title: Improved assays for xenosensor activation based on reverse transfection.
abstract: Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies.
authors.affiliation: School of Pharmacy and Biocenter Kuopio, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland. Electronic address: jenni.kublbeck@uef.fi.
authors.forename: Jenni
authors.initials: J
authors.lastname: Küblbeck
authors.affiliation: School of Pharmacy and Biocenter Kuopio, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Teemu
authors.initials: T
authors.lastname: Anttila
authors.affiliation: School of Pharmacy and Biocenter Kuopio, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Juha T
authors.initials: JT
authors.lastname: Pulkkinen
authors.affiliation: School of Pharmacy and Biocenter Kuopio, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Paavo
authors.initials: P
authors.lastname: Honkakoski
mesh_terms: D001681:Biological Assay; D045744:Cell Line, Tumor; D000090702:Constitutive Androstane Receptor; D006801:Humans; D011094:Polyethyleneimine / Q000737:chemistry; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011987:Receptors, Steroid / Q000235:genetics*; D014162:Transfection
chemical_list: D000090702:Constitutive Androstane Receptor; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011094:Polyethyleneimine
keywords: Aryl hydrocarbon receptor; Constitutive androstane receptor; Cytochrome P450; Induction; Pregnane X receptor; Reverse transfection