人降钙素基因相关肽脂质体质量标准的研究

建立完整的人降钙素基因相关肽脂质体（LipohCGRP)药物的质量标准,用家兔球膜血管扩张法测定hCGRP的生物学活性;采用RP－HPLC,等电聚焦,薄层层析等方法分别测定样品的纯度,等电点和迁移率及脂质体嵌入率的测定,并按照中国生物制品规程的要求完成了甲醇和氯仿等残留物质的分析和动物安全试验,建立了活性测定方法,能准确测定样品中不到1ng和样品活性单位,连续三批样品的各项指标均符合质量标准的要求,建立了脂质体多肽药物的质量标准。并用于质量检定。     

多肽表面修饰脂质体药物递送系统

本文通过查阅并归纳近几年相关文献,较为系统地概述了靶向活性多肽、细胞穿膜肽和靶向细胞穿膜肽等多肽表面修饰脂质体药物递送系统(drug delivery system, DDS)的研究进展。经不同活性多肽表面修饰,或可增强脂质体DDS的靶向性,或可提高药物的细胞摄取率和生物利用度。总之,多肽表面修饰的脂质体在新型DDS研究及应用中具有良好的前景。     

椒莪脂质体的制备及其质量标准的建立

目的:将椒莪油制成脂质体,优选制备工艺,建立质量标准。方法:采用薄膜超声法制备椒莪脂质体,通过正交实验优选处方和制备工艺,HPLC、GC建立其质量标准。结果:最佳处方为卵磷脂:胆固醇7:1,卵磷脂:油3.5:1;HPLC法测定脂质体中莪术油的含量,建立标准曲线,回归方程为Y=14958X+16795,r=0.9996;椒目仁油的测定方法同前文报道。得到的脂质体形态均一,包封率在75%左右。结论:建立的制备工艺简单,便于操作;检测方法的精密度、回收率均符合要求。     

肝炎治疗药苦参碱葡萄糖注射液的研制

为研制供临床直接使用的大容量苦参碱葡萄糖注射液,采用苦参碱浓配,葡萄糖先浓配除热原、后稀配混合苦参碱溶液、精滤、灌封、灭菌等制得苦参碱葡萄糖注射液,并建立质量标准。结果表明：试制3批样品均符合质量标准要求,稳定性考察未见明显变化。研制的苦参碱葡萄糖注射液处方及工艺合理可行、质量标准可控、稳定性良好。     

以苯丙氨酰-tRNA合成酶为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立结核分枝杆菌PheRS抑制剂高通量模型,并运用此模型筛选化合物和发酵液样品。【方法】克隆和表达结核分枝杆菌PheRS蛋白并优化其酶活测定方法,在此基础上建立结核分枝杆菌PheRS抑制剂高通量筛选模型,并通过耻垢分枝杆菌作为检定菌对筛选到的样品进行抗菌活性测定及细胞毒性评价。【结果】运用此模型筛选了化合物样品11 600个,发酵液样品5 200个,筛选得到阳性化合物9个,阳性发酵液37个。而后通过耻垢分枝杆菌作为检定菌的抗菌活性测定及细胞毒性评价后,得到了6个发酵液阳性样品。【结论】建立的PheRS抑制剂模型可成功用于化合物和微生物发酵液的高效筛选,得到的6个发酵液阳性样品在酶水平和抗分枝杆菌方面均具有良好活性且毒性较低,值得进一步研究。     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

胆固醇衍生物脂质体的物理稳定性和细胞相容性研究

目的：研究胆固醇衍生物（CTBBA）脂质体的物理稳定性,细胞相容性以及药物输送。方法：CTBBA组入脂质体并测定不同pH下的zeta电位。对长期保存的脂质体进行粒径分析和含磷量分析,评价脂质体的物理稳定性。通过测定包封在脂质体内的阿霉素的体外释放,来评价CTBBA对脂质体释放药物的影响。用MTT法检测CTBBA本身以及CTBBA脂质体的细胞相容性。评估了用流式细胞仪和荧光显微镜检测脂质体细胞内药物输送能力的可行性。结果：zeta电位数据表明CTBBA脂质体带有正电荷,可以改善脂质体在长期保存过程中的物理稳定性;作为胆固醇衍生物的CTBBA能有效的降低药物的渗漏;相比某些正电荷载体,CTBBA的细胞毒性较低;流式细胞仪在反映阿霉素脂质体的细胞定位上有局限,固定液的使用会改变阿霉素荧光的细胞内分布。结论：正电荷CTBBA脂质体具有改善脂质体长期保存稳定性,且细胞毒性低的特点。流式细胞仪不能完全反映载药的CTBBA脂质体在细胞内的分布。     

重组定点突变巴曲酶质量标准研究

目的 建立重组定点突变巴曲酶的质控方法和质量标准.方法 生物学活性测定采用血浆凝集活性测定法;通过胰蛋白酶消化和RP-HPLC法分析该蛋白还原型肽图;其余检测项目均按<中华人民共和国药典>2010年版(三部)相关规定进行.结果 用建立的方法对三批重组定点突变巴曲酶原液和成品进行检定,各项指标均符合2010版<中华人民共和国药典>和相应指导原则的要求.三批原液比活性均≥2000 kU/mg.肽图三批次之间一致,原液的蛋白含量、纯度、分子质量、等电点、N末端氨基酸序列等指标均符合规定.结论 建立的质控方法可有效地控制重组定点突变巴曲酶产品质量,并可用于定点突变巴曲酶原液及成品的常规检定.     

杨梅苷脂质体的制备研究

目的：制备杨梅苷脂质体。方法：采用逆相蒸发法制备杨梅苷脂质体。用冷冻离心法分离脂质体和游离药物,用高效液相色谱法测定药物含量并计算包封率。采用激光粒度仪测定平均粒径。结果：杨梅苷脂质体制备的最佳处方和工艺为：卵磷脂：杨梅6：1,卵磷脂：胆固醇2：1,有机相：水相4：1,磷酸盐缓冲溶液的pH值为6.86,浓度为0.005 mol.L-1;超声时间为5分钟。结论：最佳条件下制备的杨梅苷脂质体包封率较高,粒径分布好,质量稳定。     

pH敏脂质体对反义寡核苷酸抗流感病毒活性的影响

为了研究具有临床应用前景的 Ａ Ｓ Ｏ Ｄ Ｎ 脂质体转运系统,以临床药用大豆磷脂为主要原料制备了ｐ Ｈ 敏脂质体,并测定了脂质体体外转染活性、ｐ Ｈ 敏特性、细胞毒性和对 Ａ Ｓ Ｏ Ｄ Ｎ 抗流感病毒活性的影响 结果发现,批号为 ９８０５１９０３,９８０５１１０２ 和 ９８０５１２０２ 的脂质体具有较高转染活性,但只有ｌｉｐｏｆｅｃｔｉｎ 转染活性的 １／５０～１／１００当质粒／脂质体（ Ｗ ／ Ｗ ）为 １∶４～１∶８,转染时间为 ３～５ ｈ,质粒量为 ０５ μｇ,转染后 ２４～４８ ｈ 内检测时转染活性最高 脂质体 ９８０５１２０２ 表现明显 ｐ Ｈ值依赖溶解红细胞膜特性,而脂质体 ９８０５１１０２ 和 ９８０５１９０３ 的 ｐ Ｈ 敏特性不明显 脂质体细胞毒性明显降低,如 ９８０５１９０３、９８０５１１０２ 和 ９８０５１２０２ 的毒性分别是 ｌｉｐｏｆｅｃｔｉｎ 毒性的 １／１６、１／８ 和 １／４ｐ Ｈ 敏脂质体 ９８０５１２０２ 具有促进 Ａ Ｓ Ｏ Ｄ Ｎ 抗流感病毒作用,当 Ａ Ｓ Ｏ Ｄ Ｎ 浓度为 ０２ μｍ ｏｌ／ Ｌ 时,ｐ Ｈ 敏脂质体 ９８０５１２０２ 使其抗病毒活性提高 ５ 倍,但 Ａ Ｓ Ｏ Ｄ Ｎ 浓度较高时ｐ Ｈ 敏脂质体对 Ａ Ｓ Ｏ Ｄ Ｎ抗     

重组双功能水蛭素质量标准研究

以生色底物法测定抗凝血酶活性,比浊法测定抗血小板聚集活性,还原型SDS-PAGE测定分子量,SDS-PAGE和反相高效液相色谱测定纯度,毛细管电泳法测定等电点,胰蛋白酶酶切后进行肽图分析,其余检测项目按《中国药典》2005版三部规定进行。结果显示,用建立的方法对原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》2005版三部的要求。建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于重组双功能水蛭素产品的常规检定。     

Quality Assurance and Standardization in Immunohistochemistry. A Proposal for the Annual Meeting of the Biological Stain Commission,June, 1991

Quality assurance, quality control, proficiency testing, reagent documentation and validation are standard parts of everyday practice in clinical laboratories throughout the United States. Immunohistochemical stains employ reagents and principles in common with immunoenzyme methods utilized in the clinical laboratory. However, immunohistochemistry has not routinely been subjected to similar standardization and quality assurance procedures that manufacturers and pathologists alike have applied to essentially the same techniques in the clinical laboratory environment. The current proposal was invited by the Biological Stain Commission with the charge of incorporating the findings of previous workshops on quality control in immunohistochemistry into a practical design for implementation. The status of quality assurance, quality control and standardization in immunohistochemistry is reviewed and a phased strategy for implementation is proposed.     

Quality Assurance and Standardization in Immunohistochemistry. A Proposal for the Annual Meeting of the Biological Stain Commission, June, 1991

Quality assurance, quality control, proficiency testing, reagent documentation and validation are standard parts of everyday practice in clinical laboratories throughout the United States. Immunohistochemical stains employ reagents and principles in common with immunoenzyme methods utilized in the clinical laboratory. However, immunohistochemistry has not routinely been subjected to similar standardization and quality assurance procedures that manufacturers and pathologists alike have applied to essentially the same techniques in the clinical laboratory environment. The current proposal was invited by the Biological Stain Commission with the charge of incorporating the findings of previous workshops on quality control in immunohistochemistry into a practical design for implementation. The status of quality assurance, quality control and standardization in immunohistochemistry is reviewed and a phased strategy for implementation is proposed.     

鼠疫菌噬菌体效价噬菌斑测定法的建立

目的建立鼠疫菌噬菌体噬菌斑效价测定方法。方法通过分析细菌接种浓度、孵育吸附时间及培养温度等参数,建立鼠疫菌噬菌体效价测定方法,并分析其精密性;建立鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准。结果经优化后确定细菌接种浓度为7×108/mL,不需孵育吸附,培养温度为29℃,所建立的检测方法精密性较好,用于鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准应不低于1×106PFU/mL。结论建立了鼠疫菌噬菌体噬菌斑效价测定方法,为鼠疫菌噬菌体及疫苗质量控制奠定了基础。     

Collaborative study for the validation of alternative in vitro potency assays for human tetanus immunoglobulins

An international collaborative study to validate 2 alternative in vitro methods for the potency testing of human tetanus immunoglobulin products was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). The study, run in the framework of the Biological Standardisation Programme (BSP) under the aegis of the European Commission and the Council of Europe, involved 21 official medicines control and industry laboratories from 15 countries.Both methods, an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA), showed good reproducibility, repeatability and precision. EIA and TIA discriminated between low, medium and high potency samples. Potency estimates correlated well and both values were in close agreement with those obtained by in vivo methods. Moreover, these alternative methods allowed to resolve discrepant results between laboratories that were due to product potency loss and reporting errors.The study demonstrated that EIA and TIA are suitable quality control methods for tetanus immunoglobulin, which can be standardised in a control laboratory using a quality assurance system. Consequently, the Group of Experts on Human Blood and Blood Products of the European Pharmacopoeia revised the monograph on human tetanus immunoglobulins to include both the methods as compendial alternatives to the in vivo mouse challenge assay.     

Establishment of laboratory quality control indicators for NAT on blood donors' samples

In order to establish and screen nucleic acid test (NAT) quality control indicators for blood donor samples, C_T value of each test item were detected by NAT, internal and external quality controls, the number of unsuccessful mixed samples, the split positive rate, the rate of detection efficiency, and the equipment failure rate were collected. The "instant method" is used to establish the Levey-Jennings quality control chart, the mixed test positive rate, the rate of unqualified sample. Possion distribution is used to establish the quality control chart, and it is easily to operate and monitor NAT effectively. The results displayed that quality control charts were established, including unqualified sample Possion distribution quality control chart, the rate of equipment failure quality control chart, the failure rate of reagent batch and inefficient quality control chart, test result positive of Possion distribution quality control chart, quality control charts of NAT positive Possion distribution probability, the rate of split positive quality control charts, quality control charts of unsuccessful mixed samples, quality control chart of reagents effective rate, internal quality control chart, correctness, and NAT qualitative quality control chart. The study established quality control indicators throughout the whole NAT process, which were able to effectively improve NAT quality control and the efficacy of laboratory management.     

Using enhanced development tools offered by analytical Quality by Design to support switching of a quality control method

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE-SDS), with the aim of replacing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a conversion factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy.     

Pharmacological characterization of CGRP1 receptor subtype in the vascular system of the rat: studies with hCGRP fragments and analogs.

In order to examine whether the truncated fragments of hCGRP, hCGRP(8-37) or hCGRP(12-37), behave as competitive CGRP receptor antagonists in the vascular system of the rat, systemic blood pressure was continually monitored in pentobarbital-anesthetized Sprague-Dawley rats. The IV administration of 7.9-527 pmol hCGRP/rat caused dose-related reductions in mean arterial blood pressure that lasted, depending on the dose, about 3-10 min. In contrast, hCGRP fragments 8-37 or 12-37 proved inactive up to 60,000 pmol/rat. Pretreatment with either 10 or 30 nmol hCGRP(8-37) or 20 or 90 nmol hCGRP(12-37)/rat reduced the magnitude of the CGRP-induced hypotensive responses caused by 79 pmol hCGRP/rat; pretreatment with 10 nmol of the hCGRP fragments displaced about 3-fold the hCGRP as well as the [Cys(ACM)2.7]hCGRP dose-response curve to the right in a parallel fashion. The specificity of hCGRP(8-37) as a CGRP receptor antagonist was documented by the finding that it did not antagonize the hypotensive responses induced with bradykinin, histamine or substance P.     

Calibration of replacement international standard and European Pharmacopoeia Biological Reference Preparation for Diphtheria Toxoid, Adsorbed.

We report here the characterisation of a preparation of diphtheria toxoid, adsorbed, and its calibration by twenty laboratories in fourteen countries in terms of the Second International Standard (I.S.) for Diphtheria Toxoid, Adsorbed, coded sample A (DIXA) using the established World Health Organisation (WHO)/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 160 IU/ampoule on the basis of its calibration by in vivo bioassay. Stability was assessed within the collaborative study, and as part of candidate characterisation. Results suggest that the replacement standard will have satisfactory stability. This study also provided an opportunity to investigate serology as alternative to in vivo bioassay for potency testing of diphtheria vaccines. Six laboratories participated by performing serology according to in-house protocol. The calibration of the replacement standard in a mouse Vero cell assay gave a significantly higher results than in the established WHO/Ph Eur methods. Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Diphtheria Toxoid, Adsorbed (coded 98/560) by the WHO Expert Committee of Biological Standardization in October 1999. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 3) by the Steering Committee of the Biological Standardisation Programme of the European Directorate for the Quality of Medicines and approved by the European Pharmacopoeia Commission.