Drug Distribution and Protein Binding Studies

AAG is one of the major plasma-binding proteins and has high affinity and low-capacity properties. There are marked species and age differences in protein expression, homology, and drug binding affinity. As a result, a good understanding of whether a drug binds to AAG can be very important in the translation of PK/PD relationships from animal species to humans, healthy versus diseased populations, and adult to pediatric populations.

The total drug concentration measured in plasma or tissues may not be the concentration of pharmacologically active drug at the biological receptor site. Therefore, tissue binding will be necessary in order to obtain free drug exposure in tissues. Most screening studies employ a single dilution, eg. 3-fold to 10-fold, and the measured f_u in diluted tissues was corrected to the undiluted f_u. In late stages, the use of several dilutions of homogenates is advantageous in providing more robust data for extrapolation and confirming linearity of binding in tissue homogenates.

Determining PPB can be challenging for unstable compounds because instability can make the assay results less reliable. Several advanced methods have been reported to determine the PPB of unstable compounds, such as ultrafiltration, using inhibitors, low-temperature method, dilution method, and others. Their suitability depends on the stage of development, mechanisms of instability, and assay cost.

When a large number of cells, subcellular components, tissues, or recombinant enzyme systems are used in drug metabolism and enzyme kinetics experiments, the compounds are prone to non-specific binding with them, affecting the concentration of free drugs. Meanwhile, the apparent kinetic parameters measured in experiments will deviate from the true values, affecting the metabolic rate and inhibitory efficacy evaluated in enzyme kinetics studies. Therefore, it is necessary to determine the non-specific binding of the incubation matrix in vitro to correct the free concentration in the incubation matrix.

In vitro blood plasma ratio (BPR) experiments refer to the ratio of total drug concentration in blood to total drug concentration in plasma at equilibrium. High or low distribution of drugs in red blood cells may lead to great differences in drug concentration in blood and plasma. Therefore, understanding the distribution of drugs between plasma and blood cells is essential for the correct interpretation of kinetic parameters. However, it should be noted that the blood/plasma (B/P) ratio measured from static in vitro B/P assay could be different from in vivo setting. The in vivo blood plasma ratio and temporal profiles of the investigated drug in plasma and blood can be time-dependent on the scale of the in vivo PK-PD measurement. Therefore, it is important to carefully consider appropriate in vitro and in vivo BPR study design to reliably correct blood-to plasma parameters.