Predictive In Vitro-In Vivo Extrapolation for Time Dependent Inhibition of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 Using Pooled Human Hepatocytes, Human Liver Microsomes, and a Simple Mechanistic Static Model.

Inactivation of Cytochrome P450 (CYP450) enzymes can lead to significant increases in exposure of comedicants. The majority of reported in vitro to in vivo extrapolation (IVIVE) data have historically focused on CYP3A, leaving the assessment of other CYP isoforms insubstantial. To this end, the utility of human hepatocytes (HHEP) and human liver microsomes (HLM) to predict clinically relevant drug-drug interactions was investigated with a focus on CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Evaluation of IVIVE for CYP2B6 was limited to only weak inhibition. A search of the University of Washington Drug-Drug Interaction Database was conducted to identify a clinically relevant weak, moderate, and strong inhibitor for selective substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, resulting in 18 inhibitors for in vitro characterization against 119 clinical interaction studies. Pooled human hepatocytes and HLM were preincubated with increasing concentrations of inhibitors for designated timepoints. Time dependent inhibition was detected in HLM for four moderate/strong inhibitors, suggesting that some optimization of incubation conditions (i.e., lower protein concentrations) is needed to capture weak inhibition. Clinical risk assessment was conducted by incorporating the in vitro derived kinetic parameters maximal rate of enzyme inactivation (min-1) (kinact) and concentration of inhibitor resulting in 50% of the maximum enzyme inactivation (KI) into static equations recommended by regulatory authorities. Significant overprediction was observed when applying the basic models recommended by regulatory agencies. Mechanistic static models, which consider the fraction of metabolism through the impacted enzyme, using the unbound hepatic inlet concentration lead to the best overall prediction accuracy with 92% and 85% of data from HHEPs and HLM, respectively, within twofold of the observed value. SIGNIFICANCE STATEMENT: Coupling time-dependent inactivation parameters derived from pooled human hepatocytes and human liver microsomes (HLM) with a mechanistic static model provides an easy and quantitatively accurate means to determine clinical drug-drug interaction risk from in vitro data. Optimization is needed to evaluate time-dependent inhibition (TDI) for weak and moderate inhibitors using HLM. Recommendations are made with respect to input parameters for in vitro to in vivo extrapolation (IVIVE) of TDI with non-CYP3A enzymes using available data from HLM and human hepatocytes.

Selective Suppression of CYP3A4 mRNA and Enzyme Activity by Epidermal Growth Factor in Plated Human Hepatocytes.

BACKGROUND: Epidermal Growth Factor (EGF) is a well-known mitogen that has importance in cell proliferation and differentiation. This property has led to the common use of EGF as an additive to some cell culture media. EGF has been previously shown to modulate constitutive Cytochrome P450 (CYP) expression in vitro. OBJECTIVES: To assess the influence of EGF on the basal and induced expression of CYP3A4, CYP1A2 and CYP2B6 in plated human hepatocytes. METHODS: Human hepatocytes were treated with EGF with and without in the presence of positive control inducers. After treatment, CYP isoform mRNA expression and enzyme activity were measured. RESULTS: EGF at concentrations ranging from 0.001-500 ng/mL resulted in a concentration-dependent decrease in basal CYP3A4 catalytic activity by up to 92%. In contrast, rifampicin (RIF)-induced activity was decreased only slightly (up to 23%). CYP3A4 mRNA also decreased in an EGF concentrationdependent manner. In contrast to CYP3A4, CYP1A2 and CYP2B6 activity and mRNA were either not suppressed or suppressed to a lower extent. The preferential effect with CYP3A4 was confirmed in 4 additional donors using a single concentration of EGF (10 ng/mL) and time-dependence experiments revealed that suppression appeared after only 24h of treatment. CONCLUSION: Because of the larger effect on the basal CYP3A4 compared to the induced response, EGF as a media additive enables a higher dynamic range in a CYP3A4 induction assay, potentially expanding the range of donor hepatocytes suitable for use in induction studies. These findings also suggest that EGF may be an important regulator of CYP3A4 expression in vivo.

Evaluation of calibration curve-based approaches to predict clinical inducers and noninducers of CYP3A4 with plated human hepatocytes.

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 6ß-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 6ß-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model.

Human cytochrome p450 induction and inhibition potential of clevidipine and its primary metabolite h152/81.

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 microM. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 microM and 100 microM concentrations of clevidipine tested. Induction response for CYP3A4 to 100 microM clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC(50) values below 10 microM, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC(50) values >70 microM). The K(i) values for CYP2C9 and CYP2C19 were 1.7 and 3.3 microM, respectively, and those for CYP3A4 were 8.3 and 2.9 microM, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC(50) values >or= 69 microM). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.