Antimalarial Artemisinin Drugs Induce Cytochrome P450 and MDR1 Expression by Activation of Xenosensors Pregnane X Receptor and Constitutive Androstane Receptor

Abstract

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.Malaria, a parasitic infection by protozoans, is one of the most threatening human infections worldwide. Among the four human malaria parasites, Plasmodium falciparum causes the majority of all severe cases and deaths. Because of the extensive use of monotherapy in the past, this parasite has rapidly developed resistance to several commonly used and cost-effective antimalarial drugs, including chloroquine, sulfadoxine-pyrimethamine, and mefloquine, in many malaria-affected areas (White, 2004). Spread of resistant parasites is regarded to be a major contributor to the global resurgence of malaria in the last decades. Today, artemisinin drugs represent the only therapeutically used antimalarial drug class to which resistance has not yet been developed. Therefore, these drugs are increasingly used in the treatment of drug-resistant falciparum malaria, especially in combination therapy with a second antimalarial drug (White, 2004).Artemisinin, a sesquiterpene lactone endoperoxide, wasThis work was supported by Deutsche Forschungsgemeinschaft Grant Bu 1249/1-2, 3, and the Robert Bosch Foundation (Germany).Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.009019.ABBREVIATIONS: P450, cytochrome P450; PXR, pregnane X receptor; CAR, constitutive androstane receptor; MDR, multidrug resistance; CITCO,

Molecular Mechanism of Basal CYP3A4 Regulation by Hepatocyte Nuclear Factor 4α: Evidence for Direct Regulation in the Intestine

“…The expression plasmid pcDhHNF4␣, encoding human HNF4␣1, has been described previously (Burk et al, 2005). The expression plasmid pcDhCOUP-TFII, encoding human COUP-TFII was generated by cloning the XbaI/KpnI 1.5-kb full-length cDNA fragment of pFLCOUP-TFII (kindly provided by M. J. Tsai, Baylor College of Medicine, Houston, TX) into pcDNA3.1(Ϫ) (Invitrogen, Carlsbad, CA).…”

“…Oligonucleotides used for HNF4␣ were as follows: primers 5Ј-ATGCTTCCGGGCTGGC-3Ј (exon 3) and 5Ј-TTCGAGTGCTGATCCG-GTC-3Ј (exon 4) and probe 5Ј-CTCATTCTGGACGGCTTCCTTCTTCA-3Ј (exon 4/3, minus strand). Serial dilutions of linearized plasmid pcDhHNF4␣ (Burk et al, 2005), containing the open reading frame of human HNF4␣1, were used to create the calibration curve, ranging from 3 ϫ 10 6 to 3 copies. The CYP3A4 and CYP3A7 assays were performed as described previously (Burk et al, 2002;Wolbold et al, 2003), using respective cDNA plasmid calibration curves from 3 ϫ 10 7 to 30 copies.…”

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

“…The thermal cycling conditions for the assay were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Previously published TaqMan assays were used for the mRNA quantification of CYP2B6 (Zukunft et al, 2005), CYP2C19 (Burk et al, 2005), CYP2D6 (Toscano et al, 2006), and CYP3A4 Wolbold et al, 2003). All primers and probes used are presented in Table 2.…”

Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3)

“…9 The ABCB1 gene, one of the most extensively studied ABC transporters, is responsible for the human multidrug resistance phenotype that is a rapidly growing obstacle to the treatment of numerous infectious diseases, including human immunodeficiency 10 and malaria. 11 The properties of this transporter are also exploited in cancer pharmacological therapy where ABCB1 translocates the chemotherapeutic drugs and other molecules with a broad but defined specificity. 12 A gene duplication of ABCB1 and additional mutations selected as advantageous have created in mammals the T715I G723E L724AfsX744 A737V G954S G762X T775M G126E S320F A840D A1193T NH 2 COOH 1 2 5 4 6 7 8 12 9 11 10 EC2 EC1 ICD2 A250P Y279X A286V ICD1 R159X T175A ICD3 EC3 EC4 EC6 EC5 ICD4 ICD6 ICD5 E888X Y403H V475A A511T E558K R590Q T593A M630V 3…”