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

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 - 80degreesC for at least 60 days. M30-Apoptosense(TM) 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 - 80degreesC, while at 37degreesC it had a half- life of 80 - 100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.