title: Characterization and establishment of advanced intestinal cell culture models and the evaluation of potential new biomarkers for the prediction of drug-induced intestinal toxicity
Creators: name: Hoffmann, Stefanie
Publisher: name: UNSPECIFIED
Year: 2023

description: Drug discovery and development is a very time consuming and expensive process which requires a hugh number of in vitro and in vivo experiments (Guengerich, 2006; Paul et al., 2010; DiMasi, Grabowski and Hansen, 2016). Especially in vivo experiments are very complex and often under ethical discussion. Beside this many drug candidates fail during the development. One of the main reasons why a drug development gets stopped or the drug gets withdrawn from the market is due to enormous side effects, specifically in the GI tract. The aim of this thesis was to establish advanced intestinal cell culture models (Caco-2 2D, Caco-2 in the OrganoPlate® and colon organoids as a 3D model) and evaluate novel biomarkers, which can predict more reliable and sensitive drug-induced gastrointestinal injury. The characterization of the 3D colon organoid model showed high similarity of the human intestine in structure and function. This could be shown e.g. by polarized, connected intestinal epithelial cells in 3D structure or by immunofluorescence staining of intestine specific proteins (E-cadherin, α-tubulin, Zonula occludens-1 (ZO-1), Ezrin, CYP2C9 or Claudin7). The metabolic activity in the three cell culture models could be shown by measuring intestine specific genes (Phase I, II and III enzymes). The best predictivity of toxic effects of drugs could be observed in the 3D organoid model which recapitulate high similarity to the human colon. After 24h of treatment the colon organoids showed the strongest response to toxic compounds. This could be shown with lower IC50 values compared to the two other models with Caco-2 cells. For the prediction of toxic effects, the use of biomarkers is a reliable tool. But so far in pre-clinical studies a lack of predictive and reliable biomarkers is existing (John-Baptiste et al., 2012; Carr et al., 2017). Specifically, the 3D organoid model used in this thesis showed the possible application as tool for the study of potential biomarkers to predict drug-induced gastrointestinal injury events. After the treatment of these models with compounds which are known for the damage in the GI tract, the secreted and expressed markers were examined. This study demonstrated the suitability of some proteins and genes as potential biomarkers. In the 3D colon organoid model Lipocalin-2 (LCN-2), C-reactive protein (CRP) and Histidine decarboxylase (HDC) were higher expressed in the treated samples compared to the control, but no significant differences could be calculated here. In comparison, the Caco-2 2D and Organ-on-a-chip (OoC) model showed better application in the use of biomarkers for early detection of drug-induced damage in the intestine. The genes LCN-2 and Myosin light chain kinase (MLCK) were significantly higher expressed in treated samples compared to the control sample. Also measurable was a damage and thus decrease in enterocytes using the biomarker citrulline, so far only used in vivo. This biomarker was measured in significantly lower amounts in the treated samples of the Caco-2 models (2D and OoC) compared to the control samples. In conclusion, advanced intestinal cell culture models are a promising tool to recapitulate the human intestine and helps to evaluate the potential effects of drugs. These models can be used to study intestinal biology, metabolic and toxicologic profiles and to evaluate potential biomarkers for the prediction of drug-induced gastrointestinal injury in a more physiological environment. The models can for example be integrated into the drug development process as early screening tool and help to identify toxic side effects of drugs. The main benefit of these models is that these models can help to translate in vitro results to the human and can thereby bridge the gap between simple 2D and complex in vivo models. Overall, it could be said that not one specific model is suitable for all tested assays. Each model has his advantages and can be used for different questions.