Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors ...regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5′-upstream region of human MDR1was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about −8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about −8 kilobase pairs, to which PXR binds.
An important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the solute ...carrier family 22 (SLC22). Because interindividual variability of OCT expression may affect response to cationic drugs such as metformin, we systematically investigated genetic and nongenetic factors of OCT1/SLC22A1 and OCT3/SLC22A3 expression in human liver. OCT1 and OCT3 expression (messenger RNA mRNA, protein) was analyzed in liver tissue samples from 150 Caucasian subjects. Hepatic OCTs were localized by way of immunofluorescence microscopy. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and genome‐wide single‐nucleotide polymorphism microarray technology served to genotype 92 variants in the SLC22A1‐A3/OCT1‐3 gene cluster. Transport of metformin by recombinant human OCT1 and OCT3 was compared using transfected cells. OCT1 mRNA and protein expression varied 113‐ and 83‐fold, respectively; OCT3 mRNA expression varied 27‐fold. OCT1 transcript levels were on average 15‐fold higher compared with OCT3. We localized the OCT3 protein to the basolateral hepatocyte membrane and identified metformin as an OCT3 substrate. OCT1 and OCT3 expression are independent of age and sex but were significantly reduced in liver donors diagnosed as cholestatic (P ≤ 0.01). Several haplotypes for OCT1 and OCT3 were identified. Multivariate analysis adjusted for multiple testing showed that only the OCT1‐Arg61Cys variant (rs12208357) strongly correlated with decreased OCT1 protein expression (P < 0.0001), and four variants in OCT3 (rs2292334, rs2048327, rs1810126, rs3088442) were associated with reduced OCT3 mRNA levels (P = 0.03). Conclusion: We identified cholestasis and genetic variants as critical determinants for considerable interindividual variability of hepatic OCT1 and OCT3 expression. This indicates consequences for hepatic elimination of and response to OCT substrates such as metformin. (HEPATOLOGY 2009.)
The nuclear receptor peroxisome proliferator-activated receptor (PPAR)α is known primarily as a regulator of fatty acid metabolism, energy balance, and inflammation, but evidence suggests a wider ...role in regulating the biotransformation of drugs and other lipophilic chemicals. We investigated whether PPARα directly regulates the transcription of cytochrome P450 3A4, the major human drug-metabolizing enzyme. Using chromatin immunoprecipitation in human primary hepatocytes as well as electrophoretic mobility shift and luciferase reporter-gene assays, we identified three functional PPARα-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream sequence. Furthermore, a humanized CYP3A4/3A7 mouse model showed in vivo induction of CYP3A4 mRNA and protein by 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY14,643) in liver but not in intestine, whereas hepatic occupancy of PBRs by PPARα was ligand independent. Using lentiviral gene knock-down and treatment with WY14,643 in primary human hepatocytes, PPARα was further shown to affect the expression of a distinct set of CYPs, including 1A1, 1A2, 2B6, 2C8, 3A4, and 7A1, but not 2C9, 2C19, 2D6, or 2E1. Interestingly, the common phospholipid 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-PC), previously proposed to reflect nutritional status and shown to be a specific endogenous ligand of PPARα, induced CYP3A4 (up to 4-fold) and other biotransformation genes in hepatocytes with similar selectivity and potency as WY14,643. These data establish PPARα as a direct transcriptional regulator of hepatic CYP3A4. This finding warrants investigation of both known and newly developed PPARα-targeted drugs for their drug-drug interaction potential. Furthermore, our data suggest that nutritional status can influence drug biotransformation capacity via endogenous phospholipid signaling.
Objectives
Our objectives were to determine the content of cytochrome P450 (CYP) 3A4, CYP3A5, and P‐glycoprotein and to measure CYP3A4‐dependent catalytic activity in paired human small intestinal ...and liver specimens.
Methods
Samples of duodenum or proximal jejunum and liver wedge biopsy specimens were obtained from 15 patients undergoing a gastrointestinal operation. Enterocytes were isolated from the intestinal samples. The contents of CYP3A4, CYP3A5, and P‐glycoprotein and CYP3A4‐mediated catalytic activities were determined in homogenized enterocyte and liver samples.
Results
The CYP3A4 protein content was about 3 times (P < .01) and the P‐glycoprotein content about 7 times (P < .0001) higher in the enterocyte homogenates than in the liver homogenates. CYP3A5 protein was detected in all samples, but the levels were too low in most cases to allow quantification. The 2 cases with a quantifiable hepatic CYP3A5 content had the CYP3A5*1/*3 genotype; all other cases were homozygous for the CYP3A5*3 allele. No intraindividual correlations between the intestine and liver with respect to CYP3A4 content, P‐glycoprotein content, or the measured catalytic activities were present. Values for the maximum rate of metabolism (Vmax) of verapamil N‐dealkylation (formation of D‐617) and N‐demethylation (formation of norverapamil) activities correlated with the CYP3A4 protein content in both organs.
Conclusions
This work demonstrated a much higher content of both CYP3A4 protein and P‐glycoprotein in enterocytes isolated from human duodenal or jejunal mucosa than in paired specimens of liver tissue. These results lend support to the view that biotransformation in the gut wall substantially contributes to the overall first‐pass metabolism of many CYP3A4 substrates. Furthermore, the high content of P‐glycoprotein on the apical surface of enterocytes supports the theory that this efflux transporter may act in concert with CYP3A4 to limit oral drug bioavailability. Finally, these results indicate that neither CYP3A4 nor MDR1 (P‐glycoprotein) is coordinately regulated in the liver and intestine.
Clinical Pharmacology & Therapeutics (2004) 75, 172–183; doi: 10.1016/j.clpt.2003.10.008
The pregnane X receptor (PXR) regulates the metabolism of many xenobiotic and endobiotic substances. In consequence, PXR decreases the efficacy of many small-molecule drugs and induces drug-drug ...interactions. The prediction of PXR activators with theoretical approaches such as machine learning (ML) proves challenging due to the ligand promiscuity of PXR, which is related to its large and flexible binding pocket. In this work we demonstrate, by the example of random forest models and support vector machines, that classifiers generated following classical training procedures often fail to predict PXR activity for compounds that are dissimilar from those in the training set. We present a novel regularization technique that penalizes the gap between a model's training and validation performance. On a challenging test set, this technique led to improvements in Matthew correlation coefficients (MCCs) by up to 0.21. Using these regularized ML models, we selected 31 compounds that are structurally distinct from known PXR ligands for experimental validation. Twelve of them were confirmed as active in the cellular PXR ligand-binding domain assembly assay and more hits were identified during follow-up studies. Comprehensive analysis of key features of PXR biology conducted for three representative hits confirmed their ability to activate the PXR.
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Pregnane X receptor (PXR) is a nuclear receptor with an essential role in regulating drug metabolism genes. While the mechanism of action for ligand-mediated PXR agonism is ...well-examined, its ligand-mediated inhibition or antagonism is poorly understood. Here we employ microsecond timescale all-atom molecular dynamics (MD) simulations to investigate how our newly identified dual kinase and PXR inhibitor, compound 100, acts as a competitive PXR antagonist and not as a full agonist. We study the PXR ligand binding domain conformational changes associated with compound 100 and compare the results to the full agonist SR12813, in presence and absence of the coactivator. Furthermore, we complement our research by experimentally disclosing the effect of eight key-residue mutations on PXR activation. Finally, simulations of P2X4 inhibitor (BAY-1797) in complex with PXR, which shares an identical structural moiety with compound 100, provide further insights to ligand-induced PXR behaviour. Our MD data suggests ligand-specific influence on conformations of different PXR-LBD regions, including α6 region, αAF-2, α1-α2′, β1′-α3 and β1-β1′ loop. Our results provide important insights on conformational behaviour of PXR and offers guidance how to alleviate PXR agonism or to promote PXR antagonism.
Small-molecule protein kinase inhibitors are used for the treatment of cancer, but off-target effects hinder their clinical use. Especially off-target activation of the pregnane X receptor (PXR) has ...to be considered, as it not only governs drug metabolism and elimination, but also can promote tumor growth and cancer drug resistance. Consequently, PXR antagonism has been proposed for improving cancer drug therapy. Here we aimed to identify small-molecule kinase inhibitors of the Tübingen Kinase Inhibitor Collection (TüKIC) compound library that would act also as PXR antagonists. By a combination of in silico screen and confirmatory cellular reporter gene assays, we identified four novel PXR antagonists and a structurally related agonist with a common phenylaminobenzosuberone scaffold. Further characterization using biochemical ligand binding and cellular protein interaction assays classified the novel compounds as mixed competitive/noncompetitive, passive antagonists, which bind PXR directly and disrupt its interaction with coregulatory proteins. Expression analysis of prototypical PXR target genes ABCB1 and CYP3A4 in LS174T colorectal cancer cells and HepaRG hepatocytes revealed novel antagonists as selective receptor modulators, which showed gene- and tissue-specific effects. These results demonstrate the possibility of dual PXR and protein kinase inhibitors, which might represent added value in cancer therapy.
Artemisinin drugs are of utmost importance in the treatment of malaria, because they represent the sole class of therapeutically
used antimalarial drugs to which malaria parasites have not yet ...developed resistance. The major disadvantage of these medicines
is the comparatively high recrudescence rate, which has been attributed to the remarkable decrease of artemisinin plasma concentrations
during multiple dosing. Autoinduction of CYP2B6-mediated metabolism has been implicated as the underlying mechanism. So far,
the molecular mechanism of induction by artemisinin has not been resolved. Because the xenosensors pregnane X receptor (PXR)
and constitutive androstane receptor (CAR) have been shown to mediate induction of drug-metabolizing enzymes and drug transporters,
we investigated the hypothesis that artemisinin induces cytochrome P450 expression by activating PXR and/or CAR. By combining
in vitro transfection methods and quantitative analyses of gene expression in cell lines and primary human hepatocytes, we
here show that artemisinin drugs activate human PXR as well as human and mouse CAR and induce the expression of CYP2B6 , CYP3A4 , and MDR1 in primary human hepatocytes and in the human intestinal cell line LS174T. Furthermore, we demonstrate that artemisinin acts
as a ligand of both nuclear receptors, because it modulates the interaction of the receptors with coregulators. In conclusion,
activation of PXR and CAR and especially the resulting induction of CYP3A4 and MDR1 demonstrate that artemisinin has a higher risk of potential drug interactions than anticipated previously.
Induction of cytochrome P450 3A (CYP3A) by xenobiotics may lead to clinically relevant drug interactions. In contrast with other CYP3A family members, studies on the inducibility of CYP3A5 indicate ...conflicting results. We report the induction of CYP3A5 mRNA in 13 of 16 hepatocyte preparations exposed to rifampin. Furthermore, induction of CYP3A5 mRNA was observed in intestinal biopsies in three of eight probands following exposure to the antibiotic. The highest absolute levels of CYP3A5 transcripts were found following rifampin treatment in hepatocytes and intestines from carriers of CYP3A5*1 alleles. Elucidation of the mechanism involved in CYP3A5 induction revealed that constitutively activated receptor (CAR) and pregnane X receptor (PXR) transactivated the CYP3A5 promoter (–688 to +49) and that the transactivation was dependent on an everted repeat separated by 6 bp (ER6-dependent). Treatment with the prototypical PXR ligand rifampin led to a 2-fold induction of the CYP3A5 promoter activity. In agreement with these observations, PXR and CAR bound specifically to the ER6 motif. Hepatic expression of PXR correlated with that of CYP3A5 mRNA levels in a bank of liver samples. Taken together, studies here revealed the presence of a functional ER6 motif in the CYP3A5 promoter located –100 bp upstream from the transcription start site, suggesting that CYP3A5 is inducible by mechanisms similar to those involved in CYP3A4 induction. Enhanced expression of CYP3A5 caused by exposure to inducers may phenocopy the effects of the high expression allele CYP3A5*1. In this manner, induction of CYP3A5 may contribute to the overall importance of this P450 in drug metabolism and drug interactions.
Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug–drug ...interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5′ upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By
in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TRα1, TRβ1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional
in vitro characterization with hepatic expression analysis, we predict that TRα1, TRβ1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4
in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.
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