Comparison of Commonly Used and New Methods to Determine Small Molecule Non-Specific Binding to Human Liver...

Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (K_m), reversible inhibition (K_i), or inactivation (K_I) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (f_u,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high f_u,mic value) and significant (low f_u,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The f_u,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high f_u,mic values, all methods generated similar f_u,mic values. However, f_u,mic values varied markedly across assay formats when high binding to HLM occurred, where f_u,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed.