Investigation of differentiation conditions for canine duodenum, upper jejunum, and ileum crypt-derived cells for drug transport and metabolism

Three-dimensional spheroid cultures of crypts from the duodenum, upper jejunum, and ileum of beagle dog were established to investigate species differences in drug transport and metabolism. Four differentiation conditions were tested using jejunum-derived cells: base medium (BM; without Wnt3a, R-spondin 3, and Noggin), BM plus epidermal growth factor (BM + EGF), EGF/Noggin/R-spondin 3 (ENR) for liquid-liquid interface (LLI), and ENR for air-liquid interface (ALI). Transepithelial electrical resistance (Ω·cm2) varied among conditions (102-125 for BM and ALI vs 911-1106 for BM + EGF and ENR), whereas the apparent permeability coefficient (Papp) of Lucifer yellow was similar. LLI (BM) and ALI induced higher CYP3A, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) activities than LLI (BM + EGF and ENR). The intestinal availability (Fg) of midazolam [0.13 or 0.15 for LLI (BM) and 0.23 or 0.27 for ALI] closely matched the in vivo value (0.15). LLI (BM) and ALI also enhanced bile acid transport in ileum-derived cells. By ALI, CYP3A activity was lower in duodenum-derived cells than in jejunum- and ileum-derived cells, whereas P-gp and BCRP activities were similar. ALI culture maintained transepithelial electrical resistance values in jejunum-derived cells for 10 days. Although apparent permeability coefficient (Papp) decreased on day 10 compared with day 5, P-gp, BCRP, and CYP3A activities remained largely stable. These data demonstrate that dog spheroid-derived cells from different intestinal regions can be successfully differentiated to investigate intestinal drug-metabolizing enzymes and transporters and help address species differences in drug absorption. Further studies will assess their applicability to cells from other dogs and species. SIGNIFICANCE STATEMENT: The study successfully established an in vitro model to investigate drug transport and metabolism in differentiated cells derived from 3D spheroids of the dog duodenum, upper jejunum, and ileum, under both liquid-liquid and air-liquid interface conditions. The platform will be used to address species differences in drug absorption and pre-systemic metabolism between experimental animals and humans although their applicability to cells across other dogs and species needs to be validated in future studies.