Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4

Ryan H. Takahashi, Sheerin K. Shahidi-Latham, Susan Wong and Jae H. Chang
Drug Metabolism and Disposition June 2017, 45 (6) 581-585; DOI: https://doi.org/10.1124/dmd.116.074393
Ryan H. Takahashi
Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sheerin K. Shahidi-Latham
Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan Wong
Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jae H. Chang
Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • Fig. 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    Representative CYP3A4 stability data for human HepatoPac cocultures through the time period used for P450 degradation rate determinations. Data are mean from n = 3 replicates with error bars indicating S.D. mRNA expressions are normalized to day 10 (left axis) and CYP3A4 activity (measured as testosterone-6β-hydroxylation) and protein expression (right axis) are normalized to one million hepatocytes. Protein expressions were determined by mass spectrometry–determined abundance of peptide EVTNFLR relative to stable isotope (13C615N) labeled peptide used as an internal standard calibrator. Data shown are for one hepatocyte donor (3121A).

  • Fig. 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    P450 degradation of CYP3A4 as measured by SIL-containing peptide depletion for four donors with protein-specific peptides: (A) SLLSPTFTSGK, (B) EVTNFLR, (C) VEINGMFIPK, and (D) YWTEPEK. Labeled peptide abundances are expressed as percentages of the total measured peptide, n = 3 per time point with error bars representing S.D. Days are marked from the start of the HepatoPac cultures with SIL amino acids supplemented for days 0–10. Each line represents the linear regression for an individual donor from which the slope was used to calculate the protein t1/2.

Tables

  • Figures
  • Additional Files
    • View popup
    TABLE 1

    Degradation rates (kdeg) and half-lives (t1/2) determined using HepatoPac-SILAC for CYP3A4

    Data were calculated from four donors and are reported as mean and S.D. The average kdeg and t1/2 values determined for the three nonarginine-containing CYP3A4 peptides were 0.023 ± 0.002 h−1 and 29.7 ± 2.3 hours, respectively. The kdeg and t1/2 values for the arginine-containing peptide (EVTNFLR) were 0.033 ± 0.005 h−1 and 21.0 ± 2.6 hours, respectively. Recent kdeg and t1/2 value determinations for CYP3A4 using HepatoPac by mRNA following siRNA or interleukin 6 inhibition were 0.0240 h−1 and 28.9 hours, respectively (Ramsden et al., 2015); and by mRNA following induction they were 0.0261 h−1 and 26 hours, and 49 hours by activity following rifampicin induction (Dixit et al., 2016).

    P450Peptideakdegt1/2
    h−1h
    CYP3A4SLLSPTFTSGK0.023 ± 0.00229.6 ± 1.9
    CYP3A4DVEINGMFIPK0.023 ± 0.00129.6 ± 1.0
    CYP3A4YWTEPEK0.023 ± 0.00129.8 ± 0.8
    • ↵a The SRM parameters of the specific peptides used for LC-MS/MS protein measurements are provided in Supplemental Table 2.

Additional Files

  • Figures
  • Tables
  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Data -

      Supplemental Table 1 - Donor information for hepatocytes used for HepatoPac cultures

      Supplemental Table 2 - Summary of tryptic peptides and their selected reaction-monitoring parameters used for LC-MS/MS protein measurements

      Supplemental Table 3 - CYP3A4 stability data (mRNA, protein expression, and enzymatic activity) for four HepatoPac donors for Days 10-13 of SILAC culture

      Supplemental Table 4 - Calculated t1/2 (h) and kdeg (h-1) values from individual donors for CYP3

      Supplemental Figure 1 - Representative SRM chromatograms showing CYP3A4 peptide, SLLSPTFTSGK, for one HepatoPac donor over Days 10-13 with SILAC

PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition: 45 (6)
Drug Metabolism and Disposition
Vol. 45, Issue 6
1 Jun 2017
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Editorial Board (PDF)
  • Front Matter (PDF)
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

Utility of SILAC in HepatoPac to Determine CYP3A4 kdeg

Ryan H. Takahashi, Sheerin K. Shahidi-Latham, Susan Wong and Jae H. Chang
Drug Metabolism and Disposition June 1, 2017, 45 (6) 581-585; DOI: https://doi.org/10.1124/dmd.116.074393

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

Utility of SILAC in HepatoPac to Determine CYP3A4 kdeg

Ryan H. Takahashi, Sheerin K. Shahidi-Latham, Susan Wong and Jae H. Chang
Drug Metabolism and Disposition June 1, 2017, 45 (6) 581-585; DOI: https://doi.org/10.1124/dmd.116.074393
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Material and Methods
    • Results and Discussion
    • Acknowledgments
    • Authorship Contributions:
    • Footnotes
    • Abbreviations
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Determination of Acyl-, O-, and N-Glucuronide
  • TMDD Affects PK of IL-10 Fc-fusion Proteins
  • Uptake as the RDS in Pevonedistat Hepatic Clearance
Show more Articles

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2022 by the American Society for Pharmacology and Experimental Therapeutics