TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF METABOLIZING ENZYMES AND TRANSPORTER PROTEINS IN THE HUMAN INTESTINE AS CAUSED BY PROTOTYPICAL INDUCERS OF METABOLISM AND TRANSPORT

Drug transporters and drug metabolizing enzymes located in the small intestinal epithelia are crucial determinants in the pharmacokinetics of orally administered drugs. Nuclear receptor ligands (NRL) such as rifampicin or carbamazepine are known to influence expression of these proteins and can cause thereby unwanted drug-drug interactions. However, the available knowledge is rather limited and was predominately focused on gene expression data. Moreover, little is known about the intestinal regulation of enzymes and transporters beyond activation of nuclear receptors (e.g. epigenetic regulation by microRNAs). Thus, it was the aim of this study to comprehensively analyse the impact of NRL on gene and protein expression of clinically relevant enzymes and transporters and the potential regulatory role of microRNAs in the human intestine.

Tissue specimen (~ 2-5 mg) from duodenal mucosa were taken before and after treatment with rifampicin (600 mg, 8 days) or carbamazepine (600 mg, 18 days), respectively. Protein abundance of clinically relevant intestinal transporters was quantified using validated LC-MS/MS-based targeted proteomics assays. In parallel, the mRNA expression of 43 ADME genes and 754 microRNAs was analysed using TaqMan^® real-time RT-PCR arrays. In order to conclude on the regulatory relevance of microRNAs, correlation analysis to the respective protein amounts, complex in silico-based target gene prediction and luciferase reporter assays with appropriated in vitro systems have been performed.

Rifampicin treatment led to a significant increase in gene expression of ABCB1, ABCC2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and the UGT1A family while the protein abundance was significantly increased only for ABCB1, CYP3A4, CYP2C9 and UGT1A1. In contrast to this, pre-treatment with carbamazepine led to no significant changes on protein levels of the investigated transporters and enzymes, whereas mRNA expression of several ADME genes were found to be significantly increased. 127 microRNAs were negatively correlated to the protein content of intestinal transporters. Of these several microRNAs could be verified as regulators of some intestinal transporters in silico and in vitro.

This study investigated for the first time the impact of rifampicin and carbamazepine on the gene and protein expression of clinically relevant enzymes and transporters in the human small intestine in a comprehensive manner. Rifampicin was found to be a strong inducer of several enzymes and transporters which is in good agreement to profound DDI observed in clinical studies. In addition to the activation of nuclear receptors, the expression of intestinal enzymes and transporters may be also influenced by microRNAs.