Understand the distribution potential of your compound using our plasma protein binding assay.
Plasma protein binding is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts
1Kariv I, Cao H and Oldenburg KR. (2001) J Pharm Sci 90(5); 580-587
(at 10 %, 50 % or 100 % plasma)
|Test Article Concentration||5 μM (different concentrations available)|
|Number of Replicates||2|
|Test Article 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
3 different methods have been validated based on performing the equilibrium dialysis at different plasma concentrations (10% plasma, 50% plasma and 100% plasma). For the 10% and 50% plasma methods the fraction unbound values are scaled to a fraction unbound at 100%. The application of each method is described in the table below.
|10 % plasma||
|50 % plasma||
|100 % plasma||
Please provide an overview of Cyprotex's Plasma Protein Binding assay.
Equilibrium dialysis is used to determine the extent of binding of a compound to plasma proteins. A semi-permeable membrane separates a protein-containing compartment from a protein-free compartment. The system is allowed to equilibrate at 37°C. The test compound present in each compartment is quantified by LC-MS/MS.
The extent of binding is reported as a fraction unbound (fu) value which is calculated as detailed below;
PC = Test compound concentration in protein-containing compartment.
PF = Test compound concentration in protein-free compartment.
In addition to using whole plasma, the plasma protein binding assay can be performed using two other ratios of plasma (10% or 50% plasma in buffer v/v). It can be difficult to detect very highly bound compounds in the protein-free compartment of the 50% and 100% assay, therefore the 10% assay is recommended if it is anticipated that the compounds are likely to be highly bound. The screening costs for the 10% assay are lower as less plasma is required. The following equations are used to convert from a fraction unbound at 10% or 50% to a fraction unbound at 100%.
However, it is important to note that the fraction unbound values for compounds which are less highly bound tend to be more variable in the 10% assay and the conversion from fu10% to fu100% is less accurate. For this reason, it is recommended that the 50% plasma assay is used for less highly bound compounds. The graph below shows the effect of the scale up from a fraction unbound at 10% and 50% to a fraction unbound at 100% for low and highly bound compounds.
Why is plasma protein binding important?
The extent of binding to plasma influences the way in which a drug distributes into tissues in the body. If a compound is highly bound, then it is retained in the plasma, which results in a low volume of distribution. This may impact on the therapeutic effects of the compound by limiting the amount of free compound which is available to act at the target molecule. Extensive plasma protein binding also limits the amount of free compound available to be metabolized which can, in turn, reduce the clearance of the compound.
How do I interpret the data from the protein binding assay?
A low fraction unbound value (for example, fu < 0.1 where greater than 90% of the compound is bound to plasma proteins) represents a compound which binds extensively to plasma proteins. Clients may use results from the screen to rank the compounds. The criteria for success are client-specific and are often dependent on additional factors such as potency and therapeutic area. The plasma protein binding data can be used in conjunction with other in vitro parameters to predict the pharmacokinetics of a compound using the simulation software, Cloe PK.
What are the effects of poor solubility on the protein binding data?
Compounds are screened for plasma protein binding at a concentration of 5 µM. If a compound has a solubility value of less than 5 µM at 37°C, it is not recommended that the compound is screened in this assay as the insoluble compound will not be able to freely cross the membrane.
What are the advantages of using equilibrium dialysis over other methods such as filtration?
Equilibrium dialysis is considered to be the best method for investigating protein binding. Problems with non-specific binding are more common with filtration and can result in inaccuracies in the final measurement.
How and why is % recovery calculated?
|where||BufferF = Buffer compartment concentration after dialysis|
|PlasmaF = Plasma compartment concentration after dialysis|
|BufferI = Initial concentration in buffer|
|PlasmaI = Initial concentration in plasma|
In theory, the recovery should be 100%. If the recovery deviates from 100%, it may indicate binding to the dialysis equipment or solubility issues.
What occurs if the compound is unstable in plasma?
If the compound is unstable in plasma then it is very difficult to interpret the data. The plasma containing standards tend to have lower peak areas than the buffer standards if this is occurring. In order to overcome this problem it may be necessary to perform the assay using human serum albumin and α1-acid glycoprotein rather than whole plasma.
What is the purpose of the internal standard?
The internal standard is added at the end of the experiment just before analysis. The same quantity is added to all samples. The purpose of the internal standard is to monitor LC-MS/MS performance, and correct for minor variation in response throughout the sample run. The response of the test compound is based on the peak area ratio (peak area of test compound / peak area of the internal standard).
1 Kariv I et al. (2001) J Pharm Sci 90(5); 580-587
2 Goodman and Gilman's (1996) The Pharmacological Basis of Therapeutics