What is DMET

Abstract:Nuclear receptors (NRs), most notably the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR), regulate the transcription of several drug metabolizing enzymes and transporters (DMET) and thus represent important regulators of drug metabolism in the liver. Accordingly, the ligand dependent activation of these NRs by drugs and other xenobiotics contributes to the intra- and inter-individual variability of the drug detoxifying system. CAR and PXR were further shown to regulate the transcription of key enzymes involved in lipid and glucose metabolism. The NR peroxisome proliferator-activated receptor alpha (PPARa), a key regulator of fatty acid catabolism and target of lipid lowering fibrates, was recently identified as a direct regulator of cytochrome P450 3A4 (CYP3A4) and also potentially of other DMET genes. In this respect, CAR, PXR and PPARa are determinants of an overlapping number of liver functions including drug metabolism and energy homeostasis and are therefore associated with adverse drug reactions as well as liver disease like steatosis. Until now there have been no comparative studies investigating the transcriptomes of CAR, PXR and PPARa in humans. Therefore, a major focus of this study was to assess the genome-wide transcriptional changes provoked by these NRs in primary human hepatocytes (PHHs). To investigate human liver-specific gene expression and its regulation PHHs represent the most suitable available in vitro cell system. To identify the CAR-, PXR- and PPARa-specific genome-wide expression changes, hepatocyte cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARa (WY-14643) as well as DMSO (vehicle control). Afterwards, the mRNA expression in these samples was determined utilizing Affymetrix® microarrays. The obtained expression data were statistically evaluated to identify the genes that showed a differential expression in response to the agonist treatments and to investigate to which metabolic functions these genes contribute. The results of these experiments confirmed that CAR, PXR and PPARa regulated a highly overlapping but distinct set of genes coding for DMET. For example, according to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyzes expression of 10 DMET genes were shown to be regulated by all three NRs, whereas other DMET genes responded exclusively to the activation of one of the NRs. In addition several DMET related genes previously not shown to be regulated by CAR [like CYP2E1, sulfotransferase 1B1 (SULT1B1), UDP-glucuronosyltransferase 2B4 (UGT2B4) and cytochrome P450 reductase (POR)], PXR [like CYP2E1, alcohol dehydrogenases (ADHs) , flavin containing monooxygenase 5 (FMO5) and glutathione peroxidase 2 (GPX2)] or PPARa like UBT2B4, ADH1s and FMO5) were identified to respond to the respective agonists. For PXR and CAR, this extends the list of genes by which these NRs influence drug metabolism and potentially contribute to drug-drug interactions (DDIs). The results obtained further specify the role of PPARa as a regulator of drug metabolism in vitro by increasing expression of, e.g., CYP3A4, 2B6, 2C8 and UGT1A1, thus pointing to a potential role of PPARa in adverse drug reactions in vivo. Furthermore, several genes coding for proteins involved in energy homeostasis, were identified as differentially expressed in response to PXR activation [eg, pyruvate dehydrogenase kinase 4 (PDK4), glycogen synthase 2 (GYS2), carnitine palmitoyltransferase 2 (CPT2)], where such a relation was not reported so far. These results further expanded the knowledge of how PXR potentially impact fatty acid catabolism, gluconeogenesis and lipid de novo synthesis and provide interesting starting points to investigate how PXR activation contributes to altered glucose and lipid levels or disease like hepatic steatosis. Besides ligand-dependent regulation of nuclear receptors, post-translational modification has also been shown to influence the activity of liver-enriched NRs and expression of their target genes. In this context, protein kinase A (PKA) had been shown to repress CYP3A4 expression via PXR in a species-dependent manner, whereas the influence of PKA on the expression of other DMET genes had not been investigated in detail so far. The second part of this work therefore investigated the impact of PKA activation on the expression and activity of important drug metabolizing enzymes in a PXR as well as a CAR-dependent manner. In this work PKA activation in primary human hepatocytes was identified as a determinant of drug metabolism in vitro by repressing PXR- and CAR-mediated or reducing basal expression and activity of CYP1A1, CYP2B6, CYP2C8 and CYP3A4, but also expression of ATP-binding cassette B1 (ABCB1) and UGT1A1. Using reporter gene assays, these observed effects could be linked to PKA-mediated repression of PXR and CAR activity that may involve phosphorylation of these NRs. It could be further shown that expression of DMET genes was also repressed by the fasting hormone glucagon, a physiologically relevant activator of PKA signaling, which was not investigated in humans so far. Due to the promiscuous ligand-specificity of PXR, which includes numerous compounds, drug treatment often leads to PXR activation, even with so-called “natural” compounds like St. John's wort (SJW). It would thus be highly desirable to develop strategies in drug development to assess or circumvent the activation of NRs without compromising the pharmacological effects. Therefore, the last part of this work consists of an in vitro study to investigate synthetic acylated phloroglucinols, designed as substitutes for hyperforin, regarding their potential to activate PXR. Hyperforin the major active constituent of the plant SJW used to treat depressions was shown to exert its antidepressant properties via indirect inhibition of serotonin reuptake by selectively activating the canonical transient receptor potential channel 6 (TRPC6). In addition, hyperforin is associated with clinically relevant drug-drug interactions in patients that had taken SJW concomitantly with other drugs due to potent activation of the nuclear receptor PXR by hyperforin. The phloroglucinol derivatives investigated in this thesis had previously been evaluated for their bioactivity. It had been reported that five of the nine synthetic acylated phloroglucinols activate TRPC6 with similar potency as hyperforin. In this work, all these nine synthetic phloroglucinol derivatives were investigated in comparison to hyperforin and rifampicin for their potential to activate PXR. Hyperforin and rifampicin treatment of HepG2 cells co-transfected with a human PXR expression vector and a CYP3A4 promoter reporter construct resulted in potent PXR-dependent induction, while all TRPC6-activating compounds failed to show any PXR activation or to antagonize rifampicin-mediated CYP3A4 promoter induction. Hyperforin and rifampicin treatment of primary human hepatocytes resulted in highly correlated induction of PXR target genes, whereas treatment with the phloroglucinol derivatives elicited moderate gene expression changes that only weakly correlated to those of rifampicin treatment. The observed lack of PXR activation by the TRPC6 activating phloroglucinols was further supported by in silico pharmacophore modeling that did not indicate potent agonist or antagonist interactions for the TRPC6 activating derivatives and docking studies that suggested interaction of only one of these compounds. These in silico studies performed by Prof. Sean Ekins are published together with the results presented in this work (Kandel et al., 2014). This approach shows that strategies avoiding PXR activation are conceivable in drug development in order to prevent DDIs and improve drug safety. Taken together, these results further increase the number of genes by which CAR, PXR, and PPARa contribute to the regulation of drug metabolism and energy homeostasis. Moreover, it was demonstrated that the PKA, which is involved in the transduction of the effects of, e.g., the hormone glucagon, represents a determinant of the drug detoxifying system in humans. Furthermore, a strategy could be presented, taking the example of the hyperforin derivatives, which can be used to investigate and avoid DDIs in drug development. Such information will become imperative in future personalized medicine and the ever-present polypharmacy in order to handle intra- and inter-individual variability and to minimize drug failure or drug-drug interactions.
Nuclear receptors, above all the constitutive androstane receptor (CAR) and the pregnant X receptor (PXR), regulate the transcription of numerous drug metabolizing enzymes and transporters (DMET) and are therefore important regulators of the detoxification processes in the liver. Consequently, the ligand-dependent activation of these receptors by drugs and other exogenous substances contributes to the intra- and inter-individual variability of drug metabolism. CAR and PXR are also involved in the regulation of fat and glucose metabolism. Also for the nuclear receptor Peroxisome Proliferator-activating Receptor Alpha (PPARa), a key regulator of fatty acid degradation and starting point of fibrates, it was recently shown that this directly regulates the expression of cytochrome P450 3A4 (CYP3A4) and also regulates other important ones DMET genes is associated. In this context, CAR, PXR and PPARa represent important determinants of liver functions such as drug metabolism and energy homeostasis and are therefore associated with drug side effects and liver diseases such as steatosis. Up to now there are no comparative studies that have examined the transcriptomes of the nuclear receptors CAR, PXR and PPARa in humans. Therefore, a main aspect of this work was to investigate the genome-wide transcriptional changes that are caused by these nuclear receptors in human liver cells. These investigations were carried out with primary human hepatocytes, since these cells represent the most suitable cell model available for investigating liver-specific gene expression and its regulation. In order to determine the CAR-, PXR- and PPARa-specific, genome-wide expression changes, hepatocyte cultures from six different donors with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARa (WY-14643), as well as treated with DMSO, the vehicle control. In the following, the mRNA expression in these samples was determined using Affymetrix® microarrays. The expression data were subjected to statistical analysis to identify the genes that showed significantly altered expression by the agonist treatments; Furthermore, the metabolic functions with which these genes are associated were investigated. The results obtained in this way confirmed that CAR, PXR and PPARa regulate different, but nevertheless partially overlapping groups of DMET genes. For example, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyzes showed that a group of ten DMET genes were equally regulated by CAR, PXR and PPARa, whereas the expression of further DMET genes was exclusively through the activation of one of the three Receptors was affected. For a number of these genes, regulation by the receptors CAR [e.g. CYP2E1, sulfotransferase 1B1 (SULT1B1), UDP-glucuronosyltransferase 2B4 (UGT2B4) and cytochrome P450 reductase (POR)], PXR e.g. CYP2E1, alcohol dehydrogenases (ADHs), flavin-dependent monooxygenase 2) glut (GPO5-peroxygenase 5) (FMO-5-peroxide) and PPARa [e.g. UBT2B4, ADH1s and FMO5] shown for the first time. For CAR and PXR, this expands the list of genes by which these nuclear receptors influence drug metabolism and potentially contribute to drug interactions. The data obtained also substantiate the function of PPARa as a regulator of DMET genes in vitro, for example by increasing the expression of CYPs 3A4, 2B6, 2C8 and UGT1A1. This also suggests that PPARa are involved in drug side effects in vivo. The analyzes also showed that genes such as pyruvate dehydrogenase kinase 4 (PDK4), glycogen synthase 2 (GYS2) and carnitine palmitoyltransferase 2 (CPT2), whose proteins are involved in energy homeostasis, were differentially expressed as a result of PXR activation. Such a connection was previously unknown for these genes. These results expand the existing knowledge of the potential mechanisms by which PXR influences metabolic processes such as fatty acid breakdown, gluconeogenesis and de novo lipogenesis and thus PXR can contribute to changes in lipid and glucose levels or diseases such as hepatic steatosis. In addition to a ligand-dependent regulation of nuclear receptors, it was also shown for post-translational modifications that they influence the activity of nuclear receptors and their target gene expression. For protein kinase A (PKA), for example, repression of CPY3A4 expression as a result of PXR phosphorylation was shown. An influence of PKA on the expression of other human DMET genes, however, has hardly been investigated so far. The second part of this thesis therefore dealt with the investigation of the influence of PKA activation on the expression and activity of drug-metabolizing enzymes, depending on PXR and its closest related nuclear receptor CAR. In this work it was shown by qRT-PCR analyzes of mRNA expression and CYP activity measurements by means of a cocktail assay in primary human hepatocytes that PKA activation by 8-bromo cAMP is a determinant of drug metabolism in vitro. These analyzes showed a repression of the CAR and PXR mediated, as well as the basal expression and activity of CYP1A1, CYP2B6, CYP2C8 and CYP3A4 as well as the expression of ATP-binding cassette transporter B1 (ABCB1) and UGT1A1. Reporter gene experiments also showed that the observed effects were associated with decreased PXR and CAR activity. It was also shown that the expression of DMET genes was also repressed by the hormone glucagon, a physiologically relevant activator of the PKA signaling pathway, which had not yet been investigated in this form. Due to the broad ligand specificity of PXR, treatments with medicinal products, as well as with so-called “natural” remedies such as St. John's wort, often lead to undesired PXR activation. This PXR activation and the resulting altered expression and activity of DMET are associated with a large number of drug side effects. Such drug side effects have also been described for St. John's wort preparations that can be traced back to the potent PXR agonist Hyperforin. Hyperforin, the strongest active component of St. John's wort, which is used to treat depression, mediates its antidepressant effect through selective activation of the TRPC6 channel and, as a result, an inhibition of serotonin reuptake. In order to avoid such drug side effects, it would therefore be of great advantage if strategies were available during drug development that could prevent PXR activation without impairing the pharmacological effect. As an example of such a strategy, an in vitro study was carried out in the last part of this work to examine synthetic, acylated phloroglucinols, which were developed as substitutes for hyperforin, for their PXR activation potential in comparison to hyperforin and rifampicin. An earlier in vitro study could already show that five of these synthetic acylated phloroglucinols have a pharmacological effect comparable to that of hyperforin. Hyperforin and rifampicin treatment of HepG2 cells transfected with an expression vector for human PXR and a CYP3A4 reporter construct resulted in a potent PXR-dependent induction of the CYP3A4 promoter, while the TRPC6-activating substances showed no PXR activation and CYP3A4 promoter induction. Treatment of primary human hepatocytes with hyperforin and rifampicin resulted in a strongly correlated induction of PXR target genes; treatment with the phloroglucinol derivatives, on the other hand, produced only moderate changes in expression, which correlated only weakly with the effects mediated by rifampicin treatment. The lack of PXR activation by the TRPC6-activating phloroglucinols observed in this in vitro study was further supported by the in silico pharmacophore modeling and binding studies carried out as part of a cooperation by Prof. Ekins, which only showed weak interactions of the TRPC6-activating derivatives with PXR predicted (Kandel et al., 2014). This approach showed that strategies aimed at investigating and avoiding PXR activation offer a conceivable approach in drug development to prevent the occurrence of drug interactions and thus improve the safety of drugs. In summary, it can be said that in the genome-wide study on human hepatocytes presented here, numerous new target genes of the NRs CAR, PXR and PPARa were identified, which could contribute to the influence of these NRs on drug metabolism and energy homeostasis. In addition, it was shown that PKA, which, among other things, mediates the effects of the hormone glucagon, is an influencing factor for drug detoxification in humans. Furthermore, using the example of hyperforin derivatives, a strategy was presented that can contribute to the investigation and avoidance of drug interactions in drug development. With personalized medicine and ubiquitous polypharmacy in mind, such information will be essential in the future to account for problems caused by intra- and inter-individual variability and to minimize the incidence of therapy failure and drug interactions.