Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, β-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.

• Keywords
• cytochrome P450 3A4, gene reporter assay, mRNA expression, precision-cut liver slices, pregnane X receptor, protein expression, sesquiterpene,
• MeSH
• Aldo-Keto Reductases metabolism   MeSH
• Hep G2 Cells MeSH
• Cytochrome P-450 CYP3A metabolism   MeSH
• Farnesol pharmacology   MeSH
• Hepatocytes metabolism   MeSH
• Liver enzymology   MeSH
• Carbonyl Reductase (NADPH) metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Metabolic Clearance Rate MeSH
• Monocyclic Sesquiterpenes pharmacology   MeSH
• Polycyclic Sesquiterpenes pharmacology   MeSH
• Pregnane X Receptor agonists   metabolism   MeSH
• Receptors, Aryl Hydrocarbon agonists   metabolism   MeSH
• Cytochrome P450 Family 2 metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Sesquiterpenes pharmacology   MeSH
• Cytochrome P-450 Enzyme System metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aldo-Keto Reductases MeSH
• caryophyllene oxide MeSH Browser
• caryophyllene MeSH Browser
• Cytochrome P-450 CYP3A MeSH
• cytochrome P-450 CYP2C subfamily MeSH Browser
• Farnesol MeSH
• humulene MeSH Browser
• Carbonyl Reductase (NADPH) MeSH
• RNA, Messenger MeSH
• Monocyclic Sesquiterpenes MeSH
• nerolidol MeSH Browser
• Polycyclic Sesquiterpenes MeSH
• Pregnane X Receptor MeSH
• Receptors, Aryl Hydrocarbon MeSH
• Cytochrome P450 Family 2 MeSH
• Sesquiterpenes MeSH
• Cytochrome P-450 Enzyme System MeSH

• Publication type
• Letter MeSH

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).

• MeSH
• Biocatalysis drug effects   MeSH
• Hep G2 Cells MeSH
• Cytochrome P-450 CYP3A genetics   metabolism   MeSH
• Transcription, Genetic drug effects   MeSH
• Hepatocytes drug effects   enzymology   MeSH
• Microsomes, Liver drug effects   metabolism   MeSH
• Ketoconazole chemistry   pharmacology   MeSH
• Kinetics MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Pregnane X Receptor MeSH
• Gene Expression Regulation, Enzymologic drug effects   MeSH
• Genes, Reporter MeSH
• Stereoisomerism MeSH
• Receptors, Steroid genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochrome P-450 CYP3A MeSH
• Ketoconazole MeSH
• RNA, Messenger MeSH
• Pregnane X Receptor MeSH
• Receptors, Steroid MeSH

Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(-)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5-20× higher agonist activity (efficacy), as compared to (-)-KET; both enantiomers were AhR antagonists with equal potency (IC50). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (-)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (-)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (-)-KET are weak ligands of AhR that displaced [3H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.

• MeSH
• Antifungal Agents chemistry   pharmacology   MeSH
• Hep G2 Cells MeSH
• Candida drug effects   MeSH
• Cytochrome P-450 CYP1A1 genetics   metabolism   MeSH
• Cytochrome P-450 CYP1A2 genetics   metabolism   MeSH
• Transcription, Genetic drug effects   MeSH
• Hepatocytes drug effects   metabolism   MeSH
• Ketoconazole chemistry   pharmacology   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Guinea Pigs MeSH
• Mice MeSH
• Receptors, Aryl Hydrocarbon metabolism   MeSH
• Aged MeSH
• Signal Transduction drug effects   MeSH
• Stereoisomerism MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Guinea Pigs MeSH
• Male MeSH
• Mice MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antifungal Agents MeSH
• Cytochrome P-450 CYP1A1 MeSH
• Cytochrome P-450 CYP1A2 MeSH
• Ketoconazole MeSH
• RNA, Messenger MeSH
• Receptors, Aryl Hydrocarbon MeSH

The small/short heterodimer partner (SHP, NR0B2) is a nuclear receptor corepressor lacking a DNA binding domain. SHP is induced by bile acid-activated farnesoid X receptor (FXR) resulting in CYP7A1 gene suppression. In contrast, Pregnane X receptor (PXR) activation by its ligands was recently suggested to inhibit SHP gene transactivation to maximize the induction of PXR target genes. However, there are also conflicting reports in literature whether PXR or rodent Pxr activation down-regulates SHP/Shp expression. Moreover, the PXR-mediated regulation of the SHP gene has been studied only at the SHP mRNA and transactivation (gene reporter assay) levels. In this study, we studied the effect of rifampicin, a prototype PXR ligand, on SHP mRNA, and protein expression in three primary human hepatocyte cultures. We found that SHP mRNA is not systematically down-regulated in hepatocyte in culture after 24 h treatment with rifampicin. Consistently, we did not observe down-regulation of SHP protein in primary human hepatocytes after 24 and 48 h of incubation with rifampicin. We can conclude that although we observed slight down-regulation of SHP mRNA and protein in several hepatocyte preparations, the phenomenon is unlikely critical for PXR-mediated induction of its target genes.

• Keywords
• CYP3A4, PXR, SHP, cytochrome P450, induction,
• Publication type
• Journal Article MeSH

Metformin is widely used in the treatment of type-2 diabetes. The pleotropic effects of metformin on glucose and lipid metabolism have been proposed to be mediated by the activation of AMP-activated protein kinase (AMPK) and the subsequent up-regulation of small heterodimer partner (SHP). SHP suppresses the functions of several nuclear receptors involved in the regulation of hepatic metabolism, including pregnane X receptor (PXR), which is referred to as a "master regulator" of drug/xenobiotic metabolism. In this study, we hypothesize that metformin suppresses the expression of CYP3A4, a main detoxification enzyme and a target gene of PXR, due to SHP up-regulation. We employed various gene reporter assays in cell lines and qRT-PCR in human hepatocytes and in Pxr(-/-) mice. We show that metformin dramatically suppresses PXR-mediated expression of CYP3A4 in hepatocytes. Consistently, metformin significantly suppressed the up-regulation of Cyp3a11 mRNA in the liver and intestine of wild-type mice, but not in Pxr(-/-) mice. A mechanistic investigation of the phenomenon showed that metformin does not significantly up-regulate SHP in human hepatocytes. We further demonstrate that AMPK activation is not involved in this process. We show that metformin disrupts PXR's interaction with steroid receptor coactivator-1 (SRC1) in a two-hybrid assay independently of the PXR ligand binding pocket. Metformin also inhibited vitamin D receptor-, glucocorticoid receptor- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4 mRNA in human hepatocytes. We show, therefore, a suppressive effect of metformin on PXR and other ligand-activated nuclear receptors in transactivation of the main detoxification enzyme CYP3A4 in human hepatocytes.

• MeSH
• Transcriptional Activation MeSH
• Cytochrome P-450 CYP3A genetics   metabolism   MeSH
• Hepatocytes drug effects   metabolism   MeSH
• Hypoglycemic Agents pharmacology   MeSH
• Nuclear Receptor Coactivators metabolism   MeSH
• Constitutive Androstane Receptor MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Metformin pharmacology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Reflex, Righting drug effects   MeSH
• Pregnane X Receptor MeSH
• AMP-Activated Protein Kinases physiology   MeSH
• Receptors, Cytoplasmic and Nuclear metabolism   physiology   MeSH
• Receptors, Glucocorticoid physiology   MeSH
• Receptors, Calcitriol physiology   MeSH
• Genes, Reporter MeSH
• Signal Transduction MeSH
• Receptors, Steroid genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cyp3a11 protein, mouse MeSH Browser
• Cytochrome P-450 CYP3A MeSH
• Hypoglycemic Agents MeSH
• Nuclear Receptor Coactivators MeSH
• Constitutive Androstane Receptor MeSH
• Membrane Proteins MeSH
• RNA, Messenger MeSH
• Metformin MeSH
• nuclear receptor subfamily 0, group B, member 2 MeSH Browser
• Pregnane X Receptor MeSH
• AMP-Activated Protein Kinases MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Receptors, Glucocorticoid MeSH
• Receptors, Calcitriol MeSH
• Receptors, Steroid MeSH