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Characterization and establishment of advanced intestinal cell culture models and the evaluation of potential new biomarkers for the prediction of drug-induced intestinal toxicity

Hoffmann, Stefanie (2023)
Characterization and establishment of advanced intestinal cell culture models and the evaluation of potential new biomarkers for the prediction of drug-induced intestinal toxicity.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00023643
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

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 One of the main reasons why a drug development gets stopped or the drug gets withdrawn from the market is due to enormous side effects, specifically in the GI tract.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.

Typ des Eintrags: Dissertation
Erschienen: 2023
Autor(en): Hoffmann, Stefanie
Art des Eintrags: Erstveröffentlichung
Titel: Characterization and establishment of advanced intestinal cell culture models and the evaluation of potential new biomarkers for the prediction of drug-induced intestinal toxicity
Sprache: Englisch
Referenten: Laube, Prof. Dr. Bodo ; Süß, Prof. Dr. Beatrix
Publikationsjahr: 2023
Ort: Darmstadt
Kollation: XXXIII, 288 Seiten
Datum der mündlichen Prüfung: 19 Januar 2023
DOI: 10.26083/tuprints-00023643
URL / URN: https://tuprints.ulb.tu-darmstadt.de/23643
Kurzbeschreibung (Abstract):

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.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Die Entwicklung eines neuen Medikaments ist ein sehr zeitaufwändiger und teurer Prozess, der eine große Anzahl von in vitro und in vivo Experimenten erfordert (Guengerich, 2006; Paul et al., 2010; DiMasi, Grabowski and Hansen, 2016). Vor allem die Tierversuche sind oft sehr komplex und ethisch umstritten. Außerdem scheitern viele Arzneimittelkandidaten während der Entwicklung. Einer der Hauptgründe, warum die Entwicklung eines Medikaments gestoppt oder das Medikament vom Markt genommen wird, sind die enormen Nebenwirkungen, insbesondere im Magen-Darm-Trakt. Ziel dieser Arbeit war es, fortschrittliche Darmzellkulturmodelle (Caco-2 2D, Caco-2 in der OrganoPlate® und Dickdarmorganoide als 3D-Modell) zu etablieren und neu Biomarker zu evaluieren, die eine zuverlässigere und empfindlichere Vorhersage von arzneimittelinduzierten gastrointestinalen Schäden ermöglichen. Die Charakterisierung des 3D Dickdarmorganoid Models zeigte eine Ähnlichkeit des menschlichen Darms in Struktur und Funktion. Dies konnte z.B. durch polarisierte, verbundene Darmepithelzellen in 3D-Struktur gezeigt werden oder durch Immunfluoreszenzsfärbungen von darmspezifischen Proteinen (E-cadherin, α-Tubulin, ZO-1, Ezrin, CYP2C9 oder Claudin7). Die Stoffwechselaktivität in den drei Zellkulturmodellen konnte gezeigt werden durch die Messung von darmspezifischen Genen (Phase I, II und III Enzyme). Die beste Vorhersagbarkeit toxischer Wirkungen von Arzneimitteln konnte ebenso im 3D-Organoidmodell nachgewiesen werden. Nach 24h Behandlung zeigten die Dickdarmorganoide die stärkste Reaktion auf toxische Substanzen. Dies konnte mit niedrigeren IC50 Werten, im Vergleich zu den beiden Modellen mit Caco-2 Zellen, gezeigt werden. Für die Vorhersage toxischer Wirkungen ist die Verwendung von Biomarkern eine zuverlässige Methode. In präklinischen Studien fehlt es jedoch bisher an prädiktiven und zuverlässigen Biomarkern (John-Baptiste et al., 2012; Carr et al., 2017). Nach der Behandlung der verwendeten Zellkulturmodelle mit Substanzen, die für die Schädigung des Magen-Darm-Trakts bekannt sind, wurden die sezernierten und exprimierten Marker untersucht. Diese Studie zeigte die Eignung einiger Proteinprodukte als potenzielle Biomarker. Im 3D Dickdarmorganoid Model waren LCN-2, CRP und HDC in den behandelten Proben stärker exprimiert im Vergleich zur Kontrolle, jedoch konnten hier keine signifikanten Unterschiede berechnet werden. Im Vergleich hierzu zeigte das Caco-2 2D und OoC Model bessere Anwendungsmöglichkeiten bei der Nutzung von Biomarkern zur frühen Erkennung von medikamenteninduzierten Schäden im Darm. Die Gene LCN-2 und MLCK waren signifikant höher exprimiert in behandelten Proben im Vergleich zur Kontrollprobe. Ebenso messbar war eine Schädigung und somit Abnahme der Enterozyten mithilfe des Biomarkers Citrullin, welcher bisher nur Anwendung in Patienten findet. Dieser konnte in signifikant niedrigeren Mengen in den behandelten Proben der Caco-2 Modelle (2D und OoC) gemessen werden im Vergleich zu den Kontrollproben. Zusammenfassend lässt sich sagen, dass fortgeschrittene Darmzellkulturmodelle ein vielversprechendes Modell sind, um den menschlichen Darm in vitro abzubilden und die potenziellen Auswirkungen von Arzneimitteln zu bewerten. Diese Modelle können zur Untersuchung der Darmbiologie, der metabolischen und toxikologischen Profile und zur Bewertung potenzieller Biomarker für die Vorhersage arzneimittelinduzierten gastrointestinalen Schäden in einer physiologischeren Umgebung verwendet werden. Sie können z.B. in den Prozess der Arzneimittelentwicklung als frühes Screening-Instrument integriert werden und helfen, toxische Nebenwirkungen von Arzneimitteln zu erkennen. Der Hauptvorteil dieser Modelle besteht darin, dass sie dazu beitragen können, Ergebnisse dieser in vitro Versuche zu nutzen für die präzisere Vorhersage von Nebenwirkungen im Menschen und somit auch die Lücke zwischen einfachen 2D- und komplexen Tier-Modellen zu schließen.

Deutsch
Status: Verlagsversion
URN: urn:nbn:de:tuda-tuprints-236432
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Neurophysiologie und neurosensorische Systeme
Hinterlegungsdatum: 13 Apr 2023 13:54
Letzte Änderung: 14 Apr 2023 06:10
PPN:
Referenten: Laube, Prof. Dr. Bodo ; Süß, Prof. Dr. Beatrix
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 19 Januar 2023
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