Oliver Burk

Publications of Oliver Burk

• PXR variants and artemisinin use in Vietnamese subjects: Frequency distribution and impact on the inter-individual variability of CYP3A induction by artemisinin.

  Authors: Rita Piedade, Elke Schaeffeler, Stefan Winter, Sara Asimus, Matthias Schwab, Michael Ashton, Oliver Burk, José P Gil

  Antimicrobial agents and chemotherapy. 01/2012;

  Artemisinins induce drug metabolism through the activation of pregnane x receptor (PXR) in vitro. Herein, we report the re-sequencing and genotyping of PXR variants in 75 Vietnamese individualsArtemisinins induce drug metabolism through the activation of pregnane x receptor (PXR) in vitro. Herein, we report the re-sequencing and genotyping of PXR variants in 75 Vietnamese individuals previously characterized for CYP3A enzyme activity after artemisinin exposure. A total of 31 PXR variants were identified, including five novel SNPs, and we identified significantly different allele frequencies relative to other ethnic groups. A trend of significance was observed between the level of CYP3A4 induction by artemisinin and two PXR variants, 8118 C>T (Y328Y) and 10719 A>G.

• Evolutionary history and functional characterization of the amphibian xenosensor CAR.

  Authors: Marianne Mathäs, Oliver Burk, Huan Qiu, Christian Nusshag, Ute Gödtel-Armbrust, Dorothea Baranyai, Shiwei Deng, Kristin Römer, Dieudonné Nem, Björn Windshügel, Leszek Wojnowski

  Molecular endocrinology (Baltimore, Md.). 11/2011; 26(1):14-26.

  The xenosensing constitutive androstane receptor (CAR) is widely considered to have arisen in early mammals via duplication of the pregnane X receptor (PXR). We report that CAR emerged together withThe xenosensing constitutive androstane receptor (CAR) is widely considered to have arisen in early mammals via duplication of the pregnane X receptor (PXR). We report that CAR emerged together with PXR and the vitamin D receptor from an ancestral NR1I gene already in early vertebrates, as a result of whole-genome duplications. CAR genes were subsequently lost from the fish lineage, but they are conserved in all taxa of land vertebrates. This contrasts with PXR, which is found in most fish species, whereas it is lost from Sauropsida (reptiles and birds) and plays a role unrelated to xenosensing in Xenopus. This role is fulfilled in Xenopus by CAR, which exhibits low basal activity and pronounced responsiveness to activators such as drugs and steroids, altogether resembling mammalian PXR. The constitutive activity typical for mammalian CAR emerged first in Sauropsida, and it is thus common to all fully terrestrial land vertebrates (Amniota). The constitutive activity can be achieved by humanizing just two amino acids of the Xenopus CAR. Taken together, our results provide a comprehensive reconstruction of the evolutionary history of the NR1I subfamily of nuclear receptors. They identify CAR as the more conserved and remarkably plastic NR1I xenosensor in land vertebrates. Nonmammalian CAR should help to dissect the specific functions of PXR and CAR in the metabolism of xeno- and endobiotics in humans. Xenopus CAR is a first reported amphibian xenosensor, which opens the way to toxicogenomic and bioaugmentation studies in this critically endangered taxon of land vertebrates.

• Inferring statin-induced gene regulatory relationships in primary human hepatocytes.

  Authors: Adrian Schröder, Johannes Wollnik, Clemens Wrzodek, Andreas Dräger, Michael Bonin, Oliver Burk, Maria Thomas, Wolfgang E Thasler, Ulrich M Zanger, Andreas Zell

  Bioinformatics (Oxford, England). 07/2011; 27(18):2473-7.

  Statins are the most widely used cholesterol-lowering drugs. The primary target of statins is HMG-CoA reductase, a key enzyme in cholesterol synthesis. However, statins elicit pleitropic responsesStatins are the most widely used cholesterol-lowering drugs. The primary target of statins is HMG-CoA reductase, a key enzyme in cholesterol synthesis. However, statins elicit pleitropic responses including beneficial as well as adverse effects in the liver or other organs. Today, the regulatory mechanisms that cause these pleiotropic effects are not sufficiently understood. In this work, genome-wide RNA expression changes in primary human hepatocytes of six individuals were measured at up to six time points upon atorvastatin treatment. A computational analysis workflow was applied to reconstruct regulatory mechanisms based on these drug-response data and available knowledge about transcription factor (TF) binding specificities and protein-drug interactions. Several previously unknown TFs were predicted to be involved in atorvastatin-responsive gene expression. The novel relationships of nuclear receptors NR2C2 and PPARA on CYP3A4 were successfully validated in wet-lab experiments. Microarray data are available at the Gene Expression Omnibus (GEO) database at www.ncbi.nlm.nih.gov/geo/, under accession number GSE29868. andreas.zell@uni-tuebingen.de; adrian.schroeder@uni-tuebingen.de Supplementary data are available at Bioinformatics online.

• The unique complexity of the CYP3A4 upstream region suggests a nongenetic explanation of its expression variability.

  Authors: Huan Qiu, Marianne Mathäs, Sebastian Nestler, Christopher Bengel, Dieudonne Nem, Ute Gödtel-Armbrust, Thomas Lang, Stefan Taudien, Oliver Burk, Leszek Wojnowski

  Pharmacogenetics and genomics. 03/2010; 20(3):167-78.

  The individually variable and unpredictable expression of CYP3A4 compromises therapies with 50% of contemporary drugs. Gene variants explain only a fraction of this variability. We investigated theThe individually variable and unpredictable expression of CYP3A4 compromises therapies with 50% of contemporary drugs. Gene variants explain only a fraction of this variability. We investigated the evolution of CYP3A4 transcriptional regulation by nuclear receptors such as the xenobiotics sensors PXR and CAR. The combination of a proximal ER6 element with XREM and CLEM represents the original scheme of CYP3A regulation by nuclear receptors in placental mammals. Among human CYP3A genes, this scheme is retained only in CYP3A4, whereas non-CYP3A4 genes lost these elements to a variable extent during primate evolution. In parallel, the number of elements outside XREM and CLEM potentially responsive to PXR and CAR increased in primate CYP3A4 orthologs, which led to enhanced CYP3A4 inducibility. Additions to the other primate CYP3A genes were more restricted and specific, as exemplified by a CYP3A5 DR4 site responsive to CAR, but not to PXR. All these changes resulted in human CYP3A4 having a much more complex upstream regulatory region in comparison to its paralogs. Instead of gene variants, the intraindividual CYP3A4 expression variability in humans may be primarily caused by particular sensitivity of this gene to endogenous and exogenous PXR and CAR ligands conferred by the unique complexity of its upstream regulatory region.

• Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding.

  Authors: Monica A Istrate, Andreas K Nussler, Michel Eichelbaum, Oliver Burk

  Biochemical and biophysical research communications. 02/2010; 393(4):688-93.

  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-drugInduction 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. TRalpha1, TRbeta1, 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 TRalpha1, TRbeta1, 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.

• Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver.

  Authors: Anne T Nies, Hermann Koepsell, Stefan Winter, Oliver Burk, Kathrin Klein, Reinhold Kerb, Ulrich M Zanger, Dietrich Keppler, Matthias Schwab, Elke Schaeffeler

  Hepatology (Baltimore, Md.). 05/2009;

  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 soluteAn 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.).

• Effects of rifampicin on global gene expression in human small intestine.

  Authors: Mikael Oscarson, Oliver Burk, Stefan Winter, Matthias Schwab, Renzo Wolbold, Juergen Dippon, Michel Eichelbaum, Urs A Meyer

  Pharmacogenetics and genomics. 12/2007; 17(11):907-18.

  OBJECTIVES: The small intestinal wall serves as an important barrier for the entry of foreign substances into the organism. Of particular importance are enzymes and transporters that can inactivateOBJECTIVES: The small intestinal wall serves as an important barrier for the entry of foreign substances into the organism. Of particular importance are enzymes and transporters that can inactivate or prevent the uptake of many xenobiotics including drugs. Some of the genes encoding these proteins are transcriptionally activated by xenobiotics, a response well studied in liver but less so in the intestine. The effect of the inducer drug rifampicin on intestinal cells was therefore evaluated both in vivo and in vitro. METHODS: Seven healthy volunteers were treated with rifampicin for 9 days and the global gene expression profile was analysed in RNA from duodenal biopsies taken before and after drug treatment. The gene expression profile was also assessed in LS174T cells derived from a human colon adenocarcinoma after exposure to 10 micromol/l rifampicin for 24 h. RESULTS: We identified 32 genes that were upregulated and two genes that were downregulated by rifampicin treatment in vivo. The list of rifampicin regulated transcripts expectedly included drug metabolizing enzymes and drug transporters, but also genes involved in lipid and amino acid metabolism as well as genes not previously recognized to be part of the adaptation of intestinal cells to xenobiotic exposure. Only a limited number of these rifampicin-regulated transcripts were however also regulated by rifampicin in LS174T cells. CONCLUSION: The similarities and differences of changes in gene expression after rifampicin treatment between duodenal biopsies and cell culture provide a new assessment of the extent and diversity of systems affected by drug exposure.

• The Paneth cell alpha-defensin deficiency of ileal Crohn's disease is linked to Wnt/Tcf-4.

  Authors: Jan Wehkamp, Guoxing Wang, Irmgard Kübler, Sabine Nuding, Alex Gregorieff, Anke Schnabel, Robert J Kays, Klaus Fellermann, Oliver Burk, Matthias Schwab, Hans Clevers, Charles L Bevins, Eduard F Stange

  Journal of immunology (Baltimore, Md. : 1950). 10/2007; 179(5):3109-18.

  Ileal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence ofIleal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced alpha-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth cell differentiation and alpha-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/-) mice was sufficient to cause a significant decrease of both Paneth cell alpha-defensin levels and bacterial killing activity. Finally, the association between Paneth cell alpha-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.

• Molecular mechanism of basal CYP3A4 regulation by hepatocyte nuclear factor 4alpha: evidence for direct regulation in the intestine.

  Authors: Heike Tegude, Anke Schnabel, Ulrich M Zanger, Kathrin Klein, Michel Eichelbaum, Oliver Burk

  Drug metabolism and disposition: the biological fate of chemicals. 07/2007; 35(6):946-54.

  Cytochrome P450 3A4 plays an outstanding role in the metabolism of clinically used drugs and shows a marked interindividual variability in expression even in the absence of inducing agents. Thus,Cytochrome P450 3A4 plays an outstanding role in the metabolism of clinically used drugs and shows a marked interindividual variability in expression even in the absence of inducing agents. Thus, regulation of basal expression contributes considerably to variability. The nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) was previously shown to be associated with basal hepatic CYP3A4 expression. As how HNF4alpha regulates basal expression of CYP3A4 still remains elusive, we systematically screened 12.5 kilobase pairs (kb) of the CYP3A4 5' upstream region for activation by the receptor in the human intestinal cell line LS174T. In this study, we newly identified two widely separated regions mediating the activation by HNF4alpha: a far distal region at -9.0 kb and the proximal promoter region at approximately -0.2 kb. By gel shift experiments and transient transfections, we characterized direct repeat (DR) 1-type motifs in both regions as functional HNF4alpha response elements. Cooperation of the two regions was shown to be required for maximal activation by HNF4alpha. The effect of HNF4alpha was antagonized by chicken ovalbumin upstream promoter transcription factor II, which was shown to bind to one of the DR1 motifs. Furthermore, activation of CYP3A4 via the DR1 element in the proximal promoter depends on an additional, yet unknown, factor, which is binding at approximately -189 base pairs. Physiological relevance of this position for activation by HNF4alpha in vivo is suggested by the presence of a binding activity in small intestine similar to that in LS174T cells. In summary, we here have elucidated a molecular mechanism of direct regulation of CYP3A4 by HNF4alpha, which is probably specific for the intestine.

• Functional analysis of the polymorphism -211C>T in the regulatory region of the human ABCC3 gene.

  Authors: Ulrike Gradhand, Heike Tegude, Oliver Burk, Michel Eichelbaum, Martin F Fromm, Jörg König

  Life sciences. 04/2007; 80(16):1490-4.

  The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes.The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position -211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position -211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position -211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the -211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the -211 SNP. Taken together, we did not find any influence of the -211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

• Effects of ursodeoxycholic acid on P-glycoprotein and cytochrome P450 3A4-dependent pharmacokinetics in humans.

  Authors: Laurent Becquemont, Hartmut Glaeser, Siegfried Drescher, Monika Hitzl, Nicolas Simon, Thomas E Murdter, Georg Heinkele, Ute Hofmann, Christian Schaefer, Oliver Burk, Céline Verstuyft, Michel Eichelbaum, Martin F Fromm

  Clinical pharmacology and therapeutics. 06/2006; 79(5):449-60.

  BACKGROUND AND OBJECTIVE: On the basis of in vitro studies indicating that ursodeoxycholic acid (UDCA) is a cytochrome P450 (CYP) 3A4 inducer and a pregnane X receptor activator and because theBACKGROUND AND OBJECTIVE: On the basis of in vitro studies indicating that ursodeoxycholic acid (UDCA) is a cytochrome P450 (CYP) 3A4 inducer and a pregnane X receptor activator and because the pregnane X receptor is a transcriptional regulator of multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp), we postulated that UDCA might decrease the bioavailability of CYP3A4 and P-gp probe drugs in humans. The main objective of this study was to determine whether UDCA alters the pharmacokinetics of digoxin and midazolam. The secondary objective was to determine whether the intestinal expression of P-gp and CYP3A4 is increased by UDCA. METHODS: The effect of UDCA on MDR1 and CYP3A4 messenger ribonucleic acid (mRNA) expression was investigated in human colon carcinoma cell lines LS174T and Caco-2. Eight healthy volunteers participated in this open, nonrandomized 2-period study, in which the effects of UDCA (13 mg.kg(-1).d(-1) during 2 weeks) versus control on the pharmacokinetics of digoxin (0.5-mg single intravenous infusion), d3-digoxin (3-fold deuterated digoxin, 0.5-mg single oral dose), and midazolam (7.5-mg single oral dose) were compared. Duodenal biopsy specimens were obtained during both periods to quantify MDR1/P-gp and CYP3A4 expression. RESULTS: In vitro UDCA induced MDR1 and CYP3A4 mRNA in Caco-2 cells but not in LS174T cells. In humans UDCA significantly decreased the extent of digoxin absorption from 0.77 to 0.70 and the oral d3-digoxin area under the plasma concentration-time curve from 0 to 4 hours from 6.4 +/- 1.7 ng.h.mL(-1) to 5.3 +/- 1.5 ng.h.mL(-1) (P = .01 and P = .05, respectively). UDCA had no detectable effects on the pharmacokinetics of midazolam or the intestinal mRNA and protein expression levels of MDR1/P-gp and CYP3A4. CONCLUSION: Under the conditions in our study, UDCA only modestly decreased digoxin disposition without detectable changes in midazolam pharmacokinetics. The clinical relevance of these findings remains to be determined.

• Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor.

  Authors: Oliver Burk, Katja A Arnold, Andreas K Nussler, Elke Schaeffeler, Ekaterina Efimova, Bonnie A Avery, Mitchell A Avery, Martin F Fromm, Michel Eichelbaum

  Molecular pharmacology. 07/2005; 67(6):1954-65.

  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 yetArtemisinin 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.

• A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression.

  Authors: Oliver Burk, Katja A Arnold, Anke Geick, Heike Tegude, Michel Eichelbaum

  Biological chemistry. 07/2005; 386(6):503-13.

  MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect theMDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1 . The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.

• The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is mediated by the xenobiotic sensors pregnane X receptor (PXR) and constitutively activated receptor (CAR).

  Authors: Oliver Burk, Ina Koch, Judy Raucy, Elisabeth Hustert, Michel Eichelbaum, Jürgen Brockmöller, Ulrich M Zanger, Leszek Wojnowski

  The Journal of biological chemistry. 10/2004; 279(37):38379-85.

  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 indicateInduction 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.

• Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor.

  Authors: Katja Arnold, Michel Eichelbaum, Oliver Burk

  Nuclear receptor. 04/2004; 2(1):1.

  BACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drugBACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drug metabolizing enzymes and drug transporters. RESULTS: We identified transcripts generated by four different alternative splicing events in the human CAR gene. Two of the corresponding ligand binding domain isoforms demonstrated novel functional properties: First, CAR(SV3), which is encoded by a transcript containing an lengthened exon 7, differentially transactivated target gene promoters. Second, CAR(SV2), which results from the use of an alternative 3' splice site lengthening exon 8, showed ligand-dependent instead of constitutive interaction with coactivators. Furthermore, alternatively spliced transcripts demonstrated a tissue-specific expression pattern. In most tissues, only transcripts generated by alternative splicing within exon 9 were expressed. The encoded variant demonstrated a loss-of-function phenotype. Correct splicing of exon 8 to exon 9 is restricted to only a few tissues, among them liver and small intestine for which CAR function has been demonstrated, and is associated with the induction of CAR expression during differentiation of intestinal cells. CONCLUSION: Due to their specific activities, CAR variant proteins SV2 and SV3 may modulate the activity of reference CAR(SV1). Furthermore, we propose that transcriptional activation and regulation of splicing of exon 9 may be coupled to ensure appropriate tissue- and differentiation state-specific expression of transcripts encoding functional CAR protein. Altogether, alternative splicing seems to be of utmost importance for the regulation of CAR expression and function.

• Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens.

  Authors: Oliver von Richter, Oliver Burk, Martin F Fromm, Klaus P Thon, Michel Eichelbaum, Kari T Kivistö

  Clinical pharmacology and therapeutics. 04/2004; 75(3):172-83.

  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 intestinalOBJECTIVES: 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 (V(max)) 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.

• Genetic polymorphisms in the multidrug resistance-associated protein 3 (ABCC3, MRP3) gene and relationship to its mRNA and protein expression in human liver.

  Authors: Thomas Lang, Monika Hitzl, Oliver Burk, Esther Mornhinweg, Andrea Keil, Reinhold Kerb, Kathrin Klein, Ulrich M Zanger, Michel Eichelbaum, Martin F Fromm

  Pharmacogenetics. 03/2004; 14(3):155-64.

  AIMS: To determine the genetic variability of multidrug resistance protein 3 (MRP3). METHODS: Genomic DNA samples from 103 Caucasians were systematically screened for genetic variations to find aAIMS: To determine the genetic variability of multidrug resistance protein 3 (MRP3). METHODS: Genomic DNA samples from 103 Caucasians were systematically screened for genetic variations to find a potential relationship with hepatic MRP3 expression. Sequencing comprised all 31 exons, approximately 100 bp of the flanking intronic regions and 2 kb of the 5' UTR. RESULTS: In total, 51 mutations were identified. Fifteen SNPs were located in the coding exons of MRP3, six of which are nonsynonymous mutations. SNPs 39G>C (allele frequency: 0.5%, located in exon 1), 202C>T (1.6%, exon 2), 1037C>T (0.5%, exon 9), 1537C>A (0.5%, exon 12), 3890G>A (5.2%, exon 27) and 4267G>A (0.6%, exon 29) resulted in Lys13Asn, His68Tyr, Ser346Phe, Gln513Lys, Arg1297His and Gly1423Arg amino acid substitutions, respectively. A splice site mutation (1339-1G>T) was found at the intron 10-exon 11 boundary. To evaluate, whether mutations in the MRP3 gene correlate with human hepatic MRP3 expression, we analyzed the genetic variants in Caucasian liver samples, whose MRP3 mRNA (n = 84) and protein (n = 50) expression has been determined by real time quantitative PCR and Western Blot, respectively. We found a significant correlation of a polymorphism in the 5' promoter region (-211C>T) of MRP3 with mRNA expression. Individuals homozygous and heterozygous for the -211C>T promoter polymorphism had significantly lower MRP3 transcript levels compared to wild-type individuals (P < 0.05). Accordingly, electrophoretic mobility shift assay demonstrated that -211C>T polymorphism affected the binding of nuclear factors. CONCLUSIONS: Multiple genetic polymorphisms of MRP3 exist in Caucasians. The -211C>T promoter polymorphism appears to be associated with altered hepatic MRP3 mRNA expression.

• Sex is a major determinant of CYP3A4 expression in human liver.

  Authors: Renzo Wolbold, Kathrin Klein, Oliver Burk, Andreas K Nüssler, Peter Neuhaus, Michel Eichelbaum, Matthias Schwab, Ulrich M Zanger

  Hepatology (Baltimore, Md.). 10/2003; 38(4):978-88.

  Many drugs that are substrates of CYP3A4, the major human drug-metabolizing cytochrome P450 (CYP), show higher clearance in women than in men. Although this effect is believed to be related to drugMany drugs that are substrates of CYP3A4, the major human drug-metabolizing cytochrome P450 (CYP), show higher clearance in women than in men. Although this effect is believed to be related to drug metabolism, the underlying cause has not been elucidated. We investigated CYP3A4 in a large collection (n = 94) of well-characterized surgical liver samples and found 2-fold higher CYP3A4 levels in female compared with male samples (P <.0001) and a corresponding 50% increase in the CYP3A-dependent N-dealkylation of verapamil (P <.01). This expression difference was not due to preferential induction in women following higher drug exposure because it was even larger in a subgroup not previously exposed to drugs. Higher expression in women was also found for CYP3A4 messenger RNA (mRNA) transcripts, suggesting a pretranslational mechanism. Expression of the pregnane X receptor (PXR), which is crucially involved in CYP3A4 induction by xenobiotics, was strongly correlated to CYP3A4 at the mRNA level in all individuals as well as in the subgroup not exposed to drugs (r = 0.81; P <.0001), but no sex-dependent expression of PXR mRNA was found. The ABC transporter P-glycoprotein, which has been proposed to be implicated in the mechanism of sex-dependent drug clearance, was also not differentially expressed. The influence of drug treatment on expression was examined from patient drug histories, and strong induction of CYP3A4 by carbamazepine and St. John's wort was found. In conclusion, sex, in addition to PXR and drug exposure, is a major factor for CYP3A4 expression in humans, thus explaining many of the previous observations of sex-dependent drug clearance.

• Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA.

  Authors: Ina Koch, Regina Weil, Renzo Wolbold, Jürgen Brockmöller, Elisabeth Hustert, Oliver Burk, Andreas Nuessler, Peter Neuhaus, Michel Eichelbaum, Ulrich Zanger, Leszek Wojnowski

  Drug metabolism and disposition: the biological fate of chemicals. 11/2002; 30(10):1108-14.

  The elucidation of the individual contributions of the four CYP3A genes to the overall CYP3A activity has been hampered by similarities in their sequence and function. We investigated the expressionThe elucidation of the individual contributions of the four CYP3A genes to the overall CYP3A activity has been hampered by similarities in their sequence and function. We investigated the expression of CYP3A mRNA species in the liver and in various other tissues using gene-specific TaqMan probes. CYP3A4 transcripts were the most abundant CYP3A mRNA in each of the 63 white European livers tested and accounted on average for 95% of the combined CYP3A mRNA pool. CYP3A5 and CYP3A7 each contributed on average 2%, whereas CYP3A43 contributed 0.3% transcripts to this pool. Fourteen percent of livers exhibited an increased share of CYP3A5 transcripts (range 4-20%). These livers were either heterozygous for the marker of the CYP3A5 polymorphism, the CYP3A5*1A allele, or expressed very low levels of CYP3A4 mRNA. The CYP3A7 expression was bimodal, and it was increased in 15% livers. CYP3A4 was the dominant CYP3A in the intestine, followed by CYP3A5. CYP3A5 and CYP3A7, but not CYP3A4, were also expressed in the adrenal gland and in the prostate, whereas only CYP3A5 was detected in the kidney. These three tissues were shown to express much lower levels of pregnane X receptor mRNA than the intestine, indicating possibly a different mode of regulation of CYP3A expression. Expression of CYP3A genes was undetectable in peripheral blood lymphocytes. In summary, these assays and results should aid in our efforts to further dissect the regulation and the physiological and pharmacological significance of CYP3A isozymes.

• Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine.

  Authors: Oliver Burk, Heike Tegude, Ina Koch, Elisabeth Hustert, Renzo Wolbold, Hartmut Glaeser, Kathrin Klein, Martin F Fromm, Andreas K Nuessler, Peter Neuhaus, Ulrich M Zanger, Michel Eichelbaum, Leszek Wojnowski

  The Journal of biological chemistry. 08/2002; 277(27):24280-8.

  Human CYP3A enzymes play a pivotal role in the metabolism of many drugs, and the variability of their expression among individuals may have a strong impact on the efficacy of drug treatment. However,Human CYP3A enzymes play a pivotal role in the metabolism of many drugs, and the variability of their expression among individuals may have a strong impact on the efficacy of drug treatment. However, the individual contributions of the four CYP3A genes to total CYP3A activity remain unclear. To elucidate the role of CYP3A7, we have studied its expression in human liver and intestine. In both organs, expression of CYP3A7 mRNA was polymorphic. The recently identified CYP3A7*1C allele was a consistent marker of increased CYP3A7 expression both in liver and intestine, whereas the CYP3A7*1B allele was associated with increased CYP3A7 expression only in liver. Because of the replacement of part of the CYP3A7 promoter by the corresponding region of CYP3A4, the CYP3A7*1C allele contains the proximal ER6 motif of CYP3A4. The pregnane X and constitutively activated receptors were shown to bind with higher affinity to CYP3A4-ER6 than to CYP3A7-ER6 motifs and transactivated only promoter constructs containing CYP3A4-ER6. Furthermore, we identified mutations in CYP3A7*1C in addition to the ER6 motif that were necessary only for activation by the constitutively activated receptor. We conclude that the presence of the ER6 motif of CYP3A4 mediates the high expression of CYP3A7 in subjects carrying CYP3A7*1C.