Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds

Abstract

In vitro models based on primary human hepatocytes (PHH) have been advanced for clearance (CL) prediction of metabolically stable compounds, representing state-of-the-art assay systems for drug discovery and development. Yet, limited cell availability and large interindividual variability of metabolic profiles remain shortcomings of PHH. Upcyte human hepatocytes (UHH) represent a novel hepatic cell system derived from PHH, exhibiting proliferative capacity for approximately 35 population doublings. UHH from three donors were evaluated during culture for up to 18 days, investigating relative mRNA expression and in situ enzyme activity of cytochrome P450s (P450s), UDP-glucuronosyltransferases, and sulfotransferases. Furthermore, UHH were used for predicting hepatic CL of 21 marketed low to intermediate CL drugs. In a typical experiment, expansion from 3.9 3 10 6 up to 8.5 3 10 7 cells was achieved during subculture.When maintained at confluence, transcripts of major P450s were expressed at donor-specific levels with sustained activities for the majority of isoforms, showing generally low CYP1A2 and high CYP2B6 activity levels. For donor 151-03, CL prediction based on depletion experiments resulted in an average fold error of 2.0, and 80% of compounds being predicted within twofold to in vivo CL for a subset of 10 low CL drugs. UHH showed sustained and consistent activity of drug-metabolizing enzymes (DME), resulting in highly reproducible CL prediction performance. In conclusion, UHH show promising potential as alternative to PHH for standardized in vitro applications in discovery research based on their stable, hepatocytelike DME phenotype and virtually unlimited cell availability.

Human hepatocyte systems for in vitro toxicology analysis

“…The authors could demonstrate significant expression levels of relevant phase I and phase II enzymes, their inducibility and biotransformation activity. 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].…”

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

“…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].…”

Quantitative Expression of Hepatobiliary Transporters and Functional Uptake of Substrates in Hepatic Two-Dimensional Sandwich Cultures: A Comparative Evaluation of Upcyte and Primary Human Hepatocytes

“…Unlike HepG2, differentiated UHH exhibit key attributes of mature hepatic cells [including functional aryl hydrocarbon receptor-, constitutive androstane receptor-, and pregnane X receptormediated P450 regulation (Ramachandran et al, 2015); functional epithelial polarization and bile flow (Levy et al, 2015); and ureogenesis and albumin synthesis (Tolosa et al, 2016)], supporting their usefulness as a screening in vitro tool for DDIs and hepatotoxicity assessment (Ramachandran et al, 2015;Tolosa et al, 2016). In addition, we reported recently on the P450-, uridine 59-diphospho-glucuronosyltransferase-, and sulfotransferase-mediated metabolic capability of cultured UHH and demonstrated their utility as a hepatic model for intrinsic clearance determinations of slowly metabolized drugs (Schaefer et al, 2016). Despite this broad characterization of UHH, only limited data on the expression of drug transporters, in particular for clinically relevant hepatic transporters including organic aniontransporting polypeptide OATP1B1/1B3 (encoded by SLCO1B1/1B3), are currently available.…”

“…UHH from female Caucasian donors (151-03, 653-03, 10-03) were purchased from Upcyte Technologies GmbH at a population doubling range of 25-30. Thawing and culturing of cryopreserved UHH was performed as described previously (Schaefer et al, 2016). Briefly, after rapid thawing at 37°C, cells were suspended in 50 ml Hepatocyte Thawing Medium and sedimented at 90g for 5 minutes at room temperature.…”