title: 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 -80 degrees 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 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.
authors.affiliation: Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. jcummings@picr.man.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Cummings
authors.forename: T H
authors.initials: TH
authors.lastname: Ward
authors.forename: E
authors.initials: E
authors.lastname: LaCasse
authors.forename: C
authors.initials: C
authors.lastname: Lefebvre
authors.forename: M
authors.initials: M
authors.lastname: St-Jean
authors.forename: J
authors.initials: J
authors.lastname: Durkin
authors.forename: M
authors.initials: M
authors.lastname: Ranson
authors.forename: C
authors.initials: C
authors.lastname: Dive
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D007633:Keratins / Q000032:analysis; D009841:Oligonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011506:Proteins / Q000037:antagonists & inhibitors*; D011786:Quality Control; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: C498209:AEG 35156; D000970:Antineoplastic Agents; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011506:Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D007633:Keratins; D019311:Staurosporine