聚乙二醇修饰干扰素α2b的稳定性研究

目的研究聚乙二醇修饰干扰素α 2b的稳定性。方法以抗病毒活性为指标 ,考察单修饰干扰素α 2b、多修饰干扰素α 2b和干扰素α 2b的稳定性 (4℃、2 5℃、37℃ )、抗胰蛋白酶水解能力和 37℃人血清稳定性。结果在4℃、2 5℃和 37℃条件下 ,稳定性依次为多修饰干扰素α 2b >单修饰干扰素α 2b >干扰素α 2b。抗胰蛋白酶水解试验表明 :干扰素α 2b在 30min时抗病毒活性迅速降到原来的 7.1% ,单修饰干扰素α 2b剩余 5 0 % ,多修饰干扰素α 2b仍保留 71%。体外稳定性试验表明 ,在 37℃人血清放置 10h ,干扰素α 2b只保留 35 % ,单修饰干扰素α 2b保留 71% ,而多修饰干扰素α 2b仍保留 95 %的抗病毒活性。结论聚乙二醇修饰可以增加干扰素α 2b的稳定性 ,抗胰蛋白酶水解能力和 37℃人血清稳定性。除了抗胰蛋白酶水解能力 ,修饰度的增加能够明显增强这种作用     

聚乙二醇化尿酸酶脂质体的稳定性研究

目的 制备聚乙二醇化尿酸酶脂质体(PULPs)并考察其活性与稳定性.方法 采用逆向蒸发法制备PULPs,并考察其最适温度、最适pH、热稳定性、贮存稳定性及其抗胰蛋白酶水解的能力、抗部分金属离子及有机化合物影响的能力.结果 PULPs的最适温度为40℃,最适pH为8.5;PULPs的热稳定性、贮存稳定性、抗胰蛋白酶水解能力、抗部分金属离子及有机化合物影响的能力均优于尿酸酶.结论 PULPs在增强聚乙二醇化尿酸酶活性的同时增强了稳定性.     

重组尿酸酶部分理化性质及其初步药效学研究

产朊假丝酵母尿酸酶由基因工程菌表达，文章对重组蛋白的亚基相对分子质量、最适作用温度、最适作用pH及其热稳定性和pH稳定性等理化性质进行了考察。成功构建了小鼠高尿酸血症动物模型，并用于重组尿酸酶的初步体内药效学研究。结果：经SDS-PAGE检测重组尿酸酶亚基的表观相对分子质量为32．4k。酶的最适作用温度和pH分别为40℃和8．0，且在pH6-12，温度20℃～60℃条件下比较稳定。初步体内药效学研究结果表明，重组尿酸酶可以显著降低模型小鼠体内的血尿酸水平。     

天门冬酰胺酶聚乙二醇-透明质酸/磺丁基-β-环糊精纳米粒体外稳定性的初步研究

目的 制备含载天门冬酰胺酶(Asp)的自组装聚乙二醇-透明质酸/磺丁基-β-环糊精纳米粒(THSCD),并初步研究两者在体外的活性以及稳定性.方法 采用自组装法制备THSCD;分别从最适温度、最适pH、酸碱稳定性、热稳定性、贮存稳定性、血浆稳定性、抗胰蛋白酶水解能力、部分金属离子及有机化合物影响来考察THSCD中Asp和游离Asp的差异.结果 THSCD的最适温度为50℃,游离Asp最适温度为60℃.THSCD的最适pH为6.5,游离Asp的最适pH为7.5.THSCD的热稳定性、贮存稳定性、酸碱稳定性、血浆稳定性测定、抗胰蛋白酶水解能力和抗部分金属离子和有机化合物能力均比游离Asp好.结论 THSCD在提高了Asp在体外活性的同时,也明显地增强了游离Asp在体外的稳定性.     

载门冬酰胺酶的自组装聚乙二醇-透明质酸/二甲基-β-环糊精纳米粒体外稳定性的初步考察

采用聚乙二醇接枝透明质酸和二甲基-β-环糊精制备了含载门冬酰胺酶(1)的自组装纳米粒(THDCD),并比较其与游离1在体外的活性及稳定性.测定了THDCD和1的最适温度、最适pH值,并对比了二者的酸碱稳定性、热稳定性、贮存稳定性、血浆稳定性、抗胰蛋白酶水解能力、抗部分金属离子及有机化合物影响的能力.结果表明,THDCD的最适温度和最适pH值为50℃和pH 6.5,而1为60℃和pH 7.5.THDCD的上述稳定性和抗性均优于1.本试验表明,THDCD在提高了1体外活性的同时,也显著增强了1的体外稳定性.     

化学修饰对组织型纤溶酶原激活剂的纤溶活性和体内半衰期的影响

用肝素、右旋糖酐和聚乙二醇对重组组织型纤溶酶原激活剂(rt-PA)进行化学修饰。不同的化学修饰物对rt-PA体外纤维蛋白溶解活性的影响不同。肝素修饰小PA的纤维蛋白溶解活性提高63％;右旋糖酐和聚乙二醇修饰rt-PA的纤维蛋白溶解活性则分别降低了36％和63％。rt-PA经化学修饰后半衰期均有所延长,其中聚乙二醇修饰的小PA的半衰期由修饰前的3．4min延长至6．3mim。     

右旋糖苷及PEG—2对大肠杆菌L—天冬酰胺酶的化学修饰

本文综述了活化右旋糖苷和含有两条聚乙二醇链的ＰＥＧ－２对大肠杆菌Ｌ－天冬酰胺酶的化学修饰。修饰酶可保持５０％酶活，并且明显提高了抗胰蛋白酶及胰凝乳蛋白酶水解的能力，降低对特异的天冬酰胺酶抗体的敏感性，体内实验还表明修饰酶在动物体内循环持久性延长，使之更适于作为治疗药物。     

聚乙二醇化蛋白质多肽类药物定点修饰策略

聚乙二醇修饰具有抵抗蛋白酶降解、提高稳定性、延长体内半衰期、降低免疫原性等优点,能够有效地改善蛋白质多肽类药物的临床药效。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产物的产率,已经引起了广泛关注。本文概述近年来聚乙二醇定点修饰蛋白质多肽类药物方面的研究进展,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

弹性蛋白酶的研究(二) 右旋糖酐对弹性蛋白酶的化学修饰及其某些性质的研究

本文用溴化氰活化右旋糖酐,对弹性蛋白酶进行共价修饰,以便改变其若干性质,使之更有利于应用。实验结果表明,修饰酶不仅完全保留了天然酶的活性和抗胰蛋白酶水解能力,而且修饰酶在耐热性、耐酸性和抗胃蛋白酶水解能力上都明显地优于天然酶。修饰酶较天然酶稳定,具有较高的应用价值。     

尿酸酶-过氧化氢酶脂质体的特性分析及药效学研究

目的考察尿酸酶-过氧化氢酶脂质体(uricase and catalase liposome,UCALP)、游离尿酸酶(uricase,UAE)的最适温度和最适pH,并探究UCALP降低高尿酸血症小鼠的尿酸水平和过氧化氢水平的能力。方法采用逆向蒸发法制备UCALP,并分别测定UCALP和UAE的最适温度和最适pH;小鼠腹腔注射尿酸建立高尿酸血症模型后,分别静脉注射UCALP和UAE,用测试盒分别测定不同时间点小鼠血清中尿酸浓度和过氧化氢的浓度。结果 UCALP和UAE的最适温度均为40℃,最适pH分别为8.0和8.5;在高尿酸血症的小鼠体内,UCALP比UAE降尿酸水平更明显,且UCALP组小鼠体内的过氧化氢浓度较UAE组低。结论UCALP能有效降低高尿酸血症小鼠的尿酸水平,且能在一定程度上降低过氧化氢的浓度。     

PEGylation enhancement of pH stability of uricase via inhibitive tetramer dissociation

Objectives Previously, PEGylated uricase was demonstrated to maintain catalytic activity at pH 5.8, the isoelectric point of uricase, where native uricase ceases to function. To find out whether PEGylation could enhance pH stability of uricase, the enzyme activity to pH curve was completely characterized. Methods Complete characterization of the enzyme activity to pH curve, indicating an inverted bell‐shaped relationship not previously documented, is presented. PEGylation enhancement of uricase stability at a pH lower than that commonly found in the liver, can be explored by dynamic dissociation of uricase using ultrafiltration and size‐exclusion chromatography. Key findings The results suggest the role of PEGylation in enhanced pH stability is via inhibition of subunit disintegration. The mechanism of this effect is characterized by the wrapping of PEG chains around uricase, providing a flexible shell preventing subunit disintegration. The presence of notable PEGylation‐induced changes in uricase supports this mechanism and include improved enzyme‐substrate affinity and elevated thermal stability. Conclusions Characterization of PEGylated uricase provides a basis for the rational design of therapeutic PEGylated proteins.     

Preparation and characterization of lipid vesicles containing uricase

The study described the development of lipid vesicles as colloidal carriers for uricase, an enzyme with low activity at physicological conditions and low stability in vitro and in vivo. The lipid vesicles containing uricase (UOXLVs) were prepared and the process parameters were optimized with the indexes of entrapment efficiency, polydispersion, particle size, and zeta potential. The storage stability of uricase in lipid vesicles was significantly increased compared to that of free uricase at 4°C. The stability to proteolytic digestion was also increased obviously by entrapping the uricase in the lipid vesicles. In vitro and in vivo pharmacodynamic studies on the hyperuricemia rat model explicitly suggested that the uricase entrapped by UOXLVs possessed high uricolytic activity and distinctively decreased the uric acid level.     

Preparation and characterization of lipid vesicles containing uricase

The study described the development of lipid vesicles as colloidal carriers for uricase, an enzyme with low activity at physicological conditions and low stability in vitro and in vivo. The lipid vesicles containing uricase (UOXLVs) were prepared and the process parameters were optimized with the indexes of entrapment efficiency, polydispersion, particle size, and zeta potential. The storage stability of uricase in lipid vesicles was significantly increased compared to that of free uricase at 4°C. The stability to proteolytic digestion was also increased obviously by entrapping the uricase in the lipid vesicles. In vitro and in vivo pharmacodynamic studies on the hyperuricemia rat model explicitly suggested that the uricase entrapped by UOXLVs possessed high uricolytic activity and distinctively decreased the uric acid level.     

Uricases as therapeutic agents to treat refractory gout: Current states and future directions

Treatment of refractory gout remains a challenge on drug development. While pegloticase, a recombinant mammalian uricase modified with monomethoxyl-poly(ethylene glycol) (mPEG) is effective in treating refractory gout, after continued treatment for three months biweekly at a therapeutic dose of 0.14 mg/kg body weight, it elicits an immune response against mPEG in nearly 20% of patients. For continued treatment of refractory gout PEGylated uricases at monthly therapeutic doses below 4 μg/kg body weight have promise. To formulate uricases to achieve monthly therapeutic regimens requires pharmacodynamics simulation and experimentation including: (a) molecular engineering of uricases based on rational design and evolution biotechnology in combination to improve their inherent catalytic efficiency, thermostability and selectivity for urate over xanthine and; (b) optimization of the number and distribution of accessible reactive amino acid residues in native uricases for site-specific PEGylation with PEG derivatives with lower of immunogenicity than mPEG to retain activity, minimize immunogenicity and enhance the pharmacokinetics of the PEGylated uricase. These issues are briefly reviewed as a means to stimulate the development of safer uricase formulations for continued treatment of refractory gout.     

Stability of PEGylated salmon calcitonin in nasal mucosa

The purpose of this study was to evaluate the stabilization of salmon calcitonin (sCT) by PEGylation in nasal mucosa. Degradation of native sCT in the homogenates of rat nasal mucosa was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The initial cleavage of sCT was due to tryptic-like endopeptidase activity, and the subsequent degradation followed the sequential pattern of aminopeptidase activity. To prepare PEGylated sCT resistant to the proteolytic degradation, the lysine residues susceptible to tryptic activity were selectively PEGylated by controlling reaction pH. The PEGylated sCT showed strong resistance against enzymatic degradation in rat nasal mucosa, with 56-fold prolonged half-life compared with that of native sCT. In the MALDI-TOF MS spectrum, the PEGylated sCT did not show any degradation peak for incubation of 120 min in the homogenates of rat nasal mucosa. The improved stability may be responsible for enhancing nasal absorption of PEGylated sCT.     

A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.     

重组猪尿酸氧化酶制剂对实验鸡血尿酸浓度的影响及抗原性研究

 目的：探讨纯化重组猪尿酸氧化酶制剂对实验鸡血尿酸浓度的影响及抗原性。方法：将实验鸡随机分为两组,每组10只。实验组注射重组猪尿酸氧化酶制剂,对照组注射生理盐水,共3次。注射时间分别是实验的d 1,d 7和d 35。注射前及注射后7,24,48,72 h采集血标本,用酶比色法测定实验鸡血清尿酸浓度,间接ELISA测定实验鸡抗重组猪尿酸氧化酶抗体。结果：第1次注射后7,24 h,实验组鸡的血尿酸浓度较注射前分别下降63.38%和28.34%,差异极显著（P＜0.01）。第2次注射后7 h尿酸浓度下降52.11%,差异极显著（P＜0.01）。第3次注射后血尿酸浓度无显著性下降。对照组注射前后血尿酸浓度比较无统计学差异。3次注射后7 h都能测到尿酸酶活性,酶活性范围1.83~12.75 IU?L-1。第1,2次注射后抗体测定未达到阳性,第3次注射后7 d抗体阳性数为6/10。结论：静脉注射经纯化处理的重组猪尿酸氧化酶能降低实验鸡血清尿酸浓度。该酶可刺激实验鸡产生特异性抗体,该抗体有干扰尿酸氧化酶分解实验鸡血尿酸的作用,但未引起明显过敏反应。     

重组尿酸氧化酶对动物高尿酸血症的治疗作用及安全性研究

目的探索重组尿酸氧化酶对动物高尿酸血症的治疗作用,并评价其安全性。方法通过给予乙胺丁醇和腺嘌岭或黄嘌呤,建立小鼠高尿酸血症模型,并考察尿酸氧化酶的降血清尿酸作用：选取猕猴进行3月长期毒性研究,使用酶联免疫吸附试验（ELISA）检测猕猴毒性试验给药结束及恢复期后抗体的产生情况。结果重组尿酸氧化酶可显著降低小鼠的尿酸水平;猕猴的长期毒性试验未发现与药物相关的明显毒性反应;猕猴体内检获相关抗体,恢复期后产生抗体的比例大大降低。结论重组尿酸氧化酶对动物高尿酸血症具有显著治疗作用,猕猴的安全剂量大于3．2mg／kg。     

Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin

PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity (K_M, V_max and K_cat) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl-l-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N--benzoyl-l-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 °C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability.