聚二醇修饰牛胰核糖核酸酶

采用N－羟基珀酰亚胺活化酯法活化单甲氧基聚乙二醇,测定了乙二醇（PEG）的活化度为86．2％。以活化的PEG对牛胰核糖核酸酶进行化学修饰;分析了蛋白质被修饰程度。用毛细管电泳法给出了被修饰蛋白的修饰度与修饰蛋白分布的定量结果。比较了被修饰产物对大分子底物（酵母RNA）与小分子底物（2‘,3‘－环磷酸胞嘧啶）的降解活力,其表观酶活力分别保留了52．8％和66．3％。结合毛细管电泳定量分析得到的修正酶活力略低于表观酶活力。     

几种因素在L-天冬酰胺酶化学修饰中对酶活力及抗原性的影响

为了寻求在较好地保持酶活力的同时解除L-天冬酰胺酶抗原性的方法,采用不同分子量的乙酸酐、右旋糖酐和单甲氧基聚乙二醇,作为修饰剂和不同的修饰方法对该酶进行了化学修饰。结果表明在保持酶活性和降低抗原性方面,大分子修饰剂右旋糖酐、单甲氧基聚乙二醇优于小分子乙酸酐,底物保护修饰优于直接修饰;活化PEG,优于活化PEG₁。在底物保护下的PEG,修饰酶其抗原性完全解除的同时,酶活力保持在30%以上。     

蚯蚓纤溶酶化学修饰的研究

以三聚氰氯为活化剂，采用活化聚乙二醇(PEG)对环毛蚯蚓纤溶酶进行化学修饰，优化了修饰条件，得到了PEG-EFE的加合物(修饰酶)，并研究了PEG-EFE的部分酶学性质。修饰酶的活力为天然酶的85％，结果表明酶的活性中心基本保持不变。对修饰酶的残留氨基测定表明，70％的可滴定氨基参加了反应，且EFE的活性改变较小，可以推测PEG是连接在蛋白质表面上的。     

清蛋白作为药物载体的PEG化修饰研究进展

清蛋白（白蛋白）是一种理想的药物载体,但由于其在体内半衰期短以及易被酶降解等缺点限制了其应用,然而根据其具有多个修饰位点的结构特点,可通过PEG修饰延长循环时间,阻碍酶的作用等。目前,PEG修饰清蛋白仍处于研究阶段,已有较多关于PEG修饰清蛋白的研究,例如PEG修饰所起的作用、对清蛋白及其制剂的影响,以及修饰位点的选择等。本文对清蛋白的PEG化修饰的相关研究进行综述。     

聚乙二醇对溶菌酶和粒细胞集落刺激因子的初步化学修饰

目的考察聚乙二醇 (PEG)修饰对溶菌酶和粒细胞集落刺激因子 (G CSF)活性的影响。方法以不同方法活化的PEG修饰溶菌酶 ,通过正交试验和单因素考察确定合适的修饰条件 ;溶菌酶活力测定采用溶壁小球菌法 ,抗原性测定采用试管沉淀法 ;G CSF活性测定以小鼠血浆中中性粒细胞数目增殖情况反映。结果当偶联上一个PEG分子时 ,溶菌酶的活性保留 13% ,抗原性显著减弱 ;G CSF经PEG修饰后对小鼠中性粒细胞数目的增加有显著促进作用 ,且作用时间明显延长。结论PEG修饰对G CSF血循环半衰期的延长有显著作用     

重组L-门冬酰胺酶的聚乙二醇化学修饰及修饰后酶的稳定性研究

目的单甲氧基聚乙二醇 (mPEG)化学修饰大肠杆菌重组L 门冬酰胺酶 (L ASP) ,考察经过修饰的酶的稳定性。方法N 羟基琥珀酰亚胺 (NHS)活化酯法活化mPEG ,生成的单甲氧基聚乙二醇琥珀酰琥珀酸亚胺酯 (SS mPEG)按不同摩尔比例与L ASP偶联 ,确定适合的反应时间和反应pH值。通过聚乙二醇化学修饰后的酶 (L ASP PEG) ,酶活力和纯度通过奈氏法和丙烯酰胺凝胶电泳 (SDS PAGE)检测 ,高效液相色谱检测L ASP PEG相对分子质量并考察了L ASP PEG体外稳定性等。结果SDS PAGE显示mPEG已经偶联到L ASP分子上 ,以两者摩尔比 1 0∶1为最佳 ,反应pH条件为 8.5 ,获得的L ASP PEG平均比活单位为 6 4 .8IU/mg ,相对分子质量为 30 1 80 0 ,体外稳定性高于L ASP。结论此实验确定了mPEG化学修饰L ASP最佳反应条件为 2 5℃反应 30min ,两者投料摩尔比为 1 0∶1 ,获得的L ASP PEG比L ASP稳定性高     

聚乙二醇修饰胰高血糖素样肽1类似物反应条件的响应面法优化#br#

目的 用响应面法对马来酰亚胺活化相对分子质量40 000的聚乙二醇（polyethylene glycol,PEG）（MAL-PEG_40K）修饰胰高血糖素样肽1类似物（glucagon-like peptide-1 analog,GLP-1a）的反应条件进行优化。方法 对GLP-1a浓度、GLP-1a/MAL-PEG40K摩尔比、反应液pH值、反应温度、反应时长进行单因素试验。以前3个条件为自变量, PEG修饰率为响应值,根据中心组合试验设计原理,研究各自变量及其交互作用对PEG修饰率的影响。通过高效液相色谱法分析PEG修饰率,依据回归分析确定各反应条件的最优值。 结果 GLP-1a与MAL-PEG_40K的最佳反应条件为：GLP-1a浓度2.5 mg/ml,GLP-1a/MAL-PEG_40K摩尔比1∶1.25,反应液pH 8,反应温度4 ℃,反应时长60 min。该优化条件下,GLP-1a的PEG修饰率可达91.0%。结论 响应面法获得了GLP-1a与MAL-PEG_40K的最佳反应条件。     

聚乙二醇修饰水蛭素的分离纯化与活性分析

用活化的聚乙二醇(PEG)对水蛭素进行化学修饰，质谱分析表明，修饰产物是修饰度不同的分子质量相差5000u的水蛭素的混合物。用离子交换层析和凝胶过滤层析对反应产物进行分离纯化，离子交换层析难以达到完全分离；而采用凝胶过滤层析方法可较好地分离修饰度不同的各组份。生物学活性分析表明，不同的PEG修饰度对水蛭素的活性具有显著影响，连接1～2个PEG的水蛭素保持了原有活性，而连接3个PEG的水蛭素活性显著下降。     

聚乙二醇前药设计原理与应用研究进展

聚乙二醇（polyethylene glycol,PEG）目前被广泛应用于肿瘤药物的修饰,当与药物分子偶联时,可以将其优良性质赋予修饰后的药物分子,改变药物的溶解性,在其修饰的药物周围产生空间屏障,减少药物的酶解,避免药物在肾脏的代谢中很快被消除,同时能被动靶向肿瘤细胞,降低药物毒性。聚乙二醇是中性、无毒且具有独特理化性质和良好生物相容性的高分子聚合物,也是经美国食品药物管理局（FDA）批准的极少数能作为体内注射给药的合成聚合物之一,已得到市场的认可。该文综述了近几年聚乙二醇修饰的前药研究进展,且就聚乙二醇修饰的原理、设计、运用及面临的挑战进行了论述。     

新型定点修饰的聚乙二醇化重组人生长激素修饰位点研究

目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C₄ Column, 150 mm×2.1 mm, 1.7μm, 300?),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40℃。质谱数据采集条件为MS~E模式,一级质谱能量4 eV,二级碎裂电压30～55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH₂-CH₂-N₃中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对...     

Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease.

Bovine pancreatic ribonuclease A (RNase) was modified at various extent at the lysine residues by monomethoxypoly(ethylene glycol) (MPEG) activated as active ester. For pharmacokinetic experiments a radioactive adduct was also prepared with tritiated amino acid as spacer between polymer and protein. The modification reduced only slightly the RNase catalytic activity and Km towards the substrate cytidine-2',3'-cyclic monophosphate. On the other hand extensively modified MPEG-RNase samples, showed significant decrease in activity towards ribonucleic acid. The polymer modification did not change the pH activity profile, increased the stability to proteolytic digestion, while the behaviour towards denaturants and heat was not modified. The native and MPEG-RNase administered IV, IM and SC to rats, showed impressive differences in pharmacokinetics: the half-life of the modified enzyme, evaluated in blood by radioactivity, was increased of 40-50 folds with respect to the native form.     

The application of capillary electrophoresis for monitoring effects of excipients on protein conformation

Studies were conducted to assess the utility of free solution capillary electrophoresis (CE) for monitoring the effects of selected excipients on the thermal denaturation of a model protein (Ribonuclease A, RNase A) at low pH. Thermal denaturation/unfolding experiments were conducted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3–3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effects of selected excipients on the thermal unfolding of RNase A were then evaluated by adding either sorbitol, sucrose, polyethylene glycol 400 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conducted under the same solution conditions using circular dichroism (CD) spectropolarimetry. Using temperature-controlled CE, an increase in solution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13°C, respectively. Similar shifts in transition temperatures were observed when thermal denaturation transitions were monitored by far-UV CD. Sorbitol (0.55–1.1 M) and sucrose (0.55 M) each shifted the denaturation transition temperatures of RNase A to higher values, whereas PEG 400 and MPD had minimal effect on the unfolding transition midpoint at the concentrations evaluated (0.55 M for each). The observed changes in the transition temperatures for RNase A as a function of pH and selected excipients were similar when measured by either CE or far-UV CD. These results support the utility of CE for monitoring the effects of neutral excipients on the thermal denaturation of a model protein under selected conditions. The widespread utility of the technique may be limited by the narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition.     

Assessment of Egyptian pharmacists' attitude, behaviors, and preferences related to continuing education

Background Contemporary pharmaceutical care requires sustained pharmacist competency through maintenance and improvement of knowledge, skills, and performance. Existing continuing education (CE) models reflect a wide spectrum of international approaches to life-long learning. Objective The objective of this study was to determine CE preferences of pharmacists in Egypt before implementing a plan for compulsory annual CE activities and events for licensure renewal. Setting A questionnaire containing questions about continuing education needs and preferences of Egyptian pharmacists was distributed to 400 pharmacies in Cairo. The sample was drawn randomly from the address list in yellow pages. The survey was conducted by personal interview. Method The questionnaire was designed and validated. Questions were divided into specific domains of interest including pharmacist demographics; access to internet resources; frequency and characteristics of past CE activities; preferences for delivery and content; motivation to participation; and plans for future CE activities. All data analyses were conducted using SPSS for Windows version 18.0. All statistical tests were 2-tailed and based on a significance level of p value????0.05. Results During the six months of questionnaire distribution, 400 pharmacists (one from each randomly selected pharmacies) were asked to complete the questionnaire. The response rate was 359 out of 400 pharmacists (89.75%). Twenty three percent of respondents had held their highest pharmacy degree to practice for less than 5?years and 19% had obtained their initial degree more than 15?years ago. More than half of the respondents were female (53.3%). Topics related to therapeutics were of highest interest to 85.3%, closely followed by clinical skills topics. Pharmacists working in community pharmacies had attended less CE events (15 vs. 28%, p?=?0.034 within the past 2?years) when compared to their hospital-based counterparts. Conversely, hospital pharmacists generally reported less satisfaction with current CE (21 vs. 33%, p?=?0.021). Conclusion Respondents of the survey expressed enthusiasm towards CE activity, but cited common barriers to participation, as well, such as employer-and technology-based obstacles. These results confirm that features of a successful CE program must be flexible to meet preferences and perceived needs of Egyptian pharmacists.     

Determining of actual activities of acid and alkaline ribonuclease in human serum and urine.

Acid and alkaline ribonuclease (RNase) activities were measured in serum and urine using procedure based on assumption that all determined RNase activities, both at pH 6.5 and 7.8 represent values produced by overlapping of activities of acid leukocyte type RNase and alkaline pancreatic type RNase. The procedure requires simultaneous determining of RNase activity at pH 6.5 and 7.8 and further calculation of actual activities of acid and alkaline RNase activities using the elaborated experimental formula. Results of determining acid and alkaline RNases in human sera yielded on information on specific contribution of leukocyte type and pancreatic type RNases to increased RNase activity in such clinical conditions as terminal renal failure, myocardial infarction and chronic myelogenous leukemia. It was also found that there is in human urine a remarkably increased proportion of acid RNase activity if compared to this in serum.     

大鼠肝和心脏微粒体羧酸酯酶的诱导与抑制

用对硝基苯醋酸酯（ｐ－ＮＰＡ）为底物，测定大鼠肝和心脏敌酸醋酶（ＣＥ）的水解活性，苯巴比妥和氟贝特对肝脏ＣＥ活力有明显的诱导作用，而地塞米松（Ｄｅｘ）对心脏ＣＥ活力显示有诱导作用，表明相同药物对肝和心脏ＣＥ活力诱导是有差异的：双－对硝基苯磷酸酯钠（ＢＮＰＰ），苯甲基磺酰氟（ＰＭＳＦ）和氟磷酸二异丙酯（ＤＦＰ）对肝和心脏ＥＣ活力均有明显的抑制作用，ＢＮＰＰ，ＰＭＳＦ和ＤＦＰ对肝及心脏ＣＥ活力的Ｉ５０分别为１００，２００，５μｍｏｌ·Ｌ－１和５０．０，５．０、０１ｍｍｏｌ·Ｌ－１；对肝和心脏ＣＥ水解ｐ－ＮＰＡ的Ｋｉ分别为１．１．１．２．０．６７ｍｍｏｌ·Ｌ－１和１．０，０．８．０．５４ｍｍｏｌ·Ｌ－１，结果表明三种抑制剂对肝和心脏ＣＥ的抑制强度存在明显差异．     

Probenecid and the rat kidney: investigations by renal enzyme excretion technique.

Probenecid in doses of 640 mg/kg was administered to rats by the oral route, and the changes in five important enzymatic activities of urine were recorded thereafter for two days. The resluts exclude that probenecid impairs tubular reabsorption of low molecular weight protein, as urinary muramidase activity was not found increased. On the other hand, increased activities were encountered in those enzymatic activities in urine which derive from the renal tubular cells (ALD, G-6-PDH, LDH). These observations point towards a nephrotoxic effect of probenecid, which, however, is only of very low degree, as other "standard" enzymatic activities of urine, such as alkaline phosphatase, remained unchanged.     

Attitudes and policies about continuing education among Wisconsin hospital pharmacy directors

Pharmacy directors in Wisconsin hospitals were surveyed to determine their attitudes toward continuing professional education and to assess the status of policies and procedures regarding continuing education (CE) and funding for CE activities. A two-page questionnaire was sent to all pharmacy directors in the state. A total of 151 questionnaires were delivered and 103 (68.2%) usable responses were returned. Written policies and procedures regarding CE were available in 47.6% of pharmacy departments. Most directors (84.5%) had formal mechanisms for documenting staff participation in CE activities but few (19.6%) reported having criteria for determining who would attend CE programs. Only 64% of directors used a formal system of budgeting for CE activities, although 88% provide financial support for CE activities outside the institution. The types of CE activities considered to be most desirable were programs sponsored by pharmacy organizations, programs sponsored by schools of pharmacy, and journal reading. Many directors (72.8%) believed that CE is necessary if pharmacists are to remain competent, but few (5.9%) believed that their budgets were adequate to meet the costs of all CE activities in which their pharmacists might be interested, and few expected their budgets to increase. Based on this survey, Wisconsin pharmacy directors in both small and large hospitals believe that CE is important and that the pharmacy department should support it.     

In Vivo prophylactic effects of oleanolic acid isolated from chloroform extract of Flaveria trinervia against ethanol induced liver toxicity in rats

The prophylactic effects of oleanolic acid (OA) isolated from chloroform extract (CE) of Flaveria trinervia against ethanol induced liver toxicity was investigated using rats. CE and OA at three different doses were tested by administering orally to the ethanol treated animals during the last week of the 7 weeks study. Silymarin was used as the standard reference. The substantially elevated serum enzymatic levels of serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase and bilirubin in ethanol treated animals were restored towards normalcy by treatment of CE and OA. In vivo antioxidant and in vitro free radical scavenging activities were also positive for all the three concentrations of CE and OA. However, OA at 150 mg/kg showed significant activity when compared to the other two doses. Biochemical observations in support with histopathological examinations revealed that CE and OA possess hepatoprotective action against ethanol induced hepatotoxicity in rats.     

The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro)

Organophosphates are known primarily as neurotoxins. However, reactive oxygen species (ROS) caused by organophosphates may be involved in the toxicity of various pesticides. Therefore, in this study we aimed to examine how an organophosphate insecticide, chlorpyrifos-ethyl (CE) [0,0-diethyl 0 (3,5,6-trichloro-2-pyridyl) phosphorothioate], affects lipid peroxidation and the antioxidant defense system in vitro. For this purpose, four experiments were carried out. In experiment 1, erythrocyte packets obtained from six (three male, three female) volunteers were divided into six portions, and to each was added CE in both a high concentration range (0, 0.4, 2, 10, 50, 100 g/l) and a low concentration range (0, 0.01, 0.1 g/l). Additionally, each concentration group was divided into five tubes, and incubated at +4 degrees C for 0, 30, 60, 120, and 240 min. After incubation, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were determined in the erythrocytes in all tubes. In experiment 2, to examine the effect of CE (or its main metabolites) on the activity of purified, commercially available enzymes, CE at concentrations of 0. 0.01, 0.1, 0.4, and 10 g/l was incubated with purified SOD, GSH-Px and CAT at the concentrations observed in control group at the 0 CE concentration level in experiment 1 for 1 h at room temperature (25 degrees C). In experiment 3, the xanthine-xanthine oxidase system was used to determine whether the activities of SOD, GSH-Px and CAT were inactivated other than by CE, for example by superoxide radicals inducing lipid peroxidation in erythrocytes. Samples with xanthine and xanthine oxidase were mixed and incubated for 1 h at room temperature (25 degrees C). In experiment 4, to determine whether enzyme activities were still inhibited if lipid peroxidation was prevented by exogenous antioxidants, experiment 1 was repeated with the CE concentrations of 0.01, 0.1, 0.4, and 10 g/l by adding butylated hydroxytoluene and vitamin E to the medium. The MDA levels were determined spectrophotometrically. Enzymatic methods were used for the determination of SOD, GSH-Px, and CAT activities. The Friedman test and Wilcoxon's Signed Ranks test were used to compare paired groups. MDA values and GSH-Px activities increased with increasing CE concentration and incubation period (P<0.05), but SOD and CAT activities decreased with increasing CE concentration and incubation period (P<0.01). From these results, it can be concluded that in vitro administration of CE resulted in the induction of erythrocyte lipid peroxidation and significant changes in antioxidant enzyme activities, suggesting that ROS and/or free radicals may be involved in the toxic effects of CE.     

Nature of aggregates formed during storage of freeze-dried ribonuclease A

Ribonuclease A (RNase) has been shown to lose enzymatic activity and to form aggregates when stored in the freeze-dried form at elevated temperature. Polyacrylamide gel electrophoresis showed that the freeze-dried RNase that had lost its enzymatic activity during storage had formed aggregates that were not dissociable using both an anionic detergent and a reducing agent. Isoelectric focusing patterns of freeze-dried RNase before and after storage were quite different. The RNase that had been stored and had aggregated had become more of an acidic protein, while the RNase that was assayed immediately after freeze drying had the same pattern as non-freeze-dried RNase. This evidence indicated that the aggregates were covalently bonded together as a result of some chemical process that occurred during storage of the freeze-dried cake. Amino acid analysis of aggregates formed from RNase freeze dried in pH 10.0 phosphate buffer solutions indicated that the amino acid lysine was instrumental in the formation of the covalent bonds. Asparagine/aspartic acid and glutamine/glutamic acid may have also participated in the bonding of the aggregates.