Abstract

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC50 1.69 µM, Ki 0.92 µM for testosterone, IC50 1.46 µM, Ki 2.52 µM for midazolam; (−)-ketoconazole IC50 0.90 µM, Ki 0.17 µM for testosterone, IC50 1.04 µM, Ki 1.51 µM for midazolam).

Highlights

  • Ketoconazole is an imidazole antifungal drug that is used both systemically and topically, in the treatment of various fungal infections

  • We have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes

  • Primary human hepatocytes used in this study were obtained from two sources: (i) from multiorgan donors HH52 and LH54; the use of liver cells of donors HH52 and HH54 was approved by ‘‘Ethical committee at the Faculty Hospital Olomouc’’, and it was in accordance with Transplantation law #285/2002 Sb; ‘‘Ethical committee at the Faculty Hospital Olomouc’’ waived the authors from obtaining consent from the of kin, regarding human hepatocytes obtained from liver donors HH52 and HH54. (ii) long-term human hepatocytes in monolayer Batch HEP220770 were purchased from Biopredic International (Biopredic International, Rennes, France)

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Summary

Introduction

Ketoconazole is an imidazole antifungal drug that is used both systemically and topically, in the treatment of various fungal infections. While oral ketoconazole was discontinued in many countries, there is increasing evidence that it might be a drug of choice in the therapy of systemic infections, if the first line treatment with other antifungals fails. The mechanisms of ketoconazole-drug interactions are multiple and the most frequently, they are caused by inhibition of catalytic activity of main biotransformation enzyme CYP3A4, and CYP2C9 [3]. Ketoconazole blocks interactions between PXR and its transcriptional co-activator HNF4a [7]. The transcriptional activation of genes regulating biotransformation and transport by the liganded PXR was inhibited by ketoconazole. Mutations at the AF-2 surface of the human PXR ligand-binding domain indicated that ketoconazole may interact with specific residues outside the ligand-binding

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