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Published on: March 28, 2017
Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells
Lena C Preiss1, Ruoqi Liu1, Philip Hewitt1
1Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.).
Abstract:
Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.
Insights
HepaRG cells, including nuclear hormone receptor knockout models, effectively screen cytochrome P450 induction by oncology drug candidates. This method aids in early drug development risk assessment by elucidating CYP3A4 and CYP2B6 induction mechanisms.
Area of Science:
- Pharmacology and Toxicology
- Drug Metabolism and Pharmacokinetics
- Hepatocyte Biology
Background:
- Cytochrome P450 enzymes, particularly CYP3A4 and CYP2B6, play crucial roles in drug metabolism.
- Nuclear hormone receptors like Pregnane X receptor (PXR) and Constitutive Androstane Receptor (CAR) regulate CYP450 gene expression.
- Understanding induction mechanisms is vital for assessing drug-drug interactions and candidate safety in drug discovery.
Purpose of the Study:
- To investigate the induction mechanisms of CYP3A4 and CYP2B6 by oncology drug candidates using HepaRG cells.
- To characterize the role of Pregnane X receptor (PXR) and Constitutive Androstane Receptor (CAR) in mediating this induction.
- To evaluate the utility of HepaRG nuclear hormone receptor knockout (NHR KO) cell lines for drug discovery screening.
Main Methods:
- Utilized PXR/CAR knockout (KO) HepaRG cells and a PXR reporter gene assay.
- Assessed basal and inducible gene expression of CYP3A4 and CYP2B6.
- Tested prototypical substrates and a panel of compounds from Merck KGaA's oncology pipeline.
Main Results:
- Basal CYP3A4 expression was significantly altered in PXR and CAR KO cells, with reduced inducibility.
- CYP2B6 basal expression was elevated in PXR KO cells, showing diminished inducibility.
- Most compounds induced CYP3A4/CYP2B6 via PXR and CAR; non-NHR mechanisms were also implicated.
- A notable discrepancy was observed between HepaRG cell induction and PXR reporter gene assay activation for some compounds.
Conclusions:
- HepaRG cells, especially NHR KO variants, are highly sensitive and suitable for early screening of P450 induction in drug discovery.
- These NHR KO HepaRG models effectively deconvolute P450 induction mechanisms for novel compounds.
- The study highlights HepaRG's advantages over reporter gene assays for assessing drug candidate risks in early development.
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