Plasma Protein Binding
The degree of binding to plasma proteins can be a major determinant of a drug’s clearance rate, its efficacy, and potential for drug-drug interactions. CellzDirect offers the following two methods to measure binding to human and animal plasma proteins.
Equilibrium Dialysis
In equilibrium dialysis, human and relevant animal species (such as mouse, rat, dog and non-human primate) plasma is spiked with the test article and subjected to equilibrium dialysis at 37ºC. Prior to the definitive experiment, the time to reach equilibration and non-specific binding to the apparatus are determined. The concentration of the test article in both compartments is determined and the % protein bound is calculated.
Ultrafiltration
During ultrafiltration, the test article is pre-incubated at 37ºC in plasma (human or animal) and phosphate buffered saline (PBS) at 2 concentrations and then centrifuged through filters excluding proteins with molecular weight >12,000 Da. The filtrate is analyzed for parent compound by LC-MS/MS or HPLC and the % of plasma protein binding is estimated.
Analytical methods for detection of test articles by either HPLC, LC-MS, or LC MS/MS are developed based on available information for the compounds. Radiolabeled compounds may be detected by liquid scintillation counting or HPLC with radiochemical detection.
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