Functional ATP-Binding Cassette Drug Efflux Transporters in Isolated Human and Rat Hepatocytes Significantly Affect Assessment of Drug Disposition

Lundquist, Patrik; Englund, Gunilla; Skogastierna, Cristine; Lööf, Johan; Johansson, Jenny; Hoogstraate, Janet; Afzelius, Lovisa; Andersson, Tommy

doi:10.1124/dmd.113.054528

Cited by 46 publications

(44 citation statements)

References 53 publications

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“…In a recent study, we demonstrated retained protein levels, membrane localization, and activity for rBCRP, rP-gp, and rMRP2 in freshly isolated rat hepatocytes as well as cryopreserved human hepatocytes (Lundquist et al, 2014a). In our present study, efflux from preloaded rat hepatocytes could be inhibited with wellcharacterized ABC efflux transporter inhibitors, indicating that rBCRP and rP-gp are active in short-term cultured hepatocytes.…”

Section: Discussionmentioning

confidence: 48%

“…Some reports suggest that isolated rat hepatocytes lose part of their ABC transporters (Bow et al, 2008). Others studies have demonstrated both expression and activity of rP-gp, rBCRP, and rMRP2 in freshly isolated rat hepatocytes (Lam et al, 2006;Li et al, 2008Li et al, , 2009aLundquist et al, 2014a). The retained activity of canalicular efflux transporters in primary rat hepatocytes demonstrated by these investigators raises the possibility of predicting biliary clearance from in vitro efflux measurements.…”

Section: Introductionmentioning

confidence: 95%

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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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Section: Discussionmentioning

confidence: 48%

Section: Introductionmentioning

confidence: 95%

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

et al. 2014

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“…Many P-gp inhibitors, including elacridar, are also substrates of the transporter (Ozawa et al, 2004;Morrissey et al, 2012;Bankstahl et al, 2013). We have recently shown that ABC efflux transporters, including P-gp, are present and active in human cryopreserved hepatocytes (Lundquist et al, 2014a). For inhibitors that are efflux transporter substrates or display differences in binding between microsome and hepatocyte incubations, the higher IC 50 value measured in cryopreserved hepatocytes might be more indicative of their in vivo IC 50 .…”

Section: Discussionmentioning

confidence: 99%

Cytochrome P450 Inhibitory Properties of Common Efflux Transporter Inhibitors

et al. 2014

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Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.

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“…Epigenetic reprogramming highlights a method for direct induction of hepatocyte-like cells without the requirement of ES cells or iPS cells [14,28], but it is not clear whether this novel cell model can be used to evaluate and predict drug disposition and toxicity. Transporter expression and function are essential for xenobiotic hepatobiliary disposition and bile acid circulation in the liver [29]. In this study, we firstly characterized the expression levels and function of major transporters in novel iHep cells.…”

Section: Discussionmentioning

confidence: 99%

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

Yao²,

Ji³

et al. 2016

Cell Physiol Biochem

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Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na⁺-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CL_b,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen^2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r² = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

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Functional ATP-Binding Cassette Drug Efflux Transporters in Isolated Human and Rat Hepatocytes Significantly Affect Assessment of Drug Disposition

Cited by 46 publications

References 53 publications

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

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

Cytochrome P450 Inhibitory Properties of Common Efflux Transporter Inhibitors

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

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