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

Abstract

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.

Drug Disposition Protein Quantification in Matched Human Jejunum and Liver From Donors With Obesity

“…Although we found significant differences in some liver drugmetabolizing enzymes and transporters in our donors compared with those in donors without obesity (from the lower weight control group and published data 15,16,21 ), the differences between the groups were markedly smaller than the interindividual variability within each group. This suggests that for most of these proteins obesity plays a minor role in their regulation.…”

“…These differences in sample preparation and MS analysis methods 15,46 may affect the results. We therefore compared our data with studies using whole cell lysates, excluding data from membrane‐enriched samples as this introduces further variability 15–17 . Furthermore, mucosal isolation of jejunal samples (as used by refs.…”

“…In jejunum, these included, among others, 27 CYPs, 6 ADHs, 17 ALDHs, 4 dimethylaniline monooxygenases (N-oxide-forming) (previously known as flavin-containing monooxygenases; FMOs), 5 arylamine N-acetyltransferases (NAT), and the 2 amine oxidases (flavincontaining), MAOA and MAOB. In liver, 32 CYPs (26 of the 27 also found in jejunum, and 6 additional ones), 7 ADHs (1 additional to those found in jejunum), 16 ALDHs, 4 FMOs, 3 NATs, and MAOA and MAOB were quantified (Table 1; Data S1).…”

“… 7 , 8 , 10 , 11 , 12 , 14 Furthermore, many of these studies used membrane enrichment procedures, 9 , 10 , 11 , 12 , 13 which introduces variability to the protein quantification. 15 , 16 , 17 For example, a global proteomics study of mucosal fractions of jejunum identified up to 5,700 proteins, 7 but it only included four donors, which does not capture the interindividual variability. Meanwhile, studies of the jejunum from larger sample donor sets (up to 28 donors), have only targeted specific drug‐metabolizing enzymes and transporters.…”

“…The liver proteome has been more studied. Although membrane enrichment has also been used for liver protein quantification, 13 , 18 , 19 , 20 other studies have quantified proteins in whole liver tissue lysates, 5 , 6 , 15 , 16 , 21 both on a global level 15 , 16 and for selected drug disposition proteins. 5 , 6 , 21 However, as with the jejunum studies, sample sizes have been small (up to 15 donors) in whole liver lysate.…”