A Proposal for an Alternative Approach to Particle Size Method Development During Early-Stage Small Molecule Pharmaceutical Development

Particle size analysis in the pharmaceutical industry has long been a source of debate regarding how best to define measurement accuracy; the degree to which the result of a measurement or calculation conforms to the true value. Defining a “true” value for the size of a particle can be challenging as the output of its measurement will differ because of variations in measurement approaches, instrumental differences and calculation methods. Consequently, for “real” particles, a universal “true” value does not exist and accuracy is therefore not a definable characteristic. Accordingly, precision is then a measure of the ability to reproducibly achieve a measurement of unknown relevance.This article proposes, in place of accuracy, a means to define the “appropriateness” of a measurement in line with the critical quality attributes (CQA) of the material being characterized. The decision as to whether the measurement is correct should involve a link to the CQA; that is, correlation should be demonstrated, without which the measured particle size cannot be defined as a critical material attribute.Correspondingly, methods should also be able to provide sufficient precision to demonstrate discrimination relating to variation in the CQA. The benefits and challenges of this approach are discussed.     

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.     

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15–20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.     

脂蛋白残粒RLP-c检测的临床意义

目的 探讨脂蛋白残粒RLP-c作为新的脂质指标的临床意义。方法 采用免疫分离法测定正常对照组(NC)、冠心病组(CHD)和2型糖尿病组(T2DM)的RLP-c水平,并比较RLP-c与其他脂质指标的相关性。结果 CHD组和T2DM组RLP-c水平明显高于NC组(P<0.01),其水平与甘油三脂(TG)和极低密度脂蛋白胆固醇(VLDL-c)高度相关(P<0.01)。结论 RLP-c可用于对动脉粥样硬化的危险性评估。     

静脉输液加药后的微粒变化

本文对我院急诊病人常用的供静脉给药的22种药物、42组配伍情况,制成静脉加药输液,在超净工作台条件下,用KF—4型微粒计数器,测定了168次微粒数,结果显示输液加药后微粒虽有增加,但均在药典规定范围内。输液中加1种、2种、3种药物后所产生的微粒数分别为x_1=5.52(n_1=16);x_2=8.17(n_2=18)、x_3=10.25(n_3=8),经统计处理,三组间无显著差异(P<0.05)。本文对产生微粒的药物、注射器、操作环境等因素进行了分析。     

接枝环氧树脂水分散液的合成、分离与表征

控制不同的反应条件,采用甲基丙烯酸、苯乙烯与环氧树脂在丙二醇丁醚/正丁醇溶剂中接枝共聚,合成了粒径在87～104nm范围的环氧树脂水分散液。通过环己烷/乙醇和丙酮两步萃取,对接枝共聚产物进行分离,用FTIR表征,并估算了接枝共聚物的组成。实验结果表明,所制备的粒径为纳米级的环氧树脂水分散液,具有良好的机械稳定性、冻融稳定性及贮存稳定性。ξ电位测定表明,产物在pH>8的条件下更为稳定。     

JC病毒样颗粒可直接转运进入细胞核

目的探讨JC病毒(JCV)病毒样颗粒(VLP)是否可以直接转运进入细胞核。方法心用JCV主要外壳蛋白VP1体外表达、重组VIP，在其表面标记异硫氰基荧光素(FTTC)，同时在其内部包裹荧光染料Cy3，感染培养的HeLa细胞和SVG细胞，荧光显微镜观察VLP入核转运。结果HeLa和SVG细胞感染包裹Cy3的FTTC-VLP时，FTTC与Cy3同时出现于细胞核内相同部位；而感染FTTC—VP1与Cy3混合物时，FTTC虽可在细胞核内检测到，但Cy3信号几乎消失。包裹Cy3的VIP用SDSPAGE展开，荧光显像后行考马斯亮蓝(CBB)染色，发现Cy3和VLP移行至不同部位，证明Cy3不能与VP1结合，提示VLP以完整的颗粒形式转运进入细胞核。应用包裹外源性DNA的VLP感染培养的HeLa和SVG细胞，发现包裹的DNA在细胞浆和细胞核内均可检测到，提示JCV入核过程与VIP相同。结论VLP可以不经裂解直接转运进入细胞核，JCV入核转运可能与VLP相同。     

棉织布与无纺布尘埃粒子数及抗渗液性能比较研究

目的比较棉织布与无纺布制作的手术衣和手术洞巾等在手术铺巾时空气中的尘埃粒子数及术中抗渗液性能，为有效控制外科切口感染和预防医患交叉感染提供参考。方法将棉织布与无纺布制作的手术布类各备15包，包内内容均相同，经灭菌处理。应用尘埃粒子计数仪测定两组铺巾时、铺巾后及收巾时空气中的尘埃粒子数，同时在手术过程中观察其抗渗液性能。结果无纺布组在铺巾时、铺巾后及收巾时产生的尘埃粒子数显著少于棉织布组（P〈0．05，P〈0．01）；其抗渗液率为100％，而棉织布组为0。结论无纺布抗渗液性能优，可减少手术环境中的尘埃粒子数，从而控制外科切口感染；其阻隔防护效能对患者和医护人员具有双重保护作用。     

小柴胡汤口服液药效作用的研究

观察了小柴胡汤口服液的主要药理作用。研究表明,小柴胡汤口服液有显著抑制角叉菜胶诱发的大鼠踝关节水肿(p<0.05),保护四氯化碳所致的大鼠急性肝功能损害,有极显著降低血清SGPT及LDH的作用(P<0.01)。对家兔发热反应也有较好的抑制作用。此外,小柴胡汤口服液对小鼠兔疫反应也有一定的增强作用,可促进小鼠碳粒廓清速率,提高血清溶血素水平及增强鸡红细胞所致的迟发性过敏反应。     

大气细菌粒子浓度的时间分布特征及最佳采样时间的研究

用ＭＦ－４５型和ＨＴＫ－２０１型空气微生物采样器分别在北京、天津和沈阳三地观测了不同季节和一天中大气细菌粒子的浓度及其变化。结果表明：京、津二地春季大气细菌粒子浓度高，分别为２０５３个／ｍ３和２５５６个／ｍ３；夏季低，分别为９９５个／ｍ３和１０６４个／ｍ３。沈阳秋季大气细菌粒子浓度高，为１０１０８个／ｍ３；冬季低，为１２９４个／ｍ３。一天中，大气细菌粒子浓度呈双峰型变化，６：００～７：００和１８：００时大气细菌粒子浓度高，１１：００～１３：００和１：００～２：００时大气细菌粒子浓度低。根据大气细菌粒子浓度的季节变化和一天中大气细菌粒子浓度的时间分布特征，经过京、津、沈三地一天中分别１２次、８次、６次和４次不同采样时间组合的大气细菌粒子浓度的数理统计分析，大气细菌粒子浓度的监测拟集中在一年冬、春、夏、秋四季的中间月份，即１月、４月、７月、１０月进行；一天中采样８次的时间序列可为７：００、１０：００、１３：００、１６：００、１９：００、２２：００、１：００、４：００一天采样４次的时间序列可为５：００、１１：００、１７：００、２３：００。白天采样４次的时间序列可为春、秋季６：００、９：００、１２：００、１５：００?