Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.
Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.
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DOI：
10.1038/sj.bjc.6602363
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发表时间：
2005-02-14
影响因子：
8.8
通讯作者：
Dive, C
中科院分区：
文献类型：
--
作者：
Cummings, J;Ward, TH;LaCasse, E;Lefebvre, C;St-Jean, M;Durkin, J;Ranson, M;Dive, C
凋亡抑制蛋白XIAP经常在化疗耐药的人类肿瘤中过表达。一种针对XIAP的反义寡核苷酸（AEG 35156/GEM 640）最近在英国进入I期试验。提供了本试验期间将使用的三种药效学试验的方法验证数据。定量RT-PCR基于Taqman测定，并证实对XIAP具有特异性。测定线性扩展了四个数量级。选择稳定表达针对XIAP的RNAi载体的MDA-MB-231/U6-E1细胞和克隆X-G4作为高和低XIAP表达质量对照（QC）。循环阈值（CT）和Δ CT值（采用GAPDH和β 2微球蛋白作为管家）的精密度的日内和日间变异系数（CV）始终小于10%。使用GST-XIAP融合蛋白作为标准，HeLa细胞和SF 268（人胶质母细胞瘤）细胞作为高和低XIAP表达QC，验证蛋白质印迹技术。通过分析包括克隆X-G4的一组细胞系来评价XIAP抗体的最终选择的特异性。该测定在29倍蛋白浓度范围内呈线性，当归一化为GAPDH时，低QC和高QC的日间精密度分别为29%和23%。XIAP蛋白在-80 °C下至少可以稳定60天。M30-Apoptosense™ plasma Elisa检测细胞角蛋白18（CK 18）的半胱天冬酶切割片段，被认为是肿瘤细胞凋亡的替代标志物。通过用星形孢菌素处理X-G4细胞并收集培养基，实现了独立QC的产生。测定精密度和试剂盒间QC的测量值始终小于10%。M30抗原（CK 18-Asp 396）在−80°C下稳定3个月，而在37°C下，它在健康志愿者血浆中的半衰期为80-100小时。人们急切地等待着第一阶段试验的结果。
The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST–XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at −80°C for at least 60 days. M30-Apoptosense™ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at −80°C, while at 37°C it had a half-life of 80–100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.
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