Investigation on Drug Permeability in Control and Inflamed Human Colonoid-Derived Monolayers

Introduction and Aim Given the increasing incidence and prevalence of inflammatory bowel diseases, there is a growing need for reliable in vitro preclinical models that mimic the inflamed intestinal epithelium for preliminary pharmacokinetic studies in drug discovery programs. To date, the possible impact of inflammation on intestinal drug permeability has been scarcely investigated, indeed drug permeability is generally assessed in basal conditions on immortalized Caco-2 cells1. To overcome this limitation, the present work aims to assess the changes induced by inflammation on permeability of drugs, belonging to different BCS classes and therapeutic categories, across monolayers derived from human colonic organoids (CDMs). Methods Human colonic crypts, isolated from biopsies of 3 healthy volunteers undergoing colonoscopy for indications other than inflammatory bowel disease, were embedded in 70% Matrigel® and grown for 5-7 days. IntestiCult™ OGM was used to generate cystic-like colonoids, further expanded in suspension cultures. Single cells obtained from colonoids dissociation were seeded onto 24-well inserts coated with Matrigel®. When confluence was reached, IntestiCult™ OGM was switched to IntestiCult™ ODM. Differentiated CDMs were exposed for 24h to 25ng/mL TNF-α and IFN-γ. Cell differentiation was assessed through immunocytochemistry (ICC), and barrier integrity through TEER. The apparent permeability (Papp) of propranolol, atenolol, budesonide, and celecoxib were quantified by HPLC-MS/MS in basal conditions and after inflammatory induction. The entity of drug recovery was determined by considering also intracellular accumulation. Results Over 9-11 days, CDMs showed increasing TEER values, confirming the gradual development of epithelial barriers and the strengthening of tight junctions. The exposure to cytokines impaired significantly, although heterogeneously, the barrier integrity of CDMs. ICC staining for villin and MUC2 confirmed cell polarisation and differentiation in basal conditions, but was adversely affected in inflammation. While the steady-state flux and Papp of atenolol increased significantly under inflammatory conditions, the movement of high permeable compounds, propranolol and budesonide, was not modified. Permeability of celecoxib showed a relevant increase under inflammatory conditions, but the low drug recovery (<50%) did not allow to obtain an accurate estimate of Papp. Conclusions Exposure to an inflammatory milieu altered colonic cell morphology and compromised the epithelial barrier integrity of CDMs: as a result, the paracellular transport of low permeable compounds, as atenolol, was remarkably increased. Further investigations on drug recovery issues could help to optimise the ability of CDMs to predict oral drug permeability in inflammatory disorders. References 1 Hubatsch et al., Nat Protoc. 2007; 2(9): 2111-9. This project was financed by EU H2020 MSCA-ITN under the grant agreement 956851 COLOTAN project.