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Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6␤-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.

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.

ABSTRACT:Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6␤-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16␣-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

No abstract

Pregnane X receptor (PXR) is a receptor that regulates the transcription of genes involved in various functions, including drug metabolism and transport. Conflicting data exist as to whether meclizine, which is a histamine H1 antagonist, is an activator of human PXR (hPXR). Therefore, we conducted a detailed, systematic investigation to assess the effect of meclizine on hPXR activity by performing a cell‐based reporter gene assay, a time‐resolved fluorescence resonance energy transfer competitive ligand‐binding assay, a mammalian two‐hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. Given the pronounced species differences in PXR activation, we also compared the effect of meclizine on rat PXR (rPXR) and hPXR function. In transfected HepG2 cells, meclizine activated hPXR to a greater extent than rPXR. The drug 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 (CYP3A4) mRNA expression in cultured human hepatocytes. However, it did not increase but decreased testosterone 6β‐hydroxylation, suggesting inhibition of CYP3A catalytic activity. In conclusion, our principal findings indicate species differences in hPXR and rPXR activation by meclizine and its mechanism of hPXR activation involves receptor agonism.[Supported by CIHR and MSFHR]

Certain tissues express both pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and various target genes are cross‐regulated by these receptors. Meclizine was reported to be an inverse agonist and antagonist of human CAR (hCAR) and an activator of human PXR (hPXR) in cell‐based reporter gene assays. Therefore, activation of hPXR by meclizine may attenuate its apparent hCAR inverse agonistic effects. To test this hypothesis, we investigated whether meclizine would still be capable of suppressing hCAR target gene expression (CYP2B6) in cultured human hepatocytes, which are known to express hCAR and hPXR. Meclizine (0.03–60 μM) did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist‐mediated increase in CYP2B6 mRNA and CYP2B6‐catalyzed bupropion hydroxylation levels. These findings reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a reporter gene assay and a mammalian two‐hybrid assay in transfected HepG2 cells. Control experiment indicated that PK11195, which is a hCAR inverse agonist and antagonist, decreased constitutive and hCAR agonist‐mediated hCAR activity. In conclusion, meclizine does not act as a hCAR inverse agonist or antagonist in human hepatocytes. Therefore, it is not appropriate to use this drug as a pharmacological tool to study hCAR function in this cell type.[Supported by CIHR and MSFHR]

Ginkgo biloba extract and ginkgolide A induce CYP3A and CYP2B6 in human hepatocytes, but conflicting data exist for the effect of bilobalide on CYP3A expression. Also, it is unknown which chemicals contribute to these effects by the extract. We assessed in human hepatocytes the role of ginkgolide A and bilobalide in the modulation of CYP3A and CYP2B6 expression by G. biloba extract, and compared the effect of the extract and chemicals on these enzymes. As analyzed by HPLC, LC‐MS, and real‐time PCR, control analysis indicated that rifampicin increased CYP3A‐mediated testosterone 6β‐hydroxylation and CYP3A4 mRNA levels, and CITCO increased CYP2B6‐mediated bupropion hydroxylation and CYP2B6 mRNA levels. G. biloba extract (25–200 μg/ml) induced CYP3A to a greater extent than CYP2B6. At a level present in 100 μg/ml of extract, ginkgolide A (1.1 μg/ml) increased CYP3A but not CYP2B6 expression, whereas bilobalide (2.8 μg/ml) had no effect. Ginkgolide A (1.1–10 μg/ml) induced CYP3A to a greater extent than CYP2B6, but bilobalide (5–20 μg/ml) induced both enzymes to a similar extent. CYP3A induction by bilobalide was less than that by ginkgolide A. In summary, G. biloba extract, ginkgolide A, and bilobalide differentially induced CYP3A and CYP2B6. Among the two chemicals, only ginkgolide A contributed to CYP3A induction by the extract, whereas neither of them accounted for CYP2B6 induction. [Supported by CIHR and MSFHR]