Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.

• MeSH
• biologické modely MeSH
• buněčné sféroidy * metabolismus   MeSH
• cytochrom P-450 CYP3A metabolismus   genetika   MeSH
• hepatocyty * metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• pregnanový X receptor * metabolismus   MeSH
• regulace genové exprese * MeSH
• rifampin farmakologie   MeSH
• steroidní receptory * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochrom P-450 CYP3A MeSH
• pregnanový X receptor * MeSH
• rifampin MeSH
• steroidní receptory * MeSH

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

• MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• konstitutivní androstanový receptor * agonisté   chemie   MeSH
• lidé MeSH
• myši MeSH
• pyridiny farmakologie   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• steroidní receptory * agonisté   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• konstitutivní androstanový receptor * MeSH
• pyridiny MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidní receptory * MeSH

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

• Klíčová slova
• CAR, NR1I3, cytochrome P450, diazepam, drug interaction, gene regulation,
• MeSH
• buněčné linie MeSH
• diazepam farmakologie   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• konstitutivní androstanový receptor MeSH
• lidé středního věku MeSH
• lidé MeSH
• ligandy MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• proteinové domény účinky léků   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• reportérové geny účinky léků   genetika   MeSH
• transport proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diazepam MeSH
• konstitutivní androstanový receptor MeSH
• ligandy MeSH
• NR1I3 protein, human MeSH Prohlížeč
• Nr1i3 protein, mouse MeSH Prohlížeč
• receptory cytoplazmatické a nukleární MeSH

Cytochrome P450 (CYP) is a major group of enzymes, which conduct Phase I metabolism. Among commonly used animal models, the pig has been suggested as the most suitable model for investigating drug metabolism in human beings. Moreover, porcine CYP2A19 and CYP2E1 are responsible for the biotransformation of both endogenous and exogenous compounds such as 3-methylindole (skatole), sex hormones and food compounds. However, little is known about the regulation of porcine CYP2A19 and CYP2E1. In this MiniReview, we summarise the current knowledge about the regulation of porcine CYP2A19 and CYP2E1 by environmental, biological and dietary factors. Finally, we reflect on the need for further research, to clarify the interaction between active feed components and the porcine CYP system.

• Klíčová slova
• bioactive compounds, phase I enzymes, pig, skatole,
• MeSH
• biotransformace MeSH
• cytochrom P-450 CYP2E1 genetika   metabolismus   MeSH
• cytochrom P450 CYP2A6 genetika   metabolismus   MeSH
• krmivo pro zvířata * MeSH
• lidé MeSH
• pohlavní steroidní hormony metabolismus   MeSH
• prasata metabolismus   MeSH
• sekvenční homologie MeSH
• skatol metabolismus   MeSH
• xenobiotika metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cytochrom P-450 CYP2E1 MeSH
• cytochrom P450 CYP2A6 MeSH
• pohlavní steroidní hormony MeSH
• skatol MeSH
• xenobiotika MeSH

The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreen(TM) TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding domain (LBD) with no need to use cellular models. This in silico assay thus enables the study of direct CAR ligands and the ability to distinguish them from the indirect CAR activators that affect the receptor via the cell signaling-dependent phosphorylation of CAR in cells. For the current paper we characterized the pharmacodynamic interactions of three known CAR inverse agonists/antagonists-PK11195, clotrimazole and androstenol-with the prototype agonist CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3] thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) using the TR-FRET LanthaScreen(TM) assay. We have confirmed that all three compounds are inverse agonists of human CAR, with IC50 0.51, 0.005, and 0.35 μM, respectively. All the compounds also antagonize the CITCO-mediated activation of CAR, but only clotrimazole was capable to completely reverse the effect of CITCO in the tested concentrations. Thus this method allows identifying not only agonists, but also antagonists and inverse agonists for human CAR as well as to investigate the nature of the pharmacodynamic interactions of CAR ligands.

• MeSH
• biotest metody   MeSH
• isochinoliny farmakologie   MeSH
• klotrimazol farmakologie   MeSH
• konstitutivní androstanový receptor MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• oximy farmakologie   MeSH
• receptory cytoplazmatické a nukleární agonisté   antagonisté a inhibitory   metabolismus   MeSH
• thiazoly farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime MeSH Prohlížeč
• isochinoliny MeSH
• klotrimazol MeSH
• konstitutivní androstanový receptor MeSH
• oximy MeSH
• PK 11195 MeSH Prohlížeč
• receptory cytoplazmatické a nukleární MeSH
• thiazoly MeSH