A Novel Study Design Using Continuous Intravenous and Intraduodenal Infusions of Midazolam and Voriconazole for Mechanistic Quantitative Assessment of Hepatic and Intestinal CYP3A Inhibition.

The extent of a drug-drug interaction (DDI) mediated by cytochrome P450 (CYP) 3A inhibitors is highly variable during a dosing interval, as it depends on the temporal course of victim and perpetrator drug concentrations at intestinal and hepatic CYP3A expression sites. Capturing the time course of inhibition is therefore difficult using standard DDI studies assessing changes in area under the curve; thus, a novel design was developed. In a 4-period changeover pilot study, 6 healthy men received intraduodenal or intravenous infusions of the CYP3A substrate midazolam (MDZ) at a rate of 0.26 mg/h for 24 hours. This was combined with intraduodenal or intravenous infusion of the CYP3A inhibitor voriconazole (VRZ), administered at rates of 7.5 mg/h from 8 to 16 hours and of 15 mg/h from 16 to 24 hours, after starting midazolam administration. Plasma and urine concentrations of VRZ, MDZ, and its major metabolites were quantified by liquid chromatography-tandem mass spectrometry and analyzed by semiphysiological population pharmacokinetic nonlinear mixed-effects modeling. A model including mechanism-based inactivation of the metabolizing enzymes (maximum inactivation rate constant kinact , 2.83 h-1 ; dissociation rate constant KI , 9.33 µM) described the pharmacokinetics of VRZ well. By introducing competitive inhibition by VRZ on primary and secondary MDZ metabolism, concentration-time profiles, MDZ and its metabolites were captured appropriately. The model provides estimates of local concentrations of substrate and inhibitor at the major CYP3A expression sites and thus of the respective dynamic extent of inhibition. A combination of intravenous and intraduodenal infusions of inhibitors and substrates has the potential to provide a more accurate assessment of DDIs occurring in both gut wall and liver.

Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome.

BACKGROUND: The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. METHODS: An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC50 and Ki values via nonlinear regression. Obtained Ki values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. RESULTS: In this study, 49 IC50 experiments were conducted. In six cases, IC50 values lower than the calculated threshold for drug-drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained Ki values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained Ki values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). CONCLUSIONS: In vitro/in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations.

Development and validation of an in vitro, seven-in-one human cytochrome P450 assay for evaluation of both direct and time-dependent inhibition.

INTRODUCTION: Direct and time-dependent inhibition (TDI) of cytochrome P450 enzymes (CYP) raises drug safety concerns and has major implications in drug development. This study describes the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based screening tool to simultaneously assess both the direct and the time-dependent inhibitory potential of xenobiotics on the seven major CYPs using a two-step approach. METHODS: The in vitro cocktail of FDA recognized model substrates was incubated with human liver microsomes (HLM) and consisted of caffeine (CYP1A2), bupropion (CYP2B6), rosiglitazone (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4). Direct and time-dependent inhibitory profiles of direct and time-dependent reference inhibitors for each CYP were studied. For validation, the results were compared to those obtained with the traditional single substrate approach. Statistical uncertainty was quantified using the bootstrap method. RESULTS: The direct inhibition assay showed an acceptable fold bias of 1.35 (geometric mean fold absolute deviation, range 1.01-2.61) in the IC50 values for the cocktail assay compared to the single substrate results with no trend for under- or overestimation. Using a single point inactivation assay to assess TDI, we were able to identify all seven tested time-dependent reference inhibitors, without any false negatives. DISCUSSION: The presented design enhances throughput by assessing the seven major CYPs simultaneously and allows for detection of and discrimination between direct and time-dependent CYP inhibition via IC50 and single point inactivation experiments. For the latter, a threshold of 10% TDI is proposed for carrying out more detailed inactivation kinetic experiments.