Assessment of P‐Glycoprotein Substrate and Inhibition Potential of Test Compounds in MDR1‐Transfected MDCK Cells

Kevin C. L. Lam1, Ganesh Rajaraman1

1 NoAb BioDiscoveries, Mississauga, Ontario, Canada
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.13
DOI:  10.1002/0471141755.ph0713s58
Online Posting Date:  September, 2012
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Abstract

P‐glycoprotein (P‐gp) is the most widely studied drug transporter due to its potential role in drug disposition and efficacy, and drug‐drug interactions (DDI). It is abundantly expressed in both the intestinal wall and blood‐brain barrier where it serves as a drug permeability barrier while simultaneously facilitating drug elimination in the liver and kidney. It is also abundantly expressed in tumors where it can facilitate the elimination of chemotherapeutics, a phenomenon known as multidrug resistance (MDR). Clinically relevant DDIs involving P‐gp are well documented; for example, the P‐gp substrate, digoxin, exhibits toxicity when co‐administered with a Pgp‐inhibitor. This makes it essential to screen new chemical entities early in development for their potential to be a substrate and/or inhibitor of P‐gp. Detailed in this unit is an in vitro protocol for assessing the P‐gp substrate and inhibition potential of test compounds using the MDCK MDR1 and MDCK WT cell lines. Curr. Protoc. Pharmacol. 58:7.13.1‐7.13.17. © 2012 by John Wiley & Sons, Inc.

Keywords: cell permeability assay; MDCK; MDR1; P‐gp; substrate; inhibition; bidirectional; transport

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of P‐gp Substrate Potential Bidirectional Permeability Assay
  • Basic Protocol 2: P‐gp Inhibition Assay with MDCK MDR1 Cells
  • Support Protocol 1: Growth and Plating of MDCK MDR1 and MDCK WT Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of P‐gp Substrate Potential Bidirectional Permeability Assay

  Materials
  • MDCK WT (wild type) and MDCK MDR1 cells (see protocol 3)
  • Transport buffer (see recipe)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • 3H‐digoxin (Perkin Elmer)
  • Colchicine (Sigma)
  • 3H‐mannitol (Perkin Elmer)
  • Ultima Gold liquid scintillation cocktail (Perkin Elmer)
  • Verapamil (Sigma)
  • HTS multi‐well insert system (1.0‐µm pore size, PET membrane; BD Falcon)
  • 24‐well plates with lids (BD Falcon), sterile
  • 37°C, 5% CO 2 incubator
  • 37°C incubator shaker
  • Volt‐ohm‐meter (World Precision Instruments)
  • 96‐well, 2‐ml polypropylene plates (VWR)
  • 96‐well sample plate/liquid scintillation plate (Perkin Elmer)
  • Liquid scintillation counter (e.g.,Wallac MicroBeta Trilux, Perkin Elmer)
  • Lab‐Line Environ shaker

Basic Protocol 2: P‐gp Inhibition Assay with MDCK MDR1 Cells

  Materials
  • Test compounds
  • Dimethyl sulfoxide (DMSO, Sigma)
  • Transport buffer (see recipe)
  • 3H‐digoxin (Perkin Elmer)
  • Verapamil (Sigma)
  • MDCK Wild Type (WT) and MDCK MDR1 cells (see protocol 3)
  • Ultima Gold liquid scintillation cocktail (Perkin‐Elmer)
  • 96‐well sample plate/liquid scintillation plate (Perkin Elmer)
  • HTS multi‐well insert system (1.0‐µm pore size, PET membrane, BD Falcon)
  • 37°C, 5% CO 2 incubator
  • 37°C incubator shaker
  • Lab‐Line Environ Shaker
  • Liquid scintillation counter (Wallac MicroBeta Trilux, Perkin Elmer)

Support Protocol 1: Growth and Plating of MDCK MDR1 and MDCK WT Cells

  Materials
  • Dulbecco's modified essential medium (DMEM, see recipe)
  • DMEM‐C medium (see recipe)
  • MDCK Wild Type (WT) and MDCK MDR1 cells (NIH or ATCC)
  • 0.25% Trypsin‐EDTA (Gibco)
  • DPBS (Invitrogen)
  • 24‐well multi‐well plates with lids (BD Falcon), sterile
  • HTS multi‐well insert system (1.0‐µm pore size, PET membrane; BD Falcon)
  • Multi‐channel pipet
  • 37°C, 5% CO 2 incubator
  • 75‐cm2 NUNC easy culture flasks (Thermo Scientific)
  • 50‐ml tubes (Falcon)
  • Centrifuge
  • Hemacytometer (Bright‐line, Hausser Scientific)
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Figures

Videos

Literature Cited

Literature Cited
   Benet, L.Z. 2009. The drug transporter‐metabolism alliance: Uncovering and defining the interplay. Mol Pharm. 6:1631‐1643.
   European Medicines Agency (2010). Guideline on the Investigation of Drug Interactions. http://www.ema.euorpa.eu.
   FDA Guidance for the Industry (2012). Drug interaction studies Study design, data analysis, and implications for dosing, and labeling recommendations. U.S. Department of Health and Human Services, Food and Drug Administration. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf.
   Giacomini, K.M., Huang, S., Tweedie, D.J., Benet, L.Z., Brouwer, K.L.R., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., Hoffmaster, K.A., Ishikawa, T., Keppler, D., Kim, R.B., Lee, C.A., Niemi, M., Polli, J.W., Sugiyama, Y., Swaan, P.W., Ware, J.A., Wright, S.H., Yee, S.W., Zamek‐Gliszczynski, M.J., and Zhang, L. for The International Transporter Consortium. 2010. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9:215‐236.
   Irvine, J.D., Takahashi, L., Lockhart, K., Cheong, J., Tolan, J.W., Selick, H.E., and Grove, J.R. 1999. MDCK (Madin‐Darby canine kidney) cells: A tool for membrane permeability screening. J. Pharm. Sci. 88:28‐33.
   Pasta, I., Gottesman, M.M., Ueda, K., Lovelace, E., Rutherford, A.V., and Willingham, M.C. 1988. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P‐glycoprotein in MDCK cells. Proc. Natl. Acad. Sci. U.S.A. 85:4486‐4490.
   Rautio, J., Humphreys, J.E., Webster, L.O., Balakrishnan, W.A., Keogh, J.P., Kunta, J.R., Serabjit‐Singh, C.S., and Polli, J.W. 2006. In vitro P‐glycoprotein inhibition assays for the assessment of clinical drug interaction potential of new drug candidates: A recommendation for probe substrates. Drug Metab. Dispos. 34:786‐792.
   Tang, F., Horie, K., and Borchardt, R.T. 2002. Are MDCK cells transfected with human MDR1 gene a good model of the human intestinal mucosa? Pharm. Res. 19:765‐772.
   Taub, M.E., Podila, L., Ely, D., and Almeida, I. 2005. Functional assessment of multiple P‐glycoprotein (P‐gp) probe substrates: Influence of cell line and modulator concentration on P‐gp activity. Drug Metab. Dispos. 33:1679‐1687.
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