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ADME PK

Whole Blood Binding Assay

Understand the distribution potential of your compound using our blood binding assay.

Whole blood 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.

Binding to whole blood

  • The extent of binding to blood influences the way in which a drug distributes into tissues in the body. Extensive blood binding also limits the amount of free compound available to be eliminated from the body which can, in turn, reduce the clearance of the compound.
  • Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Understanding the extent of whole blood binding in comparison to plasma protein binding is important in identifying if differential binding to a specific component in the blood occurs and in interpreting pharmacokinetic data.
  • The method can also be used to investigate non-linear pharmacokinetics via saturation of blood binding.
  • Equilibrium dialysis is the most widely accepted method for assessing protein binding as non-specific binding effects are minimized compared with other methods such as ultrafiltration.
  • Our whole blood binding assay is performed using an equilibrium dialysis method and delivers a value of fraction of compound unbound to blood (fu).
The knowledge of binding properties of drugs is of considerable importance not so much because of possible displacement interactions with other drugs, but because it is assumed that only a small fraction of the circulating drug (the free drug concentration) is able to cross membranes.1

1Mazoit JX and Samii K (1999) Br J Clin Pharmacol 47(1); 35–42

Protocol

Whole blood binding assay protocol

Method Equilibrium Dialysis
Test Article Concentrations 5 μM (different concentrations available)
Number of Replicates 2
Test Article Requirements 120 μL of 10 mM solution in DMSO
Analysis Method LC-MS/MS quantification (both blood and buffer standards prepared)
Data Delivery Fraction unbound in blood
Recovery

Data

Data from the whole blood binding assay

The whole blood binding method has been developed using a selection of compounds with reported fu values ranging from 0.006 to 0.68% in rat and 0.01 to 0.49% in human blood2,3. The experimentally determined fu values were compared to those reported in the literature and inter-assay variability was also assessed.

 
figure 1
Figure 1
Graph showing the mean fu values for 8 compounds in rat blood and their comparison to literature values2.

The error bars represent the standard deviation of three separate experiments. The fu values have good agreement with reported literature values over a wide range of fu (r2 = 0.99 for rat). The inter-assay variability was low with a coefficient of variation equal to 15% or less.
figure2
Figure 2
Graph showing the mean fu values for 7 compounds in human blood and their comparison to literature values3.

The error bars represent the standard deviation of two separate experiments. The fu values have good agreement with reported literature values over a wide range of fu (r2 = 0.94 for human).

Q&A

Questions and answers on whole blood binding

Equilibrium dialysis is used to determine the extent of binding of a compound to blood 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.

Due to the complexities of handling undiluted blood, the assay is carried out with 50% blood that has been diluted with phosphate-buffered saline. The fraction unbound in 50% blood is then scaled to 100% using the following equation:

Why is whole blood binding important?

The extent of binding to whole blood 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 blood compartment, 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 whole blood binding also limits the amount of free compound available to be renally excreted or metabolized which can, in turn, reduce the clearance of the compound.

Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Understanding the extent of whole blood binding in comparison to plasma protein binding is important in identifying if differential binding to a specific component in the blood occurs and in interpreting pharmacokinetic data. Blood binding assays can be used to investigate non-linear pharmacokinetics due to saturation of blood binding.

How do I interpret the data from the blood binding assay?

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 blood binding data can be used in conjunction with blood to plasma ratio and plasma protein binding data to understand the distribution characteristics of a compound.

What are the effects of poor solubility on the blood binding data?

Compounds are usually screened for whole blood binding at a concentration of 5 µM. If a compound has a solubility value of less than 5 µM at 37°C, it is recommended that the compound is screened at a lower concentration 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
              BloodF = Blood compartment concentration after dialysis
              BufferI = Initial concentration in buffer
              BloodI = Initial concentration in blood

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 blood?

If the compound is unstable in blood, then it is very difficult to interpret the data. If this is occurring the blood containing standards tend to have lower peak areas than the buffer standards. To overcome this problem it may be necessary to perform the assay using human serum albumin and α1-acid glycoprotein rather than whole blood. Alternatively, if the metabolism is occurring in the red blood cells, then the compound may be stable in the plasma and the assessment of plasma protein binding may be the best approach.

How is the blood obtained and stored prior to use in the assay?

Fresh blood is drawn and stored at 4ºC for up to 24 hr (human) or 48 hr (rat) prior to use. The blood is diluted with an equal volume of phosphate buffered saline immediately before the assay.

References

1Mazoit JX and Samii K (1999) Br J Clin Pharmacol 47(1); 35–42
2Summerfield SG et al. (2006) J Pharmacol Exp Ther 316(3); 1282–1290
3Summerfield SG et al. (2008) Xenobiotica 38(12); 1518-1535

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