Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

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Abstract

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes.

In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β-d-glucuronide (E2-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly (p < 0.01) decreased the accumulation of estrone-3-sulfate and E2-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant (p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R2 = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

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.

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    1

    Equally contributed to this work.

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    Present address: Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, 171 77 Stockholm, Sweden

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