Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drugdrug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drugdrug interaction possibilities, the inducing properties of artemisinin metabolites should be considered.German Federal Ministry of Education and Research (BMBF) HepatosSys network [0313081B, 0313080F, 0313080I]; Deutsche Forschungsgemeinschaft (Germany) [KE 1629/1-1]; Robert Bosch Foundation, Stuttgart, Germanyinfo:eu-repo/semantics/publishedVersio

Transcriptional Profiling of Human Liver Identifies Sex-Biased Genes Associated with Polygenic Dyslipidemia and Coronary Artery Disease

Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease. Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5 and LDLR; reduced expression of LIPC) that is consistent with the lower female risk of coronary artery disease. Female-biased expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of 879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally, the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human hepatic sex differences are widespread and affect diverse cell metabolic processes, and may help explain sex differences in lipid profiles associated with sex differential risk of coronary artery disease

Genetic Background and Sex: Impact on Generalizability of Research Findings in Pharmacology Studies

Animal models consisting of inbred laboratory rodent strains have been a powerful tool for decades, helping to unravel the underpinnings of biological problems and employed to evaluate potential therapeutic treatments in drug discovery. While inbred strains demonstrate relatively reliable and predictable responses, using a single inbred strain alone or as a background to a mutation is analogous to running a clinical trial in a single individual and their identical twins. Indeed, complex etiologies drive the most common human diseases, and a single inbred strain that is a surrogate of a single genome, or data generated from a single sex, is not representative of the genetically diverse patient populations. Further, pharmacological and toxicology data generated in otherwise healthy animals may not translate to disease states where physiology, metabolism, and general health are compromised. The purpose of this chapter is to provide guidance for improving generalizability of preclinical studies by providing insight into necessary considerations for introducing systematic variation within the study design, such as genetic diversity, the use of both sexes, and selection of appropriate age and disease model. The outcome of implementing these considerations should be that reproducibility and generalizability of significant results are significantly enhanced leading to improved clinical translation

Studies on the expression of Cytochrome P450 3A4 and expression and transcriptional regulation of the Pregnane X Receptor in humans

Um der Exposition gegenüber Pharmaka, anderen Xenobiotika und potentiell schädigenden Metaboliten endogener und exogener Herkunft zu begegnen, besitzen viele Organismen ein Biotransformationssystem, durch welches diese Verbindungen durch chemische Modifikation oftmals unschädlich und ausscheidbar gemacht und schließlich aus dem Organismus eliminiert werden. Ein wichtiger Bestandteil des Biotransformationssystems sind die Cytochrom P450 Monooxygenasen. Cytochrom P450 3A4 (CYP3A4) ist mengenmäßig das bedeutendste P450 Isoenzym in der Leber des Menschen und ist an der Verstoffwechslung von mehr als 50% aller gegenwärtig in der Therapie eingesetzten Wirkstoffe beteiligt. Die Genexpression und enzymatische Aktivität von CYP3A4 ist von beträchtlicher interindividueller Variabilität. Eine durch Induktionsprozesse bedingte Regulation der Genexpression und genetische Polymorphismen werden als mögliche Ursachen angesehen. Die Induktion von CYP3A4 und einer Reihe anderer wichtiger Proteine der Biotransformation durch bestimmte Induktoren wird unter anderem durch den Pregnan-X-Rezeptor (PXR), ein Mitglied der Superfamilie der Kernrezeptoren, vermittelt. Diese ligandenaktivierbaren Transkriptionsfaktoren regulieren und modulieren die Transkription ihrer Zielgene durch Interaktion mit regulatorischen DNA Bereichen, anderen Transkriptionsfaktoren, Co-Aktivatoren und -Repressoren und Bestandteilen der zellulären Transkriptionsmaschinerie. Obwohl PXR als ein zentraler upstream regulator der Biotransformation an der transkriptionellen Kontrolle mehrerer xenobiotika-metabolisierender Enzyme und Transporterproteine beteiligt ist, war über Mechanismen der Regulation des menschlichen PXR Gens selbst bisher nichts bekannt. Es lagen jedoch Hinweise vor, dass die Expression des PXR Gens durch den Hepatocyte Nuclear Factor 4alpha (HNF4alpha), den Glucocorticoidrezeptor und durch PXR selbst reguliert wird. In der vorliegenden Arbeit wurde die Expression des Cytochrom P450 3A4 und des Pregnan-X-Rezeptors in der Leber und im Darm des Menschen untersucht und ein möglicher Zusammenhang zwischen beiden Parametern überprüft. Weiterhin wurde der Einfluss der individuellen PXR Expression auf die Induzierbarkeit des intestinalen CYP3A4 und P-Glykoprotein durch den PXR-Agonisten Rifampicin untersucht. Schließlich wurde die 5´ flankierende Region des PXR Gens charakterisiert, um cis-acting Elemente und trans-acting Faktoren, die an der Regulation von PXR beteiligt sind, zu identifizieren. Bei der Expressionsanalyse von CYP3A4 in menschlicher Leber anhand einer Gewebebank von 94 Patientenproben konnte die bekannte interindividuelle Variabilität sowohl auf der Protein- als auch auf der mRNA-Ebene bestätigt werden. Außerdem wurde ein Geschlechtsdimorphismus bei der Expression von CYP3A4 gefunden. In Leberproben von Frauen war die durchschnittliche CYP3A4 Protein- und mRNA Expression jeweils signifikant höher als in Proben von Männern. Auch die PXR Expression war von beträchtlicher Variabilität, sie schwankte in der Leber um einen Faktor von mehr als 17 und war mit der Expression der CYP3A4 mRNA sowohl in der Leber als auch im Darm korreliert. Bei der Untersuchung der intestinalen Induzierbarkeit von CYP3A4 und MDR1 durch Rifampicin anhand von Duodenalbiopsien wurde eine Ko-Induktion von CYP3A4 und MDR1 gefunden, die individuelle PXR Expression war jedoch nicht ausschlaggebend für das Induktionsausmaß. Für die Charakterisierung der 5´-flankierenden Region des PXR Gens wurde zunächst die Transkriptionsstartstelle identifiziert. Dabei wurden unterschiedliche Haupttranskriptionsstartstellen in der Leber und im Darm des Menschen gefunden. Eine Plasmidbibliothek von Reportergenkonstrukten mit unidirektionalen Verkürzungen des von Position –2543 bis +195 relativ zum Transkriptionsstart in der Leber subklonierten PXR-Promotors wurde generiert. Durch transiente Transfektionsexperimente mit dieser Plasmidbibliothek und ortsgerichtete Mutagenese der 5´-flankierenden Region wurde ein Kernrezeptor response element (RE) vom DR1 Typ identifiziert, das für die Aktivität des PXR Promotors in den etablierten Zellinien LS174T und HepG2 von essentieller bzw. erheblicher Bedeutung ist. In in vitro DNA Bindungsexperimenten wurde gezeigt, dass HNF4alpha spezifisch an dieses RE bindet. Das identifizierte HNF4alpha-RE war für die Responsivität des PXR-Promotors gegenüber exogenem HNF4alpha1 verantwortlich und war in der Lage, eine Responsivität gegenüber HNF4alpha1 auf einen heterologen Promotor zu vermitteln. Die Kernrezeptoren COUP-TFI und COUP-TFII, die als hemmend wirksame Faktoren auch als funktionelle Antagonisten zu HNF4alpha wirken können, inhibierten die Aktivität des PXR-Promotors in Abhängigkeit vom Vorhandensein des funktionellen HNF4alpha-RE ohne jedoch direkt an dieses zu binden, d.h. vermutlich durch eine trans-Repression. Für eine Beteiligung des Glucocorticoidrezeptors oder PXR selbst an der Regulation der Transkription von PXR wurden keine Belege gefunden.Many organisms possess xenobiotic metabolizing enzymes which, by chemical modification and transportation, are able to detoxify and eliminate drugs, xenobiotics and other potentially harmful compounds of exogenous or endogenous origin. Cytochrome P450 monooxygenases (CYP) are an important part of this biotransformation system. Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP isoform in human liver and is involved in the metabolism of more than 50% of all drugs currently in therapeutic use. Gene expression and enzymatic activity of CYP3A4 are of considerable interindividual variability, which is assumed to be caused by induction of gene expression and genetic polymorphisms. The induction of CYP3A4 and several other important drug metabolizing enzymes and transporters by distinct compounds is mediated by the pregnane x receptor (PXR) which is a member of the family of nuclear receptors. These ligand-activated transcription factors regulate and modulate their target genes´ transcription through interaction with regulatory DNA sequences, other nuclear receptors, transcription factors, and coactivators and corepressors. Although PXR plays a central role as a regulator of several xenobiotic metabolizing enzymes and transporters, and although there were indications for an involvement of the hepatocyte nuclear factor 4alpha (HNF4alpha), the glucocorticoid receptor, and PXR itself, the regulation of PXR expression itself was not well understood. In this dissertation, the expression of CYP3A4 and PXR in human liver and intestinal tissue and the relationship between these two parameters was examined. Furthermore, the impact of individual PXR expression levels on the inducibility of intestinal CYP3A4 and p-glycoprotein by the PXR agonist rifampicin was examined. Finally, the 5´ flanking region of the human PXR gene was characterized in order to identify cis-acting elements and trans-acting factors involved in the regulation of PXR transcription. Expression analysis of CYP3A4 in a tissue bank of human liver samples of 94 individuals confirmed the well-known interindividual variability of CYP3A4 expression on the protein and mRNA level. Furthermore, a sex-dependent dimorphism of CYP3A4 expression was observed. In liver samples obtained from women, the average CYP3A4 protein and mRNA expression was significantly higher than in those obtained from men. The expression of PXR was considerably variable as well, varying more than 17-fold in liver, and was correlated with CYP3A4 mRNA expression in liver as well as in intestinal tissue. Analysis of intestinal inducibility of CYP3A4 and MDR1 mRNA by rifampicin using duodenal biopsies showed that CYP3A4 and MDR1 mRNA are co-induced, but the individual PXR expression was found to be no determining factor for the extent of induction. In order to characterize the 5´ flanking region of the human PXR gene, the transcriptional start site was identified first. It was found that the major transcriptional start sites in human liver and intestinal tissue are slightly different. Subsequently, a library of reporter gene constructs with unidirectional deletions of the PXR promoter region ranging from –2543 to +195 relative to the transcriptional start site in human liver tissue was generated. Performing transient transfection experiments with this library and site-directed mutagenesis of the 5´ flanking region, a DR1 type nuclear receptor response element (RE) was identified, which was essential for or had considerable impact on promoter activity in LS174T and HepG2 cells. The RE bound HNF4alpha specifically, mediated responsivity towards exogenous HNF4alpha1 to the PXR promoter and was able to confer responsivity towards HNF4alpha1 to a heterologous promoter. The nuclear receptors COUP-TFI and COUP-TFII, which are known to exert mostly inhibitory functions and are able to act as functional antagonists of HNF4alpha, were shown to inhibit PXR promotor activity dependent on the integrity of the functional HNF4alpha-RE, but without binding directly to the RE, suggesting a trans-repression mode. No indications were found for an involvement of the glucocorticoid receptor or PXR itself in the transcriptional regulation of PXR. The observed sex-dependent dimorphism of CYP3A4 expression could provide an explanation for previously reported sex-dependent differences in the metabolism of certain drugs and should be taken into consideration in the development and evaluation of drugs which are substrates of CYP3A4. The characterization of the promoter region of the nuclear receptor PXR and the identification of factors involved in its transcriptional regulation lead to a better understanding of the complex regulatory network and signal transduction pathways of the biotransformation system. Using the promoter reporter gene construct library of the 5´ flanking region of the human PXR gene, it should be possible to further clarify the regulation of PXR transcription

Auswirkungen des kontinuierlichen Verbesserungsprozesses auf die Budgetierung im Produktionsbereich deutscher Industrieunternehmen

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