Applicability of second‐generation upcyte^® human hepatocytes for use in CYP inhibition and induction studies

Abstract

Human upcyte® hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte® hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC50 values for each compound correctly classified them as potent inhibitors. Upcyte® hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte® hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUCu/F2, and Cmax,u/Ind50. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte® hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte® hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations.

Ensartinib (X-396) Effectively Modulates Pharmacokinetic Resistance Mediated by ABCB1 and ABCG2 Drug Efflux Transporters and CYP3A4 Biotransformation Enzyme

“…Upcyte Technologies (Hamburg, Germany) prepared second generation upcyte hepatocytes, which were derived from another donor (no. 653-03) [46]. MDCKII, A431, HepG2, and A549 cells were cultivated in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).…”

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

“…Commercially available human hepatocellular carcinoma (HepG2) cells (HB-8065, ATCC, Manassas, VA, USA) were cultured under standard conditions (37 • C, 5% CO 2 ) in polystyrene-based tissue culture flasks (SARSTEDT AG & Co. KG, Nümbrecht, Germany) in Dulbecco's minimal essential medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) superior, 6 mM L-alanyl-Lglutamine and 49.2 g/L NaHCO 3 , all purchased from Biochrom GmbH (Berlin, Germany). HepaFH3 cells [29] and second generation Upcytes ® (donor 653-03, Upcyte ® technologies GmbH, Hamburg, Germany) [33] were cultivated under standard conditions on collagen I (Corning Inc., Corning, NY, USA) coated culture vessels in Hepatocyte Culture Medium (Upcyte ® technologies GmbH, Hamburg, Germany).…”

“…They show some improved liver functions compared to cell lines such as HepG2 or HepaRG, but perform still less than freshly isolated pHHs. Data on the expression and inducibility of CYP enzymes show that essential components of the phase I and phase II reactions are present and active [29][30][31][32][33].…”

Human hepatocyte systems for in vitro toxicology analysis

“…In addition, Upcyte hepatocytes, which represent non-transformed cells genetically stable over many passages, should be an interesting tool to study hepatotoxicity and clearance of xenobiotics [68][69][70]. More specifically, a panel of 11 chemicals known to induce or non-induce CYP3A4 and CYP2B6 were run on second generation Upcyte hepatocytes which proved to be a good prediction model for those CYP enzymes [71]. Mechanistically, expansion of Upcyte hepatocytes is dependent on upregulation of oncostatin M receptor during the growth phase, and differentiation into functional polarized hepatocytes upon withdrawal of oncostatin M [72].…”