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Abstract BACKGROUND
Assessment of the degree of protein-binding of drugs is highly relevant in drug development, because only the free drug fraction is pharmacologically active in-vivo. The protein-bound drug fraction serves as a depot and influences the pharmacokinetic behavior.
Determination of the degree of protein-binding of drugs is mostly done in plasma and delivers accurate results for systemically acting drugs. However, investigation of the degree of protein-binding for drugs acting in the tissue needs to be performed in the interstitial fluid (ISF) to yield reliable results. ISF is not easily accessible, and with sampling methods like suction blister or open flow micro perfusion (OFM) sample volumes of only a few microliters (up to 50 µL) can be achieved. Therefore, analytical techniques capable of handling low-volume samples of low-protein fluids are required.
Aim of this study was to assess the performance of the common methods ultrafiltration (UF), rapid equilibrium dialysis (RED) and solid phase micro extraction (SPME) when evaluating the degree of protein-binding in low-protein and low-volume samples of artificial ISF and real dermal ISF collected with open flow micro perfusion.
METHODS
The drugs lidocaine, hydrocortisone, amitriptyline, clobetasol propionate, diclofenac were used as test drugs. The degree of protein-binding for each drug was assessed with UF, RED and SPME. First, the protein-binding in human serum was examined to reproduce published data and then the ISF samples were examined. Environmental parameters (temperature and pH) were varied and different drug- and protein-concentrations were used. Quantification of the drugs was done with HPLC-MS/MS.
RESULTS
The determined free drug fractions assessed with UF, RED and SPME in serum were in good agreement with published results. Variation of the temperature within a physiological range (32°C, 37°C, 42°C) had no significant influence on the results. Change of pH (6.9, 7.4 and 7.9) only influenced the degree of protein-binding of lidocaine (pKa: 7.9). Generally, lower protein content led to an increase of the free fraction of the five investigated drugs.
SPME was not applicable with such low-volume samples, as it needs sample volumes of about 800 µL to deliver reproducible results (CV< 5% n=3). Applying UF was not feasible for low-protein samples, because adsorption to the membrane in artificial ISF was significant between 15% and 85%. RED application delivered reproducible results with the studied low-volume and low-protein ISF samples (CV <2% for n=3).
CONCLUSION
RED application was feasible to investigate the degree of protein-binding in low-protein and low-volume ISF samples
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= Discovery stage.
= Translation stage.
= Clinically available.
