Unbound proteins in plasma can greatly influence the drug distribution. They influence a drug distributes from blood into tissues as well as from tissues to action sites. To deliver high-quality and effective data for drug distribution, Creative Biolabs performs tailored plasma protein binding (PPB) assay to evaluate a value of fraction unbound in plasma.
PPB is an important factor that affects the pharmacokinetic and pharmacodynamic properties of a drug. Therapeutic compounds which are unbound to blood components are free to diffuse across the cell membranes into the action sites and then metabolized by liver. The bound drug in plasma can be served as a reservoir for free drug. After elimination, previously bound drug can be released and enter to blood circle or tissues, thus prolonging the duration of effect. In addition, PPB information is also helpful to estimate metabolism-related drug-drug interactions (DDI) if two drugs are used at the same time.
Drug Distribution and Plasma Protein Binding
Drug distribution is often measured as volume of distribution (Vdss). Vdss can be affected by a number of factors, of which PPB is the most important one. Higher volume of binding will have a lower volume of distribution, longer plasma half-life (T1/2), and lower clearance (CL). Figure 1 shows the effect of fu on Vdss.
Figure 1. Effect of PPB on Vdss
PPB Evaluation Method
The three “gold standard” methods for PPB evaluation are equilibrium dialysis, ultrafiltration and ultracentrifugation. We provide all of the three methods combined with three types of plasma concentrations (10% plasma, 50% plasma and 100% plasma) to perform PPB evaluation depending on your budget and compound characteristics.
Equilibrium dialysis is the most widely accepted method for assessing protein binding and it can be performed with the rapid equilibrium dialysis (RED) device. It is preferred by researchers as it has less binding effects to experimental artifacts. To yield accurate, reliable, and quantitative data, we use a buffer of sufficient ionic strength and prior dilution of whole plasma to avoid Gibbs-Donnan problems (particles cannot be charged near the dialysis membrane to distribute evenly on both sides of the membrane). Supernatants (compound unbound to proteins (fu)) are analyzed by LC-MS/MS.
Ultrafiltration requires very little amount of compound and it is suitable for fast screening. Compounds are first incubated in plasma for a short period (e.g. 30 min), followed by filtering the plasma-compound mixture through a low MW cut-off filter, which proteins cannot cross, by centrifugation. The supernatant is then quantitated by LC-MS/MS for unbound concentration. This method is susceptible to non-specific binding.
Ultracentrifugation is subject a drug-protein solution to a very high-speed ultracentrifuge for a long time without using membranes. The protein-bound compounds are precipitated to the bottom of the tube while the non-bound compounds are dissolved in the protein-free supernatant. The test compound present in each compartment is quantified by LC-MS/MS.
The results are usually expressed as fraction unbound (fu) or as % protein binding (PB). The formulas are shown below:
D: Free drug concentration
DP: The concentration of drug-protein complex.
Values of % PB < 90% were classified as low, values >= 90% were classified as high. Once you get to >99% bound then plasma protein binding is likely to have a significant impact.
Species and Isolated Proteins Available
We routinely measure and compare protein binding in plasma from the following species:
Protein binding can also be assessed for individual proteins:
For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
For Research Use Only.