Carboxymefloquine, the major metabolite of the antimalarial drug mefloquine, induces drug-metabolizing enzyme and transporter expression by activation of pregnane X receptor

Abstract

Malaria patients are frequently coinfected with HIV and mycobacteria causing tuberculosis, which increases the use of coadministered drugs and thereby enhances the risk of pharmacokinetic drug-drug interactions. Activation of the pregnane X receptor (PXR) by xenobiotics, which include many drugs, induces drug metabolism and transport, thereby resulting in possible attenuation or loss of the therapeutic responses to the drugs being coadministered. While several artemisinin-type antimalarial drugs have been shown to activate PXR, data on nonartemisinin-type antimalarials are still missing. Therefore, this study aimed to elucidate the potential of nonartemisinin antimalarial drugs and drug metabolites to activate PXR. We screened 16 clinically used antimalarial drugs and six major drug metabolites for binding to PXR using the two-hybrid PXR ligand binding domain assembly assay; this identified carboxymefloquine, the major and pharmacologically inactive metabolite of the antimalarial drug mefloquine, as a potential PXR ligand. Two-hybrid PXR-coactivator and -corepressor interaction assays and PXR-dependent promoter reporter gene assays confirmed carboxymefloquine to be a novel PXR agonist which specifically activated the human receptor. In the PXR-expressing intestinal LS174T cells and in primary human hepatocytes, carboxymefloquine induced the expression of drug-metabolizing enzymes and transporters on the mRNA and protein levels. The crucial role of PXR for the carboxymefloquine-dependent induction of gene expression was confirmed by small interfering RNA (siRNA)-mediated knockdown of the receptor. Thus, the clinical use of mefloquine may result in pharmacokinetic drug-drug interactions by means of its metabolite carboxymefloquine. Whether these in vitro findings are of in vivo relevance has to be addressed in future clinical drug-drug interaction studies.

FIG 1

Carboxymefloquine induces the assembly of the human PXR ligand binding domain. HepG2 cells, transiently transfected with firefly luciferase reporter gene plasmid pGL3-G5, normalization plasmid pRL-CMV, and expression plasmids carrying genes encoding GAL4-DBD/PXR-LBD (positions 132 to 188) and VP16-AD/PXR-LBD (positions 189 to 434) fusion proteins were treated with the indicated antimalarials or vehicle DMSO for 6 h. Rifampin was used at 10 μM and all antimalarials were used at 30 μM, except desbutyl-lumefantrine, which was used at 10 μM. The columns show the mean fold induction ± standard deviation (SD) of normalized firefly luciferase activities by chemical treatment, with the activities of cells treated only with DMSO designated 1. Statistically significant differences to this value were calculated by one-sample t test and P values were corrected for multiple testing by Bonferroni's method. **, P < 0.01.

FIG 2

Carboxymefloquine is an agonist of PXR. HepG2-PXR cells, transiently transfected with firefly luciferase reporter gene plasmid pGL3-G5, normalization plasmid pCMVβ, and expression plasmids carrying genes encoding VP16-AD/PXR-LBD (positions 108 to 434) and GAL4-DBD/SRC1-RID (A) or GAL4-DBD/SMRT-RID (B) were treated with 10 μM rifampin (RIF), the indicated doses of carboxymefloquine (CMQ), or 0.1% DMSO for 24 h. The columns show the mean ± SD of relative normalized firefly luciferase activities compared to the activities of cells cotransfected with empty vector pVP16-AD and respective GAL4-DBD fusion protein expression plasmid and treated with DMSO only, which was designated 1 and is indicated by the broken line. The data were analyzed by repeated-measures one-way ANOVA with Dunnett's multiple-comparison test. *, P < 0.05; **, P < 0.01.

FIG 3

Carboxymefloquine specifically activates human PXR. (A) HepG2-PXR cells were transiently transfected with CYP3A4 or CYP2B6 enhancer/promoter reporter gene plasmids, as indicated, and normalization plasmid pCMVβ. The transfected cells were treated with 0.1% DMSO, 10 μM rifampin (RIF), or the indicated doses of carboxymefloquine (CMQ) for 24 h. The columns show the mean ± SD of relative normalized luciferase activities compared to the mean activities of cells treated with DMSO only, which was designated 1. The data were analyzed by repeated-measures one-way ANOVA with Dunnett's multiple-comparison test. (B) HepG2-PXR cells were transfected as in panel A, except that the shRNA expression plasmids carrying negative-control siRNA (sh-CTR) or PXR-specific siRNA (sh-PXR#1 and sh-PXR#3) sequences were cotransfected. The cells were treated for 24 h, as indicated. The columns show the mean fold induction ± SD of normalized luciferase activities by chemical treatment, with the activities of appropriately transfected cells treated with DMSO designated 1. The data were analyzed by two-way ANOVA with Dunnett's multiple-comparison test. (C) HepG2-PXR cells were transfected with CYP3A4 enhancer/promoter reporter gene plasmid and normalization plasmid pCMVβ. The graph shows the dose response of induction by carboxymefloquine. (D) COS-1 cells were transiently cotransfected with combinations of the indicated mouse (m) or human nuclear receptor expression plasmids and the appropriate respective promoter reporter gene plasmids (as specified in Materials and Methods) and normalization plasmid pCMVβ. The transfected cells were treated with 0.1% DMSO or 100 μM carboxymefloquine for 24 h. The columns show the mean fold induction ± SD of normalized luciferase activities by carboxymefloquine, with the activities of the appropriately transfected cells treated with DMSO designated 1. The data were analyzed by one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Carboxymefloquine induces the expression of cytochrome P450 genes and ABCB1 in intestinal LS174T cells. The cells were treated for 48 h with 0.1% DMSO, 10 μM rifampin (RIF), or the indicated doses of carboxymefloquine (CMQ). mRNA expression of the indicated genes was quantified by TaqMan real-time RT-PCR and normalized to the expression levels of 18S rRNA. The columns show the mean fold induction ± SD of mRNA expression by chemical treatment, with the expression in the cells treated with DMSO designated 1. The data were analyzed by one-sample t test. *, P < 0.05.

FIG 5

Carboxymefloquine coordinately induces the expression of ADME genes in primary human hepatocytes. Primary human hepatocyte cultures from 6 donors were each treated with 0.1% DMSO, 30 μM rifampin (RIF), or the indicated doses of carboxymefloquine (CMQ) for 48 h. mRNA expression of the indicated genes was quantified by TaqMan real-time RT-PCR and normalized to the expression levels of 18S rRNA. The data are shown as the fold induction of mRNA expression by chemical treatment, with the expression in the cells treated with only DMSO designated 1; these data are presented as box and whisker plots, with boxes representing the 25th to 75th percentiles, medians indicated by horizontal lines, and whiskers showing the minimum and maximum values. The data were analyzed by Wilcoxon signed-rank test. *, P < 0.05.

FIG 6

PXR mediates induction of CYP3A4 by carboxymefloquine in primary human hepatocytes. Primary human hepatocytes of two donors (RH18 and GH25) were transfected with PXR-specific siRNA (+) or negative-control siRNA (−). The cells were treated for 24 h with 0.1% DMSO, 10 μM rifampin (RIF), or carboxymefloquine (CMQ), as indicated. mRNA expression of PXR (A) and CYP3A4 (B) was quantified by TaqMan real-time RT-PCR and normalized to the expression of 18S rRNA levels. The columns show the means ± SD from triplicate measurements. The expression data are presented relative to the mean expression of cells transfected with control siRNA and treated with DMSO, which was designated 1. The data were analyzed by two-way ANOVA with Dunnett's multiple-comparison test. Significant differences to the respective cells treated with DMSO are indicated. *, P < 0.05; ***, P < 0.001.

FIG 7

Carboxymefloquine induces the protein expression of CYP2B6. Primary human hepatocyte cultures from 3 donors were each treated with 30 μM rifampin (RIF) or the indicated doses of carboxymefloquine (CMQ) for 48 h. Cytochrome P450 and β-actin protein expression were analyzed in total protein homogenates by immunoblotting using specific antibodies. (A) Representative Western blot analysis of a single culture. (B) Fold induction of protein expression by chemical treatment was calculated compared to the expression levels in the respective cells treated with DMSO only, which was designated 1 and is indicated by the broken line. The symbols denote cultures from individual donors.