title: 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 × 10(6) up to 8.5 × 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, hepatocyte-like DME phenotype and virtually unlimited cell availability.
authors.affiliation: Department of Drug Discovery Support / Metabolism and Bioanalysis, Boehringer Ingelheim Pharma, Biberach an der Riss, Germany (M.S., G.S., D.B.); and Department of Chemistry, Technische Universität Berlin, Berlin, Germany (R.D.S.).
authors.forename: Michelle
authors.initials: M
authors.lastname: Schaefer
authors.affiliation: Department of Drug Discovery Support / Metabolism and Bioanalysis, Boehringer Ingelheim Pharma, Biberach an der Riss, Germany (M.S., G.S., D.B.); and Department of Chemistry, Technische Universität Berlin, Berlin, Germany (R.D.S.) gerhard.schaenzle@boehringer-ingelheim.com.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schänzle
authors.affiliation: Department of Drug Discovery Support / Metabolism and Bioanalysis, Boehringer Ingelheim Pharma, Biberach an der Riss, Germany (M.S., G.S., D.B.); and Department of Chemistry, Technische Universität Berlin, Berlin, Germany (R.D.S.).
authors.forename: Daniel
authors.initials: D
authors.lastname: Bischoff
authors.affiliation: Department of Drug Discovery Support / Metabolism and Bioanalysis, Boehringer Ingelheim Pharma, Biberach an der Riss, Germany (M.S., G.S., D.B.); and Department of Chemistry, Technische Universität Berlin, Berlin, Germany (R.D.S.).
authors.forename: Roderich D
authors.initials: RD
authors.lastname: Süssmuth
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D014453:Glucuronosyltransferase / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic / Q000502:physiology*; D007700:Kinetics; D008099:Liver / Q000378:metabolism / Q000502:physiology*; D008297:Male; D008657:Metabolic Clearance Rate / Q000502:physiology*; D008875:Middle Aged; D055815:Young Adult
chemical_list: D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase