TY - JOUR T1 - 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 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.117.078915 SP - dmd.117.078915 AU - John Cory Kalvass AU - Colin Phipps AU - Gary J Jenkins AU - Patricia Stuart AU - Xiaomei Zhang AU - Lance Heinle AU - Marjoleen JMA Nijsen AU - Volker Fischer Y1 - 2018/01/01 UR - http://dmd.aspetjournals.org/content/early/2018/02/05/dmd.117.078915.abstract N2 - A flux dialysis method to measure unbound fraction (fu) of compounds with high protein binding and other challenging properties was tested and validated. The 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 compound initial concentration, fu and unbound dialysis membrane permeability (Pmem). Therefore fu can be determined from initial concentration and flux rate, assuming membrane Pmem 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 fu values obtained from conventional methods (ranging from 0.000013 to 0.22) while three compounds (bedaquiline, lapatinib, and pibrentasvir) had previously qualified fu values (e.g., < 0.001). Pmem estimated from flux rates and known fu values did not meaningfully differ among compounds and were consistent with previously published values, indicating that Pmem is a constant for the dialysis membrane. This Pmem constant and the individual compound flux rates were used to calculate fu values. The flux dialysis fu values for the 11 compounds were in good agreement with their reported fu values (all within 2.5-fold; R2=0.980) confirming the validity of the method. Furthermore, the flux dialysis method allowed discrete fu to be estimated for the 3 compounds with previously qualified fu. Theoretical and experimental advantages of the flux dialysis method over other dialysis-based protein binding methods are discussed. ER -