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

Abstract

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 À801C for at least 60 days. M30-Apoptosenset 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-Asp 396 ) was stable for 3 months at À801C, while at 371C it had a half-life of 80 -100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients Undergoing Chemotherapy

“…While biomarker research is now an adjunct to most oncology drug trials, the data generated are usually exploratory, limited by the use of unvalidated assays and lacking sufficient robustness to inform on future clinical drug development. In our previous work these assays were validated and optimized to good clinical laboratory practice standard for clinical trials 8,9 and the CellSearch technology for CTCs currently dominates the technology platforms for CTC enumeration with respect to reproducibility. 15 Our current results will inform the application and interpretation of these bioassays when incorporated into upcoming trials of novel therapies for SCLC, not least those agents targeted to apoptosis regulatory components such as Bcl-2 family antagonists and inhibitors of the IAP family of proteins.…”

“…8,9 Serum samples were analyzed for nDNA [Cell Death Detection ELISA (Roche, Basel, Switzerland)] as previously described. 14 …”

“…7 nDNA release is also detected when levels of apoptosis overwhelm macrophage capacity for phagocytosis and a more necrotic cell fate ensues. 7 We have previously validated these cell death biomarkers 8,9 and optimized them for application to a busy, clinical setting. 6 Here we report on the behavior and clinical utility of these assays in patients with SCLC.…”

Docetaxel induces apoptosis in hormone refractory prostate carcinomas during multiple treatment cycles

“…This method is inexpensive and sera can be frozen and stored before analysis (Cummings et al, 2005), making the method suitable for multicentre clinical trials. Measurements of caspase-cleaved CK18 in serum may be quite useful for comparisons of the efficiencies between different treatment modalities, which is a key issue during development of novel anticancer drugs.…”

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis

“…In addition, an XIAP antisense oligonucleotide sensitised RCC to Fas/TRAIL-induced apoptosis (Mizutani et al, 2007). Antisense oligonucleotides directed against XIAP sensitise cancer cells to chemotherapeutic drugs in vitro (McManus et al, 2004) and those developed by Aegera Therapeutics Inc. are currently being evaluated in phase I and II clinical trials (Cummings et al, 2005;Schimmer et al, 2006).…”