title: Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4.
abstract: The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug interactions (DDIs) that result from mechanism-based inactivation or induction of cytochrome P450 (P450). Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently extensive uncertainty exists in steady-state predictions for DDIs. In the current investigations, the stable isotope labeled amino acids in culture technique was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4 and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. Thekdegestimate was 0.026 ± 0.005 hour-1This value was reproduced by cultures derived across four individual donors. For these cultures, the data indicated that CYP3A4 mRNA and total protein expression (i.e., labeled and unlabeled P450s), and activity were stable over the period where degradation had been determined. Thiskdegvalue for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches but also employed micropatterned primary human hepatocytes as the in vitro model.
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California.
authors.forename: Ryan H
authors.initials: RH
authors.lastname: Takahashi
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California.
authors.forename: Sheerin K
authors.initials: SK
authors.lastname: Shahidi-Latham
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California.
authors.forename: Susan
authors.initials: S
authors.lastname: Wong
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California jaechang@gmail.com.
authors.forename: Jae H
authors.initials: JH
authors.lastname: Chang
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004347:Drug Interactions / Q000502:physiology; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D007554:Isotopes / Q000378:metabolism*; D007700:Kinetics; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007554:Isotopes; D012333:RNA, Messenger; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human