The Impact of Solute Carrier (SLC) Drug Uptake Transporter Loss in Human and Rat Cryopreserved Hepatocytes on Clearance Predictions

Lundquist, Patrik; Lööf, Johan; Sohlenius-Sternbeck, Anna-Karin; Floby, E.; Johansson, Jenny; Bylund, Johan; Hoogstraate, Janet; Afzelius, Lovisa; Andersson, Tommy

doi:10.1124/dmd.113.054676

Cited by 33 publications

(29 citation statements)

References 54 publications

(74 reference statements)

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“…Lopinavir and rifampicin, however, gave less inhibition than predicted, and the inhibition by atazanavir was in the lower range of our predictions. This could indicate that the hepatic OATP1B1 expression/availability was lower in these hepatocyte batches than in the human liver tissue samples (Kimoto et al, 2012;Lundquist et al, 2014).…”

Section: Discussionmentioning

confidence: 77%

Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions

et al. 2014

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Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CL upt ) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CL upt in 12 human liver samples. The impact of inhibition on atorvastatin CL upt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CL upt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7-to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 NTCP OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CL upt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations.

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Section: Discussionmentioning

confidence: 77%

Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions

et al. 2014

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“…We recently showed that the apparent fexofenadine uptake in rat hepatocytes increased more than 4-fold when inhibiting P-gp during the uptake experiment (Lundquist et al, 2014b), supporting this hypothesis. As clearly shown in our present study, compensating for active efflux in the plated hepatocytes by calculating CL int,uptake,total (eq.…”

Section: Discussionsupporting

confidence: 61%

“…Short-term cultures of human cryopreserved hepatocytes should be investigated in future studies to help improve predictions of human biliary clearance. However, the pronounced down-regulation of OATP transporters that was recently demonstrated in human cryopreserved hepatocytes might present a limitation (Kimoto et al, 2012;Lundquist et al, 2014b).…”

Section: Discussionmentioning

confidence: 99%

Prediction of In Vivo Rat Biliary Drug Clearance from an In Vitro Hepatocyte Efflux Model

et al. 2014

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Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

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“…These results should neither be affected by individual variation in transporter protein expression, nor by variation in the hepatocyte preparation, as absolute protein abundance measurements, as well as kinetic experiments, were performed for the same batch of human hepatocytes. Thus, even if transporter protein expression in cryopreserved hepatocytes would not represent transporter expression levels in freshly isolated or human liver samples, as indicated in studies by Kimoto et al (2012) and Lundquist et al (2014), the correlation between transporter protein expression and activity should not be affected, as protein abundances and transporter activities were determined for the same lot of hepatocytes in the present study. In contrast, given the substantial variation in reported transporter protein abundances between different hepatocyte lots or liver samples, it is crucial to characterize transporter expression in hepatocytes to compare with the respective activities.…”

Section: Discussionmentioning

confidence: 99%

“…Differences in reported transporter activities and protein expression levels might be a result of substantial interindividual variation in transporter protein abundances observed in human liver samples (Nies et al, 2013;Vildhede et al, 2014). Furthermore, differences in hepatocyte isolation or transporter protein quantification procedures, as well as in the selection of reference peptides for QTAP analysis, were attributed to impact determined transporter protein abundances Lundquist et al, 2014). All studied statins are reported substrates of OATP1B1, and fluvastatin, rosuvastatin, pravastatin, and pitavastatin are substrates of OATP1B3 (Hirano et al, 2004;Neuvonen et al, 2006;Noe et al, 2007;Kalliokoski and Niemi, 2009).…”

Section: Discussionmentioning

confidence: 99%

Prediction of Organic Anion-Transporting Polypeptide 1B1- and 1B3-Mediated Hepatic Uptake of Statins Based on Transporter Protein Expression and Activity Data

Kunze¹,

Huwyler²,

Camenisch³

et al. 2014

Drug Metab Dispos

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Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 are drug transporters mediating the active hepatic uptake of their substrates. Because they exhibit overlapping substrate specificities, the contribution of each isoform to the net hepatic uptake needs to be considered when predicting drug-drug interactions. The relative contribution of OATP1B1-and OATP1B3-mediated uptake of statins into hepatocytes was estimated based on either relative transporter protein expression data or relative activity data. Therefore, kinetics of eight statins and OATP1B1-and OATP1B3-specific reference substrates was determined in OATP1B1-and OATP1B3-expressing human embryonic kidney 293 cells and in human cryopreserved hepatocytes. Absolute OATP1B1 and OATP1B3 protein abundance was determined by liquid chromatography-tandem mass spectrometry in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). Our results showed a pronounced OATP1B1 and comparatively low OATP1B3 protein expression in the investigated hepatocyte lot. Based on REF scaling, we demonstrated that the active hepatic uptake clearances of reference substrates, atorvastatin, pravastatin, rosuvastatin, and simvastatin were well predicted within twofold error, demonstrating that OATP1B1 and OATP1B3 were major contributors. For other statins, the net hepatic uptake clearance was underpredicted, suggesting the involvement of other hepatic uptake transporters. Summarized, we showed that REF-and RAF-based predictions were highly similar, indicating a direct transporter expressionactivity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporterspecific contributions to the net uptake clearance of statins in hepatocytes.

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The Impact of Solute Carrier (SLC) Drug Uptake Transporter Loss in Human and Rat Cryopreserved Hepatocytes on Clearance Predictions

Cited by 33 publications

References 54 publications

Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions

Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions

Prediction of In Vivo Rat Biliary Drug Clearance from an In Vitro Hepatocyte Efflux Model

Prediction of Organic Anion-Transporting Polypeptide 1B1- and 1B3-Mediated Hepatic Uptake of Statins Based on Transporter Protein Expression and Activity Data

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