Differential Effect of Meclizine on the Activity of Human Pregnane X Receptor and Constitutive Androstane Receptor

Abstract

Conflicting data exist as to whether meclizine is an activator of human pregnane X receptor (hPXR). Therefore, we conducted a detailed, systematic investigation to determine whether meclizine affects hPXR activity by performing a cell-based reporter gene assay, a time-resolved fluorescence resonance energy transfer competitive ligand-binding assay, a mammalian twohybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. In pregnane X receptor (PXR)-transfected HepG2 cells, meclizine activated hPXR to a greater extent than rat PXR. It bound to hPXR ligand-binding domain and recruited steroid receptor coactivator-1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene expression (CYP3A4) in human hepatocytes. However, it did not increase but decreased testosterone 6␤-hydroxylation, suggesting inhibition of CYP3A catalytic activity.Meclizine has also been reported to be an inverse agonist and antagonist of human constitutive androstane receptor (hCAR). Therefore, given that certain tissues (e.g., liver) express both hPXR and hCAR and that various genes are cross-regulated by them, we quantified the expression of a hCAR-and hPXRregulated gene (CYP2B6) in cultured human hepatocytes treated with meclizine. This drug did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist-mediated increase in CYP2B6 mRNA and CYP2B6-catalyzed bupropion hydroxylation levels. These observations reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a hCAR reporter gene assay and mammalian two-hybrid assay. In conclusion, meclizine is a hPXR agonist, and it does not act as a hCAR inverse agonist or antagonist in cultured human hepatocytes.

Isoform-Selective Activation of Human Constitutive Androstane Receptor by Ginkgo biloba Extract: Functional Analysis of the SV23, SV24, and SV25 Splice Variants

“…A hCAR-dependent mammalian two-hybrid assay was performed as described in detail previously (Lau et al, 2011). At 5 h after plating, HepG2 cells were cotransfected with pVP16-hCAR-WT-LBD (40 ng/well), pVP16-hCAR-SV23-LBD (40 ng/well), or pVP16 empty vector (40 ng/well) along with a coactivator expression plasmid (10 ng/well), pCMV6-XL4-hRXR␣ (10 ng/well), pGL4.74[hRluc/TK] internal control plasmid (10 ng/well), and pFR-luc reporter plasmid (100 ng/well).…”

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

“…pGL3-basic-CYP2B1-luc reporter was generously provided by Dr. Thomas A. Kocarek (Wayne State University, Detroit, MI) (Kocarek and Mercer-Haines, 2002). pGL3-basic-CYP2B6-PBREM/XREM-luc reporter (Lau et al, 2011b) and pGL3-basic-CYP3A4-XREM-luc reporter (Lau et al, 2010) were constructed as described previously. All constructs were sequenced by the Nucleic Acid Protein Service Unit at the University of British Columbia (Vancouver, BC, Canada), and the identity of plasmids was confirmed by comparing their sequences with published sequences.…”

“…At the end of the treatment period, cultured human hepatocytes were washed with Hanks' balanced salt solution (HBSS) and incubated for 20 min at 37°C with 0.5 ml of HBSS containing bupropion (final concentration of 500 M) dissolved in DMSO (final concentration of 0.1% v/v in HBSS). The bupropion hydroxylation assay was performed using an ultraperformance liquid chromatography-tandem mass spectrometry assay as described previously (Lau et al, 2011b).…”

CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

“…a well known peripheral benzodiazepine receptor ligand, PK11195, as a CAR inhibitor-the most potent inhibitor of CAR identified thus far (Li et al, 2008). Interestingly, all the known CAR inhibitors are also moderate-to-potent activators of PXR function, complicating expression levels of their shared target genes Moore et al, 2002;Huang et al, 2007;Li et al, 2008;Lau et al, 2011).…”

“…Clotrimazole (CLZ), meclizine, and androstanol are moderate inhibitors of CAR that function in both in vitro biochemical assays and cell-based transfection assays (Forman et al, 1998;Moore et al, 2000;Tzameli et al, 2000;Moore et al, 2002;Huang et al, 2004;Omiecinski et al, 2011). We and others, however, have observed that all these CAR inhibitors are also moderate to potent activators of PXR function Moore et al, 2002;Li et al, 2008;Lau et al, 2011).…”