Merve Ceylan: In vitro models of the human intestine for better prediction of drug absorption, pre-systemic metabolism, and bioavailability

Abstract

The human small intestine is the primary site for drug absorption, hence intestinal in vitro models can bridge the gap to in vivo studies ultimately improving the drug development processes. This PhD thesis explores the global proteome and functional characteristics of four in vitro models that recapitulate the epithelial barrier of the human small intestine.

In Project I, we analyzed the global proteome of 2D enteroid monolayer models, focusing on markers related to stem cells, differentiation, actin regulatory proteins, and ADME proteins. We assessed the maturation of these models to evaluate their potential for studying bacterial infections and drug disposition, thereby establishing their functional capacity to replicate in vivo conditions.

Project II built upon the findings from Project I by evaluating the differentiation level of human intestinal 3D enteroid models through global proteomics and functional assays. Our proteomics analysis revealed that the 3D models exhibit a higher level of differentiation compared to 2D models. We also demonstrated the functionality of some key clinically important proteins, including CYP3A4, Pgp/MDR1, and BCRP, within the enteroids. This project confirms that these advanced 3D models are well-suited for studying drug metabolism and transport.

While enteroids are derived from stem cells and differentiated under laboratory conditions, freshly isolated enterocytes come from human tissue and are already fully differentiated. This potentially makes them a more accurate representation of in vivo conditions. Leveraging this advantage, in Project III, we focus on improving the isolation method for enterocytes from fresh jejunum tissue specimens. We quantified important metabolic enzymes, performed drug metabolism assays and demonstrated the enterocytes functionality for intestinal drug metabolism studies.

In Project IV, we develop a co-culture model using two MDCK cell lines that overexpress human MDR1 and BCRP. Following global proteomics characterization, we performed drug transport studies with both specific and shared drug substrates in Transwell and the Enabling Absorption Device settings. Our findings demonstrate the potential of this co-culture model for studying the role of active efflux in the absorption of drugs that require advanced formulations.

Together, these projects enhance our understanding of intestinal in vitro models, supporting their application in drug development research.