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A novel screening strategy to identify ABCB1 substrates and inhibitors

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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

References

  • Acharya P, Tran TT, Polli JW, Ayrton A, Ellens H, Bentz J (2006) P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites. Biochemistry 45:15505–15519

    Article  PubMed  CAS  Google Scholar 

  • Acharya P, O’Connor MP, Polli JW, Ayrton A, Ellens H, Bentz J (2008) Kinetic identification of membrane transporters that assist P-glycoprotein-mediated transport of digoxin and loperamide through a confluent monolayer of MDCKII-hMDR1 cells. Drug Metab Dispos 36:452–460

    Article  PubMed  CAS  Google Scholar 

  • Adachi Y, Suzuki H, Sugiyama Y (2001) Comparative studies on in vitro methods for evaluating in vivo function of MDR1 P-glycoprotein. Pharm Res 18:1660–1668

    Article  PubMed  CAS  Google Scholar 

  • Allen JD, van Loevezijn A, Lakhai JM, van d V, van Tellingen O, Reid G, Schellens JH, Koomen GJ, Schinkel AH (2002) Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 1:417–425

    PubMed  CAS  Google Scholar 

  • Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, Pastan I (1992) Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc Natl Acad Sci USA 89:8472–8476

    Article  PubMed  CAS  Google Scholar 

  • Ambudkar SV, Kim IW, Sauna ZE (2005) The power of the pump: mechanisms of action of P-glycoprotein (ABCB1). Eur J Pharm Sci 27:392–400

    Article  PubMed  Google Scholar 

  • Balimane PV, Chong S (2005) Cell culture-based models for intestinal permeability: a critique. Drug Discov Today 10:335–343

    Article  PubMed  CAS  Google Scholar 

  • Bentz J, Tran TT, Polli JW, Ayrton A, Ellens H (2005) The steady-state Michaelis–Menten analysis of P-glycoprotein mediated transport through a confluent cell monolayer cannot predict the correct Michaelis constant K m. Pharm Res 22:1667–1677

    Article  PubMed  CAS  Google Scholar 

  • Choo EF, Leake B, Wandel C, Imamura H, Wood AJ, Wilkinson GR, Kim RB (2000) Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metab Dispos 28:655–660

    PubMed  CAS  Google Scholar 

  • Cordon-Cardo C, O’Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci USA 86:695–698

    Article  PubMed  CAS  Google Scholar 

  • Cummins CL, Jacobsen W, Benet LZ (2002) Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4. J Pharmacol Exp Ther 300:1036–1045

    Article  PubMed  CAS  Google Scholar 

  • Drescher S, Glaeser H, Murdter T, Hitzl M, Eichelbaum M, Fromm MF (2003) P-glycoprotein-mediated intestinal and biliary digoxin transport in humans. Clin Pharmacol Ther 73:223–231

    Article  PubMed  CAS  Google Scholar 

  • Essodaigui M, Broxterman HJ, Garnier-Suillerot A (1998) Kinetic analysis of calcein and calcein-acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein. Biochemistry 37:2243–2250

    Article  PubMed  CAS  Google Scholar 

  • Evers R, Kool M, van Deemter L, Janssen H, Calafat J, Oomen LC, Paulusma CC, Oude Elferink RP, Baas F, Schinkel AH, Borst P (1998) Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA. J Clin Invest 101:1310–1319

    PubMed  CAS  Google Scholar 

  • Evers R, Kool M, Smith AJ, van Deemter L, de Haas M, Borst P (2000) Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer 83:366–374

    Article  PubMed  CAS  Google Scholar 

  • Eytan GD (2005) Mechanism of multidrug resistance in relation to passive membrane permeation. Biomed Pharmacother 59:90–97

    Article  PubMed  CAS  Google Scholar 

  • Eytan GD, Regev R, Assaraf YG (1996a) Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis. J Biol Chem 271:3172–3178

    Article  PubMed  CAS  Google Scholar 

  • Eytan GD, Regev R, Oren G, Assaraf YG (1996b) The role of passive transbilayer drug movement in multidrug resistance and its modulation. J Biol Chem 271:12897–12902

    Article  PubMed  CAS  Google Scholar 

  • Fromm MF (2004) Importance of P-glycoprotein at blood-tissue barriers. Trends Pharmacol Sci 25:423–429

    Article  PubMed  CAS  Google Scholar 

  • Garrigues A, Escargueil AE, Orlowski S (2002a) The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc Natl Acad Sci USA 99:10347–10352

    Article  PubMed  CAS  Google Scholar 

  • Garrigues A, Nugier J, Orlowski S, Ezan E (2002b) A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein. Anal Biochem 305:106–114

    Article  PubMed  CAS  Google Scholar 

  • Glavinas H, Krajcsi P, Cserepes J, Sarkadi B (2004) The role of ABC transporters in drug resistance, metabolism and toxicity. Curr Drug Deliv 1:27–42

    Article  PubMed  CAS  Google Scholar 

  • Goh LB, Spears KJ, Yao D, Ayrton A, Morgan P, Roland WC, Friedberg T (2002) Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man. Biochem Pharmacol 64:1569–1578

    Article  PubMed  CAS  Google Scholar 

  • Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, von Richter O, Zundler J, Kroemer HK (1999) The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 104:147–153

    Article  PubMed  CAS  Google Scholar 

  • Homolya L, Hollo M, Muller M, Mechetner EB, Sarkadi B (1996) A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells. Br J Cancer 73:849–855

    PubMed  CAS  Google Scholar 

  • Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, Qiu W, Rahman A, Sobel S, Stifano T, Thummel K, Wei XX, Yasuda S, Zheng JH, Zhao H, Lesko LJ (2008) New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 48:662–670

    Article  PubMed  CAS  Google Scholar 

  • Klimecki WT, Futscher BW, Grogan TM, Dalton WS (1994) P-glycoprotein expression and function in circulating blood cells from normal volunteers. Blood 83:2451–2458

    PubMed  CAS  Google Scholar 

  • Kruijtzer CM, Beijnen JH, Rosing H, Bokkel Huinink WW, Schot M, Jewell RC, Paul EM, Schellens JH (2002) Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 20:2943–2950

    Article  PubMed  CAS  Google Scholar 

  • Le Grimellec C, Friedlander G, el Yandouzi EH, Zlatkine P, Giocondi MC (1992) Membrane fluidity and transport properties in epithelia. Kidney Int 42:825–836

    Article  PubMed  Google Scholar 

  • Litman T, Nielsen D, Skovsgaard T, Zeuthen T, Stein WD (1997a) ATPase activity of P-glycoprotein related to emergence of drug resistance in Ehrlich ascites tumor cell lines. Biochim Biophys Acta 1361:147–158

    PubMed  CAS  Google Scholar 

  • Litman T, Zeuthen T, Skovsgaard T, Stein WD (1997b) Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity. Biochim Biophys Acta 1361:159–168

    PubMed  CAS  Google Scholar 

  • Martin C, Berridge G, Higgins CF, Mistry P, Charlton P, Callaghan R (2000) Communication between multiple drug binding sites on P-glycoprotein. Mol Pharmacol 58:624–632

    PubMed  CAS  Google Scholar 

  • Meissner K, Sperker B, Karsten C, Zu Schwabedissen HM, Seeland U, Bohm M, Bien S, Dazert P, Kunert-Keil C, Vogelgesang S, Warzok R, Siegmund W, Cascorbi I, Wendt M, Kroemer HK (2002) Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy. J Histochem Cytochem 50:1351–1356

    PubMed  CAS  Google Scholar 

  • Nielsen PE, Avdeef A (2004) PAMPA—a drug absorption in vitro model 8. Apparent filter porosity and the unstirred water layer. Eur J Pharm Sci 22:33–41

    Article  PubMed  CAS  Google Scholar 

  • Petzinger E, Burckhardt G, Tampe R (2006) A multi-faceted world of transporters. Naunyn-Schmiedeberg’s Arch Pharmacol 372:383–384

    Article  CAS  Google Scholar 

  • Polli JW, Wring SA, Humphreys JE, Huang L, Morgan JB, Webster LO, Serabjit-Singh CS (2001) Rational use of in vitro P-glycoprotein assays in drug discovery. J Pharmacol Exp Ther 299:620–628

    PubMed  CAS  Google Scholar 

  • Sarkadi B, Price EM, Boucher RC, Germann UA, Scarborough GA (1992) Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem 267:4854–4858

    PubMed  CAS  Google Scholar 

  • Sauna ZE, Nandigama K, Ambudkar SV (2006) Exploiting reaction intermediates of the ATPase reaction to elucidate the mechanism of transport by P-glycoprotein (ABCB1). J Biol Chem 281:26501–26511

    Article  PubMed  CAS  Google Scholar 

  • Shapiro AB, Ling V (1994) ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells. J Biol Chem 269:3745–3754

    PubMed  CAS  Google Scholar 

  • Smit JW, Huisman MT, van Tellingen O, Wiltshire HR, Schinkel AH (1999) Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure. J Clin Invest 104:1441–1447

    Article  PubMed  CAS  Google Scholar 

  • Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 84:7735–7738

    Article  PubMed  CAS  Google Scholar 

  • Troutman MD, Thakker DR (2003a) Efflux ratio cannot assess P-glycoprotein-mediated attenuation of absorptive transport: asymmetric effect of P-glycoprotein on absorptive and secretory transport across Caco-2 cell monolayers. Pharm Res 20:1200–1209

    Article  PubMed  CAS  Google Scholar 

  • Troutman MD, Thakker DR (2003b) Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium. Pharm Res 20:1210–1224

    Article  PubMed  CAS  Google Scholar 

  • von Richter O, Burk O, Fromm MF, Thon KP, Eichelbaum M, Kivisto KT (2004) Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens. Clin Pharmacol Ther 75:172–183

    Article  Google Scholar 

  • Wohnsland F, Faller B (2001) High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem 44:923–930

    Article  PubMed  CAS  Google Scholar 

  • Wu CY, Benet LZ (2005) Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharm Res 22:11–23

    Article  PubMed  CAS  Google Scholar 

  • Yee S (1997) In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man—fact or myth. Pharm Res 14:763–766

    Article  PubMed  CAS  Google Scholar 

  • Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

    Article  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. Division of Drug Metabolism and Pharmacokinetics, Altana Pharma AG, Konstanz, Germany

    Oliver von Richter, Stephanie Liehner, Beate Siewert & Karl Zech

  2. Division R&D, Solvo Biotechnology, Budaörs, 2040, Hungary

    Hristos Glavinas & Peter Krajcsi

  3. Department of Pharmacokinetics and Pharmacometrics, AiCuris GmbH & Co KG, Friedrich-Ebert-Str. 475, Wuppertal, 42117, Germany

    Oliver von Richter

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  1. Oliver von Richter
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Correspondence to Oliver von Richter.

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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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