title: Evaluation of calibration curve-based approaches to predict clinical inducers and noninducers of CYP3A4 with plated human hepatocytes.
abstract: Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, Cmax/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6β-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R(2) and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6β-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model.
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.) zhangjg@corning.com.
authors.forename: J George
authors.initials: JG
authors.lastname: Zhang
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Thuy
authors.initials: T
authors.lastname: Ho
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Alanna L
authors.initials: AL
authors.lastname: Callendrello
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Clark
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Santone
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Sarah
authors.initials: S
authors.lastname: Kinsman
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Deqing
authors.initials: D
authors.lastname: Xiao
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Lisa G
authors.initials: LG
authors.lastname: Fox
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: Heidi J
authors.initials: HJ
authors.lastname: Einolf
authors.affiliation: Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
authors.forename: David M
authors.initials: DM
authors.lastname: Stresser
mesh_terms: D019540:Area Under Curve; D002138:Calibration; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000235:genetics* / Q000378:metabolism*; D004790:Enzyme Induction / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000008:administration & dosage / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013739:Testosterone / Q000378:metabolism; D014481:United States; D014486:United States Food and Drug Administration
chemical_list: D004364:Pharmaceutical Preparations; D012333:RNA, Messenger; D013739:Testosterone; D006899:Mixed Function Oxygenases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human