Figure 1.
Effects of ketoconazole cis-enantiomers on CYP3A4 mRNA expression in HepG2 cells and human hepatocytes.
(i) HepG2 cells were seeded in 6-well plates and stabilized for 16 h. Experiments were performed in four consecutive cell passages. (ii) Primary human hepatocytes from three different donors (HH52, HH54 and Hep220770) were used. Cells were incubated for 24 h with RIF (10 µM), vehicle (DMSO; 0.1% v/v) and ketoconazole ((+), (−), (rac); 1 µM, 30 µM, 50 µM). RT-PCR analyses of CYP3A4 mRNA are shown. The data are the mean ± SD from triplicate measurements and are expressed as a fold induction over vehicle-treated cells. The data were normalized to GAPDH mRNA levels. An asterisk (*) indicates that the value is significantly different from the activity of vehicle-treated cells.
Figure 2.
Effects of ketoconazole cis-enantiomers on CYP3A4 protein expression in HepG2 cells and human hepatocytes.
Western blots of CYP3A4 and β-actin from three different human hepatocytes cultures (HH52, HH54 and Hep220770) and from two consecutive passages of HepG2 cells are shown. Cells were incubated for 48 h with RIF (10 µM), vehicle (DMSO; 0.1% v/v) and ketoconazole ((+), (−), (rac); 1 µM, 30 µM, 50 µM). Density of bands was quantified by densitometry.
Figure 3.
Effect of ketoconazole cis-enantiomers on transcriptional activity of pregnane X receptor PXR in transiently transfected LS174T cells.
LS174T cells, transiently transfected with p3A4-luc reporter, were seeded in 24-well plates, stabilized for 16 h and then incubated for 24 h with (+)-KET, (−)-KET and rac-KET at concentrations ranging from 0.1 µM to 100 µM. The vehicle was DMSO (0.1% v/v). Model activator of PXR was rifampicin (RIF; 10 µM). Treatments were performed in triplicates. Upper panel: MTT test; The data are the mean from experiments from three consecutive passages of cells and are expressed as a percentage of viability of control cells. The values of IC50 were calculated and are indicated in a figure. Middle panel: Agonist mode - Transfected LS174T cells were incubated with KET in the absence of RIF (10 µM). The data are the mean from experiments from two consecutive passages of cells and are expressed as a fold induction of luciferase activity over control cells. Lower panel: Antagonist mode - Transfected LS174T cells were incubated with KET in the presence of RIF (10 µM). The data are the mean from experiments from two consecutive passages of cells and are expressed as a percentage of maximal induction attained by RIF. The values of IC50 were calculated and the average values are indicated in figures.
Figure 4.
Effect of ketoconazole cis-enantiomers on catalytic activity of CYP3A4/5 in human liver microsomes.
Inhibition of the CYP3A4/5 catalytic activity in assay with specific substrate testosterone (upper panel) and midazolam (lower panel) in human liver microsomes. Racemate, (+)-KET and (−)-KET were used in concentrations 0.3 µM, 1 µM, 2 µM, 3 µM, 5 µM and 10 µM. Inhibition of activity is determined as the mean ± SD and expressed in per cent as activity remaining relative to control (100%, without ketoconazole).
Table 1.
Enzyme kinetics parameters for in vitro biotransformation two prototypic CYP3A4/5 substrates, testosterone and midazolam, with ketoconazole and two cis enantiomers, (+)-KET and (−)-KET, as inhibitors.