Quantitative Cytochrome P450 3A4 Induction Risk Assessment Using Human Hepatocytes Complemented with Pregnane X Receptor-Activating Profiles.

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance.

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.

Indirect evolution of social fitness inequalities and facultative social exploitation.

Microbial genotypes with similarly high proficiency at a cooperative behaviour in genetically pure groups often exhibit fitness inequalities caused by social interaction in mixed groups. Winning competitors in this scenario have been referred to as 'cheaters' in some studies. Such interaction-specific fitness inequalities, as well as social exploitation (in which interaction between genotypes increases absolute fitness), might evolve due to selection for competitiveness at the focal behaviour or might arise non-adaptively due to pleiotropy, hitchhiking or genetic drift. The bacterium Myxococcus xanthus sporulates during cooperative development of multicellular fruiting bodies. Using M. xanthus lineages that underwent experimental evolution in allopatry without selection on sporulation, we demonstrate that interaction-specific fitness inequalities and facultative social exploitation during development readily evolved indirectly among descendant lineages. Fitness inequalities between evolved genotypes were not caused by divergence in developmental speed, as faster-developing strains were not over-represented among competition winners. In competitions between ancestors and several evolved strains, all evolved genotypes produced more spores than the ancestors, including losers of evolved-versus-evolved competitions, indicating that adaptation in non-developmental contexts pleiotropically increased competitiveness for spore production. Overall, our results suggest that fitness inequalities caused by social interaction during cooperative processes may often evolve non-adaptively in natural populations.

The fitness cost of rifampicin resistance in Pseudomonas aeruginosa depends on demand for RNA polymerase.

Bacterial resistance to antibiotics usually incurs a fitness cost in the absence of selecting drugs, and this cost of resistance plays a key role in the spread of antibiotic resistance in pathogen populations. Costs of resistance have been shown to vary with environmental conditions, but the causes of this variability remain obscure. In this article, we show that the average cost of rifampicin resistance in the pathogenic bacterium Pseudomonas aeruginosa is reduced by the addition of ribosome inhibitors (chloramphenicol or streptomycin) that indirectly constrain transcription rate and therefore reduce demand for RNA polymerase activity. This effect is consistent with predictions from metabolic control theory. We also tested the alternative hypothesis that the observed trend was due to a general effect of environmental quality on the cost of resistance. To do this we measured the fitness of resistant mutants in the presence of other antibiotics (ciprofloxacin and carbenicillin) that have similar effects on bacterial growth rate but bind to different target enzymes (DNA gyrase and penicillin-binding proteins, respectively) and in 41 single-carbon source environments of varying quality. We find no consistent effect of environmental quality on the average cost of resistance in these treatments. These results show that the cost of rifampicin resistance varies with demand for the mutated target enzyme, rather than as a simple function of bacterial growth rate or stress.

Transporter-mediated drug interactions: clinical implications and in vitro assessment.

Although they are less frequently compared with the reported cases of CYP-mediated drug interactions, clinically significant transporter-mediated drug interactions, which are mainly based on efflux transporter or P-glycoprotein data, have been reported. Unlike the CYP-mediated drug interactions that can be readily defined by inhibition or induction of CYP enzymes, the evidence for the so-called transporter-mediated drug interactions is often less conclusive. The difficulty in defining transporter-mediated drug interactions is due mainly to the interplay between transporters and drug-metabolizing enzymes in drug disposition, and the lack of specific and potent inhibitors for each transporter and enzyme. An important lesson learned from animal studies is that transporter inhibition has a much greater impact on the tissue distribution of drugs than on the systemic exposure of drugs measured in plasma. The potential risk of transporter-mediated drug interactions might be underestimated if only plasma concentrations are monitored.

Assessment of presystemic factors on the oral bioavailability of rifampicin following multiple dosing.

This study was carried out to elucidate the possible mechanism(s) responsible for reduced oral rifampicin bioavailability after multiple dosing. In addition to autoinduction, the relative contribution of the two possible controlling factors, e.g., intestinal metabolism and microbial degradation, was investigated using a rat model. Pharmacokinetic studies were carried out to assess the absolute rifampicin bioavailability by both oral and intravenous drug administration before and after 8 daily doses of 25 mg/kg. To estimate the possible involvement of microbial degradation, rifampicin kinetics were also assessed in rats on day 8 after receiving multiple oral dosing and concurrent administration of nonabsorbable triple antibiotics for gut sterilization 3 days prior to the study day. Pharmacokinetic parameters were generated by noncompartmental analysis. The results revealed a significant decrease in rifampicin levels for rats after multiple exposure, compared to single dosing; the mean clearance determined by intravenous dosing increased by 43% from 3.7 ml/min/kg and the half-life decreased by 24% from 238 min. However, the extent of decrease in rifampicin exposure following multiple dosing was substantially greater for rats dosed orally than intravenously; estimated absolute oral bioavailability decreased by 15% from 0.89 on day 1 to 0.76 on day 8. No apparent alterations in any of the pharmacokinetic parameters were observed after gut sterilization, suggesting minimal contribution of microbial degradation to the reduction in oral rifampicin absorption after multiple dosing. In addition to hepatic enzyme autoinduction, these results strongly suggest the involvement of enhanced intestinal metabolism as a contributing factor to the decrease in oral rifampicin bioavailability following prolonged exposure.