Assessment of P-glycoprotein function using canine intestinal organoid-derived epithelial interfaces

P-glycoprotein (P-gp), a multidrug efflux pump encoded by the <i>ABCB1</i> (formerly <i>MDR1</i>) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored.We generated canine colonoid-derived 2D monolayers to investigate <i>ABCB1</i> gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture.A significant increase in gene expression of P-gp encoded by the <i>ABCB1</i> was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model.Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research. P-glycoprotein (P-gp), a multidrug efflux pump encoded by the <i>ABCB1</i> (formerly <i>MDR1</i>) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored. We generated canine colonoid-derived 2D monolayers to investigate <i>ABCB1</i> gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture. A significant increase in gene expression of P-gp encoded by the <i>ABCB1</i> was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model. Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.

P-糖蛋白（P-glycoprotein, P-gp）是由ABCB1（原名MDR1）基因编码的多药外排泵，在人类与犬体内均发挥着限制药物吸收、清除毒性化合物的关键作用。然而，P-gp底物存在物种特异性差异，这使得开发犬专用的药物评价系统成为必要需求。犬源肠道类器官衍生单层细胞为研究药物转运提供了极具前景的平台，但此类模型中P-gp介导的药物转运仍未得到探索。本研究构建了犬结肠类器官来源的2D单层细胞模型，用以探究ABCB1基因表达与P-gp功能。我们在细胞培养的第5天与第10天，使用了公认的P-gp底物罗丹明123（Rhodamine 123）、阿霉素（Doxorubicin），并联合P-gp抑制剂PSC833开展实验。相较于培养第5天，培养第10天时ABCB1编码的P-gp基因表达水平出现显著上调。尽管基因表达存在上述差异，但通过罗丹明123与阿霉素在PSC833抑制条件下的外排实验评估P-gp转运活性后发现，两个时间点的转运活性并无显著差异。不过，PSC833对P-gp功能的抑制作用证实了本模型中存在具有活性的P-gp。犬肠道类器官衍生单层细胞为研究P-gp介导的药物转运提供了极具价值的研究工具。本研究结果可为兽医学中预测药物生物利用度与不良反应提供潜在依据，同时契合伦理与可持续性研究的原则。P-糖蛋白（P-glycoprotein, P-gp）是由ABCB1（原名MDR1）基因编码的多药外排泵，在人类与犬体内均发挥着限制药物吸收、清除毒性化合物的关键作用。然而，P-gp底物存在物种特异性差异，这使得开发犬专用的药物评价系统成为必要需求。犬源肠道类器官衍生单层细胞为研究药物转运提供了极具前景的平台，但此类模型中P-gp介导的药物转运仍未得到探索。本研究构建了犬结肠类器官来源的2D单层细胞模型，用以探究ABCB1基因表达与P-gp功能。我们在细胞培养的第5天与第10天，使用了公认的P-gp底物罗丹明123（Rhodamine 123）、阿霉素（Doxorubicin），并联合P-gp抑制剂PSC833开展实验。相较于培养第5天，培养第10天时ABCB1编码的P-gp基因表达水平出现显著上调。尽管基因表达存在上述差异，但通过罗丹明123与阿霉素在PSC833抑制条件下的外排实验评估P-gp转运活性后发现，两个时间点的转运活性并无显著差异。不过，PSC833对P-gp功能的抑制作用证实了本模型中存在具有活性的P-gp。犬肠道类器官衍生单层细胞为研究P-gp介导的药物转运提供了极具价值的研究工具。本研究结果可为兽医学中预测药物生物利用度与不良反应提供潜在依据，同时契合伦理与可持续性研究的原则。