Abstract
A novel flux dialysis method to measure unbound fraction (fu) of compounds with high protein binding and other challenging properties was developed. The method originates from derived relationships from a dialysis kinetic model which showed 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 of 14 compounds had known fu values obtained from conventional methods (ranging from 0.000013 to 0.22) while 3 compounds had previously unmeasurable fu (bedaquiline, lapatinib, and pibrentasvir). Pmem estimated from flux rates and known fu values did not meaningfully differ among compounds, indicating that Pmem was constant for the dialysis membrane and was not compound dependent. This Pmem constant and the individual compound flux rates were used to calculate fu values. The flux dialysis fu values for the 11 known compounds were in good agreement with historical fu values (all within 2.5-fold; R2=0.980) confirming the validity of the method. Furthermore, the flux dialysis method allowed fu to be estimated for the 3 compounds with previously unmeasurable fu. Theoretical and experimental advantages of the flux dialysis method are discussed in context of other dialysis-based protein binding methods.
- The American Society for Pharmacology and Experimental Therapeutics