Rapid maturation of the hepatic cell line Huh7 via CDK inhibition for PXR dependent CYP450 metabolism and induction

Beyza Bulutoglu,Camilo Rey-Bedón,Martin L Yarmush,Safak Mert,Sarah L Deng,O Berk Usta

doi:10.1038/s41598-019-52174-w

Abstract

CYP3A4, a cytochrome P450 enzyme regulated by the nuclear receptor PXR, is involved in most of the drug metabolizing pathways. Studying the regulation/induction of CYP3A4 and PXR is critical in toxicology and drug-drug interaction (DDI) studies. Primary human hepatocytes constitute the preferred in vitro platform for drug development efforts. However, they are expensive, scarce and heterogeneous. Hepatic cell lines, such as Huh7, could provide a cost-effective alternative, however, they express negligible amounts of CYP450s and PXR. In this study, we show that dinaciclib, a potent cyclin dependent kinase inhibitor, significantly increases the basal CYP3A4 and PXR levels in 24 hours. We also demonstrated that matured Huh7s can be used for drug induction studies, where CYP3A4, CYP1A2, CYP2C9, and CYP2C19 inductions were achieved following rifampicin treatment. More importantly, through a direct demonstration using amiodarone and rifampicin as model drugs, we showed that matured Huh7s present a suitable platform for DDI studies.

Highlights

Liver is the primary organ responsible for the metabolism and detoxification of the vast majority of xenobiotics, including pharmaceuticals[1,2]
This study found that the reduction in CDK2 expression correlated well with increases observed in both pregnane X receptor (PXR) and CYP3A4 expression during the 4-week confluent maturation of the Huh[7] cells
Basal CYP3A4 and PXR levels increase upon dinaciclib treatment

Summary

Introduction

Liver is the primary organ responsible for the metabolism and detoxification of the vast majority of xenobiotics, including pharmaceuticals[1,2]. PXR expression - the key regulator of CYP3A4 expression - is vital for DDI and toxicology studies[14,15,16,17] To this end, in vitro cultures of primary human hepatocytes (PHHs) are the preferred platform for drug development, toxicology, and DDI studies. It has been shown that the low to negligible PXR and CYP3A4 expression in Huh7s can be remarkably increased by culturing them over-confluently for 4 weeks[27] While this is a substantial improvement for the use of Huh7s in drug metabolism studies, this process is time-consuming and not well suited to many work-flows. A more recent study applied siRNA mediated CDK2 silencing on Huh7s29, albeit with limited success in CYP3A4 induction

Methods

Results

Conclusion