Determination of Compound Binding to Plasma Proteins

Natasha Dow1

1 Covance Laboratories, Inc., Madison, Wisconsin
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.5
DOI:  10.1002/0471141755.ph0705s34
Online Posting Date:  October, 2006
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Abstract

The pharmacokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding properties of a compound is essential during drug development and is increasingly required during lead prioritization. The protocols described in this unit detail the techniques of ultrafiltration and equilibrium dialysis for determination of plasma protein binding. Equilibrium dialysis is the more robust of the two techniques, whereas ultrafiltration is the more rapid. Two versions of the equilibrium dialysis technique are provided: the traditional approach and a higher‐throughput 96‐well plate version.

Keywords: protein binding; ultrafiltration; equilibrium dialysis; ADME screening

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Plasma Protein Binding by Ultrafiltration
  • Basic Protocol 2: Determination of Plasma Protein Binding by Traditional Equilibrium Dialysis
  • Alternate Protocol 1: Determination of Plasma Protein Binding by 96‐Well Plate Equilibrium Dialysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Plasma Protein Binding by Ultrafiltration

  Materials
  • Test article (compound of interest)
  • Solvent (e.g., acetonitrile, methanol, DMSO)
  • Isotonic phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • Plasma samples from all species to be investigated, fresh or frozen
  • Sodium phosphate, monobasic (NaH 2PO 4), solid
  • Sodium phosphate, dibasic (Na 2HPO 4), solid
  • 2‐ml microcentrifuge tubes
  • Fixed‐angle rotor
  • Long, glass Pasteur pipet
  • Micropartition devices containing membrane filters with a 10 to 30 kDa MWCO (e.g., Centrifree; Millipore)
  • Additional reagents and equipment for liquid scintillation counting (LSC; radiolabeled test articles only), determining protein concentration ( appendix 3B), and HPLC or LC/MS analysis (optional)

Basic Protocol 2: Determination of Plasma Protein Binding by Traditional Equilibrium Dialysis

  Materials
  • Isotonic phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • Test article (compound of interest)
  • Solvent (e.g., acetonitrile, methanol, DMSO)
  • Plasma samples from all species to be investigated, either fresh or frozen
  • Sodium phosphate, monobasic (NaH 2PO 4), solid
  • Sodium phosphate, dibasic (Na 2HPO 4), solid
  • 47‐mm‐diameter precut Spectra/Por 4 membrane discs with 12 to 14 kDa MWCO (Spectrum Laboratories)
  • Spectra/Por equilibrium dialyzer with stopper plugs and semi‐micro Teflon cells (Spectrum Laboratories) labeled 1 to 20 in indelible ink
  • 37°C acrylic water bath (e.g., Spectrum Laboratories or equivalent)
  • Blunt‐nose 22‐G needles and syringes
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)

Alternate Protocol 1: Determination of Plasma Protein Binding by 96‐Well Plate Equilibrium Dialysis

  • 20% ethanol
  • Dialysis membrane strips (HTDialysis)
  • 96‐well, deep‐well plate
  • HTD96b 96–well equilibrium dialysis block (HTDialysis)
  • Multichannel pipettor
  • Adhesive sealing film (Packard TopSeal‐A, Packard)
  • Shaker platform
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Figures

Videos

Literature Cited

Literature Cited
   Banker, M.J., Clark, T.C., and Williams, J.A. 2003. Development and validation of a 96‐well equilibrium dialysis apparatus for measuring plasma protein binding. J. Pharm. Sci. 92:967‐974.
   Lindup, W.E. and Orme, M.C. 1981. Plasma protein binding of drugs. Br. Med. J. 282:212‐214.
   McEnlay, J.C. and D'Arcy, P.F. 1983. Protein binding displacement interactions and their clinical importance. Drugs 25:495‐513.
   Paxton, J.W. and Calder, R.L. 1983. Propranolol binding in serum: Comparison of methods and investigation of effects of drug concentration, pH, and temperature. J. Pharmacol. Meth. 10:1‐11.
   Rolan, P.E. 1994. Plasma protein binding displacement interactions—why are they still regarded as clinically important? Br. J. Clin. Pharmacol. 37:125‐128.
   Sansom, L.N. and Evans, A.M. 1995. What is the true clinical significance of plasma protein binding displacement interactions? Drug Safety 12:227‐233.
   Sebille, B., Zini, R., Madjar, C.V., Thaud, N., and Tillement, J.P. 1990. Separation procedures used to reveal and follow drug‐protein binding. J. Chromatogr. 531:51‐77.
   Tozer, T.N., Gambertoglio, J.G., Furst, D.E., Avery, D.S., and Holford, N.H.G. 1983. Volume shifts and protein binding estimates using equilibrium dialysis: Application to prednisolone binding in humans. J. Pharm. Sci. 72:1442‐1446.
   Whitlam, J.B. and Brown, K.F. 1981. Ultrafiltration in serum protein binding determinations. J. Pharm. Sci. 70:146‐150.
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