of PEG-rUOX.
RESULTS:
The limit of quantification was found to be 0.5ng.ml~(-1) and the detection range was from 0.5 to 400 ng·ml~(-1). The intra- and inter-day precision (CV) of analysis were <15%, and accuracy were within±0.53%. The recovery of urate oxidase in different animal plasma were all >80%.
After intravenous administration of urate oxidase at dose of 0.15, 0.6, and 2.4mg·kg~(-1) and intravenous administration of Fasturtec at dose of 0.15 mg·kg~(-1), the plasma level of urate oxidase decreased with time and was down to the base at 24h after intravenous administration. The mean AUC_(0-t) values were 5741±2297, 15528±5076, 61099±22635 ng·h·mL~(-1), respectively. T_(1/2) values for urate oxidase at dose of 0.15, 0.6, 2.4 mg·kg~(-1) were 2.1±0.4h, 1.9±0.2h, 2.2±0.2h . The total body clearance (CL) were 30.0±14.7, 41.5±13.3, 42.5±13.1mL·kg~(-1)·h~(-1), and the volume of distribution (Vd) were 41.9±16.2, 81.0±34.3, 66.7±12.6mL·kg~(-1) at three doses respectively. The AUC increased with the increasing doses for intravenous administration, and had good linearity(r>0.99972, P<0.05). Whatever the dose, the CL,
T_(1/2) and V were roughly invariant. There was no significantly different between low dose of urate oxidase and low dose of Fasturtec on the trend of kinetics and on the plasma concentrations by T test. Moreover, there was no accumulation of urate oxidase in rhesus monkeys.
The levels of PEG-rUOX antibody were significantly lower than that of rUOX in SD rats.
The bioactivity of PEG-rUOX and rUOX were all lower than that of Rasburicase.
CONCLUSION:
The IRMA assay was sensitive, and specific for the pharmacokinetics study of urate oxidase and successfully to determine urate oxidase pharmacokinetic parameters after intravenous administration in rhesus monkeys. The kinetic characteristic of urate oxidase in rhesus monkeys was linearity kinetics.
The PEG- rUOX was more excellent than rUOX in clinical application.We have to carry further study of the bioactivity of PEG- rUOX.
DISCUSSION:
To evaluate urate oxidase pharmacokinetic parameters, a precise, sensitive, and specific method allowing the measurement of plasma concentrations of the enzyme was required. Here we describe the selection of monoclonal antibodies (m- Abs) directed against the recombinant urate oxidase to develop a sensitive and specific immunometric assay in plasma. The general advantages of such a IRMA performed in microtiter plates compared to other classical methods are the easy handling of large amount of samples at the same time, the possibility of automation, the need of less material, simple and convenient to operate.
For rhesus monkey, endogenous urate oxidase had no effect on pharmacokinetic experiment for absence of urate oxidase because of making some measures, such as diluting the plasma sample by blank plasma.
As a demonstration of the potential utility of the radiommunometric assay, pharmacokinetic studies of urate oxidase in rhesus monkeys were performed after intravenous administration. Whatever the dose, the CL, T~(1/2) and V were roughly invariant, demonstrating the linearity of the pharmacokinetics within the dose in rhesus monkeys. It can be seen that the small values of CL and V after urate oxidase was intravenously administered to thesus monkeys at the dose of 0.15, 0.6, and 2.4 mg·kg~(-1). These results suggested urate oxidase had a low clearance and a low volume of distribution, similar to Dussossoy D et al reported that the small values of CL and V after Fasturtec was intravenously administered to baboons at the dose of 0.15,0.45,1.5mg·kg~(-1). V was approximately equal to plasma volume by assessing. This suggests that the tissue distribution of urate oxidase is very restricted. It was important that the pharmacokinetic parameter of testing rUOX is similar to that of Fasturtec. These studies have shown the linear pharmacokinetics characteristic of urate oxidase and can provide reference for clinical administraton. The PEG- rUOX was more excellent than rUOX in clinical application by assessing the immunogenicity verus that of non-PEG-rUOX. Although the results showed the bioactivity of PEG- rUOX and rUOX, we could not deny the clinical application feasibility. We have to carry further study of the bioactivity of PEG- rUOX.
引文
[1] M. Oda, Y. Satta, O. Takenaka, et al. Loss of urate oxidase activity in hominoids and its evolutionary implications. Mol. Biol. Evol, 2002, 19:640-653.
[2] A. V. Yekdandi, V. Yeldandi, S. Kumar, et al. Molecular evolution of the urate oxidase-encoding gene in hominoid primates:nonsense mutations. Gene,1991,109:281-284.
[3] P M Navolanic, CH Pui, R A Larson, et al. Elitek-rasburicase: an effective means to prevent and treat hyperuicemia associated with tumor lysis syndrome. Leukemia,2003,17(3): 499-514.
[4] Ames BN, Cathcart R, Schwiers E, et al. Proc.Nalt.Acad.Sci.USA. 1981,78:6858-6862.
[5] Proctor P. Similar function of uric acid and ascorbate in man? Nature.1970,228:868.
[6] Keilin. J.Biol.Rev.1959,34:265-296.
[7] Davidson MB, Thakkar S, Hix JK, et al. Pathophysiology, clinical consequences,and treatment of tumor lysis syndrome tumor lysis syndrome. Am J Med,2004,116(8):546-549.
[8]EK Hani, VL Chan EK. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli. J Bacteriol.1995,177(9): 2396-2402.
[9]Motajima K, Kanaga S,Goto S, et al. Nucleotide sequence of cDNA and predicted amino acid sequence of rat liver uricase. Eur J Biochem. 1988,173: 459-463.
[10] Koyama Y, Ichikawa T, Nakana E, et al. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem. 1996,120(5):969-973.
[11] Nguyen T, Zelechowska M, Foster V, et al. Primary structure of the soybean nodulin 235 gene encoding uricase II localized in the peroxisomes of uninfected cell of nodules. Proc Natl Sci USA. 1985, 82(15):5040-5044.
[12] Jianmin Li, Zhao Chen, Lihua Hou, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli.Protein Expression and Purification. 2006,49: 55-59.
[13] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of protein and polyethylene glycol: potent tools in biotechnological processes. Journal of Molecular Catalysis B: Enzymatic. 1996, 2: 1-17.
[1]汤仲明，刘秀文，宋海峰，朱宝珍．重组蛋白多肽药物的临床药代动力学．中国新药杂志．2002，11(10)：750-754．
[2] http://www. bio. hbnu. edu. cn/immuno/application/appli-4-2
[3] http://www. bjkp. gov.cn/lnkpwk/k30328-04
[4] D. Dussossoy, ML. Py, G. Pastor, et al. Development of a two-site immunoassayof recombinant urate oxidase(SR 29142) and its use for determination ofpharmacokinetic parameters in rats and baboons. Journal of Pharmaceutical Sciences.1996,85(9):955-959.
[5] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of proteinand polyethylene glycol: potent tools in biotechnological processes. Journal ofMolecular Catalysis B: Enzymatic. 1996,2: 1-17.
[1] M. Oda, Y. Satta, O. Takenaka, et al. Loss of urate oxidase activity in hominoidsand its evolutionary implications. Mol. Biol. Evol, 2002, 19:640-653.
[2] A. V. Yekdandi, V. Yeldandi, S. Kumar, et al. Molecular evolution of the urateoxidase-encoding gene in hominoid primates:nonsense mutations. Gene,1991,109:281-284.
[3] P M Navolanic, CH Pui, R A Larson, et al. Elitek-rasburicase: an effective meansto prevent and treat hyperuicemia associated with tumor lysis syndrome. Leukemia,2003,17(3): 499-514.
[4] Sutaria S, Katbamna R, Underwood, et al. Effectiveness of interventions for thetreatment of acute and prevention of recurrent gout-a systematic review.Rheumatology,2006,21.
[5] Davidson MB, Thakkar S, Hix JK, et al. Pathophysiology, clinical consequences,and treatment of tumor lysis syndrome tumor lysis syndrome. Am J Med,2004,116(8):546-549.
[6] Cohen LF, Balow JE, Macgrath IT, et al. Acute tumor lysis syndrome review of 37patients with Burkitt's lymphoma. Am J Med, 1980,68: 486-491.
[7] Cossin M, Piovanetti O, Stark WJ, et al. Urate deposition in the iria and anteriorchamber Ophthalmology. 2006,113(3): 462-465.
[8] Tsimberidou AM, Keating MJ. Hyperuricemic syndromes in cancer patients.Contrib Nephrol. 2005,147:47-60.
[9] Bessmertny O, Robitaille LM, Cairo MS. Rasburicase: a new approach forpreventing and/or treating tumor lysis syndrome. CurrPharm Des. 2005,11(32):4177-4185.
[10] http://www.fda.gov/cder/biologics/review
[11]吴伟立，李彦英，苗林等．尿酸氧化酶的表达纯化及活性形式鉴定．军事医学科学院院刊，2005，29(2)：124-126．
[12] Legoux R, Delpech B, Dumont X. Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. J Biol Chem. 1992,267(12): 8565-8570.
[13] Hongoh Y, Sasaki T, Ishikawa H. Cloning, sequence analysis and expression in Eschrichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens. Insect Biochem Mol Biol. 2000,30(2):173-182.
[14] Jianmin Li, Zhao Chen, Lihua Hou, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli.Protein Expression and Purification. 2006,49: 55-59.
[15] Kalckar HM. Differential spectrophotometry of purine compounds by means of s- pecific enzymes. J Biol Chem 1947;167:429.
[16] Priest DG, Pitts OM. Reaction intermediate effects on the spectrophotometric uri-case assay. Anal Biochem 1972;50:195 -205.
[17] Sanders GTB, Pasman AJ, Hoek FJ. Determination of uric acid with uricase and peroxidase. Clin Chim Acta 1980;101:299 -303.
[18] Majkic-Singh M, Stojanov M, Spasic S , Berkes I. Spectrophotometric determin-ation of serum uric acid by an enzymatic method with 2.2'-azino-di(3-ethylbenz-thiazoline -6-sulfonate). Clin Chim Acta 1981;116:117 -123.
[19] Ajay K, Bhargava, Harbans LAL, Chandra S. Discrete analysis of serum uric acid with immobilized uricase and peroxidase. J Biochem Biophys Methods 1999;39:125-136.
[20] Artiss JD, Willa ME. The application of a sensitive uricaseperoxidase coupled re-action to a centrifugal fast analyzer for the determination of uric acid. Clin Chim Acta 1981;116:301-309.
[21] Domagk GF, Schlicke HH. A Colorimetric method using uricase and peroxidase for the determination of uric acid. Anal Biochem 1968;22:219 -224.
[22] Fraisse L, Bonnet MC, Farcy JP, Agut C, Dersigny D, Bayol A. A colorimetric 96 -well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters. Analytical Biochemistry 2002;309: 173 -179.
[23] D. Dussossoy, ML. Py, G. Pastor, et al. Development of a two-site immunoassay of recombinant urate oxidase(SR 29142) and its use for determination of pharmacokinetic parameters in rats and baboons. Journal of Pharmaceutical Sciences.1996,85(9):955-959.
[24] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of protein and polyethylene glycol: potent tools in biotechnological processes.Journal of Molecular Catalysis B: Enzymatic. 1996, 2: 1-17.
[25] EK Hani, VL Chan EK. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli. J Bacteriol.1995,177(9): 2396-2402.
[26] Motajima K, Kanaga S,Goto S, et al. Nucleotide sequence of cDNA and predicted amino acid sequence of rat liver uricase. Eur J Biochem. 1988,173:459-463.
[27] Koyama Y, Ichikawa T, Nakana E, et al. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem. 1996,120(5):969-973.
[28] Nguyen T, Zelechowska M, Foster V, et al. Primary structure of the soybean nodulin 235 gene encoding uricase II localized in the peroxisomes of uninfected cell of nodules. Proc Natl Sci USA. 1985, 82(15):5040-5044.
[29] Ganson NJ, Kelly SJ, Scarlett E, et al. Control of hyperuricemia in subjects with fefractory gout, and induction of antibody against poly (ethylene) glycol (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase. Arthritis Res Ther.2005,8(1):R12.
[30]Wortmann, Robert L. Recent advances in the management of gout and hyperuricemia. Crystal deposition diseases Current Opinion in Rheumatology.2005,17(3):319-324.
[2] A. V. Yekdandi, V. Yeldandi, S. Kumar, et al. Molecular evolution of the urate oxidase-encoding gene in hominoid primates:nonsense mutations. Gene,1991,109:281-284.
[3] P M Navolanic, CH Pui, R A Larson, et al. Elitek-rasburicase: an effective means to prevent and treat hyperuicemia associated with tumor lysis syndrome. Leukemia,2003,17(3): 499-514.
[4] Ames BN, Cathcart R, Schwiers E, et al. Proc.Nalt.Acad.Sci.USA. 1981,78:6858-6862.
[5] Proctor P. Similar function of uric acid and ascorbate in man? Nature.1970,228:868.
[6] Keilin. J.Biol.Rev.1959,34:265-296.
[7] Davidson MB, Thakkar S, Hix JK, et al. Pathophysiology, clinical consequences,and treatment of tumor lysis syndrome tumor lysis syndrome. Am J Med,2004,116(8):546-549.
[8]EK Hani, VL Chan EK. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli. J Bacteriol.1995,177(9): 2396-2402.
[9]Motajima K, Kanaga S,Goto S, et al. Nucleotide sequence of cDNA and predicted amino acid sequence of rat liver uricase. Eur J Biochem. 1988,173: 459-463.
[10] Koyama Y, Ichikawa T, Nakana E, et al. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem. 1996,120(5):969-973.
[11] Nguyen T, Zelechowska M, Foster V, et al. Primary structure of the soybean nodulin 235 gene encoding uricase II localized in the peroxisomes of uninfected cell of nodules. Proc Natl Sci USA. 1985, 82(15):5040-5044.
[12] Jianmin Li, Zhao Chen, Lihua Hou, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli.Protein Expression and Purification. 2006,49: 55-59.
[13] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of protein and polyethylene glycol: potent tools in biotechnological processes. Journal of Molecular Catalysis B: Enzymatic. 1996, 2: 1-17.
[1]汤仲明，刘秀文，宋海峰，朱宝珍．重组蛋白多肽药物的临床药代动力学．中国新药杂志．2002，11(10)：750-754．
[2] http://www. bio. hbnu. edu. cn/immuno/application/appli-4-2
[3] http://www. bjkp. gov.cn/lnkpwk/k30328-04
[4] D. Dussossoy, ML. Py, G. Pastor, et al. Development of a two-site immunoassayof recombinant urate oxidase(SR 29142) and its use for determination ofpharmacokinetic parameters in rats and baboons. Journal of Pharmaceutical Sciences.1996,85(9):955-959.
[5] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of proteinand polyethylene glycol: potent tools in biotechnological processes. Journal ofMolecular Catalysis B: Enzymatic. 1996,2: 1-17.
[1] M. Oda, Y. Satta, O. Takenaka, et al. Loss of urate oxidase activity in hominoidsand its evolutionary implications. Mol. Biol. Evol, 2002, 19:640-653.
[2] A. V. Yekdandi, V. Yeldandi, S. Kumar, et al. Molecular evolution of the urateoxidase-encoding gene in hominoid primates:nonsense mutations. Gene,1991,109:281-284.
[3] P M Navolanic, CH Pui, R A Larson, et al. Elitek-rasburicase: an effective meansto prevent and treat hyperuicemia associated with tumor lysis syndrome. Leukemia,2003,17(3): 499-514.
[4] Sutaria S, Katbamna R, Underwood, et al. Effectiveness of interventions for thetreatment of acute and prevention of recurrent gout-a systematic review.Rheumatology,2006,21.
[5] Davidson MB, Thakkar S, Hix JK, et al. Pathophysiology, clinical consequences,and treatment of tumor lysis syndrome tumor lysis syndrome. Am J Med,2004,116(8):546-549.
[6] Cohen LF, Balow JE, Macgrath IT, et al. Acute tumor lysis syndrome review of 37patients with Burkitt's lymphoma. Am J Med, 1980,68: 486-491.
[7] Cossin M, Piovanetti O, Stark WJ, et al. Urate deposition in the iria and anteriorchamber Ophthalmology. 2006,113(3): 462-465.
[8] Tsimberidou AM, Keating MJ. Hyperuricemic syndromes in cancer patients.Contrib Nephrol. 2005,147:47-60.
[9] Bessmertny O, Robitaille LM, Cairo MS. Rasburicase: a new approach forpreventing and/or treating tumor lysis syndrome. CurrPharm Des. 2005,11(32):4177-4185.
[10] http://www.fda.gov/cder/biologics/review
[11]吴伟立，李彦英，苗林等．尿酸氧化酶的表达纯化及活性形式鉴定．军事医学科学院院刊，2005，29(2)：124-126．
[12] Legoux R, Delpech B, Dumont X. Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. J Biol Chem. 1992,267(12): 8565-8570.
[13] Hongoh Y, Sasaki T, Ishikawa H. Cloning, sequence analysis and expression in Eschrichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens. Insect Biochem Mol Biol. 2000,30(2):173-182.
[14] Jianmin Li, Zhao Chen, Lihua Hou, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli.Protein Expression and Purification. 2006,49: 55-59.
[15] Kalckar HM. Differential spectrophotometry of purine compounds by means of s- pecific enzymes. J Biol Chem 1947;167:429.
[16] Priest DG, Pitts OM. Reaction intermediate effects on the spectrophotometric uri-case assay. Anal Biochem 1972;50:195 -205.
[17] Sanders GTB, Pasman AJ, Hoek FJ. Determination of uric acid with uricase and peroxidase. Clin Chim Acta 1980;101:299 -303.
[18] Majkic-Singh M, Stojanov M, Spasic S , Berkes I. Spectrophotometric determin-ation of serum uric acid by an enzymatic method with 2.2'-azino-di(3-ethylbenz-thiazoline -6-sulfonate). Clin Chim Acta 1981;116:117 -123.
[19] Ajay K, Bhargava, Harbans LAL, Chandra S. Discrete analysis of serum uric acid with immobilized uricase and peroxidase. J Biochem Biophys Methods 1999;39:125-136.
[20] Artiss JD, Willa ME. The application of a sensitive uricaseperoxidase coupled re-action to a centrifugal fast analyzer for the determination of uric acid. Clin Chim Acta 1981;116:301-309.
[21] Domagk GF, Schlicke HH. A Colorimetric method using uricase and peroxidase for the determination of uric acid. Anal Biochem 1968;22:219 -224.
[22] Fraisse L, Bonnet MC, Farcy JP, Agut C, Dersigny D, Bayol A. A colorimetric 96 -well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters. Analytical Biochemistry 2002;309: 173 -179.
[23] D. Dussossoy, ML. Py, G. Pastor, et al. Development of a two-site immunoassay of recombinant urate oxidase(SR 29142) and its use for determination of pharmacokinetic parameters in rats and baboons. Journal of Pharmaceutical Sciences.1996,85(9):955-959.
[24] Ayako Matsushima, Yoh Kodera, Misao Hiroto, et al. Bioconjugates of protein and polyethylene glycol: potent tools in biotechnological processes.Journal of Molecular Catalysis B: Enzymatic. 1996, 2: 1-17.
[25] EK Hani, VL Chan EK. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli. J Bacteriol.1995,177(9): 2396-2402.
[26] Motajima K, Kanaga S,Goto S, et al. Nucleotide sequence of cDNA and predicted amino acid sequence of rat liver uricase. Eur J Biochem. 1988,173:459-463.
[27] Koyama Y, Ichikawa T, Nakana E, et al. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem. 1996,120(5):969-973.
[28] Nguyen T, Zelechowska M, Foster V, et al. Primary structure of the soybean nodulin 235 gene encoding uricase II localized in the peroxisomes of uninfected cell of nodules. Proc Natl Sci USA. 1985, 82(15):5040-5044.
[29] Ganson NJ, Kelly SJ, Scarlett E, et al. Control of hyperuricemia in subjects with fefractory gout, and induction of antibody against poly (ethylene) glycol (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase. Arthritis Res Ther.2005,8(1):R12.
[30]Wortmann, Robert L. Recent advances in the management of gout and hyperuricemia. Crystal deposition diseases Current Opinion in Rheumatology.2005,17(3):319-324.