Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes

Human liver microsomes (HLM) and human hepatocytes (HHEP) are two common in vitro systems used in metabolic stability and inhibition studies. The comparison between the assays using the two systems can provide mechanistic insights on the interplay of metabolism, passive permeability and transporters. This study investigated the critical factors impacting the unbound intrinsic clearance (CL_int,u) and IC₅₀ of CYP3A inhibition between HLM and HHEP. The HLM/HHEP CL_int,u ratio and HHEP/HLM IC₅₀ ratio are inversely correlated to passive permeability, but have no correlation with P-gp efflux ratio. Cofactor-supplemented permeabilized HHEP (MetMax™) collapses the IC₅₀ differences between HHEP and HLM. P-gp inhibitor, encequidar, shows minimal impact on CL_int,u and IC₅₀ in HHEP. This is the first study that is able to separately investigate the effects of passive permeability and efflux transport. These data collectively show that passive permeability plays a critical role in metabolism and enzyme inhibition in HHEP, while P-gp efflux has a minor role. This may be due to low functional P-gp activity in suspension HHEP under the assay conditions. Low passive permeability may limit metabolism and enzyme inhibition in HHEP, leading to lower CL_int,u and higher IC₅₀ in HHEP compared to HLM. When liver microsomes give higher CL_int,u than hepatocytes, microsomes are more predictive of in vivo clearance than hepatocytes.