Comparison of commonly used and new methods to determine small molecule non-specific binding to human liver microsomes.

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 (Km ), reversible inhibition (Ki ), or inactivation (KI ) 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 (fu,mic ) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high fu,mic value) and significant (low fu,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The fu,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 fu,mic values, all methods generated similar fu,mic values. However, fu,mic values varied markedly across assay formats when high binding to HLM occurred, where fu,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.