This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions ...include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug‐induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).
The mechanistic understanding of bile salt disposition is not well established in suspension human hepatocytes (SHH) because of the limited information on the expression and function of bile salt ...export protein (BSEP) in this system. We investigated the transport function of BSEP in SHH using a method involving in situ biosynthesis of bile salts from their precursor bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Our data indicated that glycine- and taurine-conjugated CA and CDCA were generated efficiently and transported out of hepatocytes in a concentration- and time-dependent manner. We also observed that the membrane protein abundance of BSEP was similar between SHH and sandwich-cultured human hepatocytes. Furthermore, known cholestatic agents significantly inhibited G-CA and G-CDCA efflux in SHH. Interestingly, cyclosporine A, troglitazone, itraconazole, loratadine, and lovastatin inhibited G-CA efflux more potently than G-CDCA efflux (3- to 5-fold). Because of these significant differential effects on G-CA and G-CDCA efflux inhibition, we determined the IC
values of troglitazone for G-CA (9.9 µM) and for G-CDCA (43.1 µM) efflux. The observed discrepancy in the IC
was attributed to the fact that troglitazone also inhibits organic anion transporting polypeptides and Na
/taurocholate cotransporting polypeptide in addition to BSEP. The hepatocyte uptake study suggested that both active uptake and passive diffusion contribute to the liver uptake of CA, whereas CDCA primarily undergoes passive diffusion into the liver. In summary, these data demonstrated the expression and function of BSEP and its major role in transport of bile salts in cryopreserved SHH. SIGNIFICANCE STATEMENT: BSEP transport function and protein abundance was evident in SHH in the present study. The membrane abundance of BSEP protein was similar between SHH and sandwich-cultured human hepatocytes. The study also illustrated the major role of BSEP relative to basolateral MRP3 and MRP4 in transport of bile salts in SHH. Understanding of BSEP function in SHH may bolster the utility of this platform in mechanistic understanding of bile salt disposition and potentially in the assessment of drugs for BSEP inhibition.
Predicting transporter-based drug clearance (CL) and tissue concentrations (TC) in humans is important to reduce the risk of failure during drug development. In addition, when transporters are ...present at the tissue:blood interface (e.g., in the liver, blood-brain barrier), predicting TC is important to predict the drug's efficacy and safety. With the advent of quantitative targeted proteomics, in vitro to in vivo extrapolation (IVIVE) of transporter-based drug CL and TC is now possible using transporter-expressing models (cells lines, membrane vesicles) and the in vivo to in vitro relative expression of transporters (REF) as a scaling factor. Unlike other approaches based on physiological scaling, the REF approach is not dependent on the availability of primary cells. Here, we review the REF approach and compare it with other IVIVE approaches such as the relative activity factor approach and physiological scaling. For each of these scaling approaches, we review their underlying principles, assumptions, methodology, predictive performance, as well as advantages and limitations. Finally, we discuss current gaps in IVIVE of transporter-based CL and TC and propose possible reasons for these gaps as well as areas to investigate to bridge these gaps.
The sodium/bile acid co-transporter ASBT apical sodium-dependent bile acid transporter; SLC10A2 (solute carrier family 10 member 2) plays a key role in the enterohepatic recycling of the bile acids ...and indirectly contributes to cholesterol homoeostasis. ASBT inhibitors reportedly lower plasma triglyceride levels and increase HDL (high-density lipoprotein) cholesterol levels. RSV (resveratrol), a major constituent of red wine, is known to lower LDL (low-density lipoprotein) cholesterol levels, but its mechanism of action is still unclear. In the present study, we investigated the possible involvement of ASBT in RSV-mediated cholesterol-lowering effects. We demonstrate that RSV inhibits ASBT protein expression and function via a SIRT1 (sirtuin 1)-independent mechanism. The effect was specific to ASBT since other transporters involved in cholesterol homoeostasis, NTCP (SLC10A1), OSTα (SLC51A) and ABCG1 (ATP-binding cassette G1), remained unaffected. ASBT inhibition by RSV was reversed by proteasome inhibitors (MG-132 and lactacystin) and the ubiquitin inhibitor LDN57444, suggesting involvement of the ubiquitin-proteasome pathway. Immunoprecipitation revealed high levels of ubiquitinated ASBT after RSV treatment. Phosphorylation at Ser335 and Thr339 was shown previously to play a role in proteosomal degradation of rat ASBT. However, mutation at corresponding residues in rat ASBT revealed that phosphorylation does not contribute to RSV-mediated degradation of ASBT. Combined, our data indicate that RSV promotes ASBT degradation via the ubiquitin-proteasome pathway without requiring phosphorylation. We conclude that regulation of ASBT expression by RSV may have clinical relevance with regard to the observed cholesterol-lowering effects of RSV.
The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has ...been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.
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•Formation of dimeric or higher order multimeric assemblies of hASBT is proposed.•Dimers and oligomers of hASBT are functionally active.•Self-association of this protein does not rely on cysteine residues.•Cysteine-free hASBT acts as a dominant negative construct, but associates with wild-type hASBT at the membrane surface.
Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We ...noted that the rifampicin EC
generated using the HepatoPac model corresponded better to the EC
based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC
difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC
values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC
shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.
On behalf of all the authors, I am pleased to share our third annual review on drug transporter science with an emphasis on articles published and deemed influential in signifying drug transporters' ...role in drug disposition in the year 2022. As the drug transporter field is rapidly evolving several key findings were noted including promising endogenous biomarkers, rhythmic activity, IVIVE approaches in transporter-mediated clearance, new modality interaction, and transporter effect on gut microbiome. As identified previously (Chothe et Cal. 2021, 2022) the goal of this review is to highlight key findings without a comprehensive overview of each article and to this end, each coauthor independently selected 1-3 peer-reviewed articles published or available online in the year 2022 (Table 1). Each article is summarized in synopsis and commentary with unbiased viewpoints by each coauthor. We strongly encourage readers to consult original articles for specifics of the study. Finally, I would like to thank all coauthors for their continued support in writing this annual review on drug transporters and invite anyone interested in contributing to future versions of this review.
The use of in vitro data for the quantitative prediction of transporter-mediated clearance is critical. Central to this evaluation is the use of hepatocytes, since they contain the full complement of ...transporters and metabolic enzymes. In general, uptake clearance (CLuptake) is evaluated by measuring the appearance of compound in the cell. Passive clearance (CLpd) is often determined by conducting parallel studies at 4 °C or by attempting to saturate uptake pathways. Both approaches have their limitations. Recent studies have proposed the use of Rifamycin-SV (RFV) as a pan-inhibitor of hepatic uptake pathways. In our studies, we confirm that transport activity of all major hepatic uptake transporters is inhibited significantly by RFV at 1 mM (OATP1B1, 1B3, and 2B1 = NTCP (80%), OCT1 (65%), OAT2 (60%)). Under these incubation conditions, we found that the free intracellular concentration of RFV is ∼175 μM and that several major CYPs and UGTs can be reversibly inhibited. Using this approach, we also determined CLuptake and CLpd of nine known OATP substrates across three different lots of human hepatocytes. The scaling factors generated for these compounds at 37 °C with RFV and 4 °C were found to be similar. The CLpd of passively permeable compounds like metoprolol and semagacestat were found to be higher at 37 °C compared to 4 °C, indicating a temperature effect on these compounds. In addition, our data also suggests that incorporation of medium concentrations into CLuptake and CLpd calculations may be critical for highly protein bound and highly lipophilic drugs. Overall, our data indicate that RFV, instead of 4 °C, can be reliably used to measure CLuptake and CLpd of drugs.
On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In ...compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (
Table 1
).
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