Human PCIS as ex vivo model to study the interplay between P-gp and CYP3A4 in the intestine

Introduction: Both efflux transporters and metabolic enzymes are expressed in the intestinal epithelium, and they work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. The drug-drug interactions related to this are of clinical importance and require investigation in the pre-clinical phase and preferably with human tissue. However, proper and efficient in vitro or ex vivo models of the human intestine that express drug transporters and metabolizing enzymes at physiological levels in each region are lacking.The Precision-Cut Intestinal Slice (PCIS) model has been established to investigate drug metabolism, toxicity, and more recently transport in both human and animal tissue. The aim of this study is to show its application toinvestigate the interplay between P-gp and CYP3A4 in human intestine by studying the transport and metabolism of model compound simultaneously.



Methods: Human PCIS were prepared from the explant of human intestine. Quinidine was used as duo-substrate of P-gp and CYP3A4. CP100356 and PSC 833 were employed as selective P-gp inhibitors,while verapamil and ketoconazole were used as inhibitors of both transporters and metabolic enzymes. The metabolite, 3OH-quinidine, was measured by LC-MS in both medium and tissue in the presence or absence of the inhibitors. Furthermore, the tissue content of quinidine was also quantified by LC-MS to investigate the influence of transporter inhibition.



Results: In human jejunum,the intracellular concentration of quinidine was limited by P-gp effluxat low concentrations (below 5 µM), whereas at concentrations between 5 to 50 µM CYP3A metabolism became limiting. The selective P-gp inhibitors increased the intracellular concentrations of both quinidine (2.3-2.7fold) and 3-OH-quinidine (9.4-11.4 fold), and the total metabolite production (1.9-2.3fold).Verapamil and ketoconazole increased the intracellular quinidine concentrations but decreasedthe metabolite production significantly. As expected,the effect of this interplay was different in PCIS of the different regions of the intestine.



Conclusion:This study showed that human PCIS is a reliable, simple and fast ex vivomodel to study the intestinal transport and metabolism together. The results indicate that the interplay between P-gp and CYP3A4is dependent on the concentration of the drug and the selectivity of the inhibitors. Furthermore, it exhibits regional differences, probably due to the different expression profiles of P-gp and CYP3A4.In future studies, human PCIS can be used to predict the interplay between transport and metabolism to decrease the risk of drug-drug interactions and inadequate pharmacotherapeutic effects of newly developed compounds.