Small volume rapid equilibrium dialysis (RED) measures effects of interstitial parameters on the protein-bound fraction of topical drugs

The importance of plasma protein binding in the early stages of drug development is well recognized. Free and bound drug fractions in plasma are routinely determined with well-established methods. However, for physiological fluids with a small accessible volume and low protein concentrations, such as dermal interstitial fluid (dISF) validated methods are currently missing. Due to the low protein concentration and highly dynamic processes in the dermis, protein binding data obtained from plasma samples may underestimate in-vivo efficacy. This study aimed to validate a small volume rapid equilibrium dialysis (RED) for low protein samples, as a tool to examine drug-protein binding directly in the biological fluid at the site of action. The sample volume required for RED was successfully downscaled to 50 & mu;l and plasma protein binding values of the four model drugs were consistent with previous studies with an average recovery of 88 & PLUSMN; 8% which makes all tested drugs suitable for small volume RED. Inter- and intra-batch variability showed sufficient reproducibility across RED plates. Small volume RED was successfully applied to assess the effects of interstitial parameters, including the evaluation of the major binding protein and the effects of binding protein concentration, drug concentration, and pH on the protein-bound drug fraction using 2% HSA and/or diluted human plasma as a surrogate for dISF.

Automated summary

The importance of plasma protein binding in drug development is recognized. However, validated methods for assessing protein binding in physiological fluids such as dermal interstitial fluid (dISF) are currently missing. This study successfully validated small volume rapid equilibrium dialysis (RED) for low protein samples, providing a tool to directly examine drug-protein binding in the biological fluid at the site of action. Small volume RED was applied to assess the effects of interstitial parameters on protein-bound drug fraction using surrogate dISF samples.