Harnessing human colonoid-derived monolayers as an in vitro platform for investigating accumulation, toxicity, and pharmacodynamics of small molecules

The purpose of this study was to evaluate differentiated human colonoid-derived monolayers (hCDMs) as an in vitro platform for investigating small molecule accumulation, toxicity, and pharmacodynamics (PD), in comparison to Caco-2 monolayers. Differentiated hCDMs formed polarized monolayers with a physiological barrier function (332.1 ± 52.02 Ω × cm 2 ). In toxicity assays for drug-induced diarrhea, hCDMs demonstrated enhanced sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) erlotinib and gefitinib, evidenced by lower concentrations inducing 20% inhibition (IC 20 ) of cell viability and IC 20 /30× maximal plasma concentration ( C max ) ratios, and decreased sensitivity to SN-38, indicated by higher IC 20 and IC 20 /30× C max , compared to Caco-2 monolayers. Baseline eicosanome profiling showed that hCDMs provided a colon-like representation of the prostaglandin (PG) pathway, whereas Caco-2 monolayers displayed cancer-associated profiles, such as elevated PGF 2α and 15k PGF 2α . Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) revealed that celecoxib elicited a dose-dependent reduction in the abundance of a subset of PGs within hCDMs, an effect not observed in the Caco-2 system. In conclusion, hCDMs present a more physiologically relevant alternative to conventional Caco-2 monolayers for integrated studies of cellular accumulation, toxicity, and PD in the colon. Incorporating additional donors and a broader compound panel will further enhance understanding of the model’s clinical relevance. • hCDMs enable simultaneous assessment of small molecule accumulation, toxicity, and PD. • hCDMs are more sensitive to erlotinib- and gefitinib-induced toxicity than Caco-2 monolayers. • hCDMs show colon-like prostaglandin levels, in contrast to cancer-like Caco-2 profiles. • Celecoxib reduces selected prostaglandin abundance in hCDMs concentration-dependently. • Extra donors and a wider drug panel may offer deeper insight into clinical relevance.