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

Human CYP3A4, the major human, intestinal, drug metabolizing cytochrome P45 0, has been introduced into three mammalian cell lines (Caco-2, MDCK and LL C-PK1) suitable for making drug permeability measurements. The levels and s tability of expression were analyzed by enzyme assays (testosterone 6 beta -hydroxylase and nifedipine oxidase). Long term, stable CYP3A4 expression/c ell 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 obtaine d. The highest level of CYP3A4 catalytic activity was obtained in LLC-PK1 c ells. In this system, CYP3A4 activity levels appeared comparable to median level human intestinal microsomes. Metabolite formation and inhibition kine tics were examined in cell monolayers. Nifedipine was found to be extensive ly metabolized (19%) during passage across cell monolayers. In general, aff inity related parameters (apparent K-m and apparent K-i) were 1.5- to three -fold higher under conditions of Aux through the monolayers relative to ste ady-state conditions. These systems should be useful for examining the role of intestinal CYP3A4 in first-pass metabolism and drug-drug interactions. (C) 2000 Elsevier Science B.V. All rights reserved.