PhD Scientific Days 2024

Budapest, 9-10 July 2024

Poster Session B - Pharmaceutical Sciences and Health Technologies 1.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction


Emőke Sóskuti1,2, Nóra Szilvásy1, Csilla Temesszentandrási-Ambrus1, Éva Pusztai3, György Várady2, Zsuzsanna Gáborik1
1: Charles River Laboratories Hungary Kft
2: Institute of Molecular Life Sciences
3: Department of Chemical and Environmental Process Engineering, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary

Text of the abstract

Introduction: Using preclinical animals to predict human brain penetration of substrates of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) is challenging because of animal welfare issues, cost, and existing species differences in transporter affinity and expression. Thus, development of high-fidelity in vitro transporter assays to characterize the compound interactions with MDR1 and BCRP is critical to understand their impact on brain penetration.
Aims: Our aim was to develop and validate in vitro assays for substrate and inhibitor screen to provide comparable MDR1 data across preclinical species and human, and to analyse its performance in IVIVC.
Methods: Endogenous MDR1 KO MDCKII cells overexpressing mouse, rat, cynomolgus or human MDR1 were developed for bidirectional permeability assays. To reveal species-differences in inhibitory potency, 21 interactors were tested using digoxin and quinidine as substrates. Substrate properties of 83 commercially available molecules were assessed and used for comparison between species. Transporter abundance was determined by targeted proteomics. Performance metrics were calculated of the investigated cell lines, and quantitative brain penetration was predicted from in vitro data.
Results: Good correlation was observed between Efflux Ratios (ER) of 83 drugs in each MDR1 cell line, suggesting limited species differences. For CNS penetration classification, ERs were compared to in vivo rodent unbound brain-to-plasma partition coefficient (Kp,uu,brain) and the sensitivity was >80% in all species. Kp,uu,brain was also calculated from in vitro ERs using various models. Prediction performance improved using the total ER of human MDR1 and BCRP, and optimized scaling factors. Comparing IC50 values between digoxin and quinidine for each MDR1 ortholog highlighted substrate-specific differences, especially for hMDR1.
Conclusion: This is the first work to investigate MDR1 activity in human as well as three preclinical species in the context of BBB penetration using overexpressing cell lines, allowing side-by-side study of individual MDR1 orthologs. Our in vitro assays are ready to be integrated into CNS drug discovery screening programs to predict BBB penetration or reveal potential species differences in transporter susceptibility, improving the translatability of in vivo preclinical data.