Quantification of the drug concentration at the site of action is of major importance during drug development. In dermatology, this site is located in the dermis, where the pharmacological effect of the drug is also influenced by protein binding. Dermal open flow microperfusion dOFM allows direct access to the ISF in skin and can be used to assess the rate and extent to which a topical drug becomes available in the dermis. The aim of the study was to develop a dOFM method for in-vivo sampling of undiluted ISF in dermal tissue and subsequently quantify total, bound and unbound drug concentrations.
Two dOFM strategies, namely recirculation and suction were evaluated to sample undiluted ISF from the skin of anesthetized pigs (n = 5). Albumin concentration determined with BioTek Reader was used to compare undiluted ISF, lymph and plasma. Two pigs received an intravenous infusion of diclofenac and lidocaine followed by sampling of undiluted ISF, lymph and plasma. Equilibrium dialysis was performed with all body fluids. Total, bound and unbound concentrations were determined by HPLC in all samples.
Recirculation resulted in an ISF with an ISF-to-plasma ratio of 0.42 ± 0.12. Suction resulted in an ISF with an ISF-to-plasma ratio of 0.40 ± 0.11. Lymph showed a significantly higher albumin concentration than ISF with a lymph-to-plasma ratio of 0.67 ± 0.22. Diclofenac reached 30% and lidocaine reached 50% of the plasma levels in the dermis. For both drugs, protein binding was reduced in dermis (Diclofenac: Plasma 100% - ISF 98 ± 0,5%. Lidocain: Plasma 67 ± 2% - ISF 35 ± 9%). 
Both dOFM strategies successfully sampled ISF from the dermis of anesthetized pigs. Undiluted ISF had a significantly lower albumin concentration than lymph, indicating that lymph may not be an appropriate ISF representative. Protein binding analysis revealed difference in protein binding between plasma and dermal ISF, demonstrating the value of the herein established dOFM strategies.

Investigation of Protein Binding in Skin with Dermal Open Flow Microperfusion (dOFM)