Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes

[Image: see text] Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CL(int)) in the liver. However, the CL(int) values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kp(uu)) effects on the CL(int) were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CL(int). Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CL(int) with CYP amounts and Kp(uu) could only partly explain the discordance in absolute values of CL(int) for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.