Strategy for Determining the Free Fraction of Labile Covalent Modulators in Plasma Using Equilibrium Dialysis

Determination of free drug fraction (f_u) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in f_u. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of f_u if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of f_u. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on f_u. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of f_u in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on f_u. Additionally, this strategy can be used for determining f_u of other liable compounds.