HLM-beads: Rapid assessment of non-specific binding to human liver microsomes using magnetizable beads

In early drug development, drug-drug interaction (DDI) risk is routinely assessed using human liver microsomes (HLM). Non-specific binding of drugs to HLM can affect the determination of accurate enzyme parameters (Km,Ki, KI). Previously, we described a novel in vitro model consisting of HLM bound to magnetizable beads (HLM-beads). The HLM‑beads enable rapid separation of HLM from incubation media by applying a magnetic field. Here, HLM-beads were further characterized and evaluated as a tool to assess HLM non‑specific 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 to 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 ten minutes and compound apparatus adsorption. Significance Statement This work introduces a new method of rapidly assessing non-specific microsomal binding using human liver microsomes bound to magnetizable beads.