Evaluating the Utility of Canine Mdr1 Knockout Madin-Darby Canine Kidney I Cells in Permeability Screening and Efflux Substrate Determination

Abstract

Permeability assays are commonly conducted with Madin-Darby canine kidney (MDCK) cells to predict the intestinal absorption of small-molecule drug candidates. In addition, MDCK cells transfected to overexpress efflux transporters are often used to identify substrates. However, MDCK cells exhibit endogenous efflux activity for a significant proportion of experimental compounds, potentially leading to the underestimation of permeability and confounded findings in transport studies. The goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI) cells in permeability screening and human MDR1 substrate determination in a drug discovery setting. The gMDCKI cells were established by CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype (wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and gMDCKI showed that out of 135 compounds tested, 38% showed efflux activity in MDCKI wt, while no significant efflux was observed in gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral (A-to-B) and basolateral-to-apical were near unity in gMDCKI cells, which approximated passive permeability, and 17% of compounds demonstrated increases in their Papp A-to-B values. Overexpression of human MDR1 in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without the contribution of endogenous efflux, and the assay was able to deconvolute ambiguous results from MDCKI-MDR1 and identify species differences in substrate specificity. An analysis of 395 and 474 compounds in gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties that were associated with low permeability correlated with MDR1 recognition. Poorly permeable compounds and MDR1 substrates were more likely to be large, flexible, and more capable of forming external hydrogen bonds. On the basis of our evaluation, we concluded that gMDCKI is a better cell line for permeability screening and efflux substrate determination than the MDCK wt cell line.

Understanding the Effect of Hydroxypropyl‐β‐Cyclodextrin on Fenebrutinib Absorption in an Itraconazole–Fenebrutinib Drug–Drug Interaction Study

“…CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR associated protein 9) was used to knock out the endogenous canine Mdr1 gene as previously reported. 5 Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% pen-strep and 5 µg/mL plasmocin (InvivoGen, San Diego, CA) before seeding on Millipore Millicell-24 well plates with polyester membrane and 1 µm pores (EMD Millipore, Darmstadt, Germany) at 2.5 × 10 5 cells/mL and allowed to grow for 5 days. Prior to the permeability experiment cell monolayers were (1)…”

Estudo in silico de compostos fenólicos isolados de Inga laurina

“…Assim, pode-se inferir que as moléculas em questão possuem um índice de permeabilidade baixo corroborando o demonstrado por suas predições expostas (Tabela 1). Além disso, considera-se que moléculas com baixa absorção pelas células MDCK estão mais propensas a possuírem uma grande massa molecular, bem como são mais capazes de formar ligações externas de hidrogênio dificultando sua absorção epitelial (Chen et al, 2018).…”

“…Assim, sua utilização possibilita uma avaliação acerca da permeabilidade de compostos principalmente no que diz respeito a substâncias transportadas passivamente (Chen et al, 2018;Dezani, 2017). Diferente ao observado para as células MDCK, todas as substâncias analisadas possuem uma média absorção (Tabela 2).…”

Expanding the Efflux In Vitro Assay Toolbox: A CRISPR-Cas9 Edited MDCK Cell Line with Human BCRP and Completely Lacking Canine MDR1

“…However, determining BCRP-mediated transport using cell models, including MDCK, has proven difficult due to significant endogenous background expression of other ABC transporters, especially MDR1. Chen et al 47 show that as many as 38% of investigated compounds have background efflux activity in an MDCK I wildtype cell line. This is especially noticeable for low permeable compounds of which more than 80% show endogenous efflux activity.…”