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Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations.

ABSTRACT: An in vitro observation of time-dependent inhibition (TDI) of metabolic enzymes often results in removing a potential drug from the drug pipeline. However, the accepted method for predicting TDIs of the important drug metabolizing cytochrome P450 enzymes often overestimates the drug interaction potential. Better models that take into account the complexities of the cytochrome P450 enzyme system will lead to better predictions. Herein we report the use of our previously described models for complex kinetics of podophyllotoxin. Spectral characterization of the kinetics indicates that an intermediate MI complex is formed, which slowly progresses to an essentially irreversible MI complex. The intermediate MI complex can release free enzyme during the time course of a typical 30 min TDI experiment. This slow rate of MI complex conversion results in an overprediction of the kinact value if this process is not included in the analysis of the activity versus time profile. In vitro kinetic experiments in rat liver microsomes predicted a lack of drug interaction between podophyllotoxin and midazolam. In vivo rat pharmacokinetic studies confirmed this lack of drug interaction.

SUBMITTER: Barnaba C

PROVIDER: S-EPMC5059843 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations.

Barnaba Carlo C Yadav Jaydeep J Nagar Swati S Korzekwa Ken K Jones Jeffrey P JP

Molecular pharmaceutics 20160701 8

An in vitro observation of time-dependent inhibition (TDI) of metabolic enzymes often results in removing a potential drug from the drug pipeline. However, the accepted method for predicting TDIs of the important drug metabolizing cytochrome P450 enzymes often overestimates the drug interaction potential. Better models that take into account the complexities of the cytochrome P450 enzyme system will lead to better predictions. Herein we report the use of our previously described models for compl ...[more]

PMID: 27336918

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Project description:BackgroundMulti-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity.ResultsEffective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells.ConclusionThis study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.

Project description:PurposeVariability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4β-hydroxycholesterol (4βOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4βOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range.MethodsThe patients (n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4βOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam.Results4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (ρ = 0.53, p < 0.001), and hepatic CYP3A4 concentrations (ρ = 0.30, p = 0.027), but not with intestinal CYP3A4 concentrations (ρ = 0.18, p = 0.28) or intestinal microsomal CYP3A4 activity (ρ = 0.15, p = 0.53). 4βOHC concentrations correlated weakly with midazolam absolute bioavailability (ρ = - 0.23, p = 0.027) and apparent oral clearance (ρ = 0.28, p = 0.008), but not with systemic clearance (ρ = - 0.03, p = 0.81).ConclusionThese findings suggest that 4βOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4βOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs.Trial registrationClinical.Trialsgov identifier: NCT02386917.

Dataset Information

Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations.

Publications

Mechanism-Based Inhibition of CYP3A4 by Podophyllotoxin: Aging of an Intermediate Is Important for in Vitro/in Vivo Correlations.

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