Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans

Abstract

Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans

“…Results from PXR reporter gene assays were recently successfully used to classify clinically used drugs as CYP3A inducers or noninducers (Persson et al, 2006;Sinz et al, 2006). However, because induction of P450 enzymes by drug molecules does not only depend on a single transcription factor but is mediated by a combination of several transcription factors in the cell, a human hepatocyte-like cell line would be a superior in vitro model for evaluation of P450 induction.…”

“…Real-time PCR for human P450 mRNA levels was performed using a 7500 Sequence Detector (Applied Biosystems, Foster City, CA) and manufacturer-designed Assay on Demand for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and glyceraldehyde-3-phosphate dehydrogenase. For human acidic ribosomal phosphoprotein (huPO), gene-specific double fluorescent labeled probes were used as reported previously (Persson et al, 2006). The reaction mixture (25 l/well) contained 30 ng of cDNA, 1ϫTaqman Universal Master Mix, optimized concentrations of primers and probes (huPO), or 1.25 l of Assay on Demand and RNase free water.…”

“…To investigate the relationship between P450 induction in HepaRG cells and induction in vivo, the same approach as for the previously reported PXR assay was applied (Persson et al, 2006), in which the in vivo exposure of the test compound was related to the EC 50 from the PXR reporter gene assay (AUC/EC 50 ). This method correctly identified compounds known to induce CYP3A in vivo.…”

Sensor systems for medical application based on hemoproteins and nanocomposite materials

“…Изолированные срезы печени и выделенные из них микросомы использовались для исследования индукции экспрессии генов цитохромов Р450 при инкубации с лекарственными препаратами: карбамазепином, клотримазолом, дексаметазоном, гиперфолином, ловастатином, омепразолом, фенобарбиталом, фенитоином, примакином, рифампицином, TCDD, троглитазоном. После введения коктейля маркерных субстратов (фенацетин, диклофенак, мидазолам) проводился LC/MS анализ активности по регистрации метаболитов: парацетамол (CYP1A2), 4-OH-диклофенак (CYP2C9), и 1-OH-мидазолам (CYP3A) [111].…”

“…Такой подход получил название "cocktail" или "N-in-1 assay" [103,111,112]. Исследовались микросомы печени человека и бакулосомы с экспрессированными человеческими цитохромами CYP1A2, 2A6, 2B6, 2C8, 2С9, 2C19, 2D6, 2E1, 3A4 3A5.…”