PT  - JOURNAL ARTICLE
AU  - Ryan H. Takahashi
AU  - Sheerin K. Shahidi-Latham
AU  - Susan Wong
AU  - Jae H. Chang
TI  - Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4
AID  - 10.1124/dmd.116.074393
DP  - 2017 Jun 01
TA  - Drug Metabolism and Disposition
PG  - 581--585
VI  - 45
IP  - 6
4099  - http://dmd.aspetjournals.org/content/45/6/581.short
4100  - http://dmd.aspetjournals.org/content/45/6/581.full
SO  - Drug Metab Dispos2017 Jun 01; 45
AB  - 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. The kdeg estimate was 0.026 ± 0.005 hour−1. This 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. This kdeg value 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.