Background:
The pharmacokinetic and pharmacodynamic properties of drugs are influenced by the extent of binding to plasma proteins, as only unbound “free” drug has the ability to partition across membranes, undergo metabolism and clearance by the kidney, as well as interact with targets to exert an effect.
Drugs generally bind reversibly to plasma proteins (mostly albumin, a1-acid glycoprotein and lipoproteins) with different kinetics and affinity.  Highly protein bound drugs are retained in the plasma compartment, with restricted distribution into tissues, decreased metabolism, clearance (low extraction ratio drugs), prolonged half-lives and limited brain penetration.
The degree to which a compound binds to plasma proteins is determined across species during early drug discovery. Equilibrium dialysis is a commonly used technique to measure plasma protein binding (PPB).
  
 
   Assay description
   Technique
equilibrium dialysis
   Species
mouse, rat , human, dog,  rabbit,  monkey
   Compound concentration
5µM (0.5% DMSO)
   Compound requirements
50µl of 10mM stock solution or
1-2 mg of dry matter
   Detection method
LC-MS/MS with internal standard
   Results
%fraction bound (%Fb)
recovery
Figure 1. %Fb values obtained for
reference compounds in 4 different species

 

Figure 2. %Fb values of commercial compounds obtained
in human plasma in 3 separate experiments

Assay controls
   protein contamination check
   reference compounds:  acebutolol, propranolol, verapamil and nicardipine  (Figure 1)
   data obtained for 10 commercial  compounds (Figure 2)

Assay details adjustable to client’s and/or project specific requests

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