Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes

doi:10.1016/j.ejps.2006.11.003

European Journal of Pharmaceutical Sciences

Volume 30, Issue 2, February 2007, Pages 181-189

https://doi.org/10.1016/j.ejps.2006.11.003 Get rights and content

Abstract

Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein 1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of different culture conditions (suspension, conventional culture and sandwich culture) on the uptake of [³H]-taurocholate ± probenecid and the efflux of [³H]-vinblastine ± ketoconazole; mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was assessed by immunocytochemistry. The uptake of [³H]-taurocholate was higher in cryopreserved rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A significant time dependent decline in the uptake of [³H]-taurocholate was noticed from day 2 to day 4 in conventional and sandwich cultures. [³H]-taurocholate uptake was significantly reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspension was similar to that of liver, whereas much lower levels were detected in the cultures; this was in accordance with [³H]-taurocholate uptake results. Mdr1 activity was assessed by accumulation of the Mdr1 selective substrate, [³H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity.

Introduction

Drugs given by the most convenient administration route, oral administration, are absorbed through the gastrointestinal tract. Thereafter, they reach the liver via the portal vein before entering the systemic circulation. For some drugs, transport proteins present in the gastrointestinal tract and in the liver play a significant role in drug absorption and drug bioavailability (Diaz, 2000). Multidrug resistance proteins (Mdr, also known as P-glycoprotein) and organic anion transporting polypeptides (Oatp) are major members in the group of transport proteins that regulate the disposition of orally administered drugs (Kim, 2002, Beringer and Slaughter, 2005). Oatp participates in the uptake of drugs from the blood into the hepatocytes where they may undergo significant phase I and/or phase II metabolism. Oatp has also been suggested to function as an excreting protein capable of transporting drug and drug metabolites back into the systemic circulation (Chandra and Brouwer, 2004, Beringer and Slaughter, 2005). The interplay between Oatp uptake of drugs into hepatocytes, their subsequent metabolism by hepatic enzymes and further excretion into bile through Mdr efflux system is a powerful detoxification pathway in the body. Besides their presence in the liver and the intestine, Oatp and Mdr are widely distributed in the human body where they play an important role in the disposition and distribution of certain drugs. In the kidneys, Oatp and Mdr1 are involved in renal secretion of xenobiotics; at the blood–brain barrier a range of drugs have limited brain penetration due to Mdr1-mediated efflux. Oatp is also expressed in the blood–brain barrier; this suggests its role in delivery of drugs into the brain and removal of metabolites from the brain (Ayrton and Morgan, 2001, Kim, 2002, Beringer and Slaughter, 2005).

Since the important role played by transport proteins in xenobiotic disposition and distribution is now understood, a great body of investigations of their effect on the disposition of new drugs are made by pharmaceutical industries as part of drug discovery screening. Investigations of Oatp activity have mostly been performed in vitro using freshly isolated hepatocytes. There are only very few reports of studies using cryopreserved hepatocytes for the assessment of Oatp transport activity. These studies show that some transport activity is retained in cryopreserved rat and human hepatocytes in suspension (Houle et al., 2003, Shitara et al., 2003). The use of cryopreserved rat hepatocytes for investigation of the functional activity of Mdr1 has not been reported. Moreover, sandwich culture is commonly used to assess the transport activity of Mdr1 and other canalicular transport proteins in freshly isolated rat and human hepatocytes by measurements of canalicular accumulation (Annaert et al., 2001, Hoffmaster et al., 2004, Annaert and Brouwer, 2005). The function of Mdr1 using freshly isolated rat hepatocytes has been demonstrated in conventional culture in a single report (Le Bot et al., 1994). However, this study assessed the transport activity as a decline in doxorubicin accumulation over time in culture as an indication of increased efflux of doxorubicin by Mdr1. The present study is the first, to our knowledge, to investigate the transport activity of Oatp and Mdr1 using cryopreserved rat hepatocytes in suspension, in conventional culture and in sandwich culture by the use of both substrates and inhibitors. Additionally, the protein expression and mRNA expression were investigated. A recent paper published by Bi et al. (2006) investigated MDR1 protein in cryopreserved human hepatocytes.

The presence of functional transport proteins in cryopreserved rat hepatocytes will make it possible to investigate these transport proteins for a number of scientists with limited access to freshly isolated hepatocytes. It also allows standardising these studies by using cells from same donors in several experiments and will reduce considerably the number of animals used in these studies.

Section snippets

Materials

Dexamethasone, dimethyl sulfoxide (DMSO), ehthyleneglycol-bis(b-amino ethylether)-N,N,N′,N′-tetraacetic acid (EGTA), KCl, ketoconazole, Percoll, probenecid, rat tail collagen (type I), triton X-100, trypan blue and β-mercaptoethanol were obtained from Sigma–Aldrich (Bröndby, Denmark). Calcium and magnesium free Hanks’ balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), ITS (insulin (1.0 g/L), transferrin (0.55

Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression

The uptake of taurocholate using cryopreserved hepatocytes in suspension (day 0), in conventional culture (days 2–4) and in sandwich culture (days 2–4) is shown in Fig. 1. The results are presented as uptake of taurocholate after 15 min as it was shown that the uptake was not linear in the range of 0–15 min (data not shown). The highest uptake was seen in suspension treated few hours after thawing. Compared with the initial value in suspension (day 0), the uptake of taurocholate decreased, when

Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression

The present study documented that the uptake of taurocholate into cryopreserved rat hepatocytes in suspension was mediated by transport proteins; the transport activity declined when rat hepatocytes were cultured (Fig. 1). The results from Oatp mRNA investigation correlated with taurocholate update data. mRNA expressions for Oatp1a1 and Oatp1a4 were lower in cryopreserved rat hepatocytes in culture after 4 days as compared to data from cryopreserved rat hepatocytes in suspension (day 0).

It is

Conclusions

In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport. Cryopreserved rat hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to cryopreserved rat hepatocytes in culture. The presence of Mdr1 in cryopreserved rat hepatocytes cultured in both the conventional and the sandwich culture was confirmed at mRNA level, by protein expression

Acknowledgement

The study was supported by H. Lundbeck A/S, Valby, Denmark.

References (21)

K.N. Faber et al.
Drug transport proteins in the liver
Adv. Drug Deliv. Rev.
(2003)
R.B. Kim
Transporters and xenobiotic disposition
Toxicology
(2002)
M.A. Le Bot et al.
P-glycoprotein expression and function in rat hepatocytes in culture
Biochem. Pharmacol.
(1994)
A. Le Cam et al.
Ultrastructual and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions, cryopreservation of hepatocytes
Exp. Cell Res.
(1976)
O. Luttringer et al.
Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes
Biochem. Pharmacol.
(2002)
Y. Shitara et al.
Function of uptake transporters for taurocholate and estradiol 17beta-d-glucuronide in cryopreserved human hepatocytes
Drug Metab. Pharmacokinet.
(2003)
A.Q. Sun et al.
The rat liver Na+/bile acid cotransporter: importance of the cytoplasmic tail to function and plasma membrane targeting
J. Biol. Chem.
(2001)
M. Achira et al.
Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P450 3A4
AAPS Pharmsci.
(1999)
P.P. Annaert et al.
P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes
Drug Metab. Dispos.
(2001)
P.P. Annaert et al.
Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes
Drug Metab. Dispos.
(2005)

There are more references available in the full text version of this article.

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A change in the function or expression of hepatic drug transporters may have significant effect on the efficacy or safety of orally administered drugs. Although a number of clinical drug–drug interactions associated with hepatic transport proteins have been reported, in practice it is not always straightforward to discriminate other pathways (e.g. drug metabolism) from being involved in these interactions.
The present study was designed to assess the interactions between organic anion transporting polypeptide (Oatp) substrates (pravastatin or repaglinide) and inhibitors (spironolactone or diphenhydramine) in vivo in rats. The mechanisms behind the interactions were then investigated using in vitro tools (isolated hepatocytes and rat liver microsomes). The results showed a significant increase in the systemic exposures of pravastatin (2.5-fold increase in AUC) and repaglinide (1.8-fold increase in AUC) after co-administration of spironolactone to rats. Diphenhydramine increased the AUC of repaglinide by 1.4-fold. The in vivo interactions observed in rats between Oatp substrates and inhibitors may a priori be classified as transport-mediated drug–drug interactions. However, mechanistic studies performed in vitro using both isolated rat hepatocytes and rat liver microsomes showed that the interaction between pravastatin and spironolactone may be solely linked to the inhibition of pravastatin uptake in liver. On the contrary, the inhibition of cytochrome P450 seemed to be the reason for the interactions observed between repaglinide and spironolactone.
Although the function and structure of transport proteins may vary between rats and humans, the approach used in the present study can be applied to humans and help to understand the role of drug transport and drug metabolism in a given drug–drug interaction. This is important to predict and mitigate the risk of drug–drug interactions for a candidate drug in pre-clinical development, it is also important for the optimal design of drug–drug interactions studies in the clinic.
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Human African trypanosomiasis (HAT) is a parasitic disease affecting sub-Saharan Africa. The parasites are able to traverse the blood–brain barrier (BBB), which marks stage 2 (S2) of the disease. Delivery of anti-parasitic drugs across the BBB is key to treating S2 effectively and the difficulty in achieving this goal is likely to be a reason why some drugs require highly intensive treatment regimes to be effective. This study aimed to investigate not only the drug transport mechanisms utilised by nifurtimox at the BBB, but also the impact of nifurtimox–eflornithine combination therapy (NECT) and other anti-HAT drug combination therapies (CTs) on radiolabelled-nifurtimox delivery in an in vitro model of drug accumulation and the human BBB, the hCMEC/D3 cell line. We found that nifurtimox appeared to use several membrane transporters, in particular breast-cancer resistance protein (BCRP), to exit the BBB cells. The addition of eflornithine caused no change in the accumulation of nifurtimox, nor did the addition of clinically relevant doses of the other anti-HAT drugs suramin, nifurtimox or melarsoprol, but a significant increase was observed with the addition of pentamidine. The results provide evidence that anti-HAT drugs are interacting with membrane transporters at the human BBB and suggest that combination with known transport inhibitors could potentially improve their efficacy.
Time-course activities of Oct1, Mrp3, and cytochrome P450s in cultures of cryopreserved rat hepatocytes
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Activities of CYP2B1/2, 2D1, and 3A1 were also observed to decrease over time in cultures. The present data together with previous observations (Jørgensen et al., 2007; Kern et al., 1997) suggest that basolateral transporters are down regulated in primary cultures of rat hepatocytes, and that CYP activities are isoform dependent. The results obtained in this study stress the need to optimize cell cultures in order to preserve active transport of basolateral membrane proteins and CYPs in in vitro models for drug metabolism or cytotoxicity.
The organic cation transporter 1 (Oct1) has been shown to be one of the most abundant uptake transporters responsible for the uptake of xenobiotics from the sinusoidal blood across the basolateral membrane of hepatocytes. On the same membrane the multidrug resistance-associated protein 3 (Mrp3) mediates the efflux of xenobiotics or their metabolites from the hepatocytes to the blood allowing their systemic exposure. In the present study we investigated the expression and activity of Oct1 and Mrp3 in suspensions and in monolayer- and sandwich cultures, and activities of CYP2B1/2, 2D1, and 3A1 in monolayer- and sandwich cultures of cryopreserved rat hepatocytes. Oct1-mediated active uptake of 10 μM [³H]-1-methyl-4-phenylpyridinium (MPP+) into hepatocytes was assessed in the presence of quinidine (1 mM). The results showed the presence of active uptake of MPP+ in suspended hepatocytes (∼91 pmol/min/mg protein). In hepatocytes in cultures (monolayer and sandwich) a time-dependent decrease in MPP+ uptake was observed from day 0 to 4, from 80 to 90 pmol/min/mg protein at day 0 to ca. 17 pmol/min/mg protein at day 4. Mrp3 activity in suspensions and in monolayer- and sandwich cultures were investigated by measuring the efflux of [³H]-taurocholate from hepatocytes in the presence of the Mrp3 inhibitor taurolithocholate-3-sulfate (TLC-S) (500 μM). Cells in suspensions showed efflux of taurocholate by an active transport mechanism indicating Mrp3 activity. Experiments in monolayer- and sandwich cultures also showed Mrp3 activity at day 0 and 1 in culture whereas experiments performed at day 2–4 showed no difference in efflux of taurocholate in the presence or absence of TLC-S, suggesting an absence of Mrp3 activity. The time-dependent decrease in Oct1 activity from day 0 to day 4 in cultures was confirmed by qPCR data also showing a time-dependent decrease in mRNA expression, whereas qPCR data did not support the observed time-dependent decrease in Mrp3 activity in cultures. Time-course activities of CYP2B1/2, 2D1, and 3A1 were also investigated by using bupropion, bufuralol, and midazolam as respective substrates. Activities of CYP2D1 and 3A1 were reduced by ∼75% and ∼80%, respectively, from day 0 to day 4 in cultures, whereas activity of CYP2B1/2 was reduced by ∼50% from day 0 to day 4.
Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes
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The post thawed viabilities (using percoll gradient centrifugation) determined using the trypan blue exclusion method were ≥80%. Uptake experiments were performed according to the protocol described by Jørgensen et al. [11] for suspended hepatocytes with slight modifications. Hepatocytes were suspended in uptake buffer to a final density of ca. 2 × 106 viable cells/mL.
Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [³H]-estradiol-17β-glucuronide (E₂17βG, substrate of OATP1B1/3/SLCO1B1/3) and [³H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E₂17βG and MPP+. While the affinity of E₂17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p = 1), V_max and CL_uptake values decreased in average by 47% (p = 0.06). The passive diffusion of E₂17βG decreased significantly after cryopreservation (p = 0.03).
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Drug–drug interactions involving hepatic drug transporters may have clinical consequences and jeopardize development of promising drug candidates. Organic anion transporting polypeptides (OATP/Oatp) and the organic cation transporters (OCT/Oct) are among the most important transporters involved in xenobiotic uptake in the liver. In the present study, 179 molecules have been tested as inhibitors of the uptake of estradiol-17βD-glucuronide (E₂17βG), substrate of OATP1B1/3 (rOatp), or 1-methyl-4-phenylpyridinium (MPP+), substrate of OCT1 (rOct1), into suspended cryopreserved hepatocytes from humans and rats. Uptake was assessed in 96-well plates by measuring intracellular accumulation of radioactive substrate in hepatocytes in presence or absence of inhibitor.
In rat hepatocytes 140 compounds were identified as inhibitors (inhibition at 20 μM ≥ 30%) of E₂17βG uptake and 77 compounds inhibitors of MPP+ uptake. The most potent inhibitors of rOatp and rOct1 were dantrolene sodium (K_i = 2 ± 9 μM) and bepridil (K_i = 14 ± 2 μM), respectively. In human hepatocytes, the most potent inhibitors of E₂17βG and MPP+ uptake were capsazepine (K_i = 14 ± 5 μM) and cyproheptadine (K_i = 19 ± 3 μM), respectively.
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The method used here is a simple tool to screen large number of compounds as inhibitors of the uptake of substrates into suspended hepatocytes.

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Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes

Abstract

Introduction

Section snippets

Materials

Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression

Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression

Conclusions

Acknowledgement

Adv. Drug Deliv. Rev.

Toxicology

Biochem. Pharmacol.

Exp. Cell Res.

Biochem. Pharmacol.

Drug Metab. Pharmacokinet.

J. Biol. Chem.

Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P450 3A4

AAPS Pharmsci.

P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes

Drug Metab. Dispos.

Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes

Drug Metab. Dispos.