HLM-beads: Rapid Assessment of Nonspecific Binding to Human Liver Microsomes Using Magnetizable Beads

Abstract

In early drug development, drug-drug interaction risk is routinely assessed using human liver microsomes (HLMs). Nonspecific binding of drugs to HLMs can affect the determination of accurate enzyme parameters (Km, Ki, KI). Previously, we described a novel in vitro model consisting of HLMs bound to magnetizable beads [HLM-magnetizable-beads system (HLM-beads)]. The HLM-beads enable rapid separation of HLMs from incubation media by applying a magnetic field. Here, HLM-beads were further characterized and evaluated as a tool to assess HLM nonspecific binding of small molecules. The free fractions (fu,mic) of 13 compounds (chosen based on their pKa values) were determined using HLM-beads under three HLM concentrations (0.025, 0.50, and 1.0 mg/ml) and compared with those determined by equilibrium dialysis. Most fu,mic values obtained using HLM-beads were within 0.5- to 2-fold of the values determined using equilibrium dialysis. The highest fold difference were observed for high binders itraconazole and BIRT2584 (1.9- to 2.9-fold), as the pronounced adsorption of these compounds to the equilibrium dialysis apparatus interfered with their fu,mic determination. Correlation and linear regression analysis of the fu,mic values generated using HLM-beads and equilibrium dialysis was conducted. Overall, a good correlation of fu,mic values obtained by the two methods were observed, as the r and R2 values from correlational analysis and linear regression analysis were >0.9 and >0.89, respectively. These studies demonstrate that HLM-beads can produce comparable fu,mic values as determined by equilibrium dialysis while reducing the time required for this type of study from hours to only 10 minutes and compound apparatus adsorption. SIGNIFICANCE STATEMENT: This work introduces a new method of rapidly assessing nonspecific microsomal binding using human liver microsomes bound to magnetizable beads.