Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes
Introduction
Since the liver is the main drug eliminating organ, it is of great importance to have preclinical tools available which accurately predict hepatic drug clearance at an early stage of drug discovery. In the last decennia, different cell-based models have been applied to study human hepatic drug metabolism (Vermeir et al., 2005). In this context, human hepatocytes remain the gold standard because in vitro drug metabolic clearance values obtained in freshly-isolated or even cryopreserved human hepatocytes in suspension have been shown to reasonably predict in vivo metabolic clearance (Blanchard et al., 2005, McGinnity et al., 2004, Li et al., 1999, Jouin et al., 2006, Hallifax et al., 2010). In addition, cultured human hepatocytes have also been used to detect enzyme induction potential (Roymans et al., 2005). However, Riley et al. and Soars et al. have shown that underprediction of in vivo metabolic formation rates can occur (Soars et al., 2007, Riley et al., 2005). They suggested that this could be partly due to the impact of drug uptake transporters.
Drug transport across the sinusoidal membrane of hepatocytes indeed influences the total hepatic clearance of xenobiotics. Several transporters in the sinusoidal (basolateral) membrane of hepatocytes have been identified to mediate entrance of endogenous and exogenous compounds in the liver. The sodium taurocholate co-transporting polypeptide [NTCP (human), Ntcp (rat); SLC10A1/Slc10a1] is generally known as the main transporter of conjugated and unconjugated bile salts (Hagenbuch and Meier, 1994). However, drugs like rosuvastatin (Ho et al., 2006) and micafungin (Yanni et al., 2010) have also been shown to have affinity for NTCP/Ntcp. Whereas the uptake of bile salts by NTCP is sodium-dependent, members of the organic anion-transporting polypeptide [OATP (human), Oatp (rat); SLCO/Slco gene family] mediate the transport of bile salts (e.g. taurocholate, cholate) in a sodium-independent way. In addition, OATPs are also involved in the hepatic uptake of neutral compounds and organic cations and anions (Bossuyt et al., 1996). Among the OATPs present in human liver, OATP1B1 and OATP1B3 are predominantly expressed in the liver and important for hepatic uptake clearance of xenobiotics. In addition to OATPs, organic anions are also transported by the organic anion transporter (OAT/Oat; SLC/SlcOat22a7) and organic cations by the organic cation transporter family (hOCT1 and hOCT3; SLC22A1/SLC22A3) (Ciarimboli, 2008).
It has been shown that these hepatic uptake transporters can constitute the rate-limiting step in hepatobiliary drug clearance (Yamazaki et al., 1996, Parker and Houston, 2008) and contribute to transporter-based drug-drug interactions (Shitara et al., 2006). As there are interspecies-differences in drug metabolizing enzymes and in the substrate affinity profiles of transporters (Ishizuka et al., 1999, Ye et al., 2010), hepatocytes of human origin are required to make relevant extrapolations from in vitro drug transport data towards the in vivo situation in human. A few studies have been conducted illustrating the feasibility of plated and suspended human hepatocytes to study hepatic drug transport activities (Sandker et al., 1994, Olinga et al., 1998, Shitara et al., 2003, Yamashiro et al., 2006, Bi et al., 2006). However, the chronic limitation in availability of freshly-isolated human hepatocytes may have precluded more systematic studies on transporter activities in these cells. Shortage of freshly-isolated hepatocytes has led to the development of cryopreservation protocols of surplus hepatocytes (Li, 2008), which offer the unique advantage that experiments can be planned in advance and all human donor material utilized as efficiently as possible over an extended period of time. For instance, Badolo et al. recently reported an in vitro assay to screen for OATP1B1/3 and OCT1 inhibitors using human cryopreserved hepatocytes in suspension (Badolo et al., 2010). This illustrates that the more widespread application of suspended human hepatocytes from cryopreserved sources for the purpose of drug transport and interaction studies is justified. However, this requires that accurate knowledge about the function of uptake transporters in human cryopreserved hepatocytes should be systematically expanded.
The aim of the present study was to conduct a systematic comparative evaluation of transport activities of cryopreserved human hepatocyte suspensions (CHHS) from different donors. The accumulation of 5 different substrates was assessed in 14 individual batches of cryopreserved human hepatocytes and known inhibitors were used to verify the contribution of active processes to overall cellular accumulation. As a result, more insight is obtained in the inter-donor variability with respect to transporter activities, which also supports rational selection of human hepatocyte batches from different donors for the purpose of particular uptake experiments. Also, in order to test the potential effect of pooling procedures (i.e. an additional freeze–thaw cycle) on transporter activity, four pooled batches of cryopreserved human hepatocytes were characterized for transporter function and the results compared to the individual batches. The accumulation data obtained in suspended hepatocytes were compared to literature values as well as to accumulation rates observed in sandwich-cultured human hepatocytes, an established in vitro model for hepatobiliary drug disposition (Liu et al., 1998, Liu et al., 1999, Annaert and Brouwer, 2005). Finally, OATP1B1 and OATP1B3 mRNA levels were measured in selected batches of CHHS in order to reveal a possible relationship to accumulation rates of OATP substrates.
Section snippets
Materials
[3H]Taurocholic acid (specific activity, 4.6 Ci/mmol), [3H]digoxin (specific activity, 21.8 Ci/mmol), [3H]estrone-3-sulfate (specific activity, 57.3 Ci/mmol), [3H]estradiol-17β-d-glucuronide (specific activity, 45.2 Ci/mmol), [3H]methyl-4-phenylpyridinium (MMP+) acetate (specific activity, 83.0 Ci/mmol) and scintillation cocktail (Ultima Gold) were obtained from PerkinElmer Life Science Inc. (Boston, MA). Taurocholic acid, digoxin, estrone-3-sulfate, estradiol-17β-d-glucuronide, MPP+, rifampicin,
Validation of the procedure to measure saturable hepatic accumulation of probe substrates in one batch of CHHS
In one batch of cryopreserved human hepatocytes (Batch Hu4050), the accumulation of 5 different substrates was determined at 37 °C (control condition), at 0 °C (inhibition of all active transport processes) and in the presence of known inhibitors (Fig. 1). Compared to the control condition, the accumulation of taurocholate, a known substrate of human Na+-taurocholate co-transporting polypeptide (NTCP, SLC10A1) and human organic anion transporting polypeptide (OATP, SLCO), decreased by
Discussion
The aim of the present study was to assess the utility of cryopreserved human hepatocyte suspensions to study transporter-mediated in vitro hepatic drug uptake and interaction. The accumulation rates of five different substrates were determined in 14 individual batches of CHHS. The involvement of the transporters of interest was also verified by measuring uptake rates in the presence of known inhibitors. In addition, to test the possible effect of pooling procedures (which involves an
Acknowledgements
Tom De Bruyn received a PhD scholarship from the Agency for Innovation by Science and Technology, Flanders. This study was supported by grants from the following independent funders: ‘Fonds voor Wetenschappelijk Onderzoek’, Flanders and ‘Onderzoeksfonds’ of the Katholieke Universiteit Leuven, Belgium.
References (42)
- et al.
Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1
J. Biol. Chem.
(1999) - et al.
Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension
Eur. J. Pharm. Sci.
(2010) - et al.
Multispecific amphipathic substrate transport by an organic anion transporter of human liver
J. Hepatol.
(1996) - et al.
Drug and acid transporters in rosuvastatin hepatic upatke: function, expression and pharmacogenetics
Gastroenterology
(2006) - et al.
Cryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance
Eur. J. Pharm. Biopharm.
(2006) - et al.
Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel
Chem. Biol. Interact.
(1999) - et al.
Coordinate induction of PPAR alpha and SREBP2 in multifunctional protein 2 deficient mice
Biochim. Biophys. Acta
(2008) - et al.
Characterization of transport in isolated human hepatocytes. A study with the bile acid taurocholic acid, the uncharged ouabain and the organic cations vecuronium and rocuronium
Biochem. Pharmacol.
(1994) - et al.
Transporters as a determinant of drug clearance and tissue distribution
Eur. J. Pharm. Sci.
(2006) - et al.
Function of uptake transporters for taurocholate and estradiol 17beta-d-glucuronide in cryopreserved human hepatocytes
Drug Metab. Pharmacokinet.
(2003)
Species-specific interaction of HIV protease inhibitors with accumulation of cholyl-glycylamido-fluorescein (CGamF) in sandwich-cultured hepatocytes
J. Pharm. Sci.
Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes
Drug. Metab. Dispos.
Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport
Drug Metab. Dispos.
Comparison of clearance predictions using primary cultures and suspensions of human hepatocytes
Xenobiotica
Organic cation transporters
Xenobiotica
Membrane transporters in drug development
Nat. Rev. Drug Discov.
Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter
J. Clin. Invest.
Prediction of human metabolic clearance from in vitro systems: retrospective analysis and prospective view
Pharm. Res.
P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to hepatobiliary disposition of a model opioid peptide
Pharm. Res.
Species differences in the transport activity for organic anions across the bile canalicular membrane
J. Pharmacol. Exp. Ther.
Characterization of digoxin uptake in sandwich-cultured human hepatocytes
Drug Metab. Dispos.
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- 1
Equally contributed to this work.
- 2
Present address: Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, 171 77 Stockholm, Sweden