Abstract
We tested the hypothesis whether data on ABCB1 ATPase activity and passive permeability can be used in combination to identify ABCB1 substrates and inhibitors. We determined passive permeability using an artificial membrane permeability assay (HDM-PAMPA) and ABCB1 function, i.e., vanadate-sensitive ATPase activity for a training set (40 INN drugs) and a validation set (26 development compounds). In parallel experiments, we determined ABCB1 function, i.e., vectorial transport in a Caco-2 cell monolayer, and ABCB1 inhibition, i.e., calcein AM extrusion out of K562-MDR cells, to cross-validate the results with cellular assays. We found that compounds that did not modulate ABCB1-ATPase did also not affect calcein AM extrusion and were not actively transported by ABCB1 in Caco-2 cell monolayers. The results corroborated the effect of passive permeability as an important covariate of active transport: active transport in Caco-2 monolayer was only apparent for compounds showing low passive permeability (<5.0 cm × 10−6/s) in the HDM-PAMPA assay whereas compounds with high passive permeability (>50 cm × 10−6/s) were shown to inhibit calcein AM efflux with IC50 values close to their respective K m value obtained for ABCB1-ATPase. The use of HDM-PAMPA in combination with ABCB1-ATPase offers a simple, inexpensive experimental approach capable of identifying ABCB1 inhibitors as well as transported substrates.
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Abbreviations
- Calcein AM:
-
calcein acetoxymethylester
- DMSO:
-
dimethyl sulfoxide
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- INN:
-
international nomenclature name
- HBSS:
-
Hanks’ balanced salt solution
- HPLC:
-
high-performance liquid chromatography
- GF120918:
-
GG918 elacridar
- LLOQ:
-
lower limit of quantification
- MDCK:
-
Madin–Darby canine kidney cells
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Acknowledgements
We are indebted to Silke Müller, Altana Pharma, Konstanz, Germany, for the excellent technical assistance in generating the Western blots. The authors are grateful to Dr. Michel Eichelbaum, Dr. Margarete Fischer-Bosch-Institute for Clinical Pharmacology, Stuttgart, Germany, for providing the human enterocytes. We would like to thank Dr. Bálazs Sarkadi, Budapest, Hungary and Dr. Geoff Tucker, Sheffield, UK for the fruitful and challenging discussion of the manuscript. Altana Pharma AG, Konstanz, Germany and Solvo Biotechnology, Budapest, Hungary supported this study. Work at Solvo Biotechnology was further supported by Hungarian Grants Asbóth KF208318/2005, GVOP-2004-3.3.2.-2004-04-0001/3.0, GVOP-3.1.1.-2004-05-0506/3.0, EEF-Munka 00034/2003, and European Community grants FP6-NoE005137, FP6-2004-LIFESCIHEALTH-5; FP6-2004-LIFESCIHEALTH-5; Proposal No. 518246.
Conflict of interest statement
Oliver von Richter, Stephanie Liehner, Beate Siewert, and Karl Zech are employees of Altana Pharma. Hristos Glavinas and Peter Krajcsi are employees of Solvo Biotechnology.
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von Richter, O., Glavinas, H., Krajcsi, P. et al. A novel screening strategy to identify ABCB1 substrates and inhibitors. Naunyn-Schmied Arch Pharmacol 379, 11–26 (2009). https://doi.org/10.1007/s00210-008-0345-0
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DOI: https://doi.org/10.1007/s00210-008-0345-0