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Equilibrium gel filtration to measure plasma protein binding of very highly bound drugs

Weiss, Markus and Gatlik, Ewa (2013) Equilibrium gel filtration to measure plasma protein binding of very highly bound drugs. Journal of Pharmaceutical Sciences.


For very highly bound drugs (fu<2%) the determination of the unbound fraction in plasma (fu) and a reliable estimation of protein binding differences across species, populations, or concentrations is challenging. The difficulty is not mostly assay sensitivity but rather experimental bias. In equilibrium gel filtration (EGF) - opposite to the commonly used methods - the amount bound at a set free concentration is determined. Therefore, signals and differences are bigger for more highly protein bound drugs. We describe here a new experimental set-up developed to investigate binding in plasma and compare results with those obtained with standard methods for 9 Novartis compounds. The method was then applied for two drugs for which it was challenging to obtain precise data with standard methods: midostaurin and siponimod. Despite the very high binding (fu≤0.1%) precise estimation of up to 10-fold species differences relevant for safety assessments was possible. Evidence for the meaningful data by comparison with other PK parameters is provided. Sensitivity to potential sources of experimental bias is compared to standard methods and advantages and disadvantages of the method are discussed. In conclusion EGF allows accurate determination of fu for very highly bound drugs and differentiation even above 99.9% of binding.

Item Type: Article
Date Deposited: 13 Oct 2015 13:13
Last Modified: 13 Oct 2015 13:13