Pharmacokinetic characterization of transcellular transport and drug interaction of digoxin in Caco-2 cell monolayers.

Abstract

To characterize the intestinal absorption of digoxin, its transcellular transport and drug interaction activity was investigated using Caco-2 cell monolayers. We examined digoxin transport in the presence and absence of ouabain to determine whether digoxin binding to Na+,K(+)-ATPase affects its transcellular digoxin transport, and evaluated its influx and efflux clearance by model-dependent pharmacokinetic analysis. Transcellular transport in the basal-to-apical direction was greater than that in the opposite direction. In addition, ouabain decreased the cellular accumulation of digoxin, but it did not alter its transcellular transport profile. The observations for transcellular transport and cellular accumulation in the presence of ouabain were used for the pharmacokinetic analysis, which showed that the efflux clearance of digoxin on the apical side of the monolayer was 15 times greater than that on the basal side. Apical-to-basal transport was increased by carvedilol and pimobendan, and these compounds suppressed the efflux clearance on the apical side and the influx clearance on the basal side. These findings indicate that the intestinal absorption of digoxin is primarily dominated by the efflux process on the luminal side of the intestine, and that carvedilol and pimobendan may vary the rate of intestinal digoxin absorption mainly by inhibiting its exsorptive transport.

Full text

Page 1

The cardiac glycoside digoxin is one of the most com-
monly used compounds for treating congestive heart failure,
and its treatment efficacy is maximal at a serum concentra-
tion of 0.8—2.0ng/ml.1,2)However, the margin between its
effective and toxic doses is narrower and less well defined
than those of other therapeutic compounds.1—3)In some
cases, digoxin toxicity is readily perceived even at a concen-
tration of less than 2ng/ml. It is therefore recommended that
the digoxin dosage should be carefully determined according
to the patient’s clinical conditions and other medications si-
multaneously being administered with the digoxin treatment.
The serum digoxin concentration is increased by various
therapeutic compounds. Some such as carvedilol interact
with digoxin,4)whereby the plasma digoxin concentration is
increased when carvedilol and digoxin are orally adminis-
tered, while carvedilol does not affect the digoxin concentra-
tion when digoxin is intravenously administered.4)Ery-
thromycin,5)clarithromycin5—7)and talinolol8)also interact in
a similar way with digoxin. Together, these observations sug-
gest that the increased digoxin concentration is not due to a
decrease in its renal excretion, but rather an increase in
bioavailability, and that the increased bioavailability is proba-
bly a consequence of these compounds enhancing the intesti-
nal digoxin absorption.9,10)Therefore, in the present study we
characterized the intestinal absorption of digoxin to elucidate
the factor which is primarily responsible for digoxin bioavail-
ability. We also examined whether cardiovascular compounds
such as the b-blocking agent carvedilol and the inotropic
agent pimobendan affect its intestinal absorption.
During intestinal drug absorption, therapeutic compounds
first enter intestinal epithelial cells from their apical side,
then pass through the epithelia to the basal side, and finally
appear in the blood stream. Therefore, to investigate intesti-
nal drug absorption, it is important to separately assess these
sequential processes. One useful approach to studying in-
testinal drug absorption is to use Caco-2 cells monolayers,
which can be prepared on a porous filter as a polarized
monolayer.11—15)The drug concentrations on the apical and
basal sides of the monolayer can be readily determined along
with the intracellular drug accumulation. The directional
specificity of transcellular digoxin transport in Caco-2 cells
has been mentioned in various reports.16,17)However, in many
cases, digoxin transport on the apical and basal sides of the
monolayer were not separately examined, and the influx and
efflux clearance rates of digoxin were not evaluated.16,17)
For the characterization of transcellular drug transport, a
pharmacokinetic approach is useful.18—20)Transcellular drug
transport in LLC-PK1and OK cell monolayers can be ana-
lyzed in detail in a model-dependent manner,18,19)where their
drug concentration–time profiles on both sides of the mono-
layer can be assessed by curve fitting calculations and the in-
flux and efflux clearance of the monolayer separately evalu-
ated. Thus, when transcellular drug transport is examined
under the condition where the unlabeled drug concentration
in the monolayer is equilibrated with that of the incubation
medium in the apical and basal chambers, the transport data
for a small amount of radio-labeled drug can be analyzed
using a linear pharmacokinetic model.15,18)
MATERIALS AND METHODS
Materials
TBq/mmol)
GBq/mmol) were purchased from PerkinElmer (Boston, MA,
U.S.A.) and Moravek Biochemicals (Brea, CA, U.S.A.), re-
spectively, and unlabeled digoxin and ouabain were obtained
from Nacalai Tesque (Kyoto, Japan). Quinidine was obtained
from Sigma (St. Louis, MO, U.S.A.), and carvedilol was sup-
plied by Daiichi Pharmaceutical (Tokyo, Japan), and pi-
mobendan (Acardi Capsule®1.25) was purchased from Nip-
Radio-labeled digoxin ([3H]-digoxin, 1.37
and mannitol ([1-14C]-D-mannitol, 1.96
114
Biol. Pharm. Bull. 28(1) 114—119 (2005) Vol. 28, No. 1
∗ To whom correspondence should be addressed.e-mail: yukiya@ms.toyama-mpu.ac.jp© 2005 Pharmaceutical Society of Japan
Pharmacokinetic Characterization of Transcellular Transport and Drug
Interaction of Digoxin in Caco-2 Cell Monolayers
Tetsuya AIBA, Kazuya ISHIDA, Mariko YOSHINAGA, Marie OKUNO, and Yukiya HASHIMOTO*
Graduate School of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University; Sugitani, Toyama
930–0194, Japan.
Received March 24, 2004; accepted October 12, 2004
To characterize the intestinal absorption of digoxin, its transcellular transport and drug interaction activity
was investigated using Caco-2 cell monolayers. We examined digoxin transport in the presence and absence of
ouabain to determine whether digoxin binding to Na?,K?-ATPase affects its transcellular digoxin transport, and
evaluated its influx and efflux clearance by model-dependent pharmacokinetic analysis. Transcellular transport
in the basal-to-apical direction was greater than that in the opposite direction. In addition, ouabain decreased
the cellular accumulation of digoxin, but it did not alter its transcellular transport profile. The observations for
transcellular transport and cellular accumulation in the presence of ouabain were used for the pharmacokinetic
analysis, which showed that the efflux clearance of digoxin on the apical side of the monolayer was 15 times
greater than that on the basal side. Apical-to-basal transport was increased by carvedilol and pimobendan, and
these compounds suppressed the efflux clearance on the apical side and the influx clearance on the basal side.
These findings indicate that the intestinal absorption of digoxin is primarily dominated by the efflux process on
the luminal side of the intestine, and that carvedilol and pimobendan may vary the rate of intestinal digoxin ab-
sorption mainly by inhibiting its exsorptive transport.
Key words
digoxin; intestinal absorption; carvedilol; pimobendan; Caco-2 cell monolayer

Page 2

pon Boehringer Ingelheim (Kawanishi, Japan) and further
purified by ethanol extraction. All other chemicals were of
the finest grade available.
Cell Culture and Preparation of Monolayers
cells at passage number 40 were obtained from the Riken
Bioresource Center (Tsukuba, Japan), and were maintained
in plastic dishes with Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum (Valley Bio-
chemical Inc., Winchester, VA, U.S.A.) in an atmosphere of
5% CO2–95% air at 37°C. All experiments were carried out
with cells between passages 48—73. The medium was
changed every second or third day, and when the cells
reached 80—90% confluence they were removed using a
0.05% trypsin/0.02% EDTA solution. They were then
washed with phosphate buffered saline and seeded at
5?105cells/cm2on a 0.9-cm2porous membrane in a Fal-
conTMcell culture insert (BD Biosciences, Bedford, MA,
U.S.A.). The pore size of the membrane was 0.4mm in diam-
eter and the pore density was 1.6?106/cm2. The seeded cells
were maintained for 3weeks to prepare the cell monolayers.
One milliliter of the culture media was supplied to the cham-
ber on the apical side of the monolayer (the apical chamber),
and 2-ml was supplied to the chamber on the other side, the
basal chamber. The culture medium was changed every other
day, and maturity of the monolayer was judged by transep-
ithelial electrical resistance (TEER). TEER was measured
using a Millicell-ERS resistance system (Millipore, Bedford,
MA, U.S.A.). Caco-2 cell monolayers whose TEER was
above 900W ·cm2were used for experiments.
Transport of Digoxin and Mannitol in Caco-2 Cell
Monolayers
The transcellular transport of digoxin in Caco-
2 cell monolayers was examined according to a previously
reported method.18,19)All transport experiments were con-
ducted in an atmosphere of 5% CO2–95% air at 37°C. In
brief, the monolayer was first pre-incubated with the experi-
ment medium in the apical and basal chambers to equilibrate
the digoxin concentration. The experiment medium was com-
posed of cell culture medium containing unlabeled digoxin at
10nM. After a 60min equilibration period, 3H-digoxin was
applied to the apical chamber to examine the apical-to-basal
transcellular transport of digoxin. The medium in the basal
chamber was then collected after 60, 120 and 180min. Fol-
lowing the last medium collection, the porous membrane on
which the Caco-2 cell monolayer was prepared was immedi-
ately washed three times with ice-cold phosphate buffer, and
then the cells were removed and collected. The Caco-2 cells
were used to evaluate digoxin accumulation, where the
amounts of 3H-digoxin were determined using a liquid scin-
tillation counter and normalized against the initially applied
doses. The profile for digoxin transport in the opposite direc-
tion (basal-to-apical) was also examined in the same manner.
To evaluate the paracellular transport of digoxin, mannitol
transport was examined using 14C-mannitol.
To determine whether digoxin binding to Na?,K?-ATPase
affects the transcellular transport of digoxin, another series of
experiments as described above was carried out in the pres-
ence of ouabain, which was dissolved in the experiment
medium in the apical and basal chambers at a final concen-
tration of 100mM 60min before the addition of 3H-digoxin.21)
The inhibitory effects of carvedilol and pimobendan on
transcellular digoxin transport were examined in the same
Caco-2
manner as described above, for which carvedilol was dis-
solved in the experiment medium in the apical and basal
chambers at a final concentration of 1 or 5mM 60min before
the addition of 3H-digoxin. Pimobendan was used at a final
concentration of 5 or 50mM. For the inhibition study, we used
quinidine as a positive inhibition control, since it potently in-
hibits P-glycoprotein.22)Quinidine was dissolved in the ex-
periment medium at a concentration of 5mM.
Estimation of Cell Volume of the Caco-2 Cell Mono-
layer
The volume of the monolayer was calculated from
the amount of sulfanilamide that accumulated in the mono-
layer by simple diffusion under equilibrium conditions.23)
Briefly, the Caco-2 cell monolayer was incubated for 60min
in 2mg/ml sulfanilamide solution, and washed three times
with ice-cold phosphate buffer, and the monolayer was col-
lected to be homogenized. The sulfanilamide concentration
in the homogenized sample was spectrophotometerically de-
termined at 550nm after diazotization.
Pharmacokinetic Analysis
of digoxin was analyzed in a model-dependent manner using
the method of non-linear least squares.18—20)Assuming that
digoxin was transported as shown in Fig. 1, the following
mass balance equations were prepared for the pharmacoki-
netic analysis:
The transcellular transport
(1)
(2)
(3)
where XA, XBand XCare the amount of digoxin in the apical
chamber, the basal chamber and the monolayer determined at
time t, respectively. VAand VBindicate the volume of the api-
dX
dt
CL
V
X
CL
V
X
CL
(
CL
V
X
CAC
A
A
BC
B
B
CBCA
C
C
???
?
⋅⋅⋅
)
dX
dt
CL
V
X
CL
V
X
CL
V
X
CL
V
X
B BC
B
B
CB
C
C
PARA
A
A
PARA
B
B
?????
⋅⋅⋅⋅
dX
dt
CL
V
X
CL
V
X
CL
V
X
CL
V
X
AAC
A
A
CA
C
C
PARA
A
A
PARA
B
B
?????
⋅⋅⋅⋅
January 2005 115
Fig. 1.
Transcellular Digoxin Transport in Caco-2 Cell Monolayers
Digoxin influx and efflux clearance on the apical side of the monolayer are desig-
nated CLACand CLCA, respectively, and influx and efflux clearance on the basal side are
designated CLBCand CLCB, respectively. CLPARAstands for the paracellular clearance of
digoxin. The volumes of the apical and the basal chambers and the intracellular volume
of the monolayer are indicated by VA, VBand VC, respectively.
Schematic of the Three-Compartment Model Used to Analyze

Page 3

cal and basal chambers, respectively, and the distribution vol-
ume of digoxin in the monolayer is designated VC. The influx
and efflux clearance of digoxin on the apical side of the
monolayer are designated CLACand CLCA, respectively, and
these clearance parameters for the basal side of the mono-
layer are designated CLBCand CLCB, respectively. Paracellu-
lar transport clearance is denoted as CLPARA.
Paracellular clearance of digoxin (CLPARA) was estimated
by analyzing the mannitol transport profile using the mass
balance equations described below:
(4)
(5)
where XMAand XMBare the amount of mannitol in the apical
and basal chambers determined at time t, respectively.
The calculations were conducted using the UCSF NON-
MEM program,24)and with the POSTHOC directive option
the clearance parameters for each experiment were obtained
by Bayesian estimation.
Data Analysis
Data were expressed as means?S.E. de-
rived from 6 experiments. For multiple comparisons against a
single control group, Bartlett’s test for homogeneity of the
variances was first performed. Then, significant differences
were evaluated by analysis of the variance followed by Dun-
nett’s test. If homogeneity of the variance could not be as-
sumed, significance was evaluated by Kruskal–Wallis non-
parametric analysis of the variance followed by Dunnett-type
test. For the comparison of two means, Student’s t-test was
used to judge the significance, where p?0.05 was considered
to be statistically significant.
RESULTS
Transcellular Transport and Cellular Accumulation of
Digoxin in Caco-2 Cell Monolayers in the Presence and
Absence of Ouabain
We first examined the transcellular
transport of digoxin in Caco-2 cell monolayers at a digoxin
concentration of 10nM in the presence and absence of
100mM ouabain, and determined whether digoxin binding to
Na?,K?-ATPase affected the transcellular digoxin transport.
As shown in Fig. 2A, digoxin was transported in a direction-
specific manner, where the amount of digoxin transported in
the basal-to-apical direction within 180min was 6—7times
greater than that transported in the opposite direction (Fig.
2A). In addition, the digoxin transport profiles for 100nM
were almost identical to those examined for 10nM, indicating
that digoxin transport was not concentration-dependent over
this concentration range (data not shown). Ouabain did not
affect the amount of digoxin transported in either direction
(Fig. 2A). However, cellular digoxin binding was displaced
in the presence of excess ouabain, and ouabain significantly
decreased (p?0.05) the amount of digoxin remaining in the
monolayer (0.145?0.019% vs. 0.091?0.010% for A-to-B,
dX
dt
CL
V
X
CL
V
X
MBPARA
A
MA
PARA
B
MB
??
⋅⋅
dX
dt
CL
V
X
CL
V
X
MAPARA
A
MA
PARA
B
MB
???
⋅⋅
116Vol. 28, No. 1
Fig. 2.
Transcellular digoxin transport was examined at a concentration of 10nM (panel A). The effects of various compounds on digoxin transport were studied using 5mM quinidine
(panel B), 1mM and 5mM carvedilol (panels C and D), and 5mM and 50mM pimobendan (panels E and F). Digoxin transport in the basal-to-apical and apical-to-basal directions is
indicated with closed circles and triangles, respectively. The digoxin profiles determined in the absence of ouabain are shown with open symbols. The solid and dotted lines indi-
cate the simulation curves obtained from the pharmacokinetic analysis for basal-to-apical and apical-to-basal digoxin transport, respectively.
Transcellular Transport Profiles of Digoxin in Caco-2 Cell Monolayers with or without Various Compounds

Page 4

and 0.162?0.017% vs. 0.058?0.006% for B-to-A) (Table 1).
These results indicate that the transcellular transport and cel-
lular accumulation data for the presence of ouabain could be
used for pharmacokinetic analysis.
Pharmacokinetic Analysis of the Transcellular Trans-
port and Cellular Accumulation of Digoxin
termined the mean distribution volume in the monolayer to
be 2.50ml/cm2to represent the clearance parameters in units
of ml/min/cm2. Then, to characterize transcellular digoxin
transport, we analyzed the digoxin profiles obtained in the
presence of ouabain. The influx and efflux digoxin clearance
parameters determined from the analysis are shown in Table
2. For the control (10nM digoxin) study, the efflux clearance
on the apical side of the monolayer (CLCA) was 15.7times
greater than that on the basal side (CLCB); 1.023?0.045 vs.
0.065?0.001ml/min/cm2(Table 2), indicating that digoxin
in the monolayer was mainly excreted through the apical
membrane. The paracellular digoxin (mannitol) clearance
(CLPARA) was 0.016?0.002ml/min/cm2, which was smaller
than any other clearance parameter for digoxin transport
(Table 2), indicating that the paracellular transport of digoxin
is insignificant compared with its transcellular transport.
Effects of Quinidine, Carvedilol and Pimobendan on
the Transcellular Transport and Cellular Accumulation
of Digoxin
To evaluate whether the cardiovascular com-
pounds carvedilol and pimobendan affect intestinal digoxin
absorption, their inhibition effects on transcellular digoxin
transport were examined and compared to those of the posi-
tive inhibition control compound quinidine. Digoxin trans-
port was affected by 5mM quinidine (Fig. 2B). In contrast,
carvedilol did not affect digoxin transport at a concentration
of 1mM, although it altered the digoxin transport profiles
when applied at 5mM (Figs. 2C, D). Pimobendan at 5mM did
not affect digoxin transport (Fig. 2E), but changed the trans-
port profiles considerably when its concentration was in-
creased to 50mM (Fig. 2F). These compounds decreased the
We first de-
basal-to-apical transport of digoxin and increased its apical-
to-basal transport, indicating that the direction specificity of
digoxin transport has been reduced. Carvedilol and pimoben-
dan also increased the intracellular accumulation of digoxin
(Table 1).
Quinidine changed the clearance parameters considerably
(Table 2). CLCAand CLAC, which were large for the control
study were decreased, while the parameter with a small value
(CLCB) was increased (Table 2). Carvedilol and pimobendan
did not significantly affect the clearance parameters at low
concentrations, as opposed to high concentrations (Table 2).
These cardiovascular compounds decreased the digoxin ef-
flux clearance on the apical side of the monolayer (CLCA) to
one-third that of the control value (Table 2). Carvedilol and
pimobendan also decreased the digoxin influx clearance on
the basal side of the monolayer (CLBC), while quinidine did
not affect it (Table 2).
DISCUSSION
To analyze the characteristics of transcellular digoxin
transport in detail, we followed a pharmacokinetic approach
in which a three-compartment model was utilized to assess
the influx and efflux of digoxin on both sides of the mono-
layer (Fig. 1).18—20)We also examined 3H-digoxin transport at
the constant concentration of 10nM unlabeled digoxin so that
the influx and efflux parameters of 3H-digoxin would not
change in a time- and concentration-dependent manner dur-
ing the experiments.18,19)Na?,K?-ATPase is expressed on the
basal side of the Caco-2 cell monolayer,25,26)and digoxin po-
tently binds to Na?,K?-ATPase. Therefore, we also used ex-
periment conditions under which digoxin binding to Na?,K?-
ATPase was minimized. As a result, 100mM ouabain signifi-
cantly decreased cellular digoxin accumulation, although it
had little effect on the digoxin transport profile (Fig. 2A,
Table 1). We then analyzed the data for transcellular digoxin
January 2005117
Table 1.Digoxin Accumulation after 180min in Caco-2 Cell Monolayers in the Presence and Absence of Ouabain
Without ouabain (%)With ouabain (%)
A-to-BB-to-A A-to-BB-to-A
Digoxin 10nM
?5mM quinidine
?1mM carvedilol
?5mM carvedilol
?5mM pimobendan
?50mM pimobendan
0.145?0.019
0.423?0.044**
N.D.
0.466?0.048**
N.D.
0.490?0.041**
0.162?0.017
0.434?0.035**
N.D.
0.508?0.035**
N.D.
0.399?0.017**
0.091?0.010
0.057?0.005
0.087?0.012
0.173?0.008**
0.074?0.012
0.111?0.006
0.058?0.006
0.099?0.006**
0.061?0.003
0.091?0.004**
0.063?0.003
0.096?0.005**
The values are expressed as means?S.E. for 6 experiments. **p?0.01; significantly different from 10nM digoxin.
Table 2.Influx and Efflux Clearance of Digoxin for Transcellular Digoxin Transport in Caco-2 Cell Monolayers in the Presence of Ouabain
Clearance (ml/min/cm2)
CLCA
CLCB
CLAC
CLBC
Digoxin 10nM
?5mM quinidine
?1mM carvedilol
?5mM carvedilol
?5mM pimobendan
?50mM pimobendan
1.023?0.045
0.369?0.011*
0.885?0.023
0.339?0.006**
0.759?0.009
0.282?0.005**
0.065?0.001
0.327?0.012**
0.076?0.001
0.102?0.003
0.120?0.003*
0.178?0.002**
0.437?0.013
0.181?0.007**
0.380?0.013
0.340?0.006
0.310?0.012
0.223?0.003**
0.279?0.011
0.309?0.007
0.250?0.004
0.176?0.003**
0.246?0.005
0.195?0.005*
The values are expressed as means?S.E. for 6 experiments. *p?0.05, **p?0.01; significantly different from 10nM digoxin.

Page 5

transport in the presence of ouabain,21)and quantitatively
evaluated digoxin transport based on the influx and efflux
clearances.
By characterizing the transcellular digoxin transport and
also calculating the rate of digoxin clearance, we found that
digoxin, which enters the monolayer from the apical side, is
excreted in large part back into the apical chamber (Fig. 1,
Table 2). In contrast, digoxin which enters the monolayer
from the basal side passed through the monolayer to the api-
cal side (Fig. 1, Table 2). The direction specificity of tran-
scellular digoxin transport can be explained by the observa-
tion that digoxin efflux clearance on the apical side of the
monolayer (CLCA) was larger than that on the basal side
(CLCB). ATP-binding cassette (ABC) transporters such as
MDR1/P-glycoprotein and MRP2 are involved in digoxin ex-
sorption,16,17,22,27)and these transporters are expressed on the
apical membrane of Caco-2 cell monolayers.28,29)Thus, they
are probably responsible for digoxin efflux on the apical side
of the monolayer.
Five micromolar carvedilol and 50mM pimobendan de-
creased transcellular digoxin transport in the basal-to-apical
direction (Figs. 2D, F). These compounds decreased the
digoxin efflux clearance on the apical side of the monolayer
(CLCA) to the greatest degree among all of the clearance pa-
rameters (Table 2). Therefore, it is likely that carvedilol and
pimobendan inhibit the ABC transporters, and suppress
digoxin efflux on the apical membrane.30)They also in-
creased the amount of digoxin transported in the apical-to-
basal direction. Furthermore, carvedilol inhibited digoxin
transport more potently than pimobendan, and its effect was
comparable to that of quinidine with respect to the clearance
parameters at 5mM (Table 2). It is unlikely that the blood
concentration of these cardiovascular compounds can rise to
such high levels in clinical situations,31,32)and therefore the
renal excretion of digoxin may not be affected by these com-
pounds. However, their concentrations might reach such high
levels in the gastrointestinal tract when they are orally ad-
ministered.
As listed in Table 2, quinidine, carvedilol and pimobendan
increased efflux clearance on the basal side of the monolayer
(CLCB) at the concentration for which they affected transcel-
lular digoxin transport, but the reason for this is unclear.
These compounds may increase the unbound digoxin fraction
in the Caco-2 cell monolayer by displacing digoxin bound to
cellular components and/or transporters. The increased un-
bound fraction may then partly contribute to increased efflux
clearance of digoxin (CLCB). On the other hand, digoxin in-
flux clearance on the apical side (CLAC) was found to be
larger than that on the basal side (CLBC): 0.437?0.013 vs.
0.279?0.011ml/min/cm2(Table 2). In addition to this, pi-
mobendan and quinidine decreased CLACmore significantly
compared with CLBC, while carvedilol significantly de-
creased CLBC(Table 2). These results suggest that the mecha-
nisms responsible for the digoxin influx on the apical side of
the monolayer are probably different from those for the basal
side. Further investigation is required to clarify the molecular
aspects of digoxin influx in Caco-2 cell monolayers and/or
intestinal tissues.
In summary, we studied the transcellular transport and
drug interaction of digoxin in Caco-2 cell monolayers to
characterize intestinal digoxin absorption. We first examined
digoxin transport in the presence and absence of ouabain,
and found that the observations for transcellular digoxin
transport in Caco-2 cell monolayers in the presence of
ouabain is appropriate for pharmacokinetic analysis. Pharma-
cokinetic analysis showed that the efflux of digoxin on the
apical side of the monolayer is greater than that on the basal
side. Carvedilol and pimobendan increased the apical-to-
basal transport of digoxin, and these compounds suppressed
digoxin efflux on the apical side and digoxin influx on the
basal side of the monolayer. These findings indicate that ef-
flux on the apical side of the monolayer is the process pri-
marily responsible for transcellular digoxin transport, and
that carvedilol and pimobendan may affect the intestinal ab-
sorption of digoxin.
Acknowledgements
Dr. Tsuneo Imanaka for their valuable discussion on cell cul-
ture techniques. This work was supported in part by a grant
from the Japan Research Foundation for Clinical Pharmacol-
ogy.
We thank Dr. Masashi Morita and
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