Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system (fup/fumic) for refined prediction of phase I mediated metabolic hepatic clearance

At the drug discovery stage, in vivo metabolic hepatic clearance (CLhep) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma (fup) to unbound fraction in the microsomal system (fumic) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fup and fumic data using ultracentrifugation. The correlation for fup/fumic between the two methods for a set of 23 data points was very good with R2 of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CLhep was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CLhep using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fup only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration.