Only unbound circulating drugs have access to targets and excretion pathways, therefore the binding of drug candidates to plasma proteins plays a significant role in efficacy, drug distribution, and establishing safety margins. For these reasons, pharmaceutical companies and regulatory agencies are interested in determining the extent of drug binding to plasma and/or tissue proteins early in the drug development process.
MicroConstants will measure the binding of test articles to plasma or tissue proteins using the following sequence:
MicroConstants employs the following methods to determine unbound drug concentration in plasma, serum, or tissue:
The plasma or tissue sample is first spiked with the test article at one or more concentrations and then incubated with the appropriate matrix for a prescribed period of time; however, samples from clinical or toxicology studies can be used instead. The unbound concentration is then determined using one of the above mentioned assays followed by LC/MS/MS analysis. Equilibrium dialysis can be performed with the RED device or a Spectrum 20. Several concentrations can be used to calculate a dissociation constant, Kd.
We routinely measure and compare protein binding in plasma or serum from the following species:
Protein binding can also be assessed for individual proteins such as:
Depending on your study needs, we will perform:
A pilot study is conducted to determine which methodology (equilibrium dialysis, ultrafiltration, or ultracentrifugation) is most appropriate for the measurement of the unbound test article. During this study we will assess stability in plasma at 37ºC, non-specific binding to the apparatus, and the estimated protein binding ratio. The results from these assessments will allow us to select the method best suited to provide robust results for the test article.
During the pilot study, we may find it necessary to stabilize the compound with protease inhibitors, or to determine non-specific drug-protein binding for each concentration so we can normalize the binding ratio. Our extensive experience with protein binding studies enables us to handle compounds with limitations of stability and/or solubility.
The compounds are spiked into plasma or serum and dialyzed overnight using the RED device. Following the incubation, the recovery samples, donor sides, and receiver sides are analyzed by time-of-flight mass spectrometry using a generic method. We then calculate the binding values and percent recovery from the relative peak heights.
When compounds with low recovery are encountered, we notify the sponsor and give them the option of investigating non-specific binding and stability in plasma.
An Excel spreadsheet with the drug-protein binding data will be submitted immediately after completion of the experiment. If desired, a formal report complete with the Abstract, Experimental, Results, and Conclusions can be provided as well. The formal report is suitable for inclusion in regulatory submissions.