Culture-period-dependent changes in the uptake of ...dmd.aspetjournals.org/content/dmd/early/2011/06/14/dmd.111.0389… · 14/06/2011 · blockers (ARBs), 3-hydroxy-3-methylglutaryl-coenzyme

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Culture-period-dependent changes in the uptake of transporter substrates in

sandwich-cultured rat and human hepatocytes

Naoki Kotani, Kazuya Maeda, Takao Watanabe, Mariko Hiramatsu, Li-kun Gong,

Yi-an Bi, Toshiaki Takezawa, Hiroyuki Kusuhara and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, the University of Tokyo, 3-1, 7-Chome

Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (N.K., K.M., T.W., M.H., H.K. and Y.S.)

Center for Drug Safety and Evaluation Research, State Key Laboratory of New Drug

Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501

Haike Road, Zhang Jiang Hi-Tech Park, Pudong, Shanghai 201203, China (L-K. G.)

Pfizer Global Research & Development, Eastern Point Road, Groton, CT 06340, USA

(Y-A. B.)

Laboratory of Animal Cell Biology, National Institute of Agrobiological Sciences,

Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan (T.T.)

DMD Fast Forward. Published on June 14, 2011 as doi:10.1124/dmd.111.038968

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on June 14, 2011 as DOI: 10.1124/dmd.111.038968

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Running title: Uptake activity in sandwich-cultured hepatocytes

Address correspondence:

Yuichi Sugiyama, Ph.D.

Department of Molecular Pharmacokinetics

Graduate School of Pharmaceutical Sciences

The University of Tokyo

7-3-1 Hongo, Bunkyo-ku, Tokyo

113-0033 JAPAN

Phone: +81-3-5841-4770

Fax: +81-3-5841-4766

Email: [email protected]

Number of text pages: 40 Number of tables: 2 Number of figures: 7 Number of references: 40 Number of words in the Abstract: 237 Number of words in the Introduction: 712 Number of words in the Discussion: 1484

Abbreviations: SCH, sandwich-cultured hepatocytes; SCRH, sandwich-cultured rat

hepatocytes; SCHH, sandwich-cultured human hepatocytes; OATP, organic

anion-transporting polypeptide; CLuptake, in vitro uptake clearance; CLH,vitro, hepatic

clearance predicted from in vitro uptake clearance; CLH,vivo, observed hepatic clearance

in vivo; CLbile,vitro, biliary clearance predicted from in vitro SCH experiments; CLbile,vivo,

observed biliary excretion clearance in vivo


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Abstract

Sandwich-cultured hepatocytes (SCH) are a useful tool for evaluating

hepatobiliary drug transport in vitro. Some studies have investigated the in vitro–in

vivo correlations of the biliary clearance of drugs using SCH. In most cases, the

biliary clearance observed in vivo correlated well with the predicted clearance, but the

predicted absolute values were underestimated when based on in vitro experiments with

SCH. We hypothesized that the downregulated function of uptake transporters is one

of the cause of this underestimation. Therefore, the uptake of taurocholate, digoxin,

pravastatin, and rosuvastatin was investigated in sandwich-cultured rat hepatocytes

(SCRH) cultured for 5, 24, 48, and 96 h, and the predicted hepatic clearance (CLH,vitro)

was calculated with a dispersion model. In SCRH cultured for 96 h, the saturable

uptake of taurocholate, digoxin, pravastatin, and rosuvastatin decreased to 7.5%, 3.3%,

64%, and 23%, respectively, of their uptake in hepatocytes cultured for 5 h, and a better

prediction of in vivo hepatic clearance (CLH,vivo) was achieved when based on CLH,vitro

of 5 h-cultured hepatocytes. These results suggest that the uptake activity is

considerably reduced in cell culture, even in a sandwich-culture format. In a similar

study, we also examined taurocholate and rosuvastatin in sandwich-cultured human

hepatocytes (SCHH). Unlike in SCRH, the saturable uptake of these compounds did

not differ markedly in SCHH cultured for 5 h or 96 h. Thus, the uptake activity in

SCHH was maintained relatively well compared with that in SCRH.


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Introduction

Together with their metabolism, many endogenous and exogenous compounds

undergo hepatic uptake and biliary excretion mediated by transporters, which facilitate

the efficient detoxification of xenobiotics. Several experimental tools have been

established to evaluate hepatic uptake, such as cell lines stably expressing human uptake

transporters and cryopreserved human hepatocytes. In contrast, it is fairly difficult to

evaluate the biliary excretion of compounds from hepatocytes to bile in humans.

Primary cultured hepatocytes are used to predict the hepatic transport and metabolism of

drugs, but a previous report suggested that their polarity was lost, followed by the

internalization of the efflux transporters a short time after the cells were isolated

(Hoffmaster et al., 2004), with a rapid reduction in the expression and activities of the

uptake transporters in long-term-cultured hepatocytes(Liang et al., 1993; Ishigami et al.,

1995; Rippin et al., 2001). This limits the utility of this experimental system for the

assessment of the biliary excretion of drugs.

In sandwich-cultured hepatocytes (SCH), the hepatocytes are repolarized and

the expression, cellular localization, and transport activities of the hepatic transporters

are well maintained (Liu et al., 1998; Hoffmaster et al., 2004), and functional

canalicular networks, called “bile pockets”, are also formed between these hepatocytes

(Swift et al.). The accumulation of compounds in the bile pockets can be measured by

opening them in Ca2+-free buffer (Liu et al., 1999b; Bi et al., 2006). Therefore, SCH

are the only in vitro experimental system in which the biliary excretion of drugs from

the blood to the bile can be evaluated. SCH can be useful for several applications such

as the prediction of overall biliary clearance of compounds whose clearance was not

dominated only by uptake process, the determination of the contribution of each efflux


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transporter to the overall efflux of drugs, and the impact of the inhibition of efflux

transporters by coadministered drugs on the change in the biliary transport of substrate

drugs (Swift et al.).

Until now, several studies have tried to demonstrate an in vitro–in vivo

correlation for the biliary clearance of drugs using SCH. In most cases, a good

correlation between the biliary clearance predicted from in vitro SCH experiments

(CLbile,vitro) and the biliary clearance observed in vivo (CLbile,vivo) has been reported, but

the absolute values for CLbile,vitro are often underestimated. For example, Fukuda et al.

(2008) showed that the absolute CLbile,vitro values for some angiotensin II receptor

blockers (ARBs), 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitors (statins),

and antibiotics were about 20- to 200-fold lower than the CLbile,vivo values, although

good correlations between CLbile,vitro and CLbile,vivo were observed (Fukuda et al., 2008).

Abe et al. (2008, 2009) compared the CLbile,vitro of some ARBs and statins with their

CLbile,vivo in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured

human hepatocytes (SCHH), and these CLbile,vitro values were also 20- to 200-fold lower

in the SCRH and about 20-fold lower in the SCHH than the CLbile,vivo values (Abe et al.,

2008; Abe et al., 2009). Li et al. (2010) tried to improve the in vitro–in vivo

correlation of the biliary clearance of drugs using SCRH by introducing an appropriate

scaling factor, because CLbile,vitro was about sixfold lower than CLbile,vivo based on a

conventional calculation method (Li et al., 2010). All of these results indicated that

the transport activity was reduced in SCH, but the exact reasons for its underestimation

were still unknown.

CLbile,vitro is usually calculated based on the drug concentration in the medium

of SCH, and corresponds to the biliary clearance based on the plasma unbound


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concentration in vivo. Based on the pharmacokinetic theory, the uptake process is the

first step in the hepatic elimination of drugs, and changes in uptake activity directly

affect the overall hepatic clearance. Therefore, we hypothesized that the

downregulation of uptake transporters is one of the major causes of the underestimation

of CLbile,vitro in the SCH system.

In the present study, the changes in transporter uptake activity in SCRH were

investigated using several transporter substrates (taurocholate, digoxin, pravastatin, and

rosuvastatin), as were these changes in SCHH (taurocholate, rosuvastatin). The

predicted hepatic clearance (CLH,vitro) values were calculated based on a dispersion

model, under the assumption that the uptake process is the rate-determining process in

hepatic clearance, and were also compared with in vivo hepatic clearance (CLH,vivo)

values in hepatocytes cultured for either 5 h or 96 h.


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Materials and Methods

Chemicals and reagents

[3H]-Pravastatin (45.5 Ci/mmol) and unlabeled pravastatin were kindly donated

by Daiichi Sankyo Co. Ltd (Tokyo, Japan). [3H]-Rosuvastatin (79 Ci/mmol) and

unlabeled rosuvastatin were donated by AstraZeneca (Macclesfield, UK).

[3H]-Taurocholate (4.6 Ci/mmol) and [3H]-digoxin (40 Ci/mmol) were purchased from

PerkinElmer Life and Analytical Sciences (Boston, MA). Triton X-100, ethylene glycol

tetraacetic acid (EGTA), and dexamethasone were purchased from Sigma-Aldrich (St.

Louis, MO). L-Glutamine, bovine serum albumin (BSA), and collagenase (type I) were

obtained from Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Williams’ medium

E (WME), hepatocyte wash medium (HWM), fetal bovine serum (FBS),

penicillin–streptomycin solution, human recombinant insulin, and standard (containing

Ca2+/Mg2+) and Ca2+/Mg2+-free Hanks’ balanced salt solution (HBSS) were purchased

from Invitrogen (Carlsbad, CA). BioCoat™ 24-well plates, Percoll, BD Matrigel™,

and ITS+ culture supplement (6.25 mg/mL insulin, 6.25 mg/mL transferrin, 6.25 μg/mL

selenious acid, 5.35 mg/mL linoleic acid, and 1.25 g/mL BSA) were purchased from

BD Biosciences (San Jose, CA). The hepatocyte thawing medium (InVitroGRO™ HT

medium), plating medium (InVitroGRO™ CP medium), incubation medium

(InVitroGRO™ HI medium), and Torpedo™ Antibiotic Mix were purchased from

Celsis In Vitro Technologies (Baltimore, MD). All other chemicals and reagents were

commercially available and of analytical grade.

Animals

Male Sprague Dawley rats (SD rats; 7–8 weeks old) were purchased from Japan


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SLC (Shizuoka, Japan). All animals were maintained under standard conditions with a

12-h dark/light cycle and were acclimatized for one week before the experiment. Food

and water were available ad libitum. All the study protocols were performed in

accordance with the guidelines provided by the Institutional Animal Care Committee,

Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

Isolation and culture of rat hepatocytes

Hepatocytes were isolated from the male SD rats with a two-step collagenase

perfusion procedure. Briefly, after anesthesia with an intraperitoneal injection of

sodium pentobarbital (64.8 mg/kg), the rat livers were perfused with Ca2+/Mg2+-free

perfusion buffer containing 1 mM EGTA for 5 min at a flow rate of 35–40 mL/min,

followed by perfusion with perfusion buffer containing 5 mM CaCl2, 20 mg/mL BSA,

and 1.0 mg/mL collagenase for 5 min at a flow rate of 20 mL/min. The hepatocytes

from the digested liver were dispersed in HWM, and were separated from the

connective tissue by filtering them through a sterile nylon mesh and from the

nonparenchymal cells by centrifugation at 50 × g for 1 min. The cell pellets were

resuspended in 45% isotonic Percoll in phosphate-buffered saline (PBS) and centrifuged

at 90 × g for 4 min. The cells were then resuspended again in HWM and washed once

by centrifugation at 50 × g for 1 min. The resultant cell pellets were resuspended in

WME containing 5% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 1 μM

dexamethasone, 4 μg/mL insulin, and 2 mM L-glutamine. Cell viability was

determined by Trypan blue exclusion. Only those hepatocytes with greater than 90%

viability were used for further experiments. The hepatocytes were seeded onto


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BioCoat™ 24-well plates at a density of 2.0 × 105 viable cells/well in 0.5 mL of

supplemented WME. The hepatocytes were allowed to attach for 5 h at 37 °C in a

humidified chamber under a 95% air/5% CO2 atmosphere. The plates were then

swirled gently and the medium containing the unattached cells was aspirated. WME

supplemented with 5% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 0.1 μM

dexamethasone, 4 μg/mL insulin, and 2 mM L-glutamine was added to each well (this

procedure was not applied to the 5-h-cultured hepatocytes). Within 18–24 h of

seeding, the cells were overlain with ice-cold 0.25 mg/mL BD Matrigel™ in WME

supplemented with 1% ITS+™ premix, 0.1 μM dexamethasone, and 2 mM L-glutamine

at 0.5 mL/well. BD Matrigel™ was only applied to the 48- or 96-h-cultured

hepatocytes. The cultures were maintained in supplemented WME, which was

changed every 24 h. Hepatocytes cultured for 5, 24, 48, or 96 h were used for the

uptake study, and cultured for 5, 24, or 96 h were used for Western blot analysis.

Western blot analysis of uptake transporters in SCRH

Crude membrane was prepared from SCRH cultured for designated time as

reported previously (Sasaki et al., 2002). After the crude membrane fraction was

suspended in phosphate buffered saline, it was immediately frozen in liquid N2 and

stored at -80°C until used. The protein concentration of the crude membrane prepared

from SCRH was determined by the method of Lowry with bovine serum albumin as a

standard (Lowry et al., 1951). The crude membrane fraction was dissolved in

EzApply containing DTT (ATTO, Tokyo, Japan) and loaded onto SDS-PAGE mini

4-12 % gel (TEFCO, Tokyo, Japan). The molecular weight was determined using a

Precision Plus Western C protein standard (Bio-Rad, Hercules, CA). Proteins were


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transferred electrophoretically to an Immuno-Blot PVDF membrane (Bio-Rad, Hercules,

CA) using a blotter (Transblot; Bio-Rad, Hercules, CA) at 100 V for 1 h. The

membrane was blocked with 10-fold diluted 10 x EzBlock (ATTO, Tokyo, Japan) by

Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature.

After washing with TTBS, the membrane was incubated overnight at 4°C in 1 x

EzBlock with 1000-fold diluted anti-Oatp1a1 (antiserum that we had produced

previously in rabbit (Aoki et al., 2008)), 120-fold diluted anti-Oatp1a4 (alpha diagnostic

international Inc., San Antonio, US), 100-fold diluted anti-Oatp1b2 (N-17 antibody,

Santa Cruz Biotechnology Inc., California, US), or Anti-Actin Clone C4 (MILLIPORE,

Billerica, MA). For the detection of Oatp1a1 and 1a4, the membrane was placed in

contact with 5000-fold diluted goat anti-rabbit IgG conjugated with horseradish

peroxidase (Bio-Rad, Hercules, CA), for the detection of Oatp1b2, the membrane was

placed in contact with 1000-fold diluted donkey anti-goat IgG conjugated with alkaline

phosphatase (Santa Cruz Biotechnology Inc., California, US), and for the detection of

β-actin, the membrane was placed in contact with 1000-fold diluted goat anti-mouse

IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA) for 1 h in 1 x

EzBlock. The immunoreactive band was detected using an AP conjugate Substrate Kit

(Bio-Rad, Hercules, CA).

Culture of cryopreserved human hepatocytes

Three batches of cryopreserved human hepatocytes were used in this study: lot

KQG was purchased from Celsis In Vitro Technologies, lot HH190 was purchased from

BD Gentest (Becton Drive, NJ), and lot Hu0930 was purchased from CellzDirect

(Durham, NC). The cryopreserved human hepatocytes were prepared according to the


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protocol suggested by Celsis In Vitro Technologies. Torpedo™ Antibiotic Mix (1.1

mL) was mixed into 50 mL of InVitroGRO™ HT medium, InVitroGRO™ CP medium,

and InVitroGRO™ HI medium, to produce complete thawing medium, plating medium,

and incubation medium, respectively. Before these media were used, they were

prewarmed in a water bath to 37 °C. The cryopreserved human hepatocytes were

incubated for about 1 min in the water bath at 37 °C and were diluted immediately with

thawing medium. The hepatocytes were then centrifuged at 50 × g for 3 min and the

supernatant was aspirated. The cell pellets were resuspended in 5 mL of plating

medium and cell viability was determined by Trypan blue exclusion. Only those

hepatocytes with greater than 85% viability were used for further experiments. The

hepatocytes were seeded onto BioCoat™ 24-well plates at a density of 7.5 × 105 viable

cells/well in 0.5 mL of plating medium and were cultured in a humidified chamber at

37 °C under a 95% air/5% CO2 atmosphere. Within 18–24 h of seeding, the medium

containing the unattached hepatocytes was removed and the hepatocytes were overlain

with ice-cold 0.25 mg/mL BD Matrigel™ in 0.5 mL/well incubation medium. BD

Matrigel™ was only applied to the 96-h-cultured hepatocytes. The cultures were

maintained in the incubation medium, which was changed every 24 h. Hepatocytes

cultured for 5 h or 96 h were used for the uptake study.

Transport assay with SCH

The uptake studies were performed according to the method described

previously (Liu et al., 1999a). Briefly, rat hepatocytes cultured in a collagen-sandwich

configuration (cultured for 96 h) were rinsed twice and preincubated at 37 °C for 10 min

with 0.5 mL of standard HBSS to maintain their tight junctions and bile canaliculi, or


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Ca2+/Mg2+-free HBSS with 1 mM EGTA to disrupt their tight junctions. Uptake was

initiated by the addition of 0.5 mL of standard HBSS containing radiolabeled

compounds with unlabeled substrates. The uptake reaction was terminated at the

designated time by addition of 1 mL of ice-cold HBSS after the removal of the

incubation buffer. The hepatocytes were then rinsed three times with 1 mL of ice-cold

HBSS. The hepatocytes were lysed with 0.5% Triton X-100 in PBS (0.5 mL/well)

with vigorous shaking for 30 min at room temperature. The radioactivity associated

with the cell lysate (500 μL) and the incubation buffer (100 μL) was analyzed with a

liquid scintillation counter (TRI-CARB 3100TR; PerkinElmer Life and Analytical

Sciences, Boston, MA) after the addition of 3 mL of scintillation cocktail (Clear-sol I;

Nacalai Tesque, Tokyo, Japan) to the scintillation vials. The remaining 25 μL of cell

lysate was used to determine the protein concentration with a BCA protein assay kit

(Thermo Scientific, Rockford, IL), with BSA as the standard. All data were corrected

for nonspecific binding by subtracting the radioactivity in hepatocyte-free wells from

that in hepatocyte-containing wells. The biliary excretion index (BEI), which is defined

as the percentage of the amount of substrates accumulated in the bile pockets in the total

accumulation in SCH after incubation for 10 min, was calculated with Eq. 1 (Liu et al.,

1999a):

100 onAccumulati

onAccumulati onAccumulatiEIB

)/MgCa(

free)-/Mg(Ca)/MgCa(

22

2222

×−

=++

++++

+

+ (Eq. 1)

where Accumulation(+ Ca2+

/Mg2+

) and Accumulation(Ca2+

/Mg2+

free) indicate the cumulative

uptake amount of compounds for 10 min in SCH preincubated with buffer in the presence

or absence of Ca2+/Mg2+ ions, respectively. Using the B-CLEAR® technology (Qualyst,

Inc., Research Triangle Park, NC), the amount of substrate secreted into the bile pockets


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can be estimated as the difference in the cumulative uptake of the compound into the SCH

in the presence and absence of Ca2+/Mg2+ ions, because these ions are required in the

medium to maintain the tight junctions that seal the bile pockets tightly. The absence of

Ca2+/Mg2+ ions causes the disruption of the tight junctions and the subsequent leakage of

the contents from the bile pockets into the medium.

Determination of the uptake clearance of the compounds using SCH

To evaluate the uptake clearance of each compound in SCH, uptake studies were

performed as described above, using only standard HBSS. The hepatocytes cultured for

the designated time periods were incubated in standard HBSS containing radiolabeled

compounds and unlabeled substrates for designated times. The uptake studies were

performed using two different concentrations of each compound to estimate the

contribution of transporter-mediated saturable uptake and passive diffusion to the overall

uptake clearance. Saturable uptake clearance was estimated as the difference between

the in vitro uptake clearance of a tracer concentration (CLuptake,tracer:μL/min/mg protein)

and an excess concentration (CLuptake,excess):

excessuptake,traceruptake, CLCLclearance) uptake (saturable −= (Eq. 2)

All data were corrected for nonspecific binding by subtracting the radioactivity in

hepatocyte-free wells from that in hepatocyte-containing wells.

Calculation method used to predict the in vivo biliary clearance from the in vitro

data

CLuptake was determined by calculating the slope of the distribution volume (Vd)

(μL/mg protein), given as the amount of radioactivity associated with the cells (dpm/mg


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protein) divided by its concentration in the incubation medium (dpm/μl), between 0.5

and x min (5 min for taurocholate, pravastatin, and rosuvastatin; 2 min for digoxin) (Eq.

3). In these ranges, the linearity of the time-dependent uptake was confirmed for each

compound.

0.5-xCL min50dmind

uptake.,,x VV −

= (Eq. 3)

The hepatic clearance (CLH,vitro) predicted from the in vitro uptake clearance was

calculated with Eqs 4–7 based on a dispersion model (Roberts and Rowland, 1986),

under the assumption that the uptake process is a rate-determining process in the overall

biliary clearance and uptake clearance is thought to be equal to the hepatic intrinsic

clearance (CLint,B = CLuptake).

)F(Q hh −= 1CL vitroH, (Eq. 4)

{ } { }N2

N2 1)/2/(aexpa)(11)/2/(aexpa)(1

4a

DDFh +−⋅−−−⋅+

= (Eq. 5)

2/1)41(a NN DR ⋅+= (Eq. 6)

hN Q

R Bint,B

CLf ⋅= (Eq. 7)

where Qh represents the hepatic blood flow, which is 67 mL/min/kg in rats (Nies et al.,

1976) and 20.7 mL/min/kg in humans (Davies and Morris, 1993); and fB represent the

blood unbound fraction of the test compounds, respectively. fB, the blood-to-plasma

concentration ratio (RB), and the in vivo hepatic clearance are cited from other reports.

Dispersion number (DN) was defined as an indicator for the degree of spreading of an

injected solute on transit through an organ in the dispersion model and set at 0.17

according to the previous reports (Roberts and Rowland, 1986; Iwatsubo et al., 1996).


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A physiological scaling factors of 40 g liver/kg, 1 × 106 cells/mg protein, and 120 × 106

cells/g liver for rat were used for scaling up in vitro clearance to the body level (Seglen,

1976; Davies and Morris, 1993; Bayliss et al., 1999). In humans, a physiological

scaling factor of 25.7 g liver/kg body weight, 1.5 × 106 cells/mg protein, and 107 × 106

cells/g liver, were used for in vivo scaling up (Davies and Morris, 1993; Ghibellini et al.,

2007).

Statistics

Statistical differences were analyzed with Student’s t-test to identify the

significant differences between two sets of data and by one-way analysis of variance

with Dunnett’s test for multiple pairwise comparisons. Differences were considered

statistically significant at P < 0.05.


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Results

Confirmation of hepatic transport function in SCH

To confirm that sandwich culture was successfully performed and that the bile

pockets were reestablished in this study, we performed an accumulation study of SCH

using standard and Ca2+/Mg2+-free HBSS. A significantly higher accumulation of

taurocholate was observed in the hepatocytes incubated with standard HBSS than in

hepatocytes incubated with Ca2+/Mg2+-free HBSS in both SCRH (Fig. 1) and SCHH

(Fig. 5). The BEI values for taurocholate, calculated with Eq. 1, in SCRH and SCHH

were 63.1 ± 5.8% and 51.0 ± 17.3%, respectively. In contrast, the absence of Ca2+ in

the incubation buffer had no effect on the accumulation of salicylic acid in the SCRH or

SCHH.

Changes in uptake activities in SCRH

The amount of each test compound taken up into the rat hepatocytes decreased

in a culture-period-dependent manner (Fig. 2). The same accumulation study was

performed three times and reproducible results were obtained. The typical data were

shown in Fig. 2 because the absolute values of the uptake amount into hepatocytes

varied among experiments. Uptake was temporarily improved only for pravastatin in

the 48-h-cultured hepatocytes (24 h after the cells were overlain with Matrigel), but it

also decreased in the 96-h-cultured hepatocytes, as did the other compounds. The

contribution of passive diffusion to the overall uptake was very small for taurocholate,

pravastatin, and rosuvastatin (≤ 10% of the overall uptake into the 5-h-cultured

hepatocytes), but was about 50% for digoxin (Fig. 3). When we compared the uptake

activities in the 5-h-cultured hepatocytes with those in the 96-h-cultured hepatocytes,


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the saturable uptake of taurocholate, digoxin, pravastatin, and rosuvastatin, calculated

with Eq. 2, decreased from 23.5 ± 1.1 to 1.79 ± 0.70, from 2.73 ± 0.86 to 0.016 ± 0.327,

from 6.40 ± 0.65 to 4.10 ± 0.99, and from 46.7 ± 4.5 to 10.9 ± 1.4 μL/min/mg protein,

respectively.

Alteration of expression levels of uptake transporters in SCRH

The expression levels of Oatp1a1/1a4/1b2 in SCRH over time in culture were

evaluated by Western blot analysis (Fig. 4). The expression levels of these

transporters hardly changed between 5-h- and 24-h-cultured hepatocytes. Oatp1a1

expression was maintained even in 96-h-cultured hepatocytes. However, the expression

levels of Oatp1a4 and 1b2 drastically decreased in 96-h-cultured hepatocytes.

Comparison of CLH,vitro and CLH,vivo in SCRH

To investigate the time-dependent changes in the uptake activities in SCRH,

we calculated the predicted hepatic clearance from the in vitro uptake clearance

(CLH,vitro) based on CLuptake for the 5-h- or 96-h-cultured hepatocytes using a dispersion

model (Eqs 4�7). CLH,vitro of taurocholate, digoxin, pravastatin, and rosuvastatin

decreased from 32.1 to 5.65, from 12.7 to 7.96, from 29.4 to 22.5, and from 16.7 to 5.21

mL/min/kg, respectively, during the long-term culture required for SCH. The CLH,vitro

values for the 5-h- and 96-h-cultured hepatocytes were compared with the observed

CLH,vivo cited in other reports (Table 1). The predicted CLH,vitro values for these

compounds, calculated from the data for the 5-h-cultured hepatocytes, were 1 to

3.6–fold lower than the CLH,vivo values, whereas those calculated from the data for the

96-h-cultured hepatocytes were very low.


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Changes in uptake activities in SCHH

A similar type of uptake study was also performed in SCHH to check whether

the downregulation of these uptake activities also occurs in human hepatocytes.

Unlike the results for SCRH, the uptake of the test compounds into three different

batches of human hepatocytes did not differ so much between the 5-h- and

96-h-cultured hepatocytes (Fig. 6). The contribution of passive diffusion to the overall

uptake of taurocholate and rosuvastatin was also very small (Fig. 7). When the uptake

activities of the 5-h-cultured hepatocytes and the 96-h-cultured SCHH were compared,

the saturable uptake calculated with Eq. 2, in each batch of human hepatocytes (KQG,

HH190, and Hu0930) changed from 18.2 ± 0.5, 15.6 ± 0.4, and 22.9 ± 0.4 μL/min/mg

protein, respectively, to 15.5 ± 0.5, 20.6 ± 0.6, and 11.9 ± 0.3 μL/min/mg protein,

respectively, for taurocholate, and from 7.47 ± 0.36, 8.71 ± 0.43, and 7.99 ± 0.28

μL/min/mg protein, respectively, to 7.58 ± 0.26, 6.79 ± 0.22, and 4.52 ± 0.19

μL/min/mg protein, respectively, for rosuvastatin.

Comparison of CLH,vitro and CLH,vivo in SCHH

The CLH,vitro values were calculated based on CLuptake in 5-h-cultured

hepatocytes from each batch of human hepatocytes, using the same method we used for

SCRH, and were compared with CLH,vivo (Table 2). The CLH,vitro values for

taurocholate and rosuvastatin were 9.68 ± 0.69 and 2.96 ± 0.17 mL/min/kg, respectively

(means ± S.E. of three batches), and both of these values were 1.9 to 3.0–fold lower

than the CLH,vivo values. CLuptake for taurocholate in the 96-h-cultured hepatocytes was

87% (KQG), 132% (HH190), or 54% (Hu0930) of the CLuptake observed in the


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5-h-cultured hepatocytes, and the CLuptake for rosuvastatin was 104% (KQG), 90%

(HH190), or 59% (Hu0930) of the CLuptake observed in the 5-h-cultured hepatocytes

(Fig. 7), so the CLH,vitro values did not differ as much in the 96-h culture in SCHH as

they did for SCRH.


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Discussion

The prediction of biliary clearance in vivo using SCH has been demonstrated in

previous reports, but in most cases, the absolute values for the biliary clearance of

various drugs have tended to be underestimated. We hypothesized that the

downregulation of uptake transporters in SCH is an important factor in this

underprediction, and investigated the dependence of the uptake clearance of several

compounds on the culture time in rat and human SCH.

The overall intrinsic biliary clearance (CLbile,int,all) is composed of several

microscopic parameters: uptake clearance from the medium or blood to the cells (PSup),

backflux clearance from the cells to the medium or blood (PSback), metabolic clearance

(PSmet), and intrinsic efflux clearance into the bile (PSeff), as shown in the following

equation (Shitara et al., 2006):

backmeteff

meteffupint,, PSPSPS

PSPSPS

+++

×=allbileCL (Eq. 8)

From this equation, CLbile,int,all is always proportional to PSup. In the present

study, the effect of the culture period on the changes in the transport activity mediated by

hepatic uptake transporters in SCH was investigated and its impact on the prediction of

biliary clearance in vivo was also evaluated.

To confirm that the sandwich-culture technique was correctly established in our

hands, we first checked the transport of typical ligands in our SCH. The accumulation

of taurocholate was significantly higher in hepatocytes incubated with standard HBSS

than in those incubated with Ca2+/Mg2+-free HBSS, and its BEI values ranged from 45

to 70% in SCRH, and from 50 to 60% in SCHH in our experiments. These values

were comparable to those in previous reports (Liu et al., 1999a; Abe et al., 2009). In


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contrast, no difference in the accumulation of salicylate in SCRH and SCHH was

observed when they were incubated with standard HBSS or Ca2+/Mg2+-free HBSS, as

previously demonstrated (Liu et al., 1999a). Therefore, our experimental

sandwich-culture technique was confirmed to be comparable to those of previous

studies.

The initial uptake of transporter substrates (taurocholate, digoxin, pravastatin,

and rosuvastatin) into SCRH was then observed to investigate the effect of the culture

period on their uptake activity. Transporter-mediated uptake activity is thought to be

well maintained for several hours after the isolation of hepatocytes (Ishigami et al.,

1995; Liu et al., 1999a). However, four-day culture is required for the optimal

formation of the canalicular networks and the expression of the efflux transporters in

SCRH (Chandra et al., 2001). Therefore, we decided to compare the uptake clearance

in 5-h- and 96-h-cultured hepatocytes in this study. The saturable uptake of all the

compounds we tested was observed clearly, and the uptake clearance of taurocholate,

digoxin, and rosuvastatin decreased through the culture period, whereas that of

pravastatin recovered temporarily in the 48-h-cultured hepatocytes (24 h after they were

overlain with Matrigel), but decreased again in the 96-h-cultured hepatocytes (Figs 2

and 3). Taurocholate is a typical substrate of Na+-taurocholate cotransporting

polypeptide (Ntcp) and its uptake clearance in 96-h-cultured SCH decreased to 13% of

that in the 5-h-cultured hepatocytes (Fig. 3). This result is similar to the reduction in

Ntcp protein expression detected with an immunoblot analysis (Liu et al., 1998). The

saturable uptake of digoxin, pravastatin, and rosuvastatin in 96-h-cultured SCRH

decreased to 3.3%, 64%, and 23% of that in the 5-h-cultured hepatocytes, respectively

(Fig. 3). Because digoxin is an Oatp1a4-specific substrate (Shitara et al., 2002), our


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result implies that the transport function of Oatp1a4 was greatly reduced during the 96-h

culture period required for SCH. In our data, half of the digoxin uptake was

dominated by active saturable transport in isolated rat hepatocytes, while previous

report indicated that 79% of its uptake was saturable in cultured rat hepatocytes (Ito et

al., 2007). The apparent discrepancy may come from the different experimental

systems and possible alteration of passive permeability by the inhibition of

Na+/K+-ATPase by 100 μM digoxin. So it is still possible that active transport of

digoxin was partly preserved at 96-h-cultured SCRH. Multiple Oatp transporters are

involved in the uptake of pravastatin and rosuvastatin (Ho et al., 2006). These data

suggest that the relative contribution of each transporter to the hepatic uptake of

rosuvastatin and pravastatin differs in rats.

We also evaluated the expression levels of Oatps in SCRH by Western blot

analysis (Fig. 4). Although the expression level of Oatp1a1 was maintained relatively

well also in 96-h-cultured SCRH compared with that in 5-h-cultured hepatocytes, that of

Oatp1a4 decreased drastically in 96-h-cultured SCRH. These results are comparable

with the previous report (Hoffmaster et al., 2004), and support our results in which the

saturable uptake of digoxin, an Oatp1a4-selective substrate, was drastically decreased in

96-h-cultured SCRH. Oatp1b2 expression was also largely decreased after long-term

culture, whereas uptake activity of pravastatin was well preserved, which suggests that

pravastatin uptake into hepatocytes is not only dominated by Oatp1b2, but also by

Oatp1a1 (Hsiang et al., 1999) and Oatp1a4 (Tokui et al., 1999).

Next, we tried to predict the hepatic clearance of drugs in vivo (CLH,vivo) from

the in vitro uptake clearance in 5-h- and 96-h-cultured hepatocytes under the assumption

that the uptake process is a rate-determining process in the overall biliary clearance


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(Table 1). Because the hepatic clearance of some of the test compounds was very

close to the hepatic blood flow rate, a dispersion model (DN = 0.17) was used to test this

prediction (Roberts and Rowland, 1986). Our results indicate that the predicted

hepatic clearance (CLH,vitro) decreased together with the reduced uptake function in the

96-h-cultured SCRH compared with that in the 5-h-cultured hepatocytes. In the

sandwich-culture format, the in vitro clearance from the medium to the bile pockets is

defined as the accumulation rate of the compound in the bile pockets divided by the

medium concentration, and corresponds to the in vivo biliary clearance from the blood

to the bile. Considering Eq. 8, the CLH,vitro value predicted from the uptake clearance

in vitro is the upper limit of the clearance calculated from SCH. Therefore, the

CLH,vitro predicted from uptake clearance in SCRH could be further underestimated

relative to CLH,vivo. These results suggest that we must consider the reduced function

of uptake transporters in SCRH when predicting the biliary clearance in vivo using

SCRH.

To understand the predictability of SCH in humans, the same type of study was

performed in SCHH. The uptake of taurocholate and rosuvastatin did not decrease as

much in the 96-h-cultured SCHH relative to that in the 5-h-cultured human hepatocytes

(Figs 6 and 7), so the function of uptake transporters was maintained relatively well in

SCHH compared with that in SCRH. NTCP also makes a large contribution to the

uptake of taurocholate in human hepatocytes (Hagenbuch and Dawson, 2004), whereas

OATP1B1 rather than OATP1B3 plays a major role in the uptake of rosuvastatin

(Kitamura et al., 2008). These results are consistent with a previous study

demonstrating that the expression levels of OATP1B1 and OATP1B3 in SCHH were

almost the same on day 1 and day 6 (Hoffmaster et al., 2004). Why the expression and


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function of uptake transporters are better maintained in SCHH than in SCRH is

unknown, but this tendency was also observed in primary cultured hepatocytes (Jigorel

et al., 2005). The predicted CLH,vitro values were 1.9 to 3.0-fold lower than the CLH,vivo

for taurocholate and rosuvastatin in both the 5-h-cultured hepatocytes and the

96-h-cultured SCHH (Table 2). (Watanabe et al., 2009) reported that a scaling factor

(x 1.7–4.3) was required to fit the calculated value to the CLH,vivo value when they

assessed the CLH,vitro value for four kinds of statins using cryopreserved human

hepatocytes in suspension, which is comparable to our situations.

In the previous report, there was 20-fold difference at least between in vitro

biliary clearance predicted by SCHH and human in vivo biliary clearance (Abe et al.,

2009), but in our results, the difference between the predicted hepatic clearance from in

vitro uptake assay and in vivo hepatic clearance was only about 2.5-fold. This

discrepancy may be due to the change in the backflux or biliary efflux intrinsic

clearance, large inter-batch difference in the uptake clearance of substrates, and partial

saturation of OATP transporters because of the substrate concentration they used was

almost similar to the Km values for OATP transporters.

In conclusion, we have investigated the changes in the uptake of transporter

substrates over several days in culture in both SCRH and SCHH. In SCRH, the uptake

activity was dramatically down-regulated throughout the culture period, and the reduced

uptake activity led to the underestimation of CLH,vitro from the in vitro data for SCRH.

One of the strategies for the prediction of in vivo biliary clearance may be available by

taking correlation between in vitro and in vivo biliary clearance of many compounds

with some scaling factors. Conversely, the uptake activity was maintained relatively

well in SCHH, so our results suggest that we can predict the hepatobiliary transport of


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compounds in the human liver well using SCHH. More data regarding the prediction

of biliary clearance in vivo from experiments in SCHH in vitro are required to confirm

the usefulness of SCHH.


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Acknowledgments

We sincerely thank AstraZeneca and Daiichi Sankyo Co., Ltd. for kindly

providing us with radiolabeled and unlabeled rosuvastatin and pravastatin, respectively.


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Authorship Contributions

Participated in research design: Kotani, Maeda, Kusuhara, and Sugiyama.

Conducted experiments: Kotani, Maeda, Watanabe, Hiramatsu, Gong and Sugiyama

Contributed new reagents or analytic tools: Bi and Takezawa

Performed data analysis: Kotani, Maeda, Watanabe, Hiramatsu, Gong, Kusuhara, and

Sugiyama

Wrote or contributed to the writing of the manuscript: Kotani, Maeda, and Sugiyama


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Footnote

This study was partly supported by Grants-in-Aid for Research on Publicly

Essential Drugs and Medical Devices from the Ministry of Health, Labor and Welfare

of Japan and a Grant-in-Aid for Scientific Research (A) [20249008] from the Japan

Society for the Promotion of Science (JSPS). B-CLEAR® is a registered trademark of

Qualyst, Inc. and is covered by US Patent No. 6,780,580, EU Patent No. 00919459.8,

and other US, Japanese, and international patents, both issued and pending.


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Legends for Figures

Fig. 1 Ca2+-dependent uptake of taurocholate and salicylic acid by SCRH

The uptake of [3H]-taurocholate (1 μM) (A) and [14C]-salicylic acid (2 μM)

(B) by rat hepatocytes cultured for 96 h in a sandwich configuration in the presence or

absence of Ca2+ ions in the incubation medium for 10 min. Each datum is a mean ±

S.E. (n = 3). ** P < 0.01.

Fig. 2 Culture-time-dependent uptake of taurocholate, digoxin, pravastatin, and

rosuvastatin by SCRH

The uptake of [3H]-taurocholate (1 μM) (A), [3H]-digoxin (0.01 μM) (B),

[3H]-pravastatin (0.1 μM) (C), and [3H]-rosuvastatin (0.1 μM) (D) was observed in rat

hepatocytes cultured on a collagen-coated dish for 5 h (closed squares) and for 24 h

(closed triangles),or cultured in a sandwich configuration for 48 h (closed diamonds)

and 96 h (closed circles). The uptake of test compounds was determined for 0.5, 2, 5,

10, and 15 min. The same accumulation study was performed three times and

reproducible results were observed. This figure shows typical data from single

preparation, which are means ± S.E. (n = 3). The same accumulation study was

performed three times and reproducible results were obtained. Where vertical bars are

not shown, the S.E. values are within the limits of the symbols.

Fig. 3 Ratio of the uptake clearance of taurocholate, digoxin, pravastatin, or

rosuvastatin in each culture period normalized by that in 5-h-cultured

hepatocytes

Ratio of the uptake clearance of [3H]-taurocholate (closed bar, 1 μM; open bar,


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1 mM) (A), [3H]-digoxin (closed bar, 0.01 μM; open bar, 100 μM) (B),

[3H]-pravastatin (closed bar, 0.1 μM; open bar, 1 mM) (C), or [3H]-rosuvastatin

(closed bar, 0.1 μM; open bar, 100 μM) (D) in 5-h-cultured hepatocytes to that in

96-h-cultured hepatocytes was shown as a percent of control. The data are means ±

S.E. (n = 9; triplicate in three independent preparations for closed bars and n = 6;

triplicate in two independent preparations for open bars). * P < 0.05, ** P < 0.01

compared with the uptake clearance in 5-h-cultured hepatocytes.

Fig. 4 Culture time-dependent alteration of protein expression levels of uptake

transporters for organic anions in SCRH

Protein expression levels of Oatp1a1/1a4/1b2 and β-actin (internal control)

in 5-h-, 24-h-, and 96-h-cultured rat hepatocytes were examined by Western blot

analysis.

Fig. 5 Ca2+-dependent uptake of taurocholate and salicylic acid by SCHH

The uptake of [3H]-taurocholate (1 μM) (A) and [14C]-salicylic acid (2 μM)

(B) by human hepatocytes (lot; KQG) cultured for 96 h in a sandwich configuration in

the presence or absence of Ca2+ ions in the incubation medium for 10 min. Each

datum is a mean ± S.E. (n = 3). * P < 0.05.

Fig. 6 Culture-time-dependent uptake of taurocholate and rosuvastatin by SCHH

The uptake of [3H]-taurocholate (1 μM) (A, B, C) or [3H]-rosuvastatin (0.1

μM) (D, E, F) was observed in human hepatocytes cultured on a collagen-coated dish


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for 5 h (closed circles) or cultured in a sandwich configuration for 96 h (open circles).

The uptake of test compounds was determined for 0.5 and 5 min. We used three

different batches of human hepatocytes: KQG (A, D), HH190 (B, E), and Hu0930 (C,

F). The data are means ± S.E. (n = 6). Where vertical bars are not shown, the S.E.

values are within the limits of the symbols.

Fig. 7 Ratio of the uptake clearance of taurocholate or rosuvastatin in

96-h-cultured SCHH normalized by that in 5-h-cultured hepatocytes

Ratio of the uptake clearance of [3H]-taurocholate (closed bar, 1 μM; open bar,

1 mM) (A) or [3H]-rosuvastatin (closed bar, 0.1 μM; open bar, 100 μM) (B) in

5-h-cultured hepatocytes to that in 96-h-cultured hepatocytes was shown as a percent

of control. Data are means ± S.E. (n = 6; triplicate in two independent preparations).

* P < 0.05, ** P < 0.01 compared with the uptake clearance in 5-h-cultured

hepatocytes.


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Table 1 Pharmacokinetic parameters for taurocholate, digoxin, pravastatin, and

rosuvastatin in rats obtained from the literature, and a comparison of rat hepatic

clearance in vivo (CLH,vivo; A) with the hepatic clearance predicted from the uptake

clearance in SCRH (CLH,vitro; B).

(A)

CLH,P

(mL/min/kg) RB CLH,vivo

(mL/min/kg)

Taurocholate 31.4a 0.6b 52.3

Digoxin 13.3d 1.04e 12.7

Pravastatin 62g

Rosuvastatin 37.6h 0.631i 59.6

(B)

fB

CLH,vitro

(5 h) (mL/min/kg)

CLH,vitro

(96 h) (mL/min/kg)

Taurocholate 0.4c 32.1 (0.615)

5.65 (0.108)

Digoxin 0.615e 12.7 (1.00)

7.96 (0.627)

Pravastatin 1.2g 29.4 (0.475)

22.5 (0.362)

Rosuvastatin 0.083i 16.7 (0.281)

5.21 (0.0874)

CLH,P: in vivo hepatic clearance based on the plasma concentration was assessed as the

difference between the total plasma clearance and the renal plasma clearance for each

compound; RB: ratio of the blood concentration to the plasma concentration of each

compound; fB: unbound fraction of each compound in the total blood.


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The CLH,vitro/CLH,vivo ratio is described in parentheses under the CLH,vitro values in

Table 1 (B).

a (Hayashi and Sugiyama, 2007)

b Blood-to-plasma concentration ratio of taurocholate in rats is assumed to be 0.6

c (Inoue et al., 1985)

d (Harrison and Gibaldi, 1976)

e (Liu et al., 2005)

f (Yamazaki et al., 1996)

g (Watanabe et al., 2010)

h (Kitamura et al., 2008)

i (Watanabe et al., 2009)


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Table 2 Pharmacokinetic parameters for taurocholate and rosuvastatin in humans, and a

comparison of human nonrenal (hepatic) clearance (CLH,vivo; A) with the biliary

clearance in vitro, predicted based on the uptake clearance in SCHH (CLH,vitro; B).

(A)

CLH,P

(mL/min/kg) RB CLH,vivo

(mL/min/kg)

Taurocholate 0.6a 20.7b

Rosuvastatin 4.83d 0.6a 8.06d

(B)

fB CLH,vitro (5 h) (mL/min/kg)

CLH,vitro (96 h) (mL/min/kg)

HH lot KQG HH190 Hu0930 KQG HH190 Hu0930

Taurocholate 0.4c 9.50 (0.459)

8.58 (0.415)

10.9 (0.529)

8.66 (0.418)

10.4 (0.502)

7.14 (0.345)

Rosuvastatin 0.18d 2.65 (0.329)

2.99 (0.372)

3.23 (0.401)

2.74 (0.341)

2.71 (0.337)

1.99 (0.247)

CLH,P: the hepatic clearance in vivo based on the plasma concentration was assessed as

the difference between the total plasma clearance and the renal plasma clearance for each

compound; RB: blood-to-plasma concentration ratio of each compound; fB: unbound

fraction of each compound in the total blood

The CLH,vitro/CLH,vivo ratio is described in parentheses under the CLH,vitro values in

Table 2 (B)

a Blood-to-plasma concentration ratio of these compounds in humans was assumed to


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be 0.6

b Biliary clearance of taurocholate was assumed to be the same as the hepatic blood

flow rate

c There are no data for the blood unbound fraction of taurocholate in humans, so we

used the value for rats (Inoue et al., 1985)

d These data were obtained from the interview form of CRESTOR®.


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75 kDa

75 kDa




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0 1 2 3 4 50

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