A Comparison of Total and Plasma Membrane Abundance of Transporters in Suspended, Plated, Sandwich-Cultured Human Hepatocytes Versus Human Liver Tissue Using Quantitative Targeted Proteomics and Cell Surface Biotinylation

Abstract

Suspended (SH), plated (PH), and sandwich-cultured hepatocytes (SCH) are commonly used models to predict in vivo transportermediated hepatic uptake (SH or PH) or biliary (SCH) clearance of drugs. When doing so, the total and the plasma membrane abundance (PMA) of transporter are assumed not to differ between hepatocytes and liver tissue (LT). This assumption has never been tested. In this study, we tested this assumption by measuring the total and PMA of the transporters in human hepatocyte models versus LT (total only) from which they were isolated. Total abundance of OATP1B1/2B1/1B3, OCT1, and OAT2 was not significantly different between the hepatocytes and LT. The same was true for the PMA of these transporters across the hepatocyte models. In contrast, total abundance of the sinusoidal efflux transporter, MRP3, and the canalicular efflux transporters, MRP2 and P-gp, was significantly greater (P < 0.05) in SCH versus LT. Of the transporters tested, only the percentage of PMA of OATP1B1, P-gp, and MRP3, in SCH (82.8% 6 7.3%, 57.5% 6 10.9%, 69.3% 6 5.7%) was significantly greater (P < 0.05) than in SH (73.3% 6 6.4%, 27.4% 6 6.4%, 53.6% 6 4.1%). If the transporters measured in the plasma membrane are functional and the PMA in SH is representative of that in LT, these data suggest that SH, PH, and SCH will result in equal prediction of hepatic uptake clearance of drugs mediated by the transporters tested above. However, SCH will predict higher sinusoidal efflux and biliary clearance of drugs if the change in PMA of these transporters is not taken into consideration.

Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach

“…Human liver frozen tissues and cryopreserved human hepatocytes, which were obtained from the same donor, were obtained from BioIVT (Westbury, NY). The demographics of the human liver donors (n = 4, ADR, FEA, JEL, and YTW) were as described before (Kumar et al, 2019 (Bi et al, 2013). After incubation, the [ 3 H]RSV solution was removed, and the cells were washed by ice-cold HBSS buffer three times (1ml each).…”

Surface Plate, A Critical Review

“…Be that as it may, when the article is put superficially plate then the genuine mistake turns out [9].…”

Toward a Consensus on Applying Quantitative Liquid Chromatography‐Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper

“…However, the biotinylation protocol is cumbersome and requires several additional steps, which leads to the requirement for additional quality controls. In addition, to date, the biotinylation method has only been reported to be applicable to cellular systems and not to tissue samples . Provided the abundance of plasma membrane transporters is not sensitive to cross‐laboratory differences in culturing techniques, surface‐labeling methods can provide valuable information for proteins in the plasma membrane of cells (e.g., hepatocytes), which could be used by others for future IVIVE without having to establish this technique in their laboratory .…”

“…First, the clearance is assumed to be directly proportional to the abundance of the “active” protein both in vitro and in vivo . Second, in the case of transporters, the proportion of transporters in the plasma membrane should either be measured in both systems or is assumed to be similar in vitro to in vivo . When transporter clearance is measured using overexpressed vesicles, the proportion of inside‐out vesicles should be measured and a correction should be incorporated prior to IVIVE.…”

“…Second, in the case of transporters, the proportion of transporters in the plasma membrane should either be measured in both systems or is assumed to be similar in vitro to in vivo. 41 When transporter clearance is measured using overexpressed vesicles, the proportion of inside-out vesicles should be measured and a correction should be incorporated prior to IVIVE. Third, the mechanism of metabolism/transport in vitro is replicated in vivo; for example, stoichiometry of DMEs and coenzymes/cofactors (CYP reductase and cytochrome b5) in metabolic clearance predictions or membrane potential in transporter studies.…”