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

Ting Wang; Andrea Whitcher-Johnstone; Young Sun Scaringella; Monica Keith-Luzzi; Juntang Shao; Mitchell E Taub; Tom S Chan

doi:10.1016/j.xphs.2024.04.004

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

J Pharm Sci. 2024 Apr 12:S0022-3549(24)00135-7. doi: 10.1016/j.xphs.2024.04.004. Online ahead of print.

Authors

Ting Wang¹, Andrea Whitcher-Johnstone², Young Sun Scaringella³, Monica Keith-Luzzi⁴, Juntang Shao⁵, Mitchell E Taub⁶, Tom S Chan⁶

Affiliations

¹ Department of Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA. Electronic address: ting_3.wang@boehringer-ingelheim.com.
² DMPK oncology, AstraZeneca Inc., 35 Gatehouse Dr., Waltham, MA 02451, USA.
³ Department of Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA.
⁴ Department of Nonclinical Drug Safety, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA.
⁵ Anhui Medical University, 1980 Meishan Road, Anhui, China.
⁶ Department of Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA.

PMID: 38615815
DOI: 10.1016/j.xphs.2024.04.004

Abstract

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.

Keywords: Equilibrium dialysis; HLM; HLM-beads; LESA; Methods comparison; Microsomal binding study; Small molecule; Transil™; Ultracentrifugation; Ultrafiltration.