Effects of atorvastatin metabolites on induction of drug‐metabolizing enzymes and membrane transporters through human pregnane X receptor

Abstract

BACKGROUND AND PURPOSEAtorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACHLigand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTSAll atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONSAtorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.

Carboxymefloquine, the Major Metabolite of the Antimalarial Drug Mefloquine, Induces Drug-Metabolizing Enzyme and Transporter Expression by Activation of Pregnane X Receptor

“…The absolute quantification of ABCB1, CYP2B6, CYP3A4, and 18S rRNA gene expression levels in LS174T cells (ATCC) was performed with cDNA corresponding to 25 ng or 25 pg (18S rRNA) total RNA using the 7500 real-time PCR system (Life Technologies), as described previously (20). The respective TaqMan assays are indicated below.…”

“…The COS-1 cells were cultivated as described previously (14). The culture of HepG2 cells, purchased from ATCC (Manassas, VA), and the generation and culture of HepG2-PXR cells stably transfected with a human PXR expression plasmid have been described (20). One day before transfection, the COS-1 cells were plated at 3 ϫ 10 4 cells per well and the HepG2 and HepG2-PXR cells at 1.5 ϫ 10 5 cells per well in 24-well plates.…”

“…Total protein homogenates were prepared by lysing cells for 30 min on ice in 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1% (vol/vol) Nonidet P-40, supplemented with Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL) and quantified using the bicinchoninic acid method. Thirty micrograms of each protein sample was analyzed by SDS-polyacrylamide gel electrophoresis and subsequent Western blotting (20), using the antibodies WB-3A4 and INHIBITION MAB-2B6 (BD Biosciences, Heidelberg, Germany) specific for CYP3A4 and CYP2B6, respectively, and ␤-actin antibody (clone AC-15) from Sigma-Aldrich. The protein bands were quantified with the digital chargecoupled-device camera Stella and AIDA software (Raytest, Straubenhardt, Germany).…”

“…The experimental procedures were performed according to the institutional guidelines for liver resections of tumor patients, which included each patient's written informed consent, and these were approved by the local ethics committee of the Eberhard-Karls University of Tübingen, Germany. For the induction experiments, the isolated cells were seeded at 1.5 ϫ 10 6 cells per well into collagen type I-coated 6-well plates and treated with chemicals, as described previously (20).…”

“…The assays were done in triplicate. The following TaqMan assays have been reported: ABCB1 and CYP2B6 (11), UGT1A3 (23), and EPHX1 and 18S rRNA (20). TaqMan gene expression assays, consisting of predesigned commercial primer-probe sets (Life Technologies), were used to quantify the other genes: Hs00166123_m1 (ABCC2), Hs00868409_s1 (CYP2A6), Hs00946140_g1 (CYP2C8), Hs00604506_m1 (CYP3A4), Hs01114267_m1 (PXR), Hs99999902_m1 (RPLP0), and Hs00272374_m1 (SLCO1B1).…”