Anja Fritz , University Medicine of Greifswald, Greifswald, Germany
Susanne Brück , Clinical Pharmacology, University Medicine of Greifswald, Greifswald, Germany
Janett Müller , University Medicine of Greifswald, Greifswald, Germany
Stefan Oswald , Department of Clinical Pharmacology, University Medicine of Greifswald, Greifswald, Germany
Intestinal metabolizing enzymes and transporter proteins are critical determinants in the oral bioavailability of drugs. Their inhibition and induction can result in serious unwanted drug-drug interactions (DDIs) and are therefore investigated in the preclinical and clinical phase of drug development. It is well established that the induction of enzymes / transporters is mediated via activation of nuclear receptors (e.g. PXR) and microRNAs. However, there is so far a lack of appropriate in vitro models which can mimic the induction of intestinal enzymes / transporters and in turn predict aforementioned DDIs. Thus, it was the aim of this study to compare the expression pattern of clinically relevant ADME genes / proteins in different intestinal cell lines and their transcriptional as well as epigenetic regulation by prototypic inducers.

Initially, the expression of clinically relevant enzymes / transporters was determined before (basal) and after incubation with rifampicin (100 µM, 72h) in Caco-2, LS180, FHs74Int and HuTu80 cells. Subsequently, the cell line which showed a similar expression pattern of ADME genes and induction properties as the human small intestine was subjected to a time-course experiment. Here, a comprehensive targeted expression analysis of enzymes and transporters (mRNA, N=44; microRNA, N=754; and proteins, N=13) was applied before and after incubation with rifampicin (100 µM) and carbamazepine (100 µM) for 24, 48, 72, 96, 120 and 144h. Gene and microRNA expression analysis was done by real time RT-PCR (TaqMan low density arrays) and protein abundance was measured by validated targeted proteomics assays.

Of the included cell lines, only the LS180 cells showed an expression of the required nuclear receptor PXR, the activation of which resulted in a significantly increased expression of respective target genes (e.g. ABCB1). Moreover, these cells expressed also clinically relevant phase I and II enzymes and transporter proteins (e.g. ABCC2, CYP2C9, CYP3A4, UGT1A1) that are known to be present in the human small intestine. Consequently, LS180 cells were used for the time-course experiment. Incubation of the aforementioned cells with the nuclear receptor ligands rifampicin and carbamazepine caused time-dependent and significant up-regulation of ABCB1, ABCG2, SLC51A / B, CYP2C9, CYP3A4 and UGT1A enzymes. Surprisingly, the highest induction has been observed in each case after incubation for 120-144h. Preliminary data indicate that activation of nuclear receptors caused also time-dependent up-regulation of several microRNAs which were in many cases negatively correlated to their controlled enzymes / transporters.

In conclusion, our data indicate that LS180 cells may be an appropriate in vitro model to investigate intestinal metabolism and transport which are especially useful for studying the induction of the respective enzymes and transporters. Moreover, preliminary data suggest that microRNAs may play an additional role in the induction of intestinal ADME genes most likely by quenching initially their overshooting up-regulation as caused by activation of nuclear receptors.