A novel non-specific binding method enables measurement of the fraction unbound of beyond rule-of-five compounds.

The accurate determination of the fraction unbound is critical for interpreting pharmacological and toxicological data and for predicting human pharmacokinetics, dose selection, and drug-drug interaction risk. Drug development is trending away from traditional rule-of-five physicochemical properties towards new chemical modalities which often have high lipophilicity. This presents the experimental scientist with additional challenges in measuring fraction unbound using traditional techniques such as dialysis systems by exacerbating practical limitations around non-specific binding, low recoveries, bioanalytical sensitivity, and poor diffusion across dialysis membranes. Whilst there is already a plethora of methods available to combat some of the challenges, based upon our experiences there is still a need to continue to add novel options to address the increasing chemical complexity of beyond rule-of-five compounds. Here, we describe a non-dialysis method that exploits the non-specific binding properties of compounds to determine fraction unbound over a broad dynamic range of concentrations. Through understanding the equilibrium between free compound in the incubation versus compound, which is non-specifically bound to labware, indirect determination of the free fraction within the matrix of interest can be calculated. The robustness of the fraction unbound measurement via this approach is increased by establishing a consistent fraction unbound value over a large range in incubation concentrations. A focused test set of compounds was used to evaluate the method and demonstrate that this approach could be used for compounds with significant non-specific binding which struggle to generate accurate data via many of the traditionally available techniques.