Protein binding

Plasma protein binding

To understand the distribution potential of tested compound a fraction of unbound in plasma compound is determined with a usage of equilibrium dialysis. The measure of binding to plasma affects the way of potential drug distribution into tissues in the body. Extensive plasma protein binding also limits the amount of free compound available to access sites of action in the cell, this may cause slower metabolism and elimination. Equilibrium dialysis is the most widely accepted method for determination of plasma protein binding due to minimized non-specific binding effects, when compared to other methods, for example ultrafiltration.

 

Standard plasma protein binding assay protocol:

Method Equilibrium Dialysis
(at 10 %, 50 % or 100 % plasma)
Test Compound Concentration 5 μM (different concentrations available)
Number of Replicates 2
Compound Requirements 150 μL of 10 mM solution
Analysis Method LC-MS/MS quantification (both plasma
and buffer standards prepared)
Data Delivery Fraction unbound in 100% plasma
Recovery

 

Whole blood binding

Protein binding has a very important effect on drug dynamics since only the free (unbound) fraction of drug interacts with receptors and may cause pharmacological effects. It is more common to determine the concentration of a drug in plasma rather than whole blood. On the other hand it is crucial for the interpretation of pharmacokinetic data if differential binding to a specific component in the blood occurs.

 

Standard whole blood protein binding assay protocol:

Test method

Rapid equilibrium dialysis (RED, Pierce)

(8K MWCO, distribution: 100% blood-buffer)

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37oC
Controls Blank sample
Positive control (2 compounds: Metoprolol; Chlorthalidone)
Number of replicates

3 samples for test compound

1 sample for controls

Analysis Method LC-MS
Output data

Fraction unbound in blood

Recovery

Brain tissue binding

Differences between plasma and brain composition are significant. Plasma contains twice as much protein and brain contains 20 fold more lipids.

 

Standard brain tissue binding assay protocol:

Test method

Rapid equilibrium dialysis (RED, Pierce)

(8K MWCO, distribution: 100% brain tissue homogenate-buffer)

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37oC
Controls Blank sample
Positive control (2 compounds, depends on selected species)
Number of replicates

3 samples for test compound

1 sample for controls

Analysis Method LC-MS
Output data

Fraction unbound in 100% brain tissue homogenate

Recovery

Microsomal binding

It is proved that observed kinetics of metabolism in the in vitro assays can be influenced by nonspecific liposomal binding causing problems with accurate prediction of clearance. Several examples proved that knowledge of microsomal binding leads to more precise understanding of connections between in vitro metabolism and in vivo pharmacokinetics.

 

Standard microsomal binding assay protocol:

Test method

Rapid equilibrium dialysis (RED, Pierce)

(8K MWCO, distribution: 100% microsomes-buffer)

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37oC
Controls Blank sample
Postive control (2 compounds: Atenolol, Propranolol)
Number of replicates

3 samples for test compound

1 sample for controls

Analysis Method LC-MS
Output data

Fraction unbound in microsome

Recovery

Blood to plasma ratio

Blood to plasma distribution is calculated using plasma protein binding assay and whole blood protein binding assay.