Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines

Abstract

Fraction unbound (f_u) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. f_u,liver and f_u,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for f_u,liver and f_u,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as f_u,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat f_u,liver. Therefore, we recommend using rat f_u,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of f_u for liver tissues, hepatocytes, and other cell types. In addition, f_u under hepatocyte stability incubation conditions should not be confused with f_u,cell, as one is a diluted f_u and the other is an undiluted f_u. Cell density also plays a critical role in the accurate measurement of f_u,cell.