title: Validation of a flow cytometry based G(2)M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition.
abstract: Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration (pharmacokinetics) and drug "effect" or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool. During recent years, flow cytometry has established itself as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry based cell cycle assays. Here we report the validation of a flow cytometry based cell cycle G(2)/M delay assay for use in evaluating the effect of investigational drug MLN8237, a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, precision, and determining the cutoff for a true drug effect based on biostatistical analysis models. Experimental results show that the intra-assay repeatability was less than 20% with an intra-donor variability of less than 40%. The robustness of the assay was less than 30%. Since this is an ex-vivo stimulation assay, variability parameters were expected to be higher. Based on biostatistical modeling, an absolute change in %G(2)M of 5.2% (95% CI) was needed in order to detect a true drug effect. Overall, the assay demonstrated acceptable variability to warrant further in vivo testing.
authors.affiliation: Millennium Pharmaceuticals, Inc. 40 Landsdowne St., Cambridge, MA 02139, USA. jose.estevam@mpi.com
authors.forename: Jose
authors.initials: J
authors.lastname: Estevam
authors.forename: Hadi
authors.initials: H
authors.lastname: Danaee
authors.forename: Ray
authors.initials: R
authors.lastname: Liu
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Ecsedy
authors.forename: William L
authors.initials: WL
authors.lastname: Trepicchio
authors.forename: Timothy
authors.initials: T
authors.lastname: Wyant
mesh_terms: D000880:Anthraquinones / Q000737:chemistry; D064127:Aurora Kinases; D001381:Azepines / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D015203:Reproducibility of Results
chemical_list: C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones; D001381:Azepines; C550258:MLN 8237; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases