Quantification of human hepatocyte cytochrome P450 enzymes and transporters induced by HIV protease inhibitors using newly validated LC-MS/MS cocktail assays and RT-PCR

Biopharm Drug Dispos. 2012 May;33(4):207-17. doi: 10.1002/bdd.1788. Epub 2012 May 2.


Human immunodeficiency virus (HIV) protease inhibitors (PIs) produce profound and unpredictable drug-drug interactions (DDIs) that cannot be explained fully by their inhibition/inactivation of CYP3A enzymes. Delineating and quantifying the CYPs and transporters inducible by PIs are crucial in developing an integrative mechanistic understanding and prediction of PI-based DDIs. To do so, two LC-MS/MS cocktail assays were modified and validated simultaneously to quantify the CYP activity of CYP3A, 2B6, 2C8, 2C9, 2C19, 1A, 2E1, 2A6 and 2D6 enzymes. These new assays were applied to evaluate the induction potential of eight PIs in microsomes isolated from PI-treated human hepatocytes. The mRNA expression of these CYPs and transporters (OATP1B1, OATP1B3, OATP1A2, MDR1, MRP2 and MRP4) was also evaluated using relative RT-PCR. The majority of PIs were net inducers of CYP3As and 2B6 at both the mRNA and activity level (> 2-fold), while ritonavir, saquinavir, nelfinavir or lopinavir did not induce CYP3A activity (< 2-fold), presumably due to CYP3A inactivation. OATP1B1 and MDR1 were the only two hepatic transporters induced (> 2-fold) by the PIs. Amprenavir was the most potent net inducer. In conclusion, our validated cocktail assays can be implemented to comprehensively quantify CYP activities in human liver microsomes and hepatocyte studies. The results also provide the much needed data on the net induction potential of the PIs for hepatic CYPs and transporters. A qualitative agreement was observed between our results and published PI-based DDIs, suggesting that human hepatocytes are a useful platform for more extensive and quantitative in vitro-in vivo prediction of PI-based DDIs.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Chromatography, Liquid / methods
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / drug effects*
  • Cytochrome P-450 Enzyme System / metabolism
  • Enzyme Induction / drug effects*
  • Female
  • Gene Expression Regulation / drug effects
  • HIV Protease Inhibitors / pharmacology*
  • Hepatocytes / drug effects*
  • Hepatocytes / enzymology
  • Humans
  • Male
  • Membrane Transport Proteins / genetics
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Middle Aged
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tandem Mass Spectrometry / methods


  • HIV Protease Inhibitors
  • Membrane Transport Proteins
  • RNA, Messenger
  • Cytochrome P-450 Enzyme System