Abstract
The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC50 data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.
Highlights
Regulatory agencies have issued guidelines and guidances for the conduct of drug-drug interaction (DDI) studies with specific sections focusing on human cytochrome P450 (P450) induction
To evaluate potential thresholds for positive or negative in vitro induction response, the variability in in vitro human hepatocyte induction experiments was interrogated by analyzing CYP3A4 mRNA and activity data generated with a negative control compound, namely, flumazenil, repeated under the same experimental conditions
Since mRNA is the recommended primary endpoint in most P450 induction experiments, subsequent data analyses focused on the variability observed in the CYP3A4 mRNA data sets
Summary
Regulatory agencies have issued guidelines and guidances for the conduct of drug-drug interaction (DDI) studies with specific sections focusing on human cytochrome P450 (P450) induction. All three agencies consider a 2-fold increase in mRNA the ABBREVIATIONS: AUC, area under the curve; AUCR, area under the curve ratio; Cmax,ss, maximum steady state concentration; Cmax,ss,u, unbound maximum steady state concentration; Cmpd, compound; Ct, cycle time; DDI, drug-drug interaction; DMSO, dimethylsulfoxide; EMA, European Medicines Agency; Emax, maximum fold increase (or induction) minus baseline of 1-fold; F2, concentration achieving 2-fold induction; FDA, Food and Drug Administration; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IVIVE, in vitro in vivo extrapolation; IQ, innovation and quality; IWG, Induction Working Group; LoB, limit of blank; LoD, limit of detection; P450, cytochrome P450; PCR, polymerase chain reaction; PMDA, Pharmaceutical and Medical Devices Agency; RIS, relative induction score. The EMA and PMDA state that an in vitro induction response of ,100% (i.e., ,2-fold) is only negative if it is ,20% of the positive control response. All three agencies agree that the in vitro donor providing the most sensitive, worst-case positive response be used to determine the clinical induction risk
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