Abstract

A flux dialysis method to measure unbound fraction (f_u) of compounds with high protein binding and other challenging properties was tested and validated. This method is based on the principle that the initial flux rate of a compound through a size-excluding dialysis membrane is proportional to the product of the compound initial concentration, f_u, and unbound dialysis membrane permeability (P_mem). Therefore, f_u can be determined from the initial concentration and flux rate, assuming membrane P_mem is known. Compound initial flux rates for 14 compounds were determined by dialyzing human plasma containing compound (donor side) versus compound-free plasma (receiver side) and measuring the rate of compound appearance into the receiver side. Eleven compounds had known f_u values obtained from conventional methods (ranging from 0.000013 to 0.22); three compounds (bedaquiline, lapatinib, and pibrentasvir) had previously qualified f_u values (e.g., <0.001)_. P_mem estimated from flux rates and known f_u values did not meaningfully differ among the compounds and were consistent with previously published values, indicating that P_mem is a constant for the dialysis membrane. This P_mem constant and the individual compound flux rates were used to calculate f_u values. The flux dialysis f_u values for the 11 compounds were in good agreement with their reported f_u values (all within 2.5-fold; R² = 0.980), confirming the validity of the method. Furthermore, the flux dialysis method allowed discrete f_u to be estimated for the three compounds with previously qualified f_u. Theoretical and experimental advantages of the flux dialysis method over other dialysis-based protein binding methods are discussed.