Abstract
Malaria is one of the oldest diseases know to mankind, still having devastating consequences; killing 1 million people/year. The efforts to control this disease have been focused on the control of its transmission and, most importantly, its clinical management by effective chemotherapy. Concerning the latter, several antimalarials have been developed in the last decades. They can be subdivided into four structural classes: aminoquinolines, antifolates, artemisinin derived compounds and naphtoquinones. Although reasonably well studied in pharmacokinetics aspects, very few data is available concerning their effects in the gene expression regulation system. This is particularly relevant concerning the xenosensors PXR and CAR. In a first part, this project aimed to elucidate the capacity of currently used antimalarials and their main known metabolites to activate the PXR/CAR system. For this purpose, mammalian two hybrid and gene reporter assays, together with induction studies in primary human hepatocytes were performed. For PXR and CAR we could observe activation, by compounds from the artemisinin family (artemisinin, arteether, artemeether, deoxy-arteether and deoxy-artemisinin) both in vitro and in primary human hepatocytes, corroborated by the induction of prototypical target genes. A repression by artesunate of key targets genes, CYP3A4 and CYP2B6 was observed by this compound in primary hepatocytes, confirming the observed inhibition in vitro of the induction of both PXR and CAR. Further activation of PXR by the aminoquinolines, lumefantrine, carboximefloquine, amodiaquine, DEAQ and chloroquine, was observed in the in vitro system. Amodiaquine also induced CAR in the in vitro system. From the aforementioned compounds only carboximefloquine, consistently induced the induction of CYP2B6 in the hepatocytes, indicating a promoter specific activity. Both amodiaquine and DEAQ showed a repression of CYP3A4 and CYP2B6, indicating that although they may induce the interaction of PXR with its co-activators, they may not be able to induce the release of co-repressors. The present data is of particular interest regarding the pivotal role of artemisinins and aminoquinolines in the current worldwide WHO supported artemisinin combination therapies (ACT), the first line treatment currently recommended. Future evidence based developments of this therapy can benefit from this data on the high probability of induction of this two gene expression systems by ACT components, and its drug-drug interactions. The second part of this project focused on the study of the genetic basis of variability of PXR dependent induction of key CYP450 upon exposure to artemisinins. The project was based on a previously study in 75 Vietnamese subject (Asimus et al., 2007), where metabolic ratios for a number of pivotal PXR/CAR inducible CYPs (e.g. CYP3A4, CYP2C19) were determined upon artemisinin compounds exposure. For this purpose the promoter region, exons and exon-intron boundaries of the PXR gene were re-sequenced for every subject. This approach allowed the identification of 32 SNPs, six of them described for the first time. Overall, we could observed an increased prevalence of the minor allele for the polymorphisms 252 /275 A>G and 10331 A>G, 10483 T>C in the group of higher inducers. Further analysis of these SNPs in primary human hepatocytes showed a higher CYP3A4 mRNA induction in the carriers of the wt/wt genotype. This is an indication that different distribution of the allele in the high and low inducers group is not influencing directly the expression of CYP3A4. Taken together we could confirm some of the data published concerning phenotypic changes induced by PXR SNPs. However no clear results were obtained for the newly identified SNPs due to a reduced number of samples, except for the SNP F420Y.
Author and committee
dc:creator, dc:contributor.*- Author
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- Piedade, Rita
Identifiers
dc:identifier.*- Identifier
- hdl:10900/49443