Canine duodenal organoids as a functional platform for intestinal CYP regulation and drug metabolism studies

Cytochrome P450 (P450) enzymes in the small intestine play a critical role in determining the systemic availability of orally administered drugs. In dogs, the major intestinal drug-metabolizing P450 enzymes are CYP3A98, an intestine-specific isoform, and CYP2B11, which are expressed in both the liver and intestines. This study aimed to establish differentiated canine duodenal organoids and evaluate the expression, inducibility, and enzymatic activity of these key intestinal P450 enzymes. Duodenal organoids were generated from healthy canine intestinal biopsies and cultured under expansion and differentiation conditions. CYP3A98 and CYP2B11 gene expression was assessed by quantitative reverse transcription polymerase chain reaction, while enzyme function was evaluated using midazolam (CYP3A98) and bupropion (CYP2B11) hydroxylation assays. To assess P450 induction, organoids were treated with rifampicin (a pregnane X receptor [PXR] selective inducer) and phenobarbital (a constitutive androstane receptor [CAR] inducer). Organoid differentiation significantly upregulated CYP3A98 and CYP2B11 mRNA expression and enzyme activity. Rifampicin (50 μM) strongly induced CYP3A98 gene expression (7.1-fold) and enzyme activity (2.5-fold) without affecting CYP2B11 expression. CYP3A98 and CYP2B11 expression were unaffected by phenobarbital treatment at a CAR-selective concentration (250 μM). However, treatment with phenobarbital at a high concentration (2 mM), known to directly bind and activate PXR, resulted in a significant increase in CYP3A98 expression (3.6-fold) and activity (1.4-fold) without substantially affecting CYP2B11 expression. Differentiated canine duodenal organoids expressed functional CYP3A98 and CYP2B11. CYP3A98 was inducible through PXR, while CYP2B11 was not regulated by CAR or PXR. This platform may provide a valuable tool for evaluating drug absorption, metabolism, and drug-drug interactions in veterinary drug development. SIGNIFICANCE STATEMENT: A physiologic canine intestinal in vitro model for drug development is lacking in veterinary medicine. The canine differentiated duodenal organoids used in this study expressed CYP3A98 and CYP2B11 enzymes and may provide a physiological platform for studying drug metabolism and drug-drug interactions during the development of veterinary pharmaceuticals.