Mathematical and Experimental Validation of Flux Dialysis Method: An Improved Approach to Measure Unbound Fraction for Compounds with High Protein Binding and Other Challenging Properties.

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; R 2 = 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.