One Assay, Two Vital Values‐Estimating Intrinsic Permeability and Efflux Using MDCK‐MDR1 Assay

Abstract

BackgroundIntestinal intrinsic permeability and transporter mediated efflux are two major determinants of oral absorption. Typically, wild type Madin‐Darby canine kidney cells (MDCK) and MDCK cells transfected with the human MDR1 gene (MDCK‐MDR1) are used to evaluate intrinsic permeability and efflux, respectively. The MDCK assay provides intrinsic permeability but lacks transporter mediated efflux. Conversely, the MDCK‐MDR1 assay provides efflux ratio and “apparent permeability”. However, the apparent permeability can differ significantly from intrinsic permeability due to efflux. Traditionally, both assays (MDCK, MDCK‐MDR1) are applied separately and sequentially, which can be time‐consuming and costly.In this study, we present a novel approach to estimate intrinsic permeability and efflux simultaneously using the MDCK‐MDR1 assay alone. Intrinsic permeability can be calculated using a scaling method by combining the 3 parameters (bi‐directional apparent permeability values and efflux ratio) determined from the MDCK‐MDR1 assay.MethodsTest compounds of varying permeability and efflux ratios were used to assess the validity of the hypothesis. Results from the MDCK, MDCK‐MDR1 and caco‐2 assays were analyzed. Efflux ratio is estimated by dividing the apparent permeability from the basolateral to apical direction (PappBA) by the apparent permeability from the apical to basolateral direction (PappAB). Different averaging methods were used to calculate intrinsic permeability from MDCK‐MDR1 assay, and the measured intrinsic permeability from the MDCK assay was used as a comparison to find the most accurate averaging method.ResultsOf the different scaling and averaging methods used for estimating intrinsic permeability from MDCK‐MDR1 assay, the best method we found was a “quotient” scheme where calculated intrinsic permeability was adjusted based on the magnitude of efflux ratio. With this adjustment, both the intrinsic permeability and efflux ratio can be reliably obtained from the MDCK‐MDR1 assay for compounds with a wide range of efflux ratios. The calculated intrinsic permeability from the MDCK‐MDR1 assays aligns within 2 fold of the permeability values determined from the MDCK assay. Results from the caco‐2 cell line were also evaluated and applicable to the method mentioned above. Several hypotheses were formulated regarding why the “quotient” method was a better fit than simple apparent permeability averaging.ConclusionThe strategy presented here provides an efficient and effective method to estimate both intrinsic permeability and efflux solely based on the MDCK‐MDR1 assay. Compared to the traditional approach of using MDCK and MDCK‐MDR1 assays separately, the method used here requires less time, cost, and compound for testing. This is particularly attractive for early drug discovery screening needs.