title: High-content micronucleus assay in genotoxicity profiling: initial-stage development and some applications in the investigative/lead-finding studies in drug discovery.
abstract: This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of "drug-like but less optimized" early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories--NEG (negative), NEG/xx μM (negative up to the solubility limit of xx μM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1-2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development-compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates.
authors.affiliation: Preclinical Safety Profiling, CPC, Novartis Institutes of Biomedical Research, Inc., Cambridge, Massachusetts 02139, USA. madhu.mondal@novartis.com
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Mondal
authors.forename: Joseph
authors.initials: J
authors.lastname: Gabriels
authors.forename: Claudia
authors.initials: C
authors.lastname: McGinnis
authors.forename: Maria
authors.initials: M
authors.lastname: Magnifico
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Marsilje
authors.forename: Laszlo
authors.initials: L
authors.lastname: Urban
authors.forename: Alan
authors.initials: A
authors.lastname: Collis
authors.forename: Dejan
authors.initials: D
authors.lastname: Bojanic
authors.forename: Scott A
authors.initials: SA
authors.lastname: Biller
authors.forename: Wilfried
authors.initials: W
authors.lastname: Frieauff
authors.forename: Hans-Joerg
authors.initials: HJ
authors.lastname: Martus
authors.forename: Willi
authors.initials: W
authors.lastname: Suter
authors.forename: Philip
authors.initials: P
authors.lastname: Bentley
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004345:Drug Industry / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D015870:Gene Expression; D020869:Gene Expression Profiling; D015162:Micronucleus Tests; D015203:Reproducibility of Results