A Multi-Endpoint Evaluation of Cytochrome P450 1A2, 2B6 and 3A4 Induction Response in Human Hepatocyte Cultures after Treatment with β-Naphthoflavone, Phenobarbital and Rifampicin†

Zhang, J. G.; Ho, Thuy; Callendrello, Alanna L.; Crespi, Charles L.; Stresser, David M.

doi:10.2174/18722102055839673128

Cited by 13 publications

(14 citation statements)

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“…3). Second, at the optimal time point selected (10 hours), induction parameters for prototypical inducers (omeprazole for CYP1A2, phenobarbital for CYP2B6, and rifampicin for CYP3A4) were found to be comparable to those reported in the literature (Fahmi et al, 2008a(Fahmi et al, ,b, 2009(Fahmi et al, , 2010Shou et al, 2008;Zhang et al, 2010). Under these validated experimental conditions, deleobuvir and deleobuvir-AG did not induce P450 enzymes.…”

Section: Tablesupporting

confidence: 54%

“…Deleobuvir and its metabolites were cytotoxic to sandwich cultured hepatocytes at concentrations above 1 mM upon incubation for 48 hours. Elevation of mRNA through upregulation by nuclear receptors is the initial event leading to increases in protein and can be detected within 4-6 hours (Zhang et al, 2010). Validation of shorter incubation times was done using a two-step approach.…”

Section: Tablementioning

confidence: 99%

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Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes

Sane

Ramsden

Sabo

et al. 2015

Drug Metabolism and Disposition

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The drug-drug interaction (DDI) potential of deleobuvir, an hepatitis C virus (HCV) polymerase inhibitor, and its two major metabolites, CD 6168 (formed via reduction by gut bacteria) and deleobuvir-acyl glucuronide (AG), was assessed in vitro. Area-under-the-curve (AUC) ratios (AUCi/AUC) were predicted using a static model and compared with actual AUC ratios for probe substrates in a P450 cocktail of caffeine (CYP1A2), tolbutamide (CYP2C9), and midazolam (CYP3A4), administered before and after 8 days of deleobuvir administration to HCV-infected patients. In vitro studies assessed inhibition, inactivation and induction of P450s. Induction was assessed in a short-incubation (10 hours) hepatocyte assay, validated using positive controls, to circumvent cytotoxicity seen with deleobuvir and its metabolites. Overall, P450 isoforms were differentially affected by deleobuvir and its two metabolites. Of note was more potent CYP2C8 inactivation by deleobuvir-AG than deleobuvir and P450 induction by CD 6168 but not by deleobuvir. The predicted net AUC ratios for probe substrates were 2.92 (CYP1A2), 0.45 (CYP2C9), and 0.97 (CYP3A4) compared with clinically observed ratios of 1.64 (CYP1A2), 0.86 (CYP2C9), and 1.23 (CYP3A4). Predictions of DDI using deleobuvir alone would have significantly over-predicted the DDI potential for CYP3A4 inhibition (AUC ratio of 6.15). Including metabolite data brought the predicted net effect close to the observed DDI. However, the static model over-predicted the induction of CYP2C9 and inhibition/inactivation of CYP1A2. This multiple-perpetrator DDI scenario highlights the application of the static model for predicting complex DDI for CYP3A4 and exemplifies the importance of including key metabolites in an overall DDI assessment.

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Section: Tablesupporting

confidence: 54%

Section: Tablementioning

confidence: 99%

Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes

Sane

Ramsden

Sabo

et al. 2015

Drug Metabolism and Disposition

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“…The testosterone 6b-hydroxylase activity assay was performed essentially as described by Zhang et al (2010). Briefly, after treatment, hepatocyte cultures were washed with culture medium and incubated with 100 ml of culture medium containing CYP3A4 probe substrate testosterone at a concentration of 200 mM for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Calibration Curve–Based Approaches to Predict Clinical Inducers and Noninducers of CYP3A4 with Plated Human Hepatocytes

Zhang

Callendrello

et al. 2014

Drug Metab Dispos

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Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R 3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, C max /EC 50 , and area under the curve (AUC)/F 2 (the concentration causing 2-fold increase from baseline of the doseresponse 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 6b-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatographytandem 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 C max 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 R 3 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 6b-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model.

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“…The drug concentration used to demonstrate metabolic responses or toxicity is usually very different from corresponding in vivo plasma concentrations achieving similar effects (22,26,33,41,47). A drug is administered as a bolus dose at a concentration often orders of magnitude higher than tissue exposure in vivo.…”

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confidence: 99%

Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro

Dash

Simmers

Deering

et al. 2013

American Journal of Physiology-Cell Physiology

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Dash A, Simmers MB, Deering TG, Berry DJ, Feaver RE, Hastings NE, Pruett TL, LeCluyse EL, Blackman BR, Wamhoff BR. Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro. Am J Physiol Cell Physiol 304: C1053-C1063, 2013. First published March 13. 2013 doi:10.1152/ajpcell.00331.2012.-In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma Cmax levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4␣ (HNF-4␣)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ϳ4-fold and urea ϳ5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 Ϯ 10.3; CYP1A2: 64.0 Ϯ 15.1; CYP2B1: 15.2 Ϯ 2.9; CYP2B2: 2.7 Ϯ 0.8; CYP3A2: 4.0 Ϯ 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 Ϯ 2.41 vs. 0.42 Ϯ 0.015; CYP1B: 3.47 Ϯ 1.66 vs. 0.4 Ϯ 0.09; CYP3A: 11.65 Ϯ 4.70 vs. 2.43 Ϯ 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A ϭ 27.33 and CYP3A ϭ 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.hemodynamics; hepatocyte; metabolism; organotype; phenotype HEPATOTOXICITY AND BIOAVAILABILITY issues comprise over 60% of drug failures during clinical trials (45) and are a major cause of postmarketing withdrawal (23), pointing to the need to develop more efficient and predictive preclinical test systems. Simple cellular and subcellular assays used to screen compound libraries offer the advantage of higher throughput but are often unable to capture complex biological effects that may require a physiological context for drug interactions with cells. Primary in vitro hepatocyte models widely used to study liver disease, drug metabolism, and toxicity are extensively reviewed in the literature (16,42). The ability to test the metabolic f...

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A Multi-Endpoint Evaluation of Cytochrome P450 1A2, 2B6 and 3A4 Induction Response in Human Hepatocyte Cultures after Treatment with β-Naphthoflavone, Phenobarbital and Rifampicin†

Cited by 13 publications

References 0 publications

Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes

Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes

Evaluation of Calibration Curve–Based Approaches to Predict Clinical Inducers and Noninducers of CYP3A4 with Plated Human Hepatocytes

Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro

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