Human ileal organoid model recapitulates clinical incidence of diarrhoea associated with small molecule drugs
Gastro-intestinal toxicity (GIT) can negatively impact dosing, thereby limiting efficacy and treatment options for patients. Specialised preclinical models can help predict the GIT potential of drug candidates early in the discovery process.
In vitro models of mouse and human GIT can be attributed to one of two categories:
- 2D primary gut epithelial cells or tumour-derived Caco-2 cells to study transport and metabolism or assess barrier function of the gut epithelium in response to pathogenic bacteria or chemical insult on transwell membranes.
- 3D culture of primary gut stem cells or small intestinal crypts via Matrigel encapsulation. Described first by Hans Clevers in 2009, the GI organoids or enteroids are useful for characterisation of the gut biology, transport and host-pathogen interaction in the gut, as well as response of the gut epithelium to pro-inflammatory or cytotoxic challenge.
Peters et al. previously established a reference set of 31 well-characterised pharmaceutical compounds to use as a benchmark and assess predictivity of in vitro gut models for drug-induced GIT. The set showed an accuracy of 83-84% for recapitulating drug-induced diarrhoea in the clinic, in an in vitro gut barrier model derived from primary human small intestinal stem cells using trans-epithelial electric resistance (TEER).
In this study, the authors assayed the same reference set of drugs on human ileal enteroids to recapitulate the drug-induced diarrhoea.
Enteroids were generated from four human donors using isolated crypt from ileal tissue, encapsulated in Matrigel. Inherent donor variability in growth rate was observed by measuring the mean enteroid diameter over time from phase contrast images.
Differentiation of the enteroids was assessed immunohistochemically for lysozyme (Paneth cells), chromogranin A (enteroendocrine cells), mucin (goblet cells) and sucrase isomaltase (enterocytes). A correlation between the size and the epithelial cell differentiation status of the enteroids was observed. MUC2 was not expressed at seven days of culture (mean diameter of 400μM) but all four cell types were present after ten days of culture (mean diameter of 600μM).
The 31-drug reference set was first screened in an eight-point dose response (10nM – 100μM) for four days resulting in reproducible data for 28 drugs. A secondary screen was performed with eight drugs from the set (three were too potent in the first screen and were re-tested in an expanded concentration range. Five were selected as exhibiting a varying potency in the first screen and to serve as control in the secondary screen). The secondary screen corroborated the findings from the first screen for the five control drugs.
Drugs were classed as non-diarrheagenic (<0-3% incidence of diarrhoea in the clinic) or diarrheagenic (>40% incidence in the clinic) with a further five sub-categories based on incidence: severe (70%-100%), moderate (50-69%), mild (40-49%), minimal (1-3%) and no incidence.
Based strictly on IC50 value, 77% showed balanced accuracy for recapitulating drug-induced diarrhoea. IC50 value for 15 out of 31 drugs was below 100μM and characterised as assay positives. The remaining 16 drugs were deemed assay negatives.
Normalising viability IC50 for the enteroid to clinical Cmax exposures (IC50/Cmax) provided a balanced accuracy of 90% for recapitulating clinical drug-induced diarrhoea incidence. This method was used with a previous dataset in a barrier function assay to determine an optimal distinction between the drugs to induce diarrhoea.
Differences in drug responses between the gut epithelial barrier model and the enteroid viability assay present an opportunity to explore the suitability of each assay for sensitivity or lack of sensitivity to known diarrhoea inducing GI toxicants.
In this study the authors described the characterisation of a human 3D enteroid culture model for assessing drug-induced GIT. The assessment of enteroid viability in response to drug exposure is a simple, reliable, and high throughput assay readout. It can reasonably be applied in preclinical drug development to determine the risk of drug-induced GIT in humans.
Further research can be performed to reverse the polarity of the enteroid (“inside-out”) with drug exposure through the basal side where it is primarily from the apical side in the gut.
Reference
David G. Belair, Richard J. Visconti, Miyoun Hong, Mathieu Marella, Matthew F. Peters, Clay W. Scott, Kyle L. Kolaja.
Human ileal organoid model recapitulates clinical incidence of diarrhea associated with small molecule drugs Toxicology in Vitro, Volume 68, 2020, 104928.
10.1016/j.tiv.2020.104928
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