Analysis of drug transport and metabolism in cell monolayer systems that have been modified by cytochrome P4503A4 cDNA-expression

Eur J Pharm Sci. 2000 Nov;12(1):63-8. doi: 10.1016/s0928-0987(00)00141-x.


Human CYP3A4, the major human, intestinal, drug metabolizing cytochrome P450, has been introduced into three mammalian cell lines (Caco-2, MDCK and LLC-PK1) suitable for making drug permeability measurements. The levels and stability of expression were analyzed by enzyme assays (testosterone 6beta-hydroxylase and nifedipine oxidase). Long term, stable CYP3A4 expression/cell growth rate was obtained in MDCK cells. In the LLC-PK1 system, shorter term, stable expression was achieved. However, in Caco-2 cells, derivatives with better properties than those previously reported could not be obtained. The highest level of CYP3A4 catalytic activity was obtained in LLC-PK1 cells. In this system, CYP3A4 activity levels appeared comparable to median level human intestinal microsomes. Metabolite formation and inhibition kinetics were examined in cell monolayers. Nifedipine was found to be extensively metabolized (19%) during passage across cell monolayers. In general, affinity related parameters (apparent Km and apparent Ki) were 1.5- to three-fold higher under conditions of flux through the monolayers relative to steady-state conditions. These systems should be useful for examining the role of intestinal CYP3A4 in first-pass metabolism and drug-drug interactions.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Atenolol / metabolism
  • Biological Transport
  • Cell Line
  • Cell Membrane Permeability
  • Cimetidine / metabolism
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • DNA, Complementary
  • Erythromycin / metabolism
  • Glucose / metabolism
  • Humans
  • Mammals
  • Mannitol / metabolism
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Pharmaceutical Preparations / metabolism*
  • Phenylalanine / metabolism
  • Propranolol / metabolism
  • Recombinant Proteins / metabolism
  • Testosterone / metabolism
  • Tumor Cells, Cultured
  • Verapamil / metabolism


  • DNA, Complementary
  • Pharmaceutical Preparations
  • Recombinant Proteins
  • Mannitol
  • Testosterone
  • Phenylalanine
  • Atenolol
  • Erythromycin
  • Cimetidine
  • Cytochrome P-450 Enzyme System
  • Propranolol
  • Verapamil
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cyp2c13 protein, rat
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Glucose