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Introduction: High patient compliance and ease of use make oral delivery as one of the most commonly adopted administration routes during drug development. Oral absorption is however highly restricted and regulated by intestinal barriers with jejunum being the most studied absorption site, affecting the absolute oral bioavailability. ABC efflux transporters, namely Multidrug Resistance protein 1 (MDR1) and Breast Cancer Resistance protein (BCRP), are recommended for assessment by the International Transporter Consortium during in vitro development. MDCK cells are commonly used as an in vitro transport model for measuring apparent permeability (P_app) and predicting the main transport mechanisms of drug candidates. Recently, MDCK cell clones overexpressing the human MDR1 and BCRP transporters but where the endogenous canine-origin MDR1 transporter has been knocked out were established. In this project these two cell clones were used for developing a more sophisticated permeability model and thereby improve in vitro permeability predictions. Purpose: The goal of this project was to establish a MDCK cell-based co-culture overexpressing BCRP and MDR1 transporters simultaneously to better mimic the in vivo intestinal epithelium. Methods: First the impact of sodium butyrate pretreatment on MDCK-hMDR1^cMDR1-KO regarding protein expression levels and MDR1’s function was investigated using global proteomics and bi-directional Transwell experiments. In parallel, a literature search for in vivo abundance of MDR1 and BCRP was conducted to select a ratio for the co-culture. The transporter-overexpressing MDCK cells were thereafter co-cultured on polycarbonate Transwell inserts for functional transport studies, whereas the co-culture expression levels of MDR1 and BCRP in the co-culture was analysed using global proteomics. Results: Pretreatment with sodium butyrate enhanced MDR1 protein concentration by two-fold without affecting its functional performance and monolayer integrity (P_app in the apical to basolateral direction (P_appAB) for enalaprilat lower than 1×10⁶ cm/s). For jejunum a ratio of 1.5 was selected for the co-culture and global proteomics could verify that a seeding ratio of 58% of MDR1 and 42% BCRP cells resulted in a co-culture with an expression ratio of 1.44 similar to human jejunum. Transepithelial electrical resistance measurements showed that the resistance over the co-culture was comparable to the monocultures. Functional study of the co-culture showed very low mass balance of less than 10% for P_appBA values. P_appAB values of MDR1 substrates in the co-culture were similar with those in monocultures; however, the same trend was not observed in BCRP substrates. 

Conclusion: This project has successfully established a preliminary MDCK cell-based co-culture model simultaneously expressing both MDR1 and BCRP. This co-culture has a similar monolayer integrity as the MDCK monocultures and can be used for permeability studies. However, results from functional studies in the co-culture model using known MDR1 and BCRP substrates are inconclusive. Thus, further optimisation and characterisation of this co-culture need to be performed to attain the closest in vitro model mimicking the in vivo human intestinal epithelium.