Acta Pharmaceutica Sinica B 2012;2(2):168–173

Institute of Materia Medica, Chinese Academy of Medical Sciences

Chinese Pharmaceutical Association

Acta Pharmaceutica Sinica B

www.elsevier.com/locate/apsb
www.sciencedirect.com

ORIGINAL ARTICLE

Determination of the enantioselectivity of six chiral

aryloxy aminopropanol drugs transport across
Caco-2 cell monolayers
Ye Tiany, Ying Hey, Haihong Hu, Lu Wang, Su Zengn

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China

Received 9 January 2012; revised 5 February 2012; accepted 12 February 2012

KEY WORDS Abstract This study aimed to determine the transepithelial transport characteristics of chiral
drug enantiomers across Caco-2 cell monolayers, a model of human intestinal epithelial
Chiral aryloxy
membrane. Six chiral aryloxy enantiomers (atenolol, sotalol, celiprolol, carvedilol, metopro-
aminopropanol drugs;
Transport; lol and propafenone) were tested in bi-directional transport studies. The separation and
Enantioselectivity; quantitation of these enantiomers were performed by reversed-phase high-performance liquid
P-gp; chromatography (RP-HPLC) using 2, 3, 4, 6-tetra-O-acetyl-b- D-glucopyranosyl isothiocya-
Caco-2 cell nate (GITC) as a pre-column derivatizing agent. Bi-directional transport studies demon-
strated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized
transport at the concentration range tested. The efflux ratio (ER) for (R)-(þ)-celiprolol was
8.96, but it was much lower for (S)-(-)-celiprolol which is 3.42 at the concentration of
96.0 mM; carvedilol had the same transport behavior as celiprolol while the difference between
the ER values of two enantiomers was not as significant as celiprolol at the concentration of
5.0 mM. They are 2.41 for (R)-(þ)-carvedilol and 1.98 for (S)-(-)-carvedilol. But in the
transport studies of racemic atenolol, sotalol, metoprolol and propafenone, no enantioselec-
tive transport were observed over the concentration range tested. Because P-glycoprotein
(P-gp) is highly expressed in Caco-2 cells, we inferred that P-gp might participate in the
transport processes of celiprolol and carvedilol in chirally discriminative ways.

& 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical
Association. Production and hosting by Elsevier B.V. All rights reserved.

n
Corresponding author. Tel./fax: þ86 571 88208407.
E-mail address: zengsu@zju.edu.cn (Su Zeng).
y
These authors contributed equally to this work.

2211-3835 & 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and
hosting by Elsevier B.V. All rights reserved.

Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.
doi:10.1016/j.apsb.2012.02.005
Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs
1. Introduction
Chiral drugs, which took more than 50% of all current drugs,
have been paid more and more attention for their clinical
applications. A pair of enantiomers would be discriminated as
different molecules by chiral environment such as enzymes,
receptors, carries, etc. Biomembrane permeability and cellular
uptake of chiral drugs may be also stereoselective if intracel-
lular macromolecules recognize the enantiomers in chirally
discriminative ways. Therefore, the pharmacodynamics, phar-
macokinetics and toxicology between the enantiomers could
be stereoselective.
Aryloxy aminopropanol drugs, such as atenolol, sotalol,
celiprolol, carvedilol, metoprolol and propafenone, were widely
used to treat cardiovascular diseases and administrated mainly by
racemes. Studies proved that the pharmacokinetics of some
aryloxy aminopropanol compounds, for example, plasma protein
binding, liver metabolism, bile excretion, were stereoselective1–3,
and the enantiomers’ pharmacological activities are different from
each other4. For example, (S)-(þ)-sotalol hydrochloride and (R)-
()-isomer have similar anti-arrhythmia activity but the compre-
hensive effect of b-adrenergic blockers is actually attributed by
(R)-()-isomer5. Due to these stereo-different pharmacological
profiles, the uncovering of the transport stereoselectivity of aryloxy
aminopropanol drugs was important in clarifying the pharmaco-
kinetics of these drugs; furthermore, the uncovering of pharma-
cokinetic behaviors of these drugs will facilitate the understanding
of the pharmacological differences.
To evaluate the transportation mechanisms and absorption
of aryloxy aminopropanol drugs and to further explain the
pharmacological differences of these drugs, we investigated the
transepithelial transport of six chiral aryloxy aminopropanol
drugs (atenolol, sotalol, celiprolol, carvedilol, metoprolol and
Figure 1 The structures of six aryloxy aminopropanol drugs (atenolol, sotalol, celiprolol, carvedilol, metoprolol and propafenone),
the chiral carbon atom was labeled by ‘‘’’.
169
propafenone) in Caco-2 cell model, which has been widely
accepted as an in vitro model for intestinal drug absorption
(Fig. 1). The chiral RP-HPLC method using 2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl isothiocyanate (GITC) as a pre-
column derivatizing agent was used to separate and assay these
enantiomers, which was previously described by our group6.
In this study, we will concentrate on the transport character-
istics of these chiral drug enantiomers across Caco-2 cells.
2. Materials and methods
2.1. Materials
Racemic atenolol and racemic metoprolol tartrate were pur-
chased from National Institutes for Food and Drug Control
(Beijing, China). Racemic sotalol hydrochloride was provided
by Dong Dong Chemical Pharmaceutical Company (Taizhou,
Zhejiang, China). Racemic celiprolol hydrochloride was pro-
vided by Hai Zheng Pharmaceutical Company (Zhejiang,
China). Racemic propafenone hydrochloride was purchased
from Sigma. Racemic carvedilol was provided by Tian Heng
Pharmaceutical Company (Ningbo, Zhejiang, China). Lucifer
yellow was purchased from Biochemika. Methanol and acet-
onitrile were of HPLC grade. Other solvents used were of
analytical grade.
2.2. Cell culture
Caco-2 cells (35 passage) obtained from Chinese Academy of
Medical Sciences (CAMS, Beijing, China) were grown in a
humidified atmosphere of 5% CO2 at 37 1C. Cells were
cultured in high-glucose Dulbecco’s modified eagle’s medium
170 Ye Tian et al.

(DMEM, Gibco, Bethesda, MD) supplemented with 10% fetal
bovine serum (Gibco), 1% nonessential amino acids (Gibco).
After reaching 90% confluence, Caco-2 cells were harvested
with 0.25% trypsin-0.02% EDTA solution and seeded in Trans-
wells inserts (catalog number 3460, Corning Coster Corp., Acton,
MA) in 12-well plates at a density of 1.0  105 cells/cm2. After
culturing for 21 days, Caco-2 cells were used in experiment7. The
integrity of the monolayer was checked by measuring the transe-
pithelial electrical resistance (TEER) value across the monolayer
using Millicell-ERS Voltohmmeter (Millipore) and monitoring the
apparent permeability coefficient (Papp) of the paracellular leakage
marker Lucifer yellow across the monolayer8,9. Only monolayers
with initial TEER values higher than 450 O/cm2 and the Papp value
lower than 0.2  106 cm/s were used.
2.3. Transport experiments
The stock solutions of racemic drugs were made in Hank’s
Balanced Salt Solution (HBSS), containing 25 mM HEPES,
pH 7.4, and were diluted to a series of concentration (Table 1).
The solutions were filtered through 0.22 mm filter membrane
for sterilizing. To ensure the transport experiments were
conducted in the sink conditions, the incubation time in
Caco-2 cell monolayers of each drug was determined by
preliminary experiments. The transport studies were com-
pleted before the receiver concentration exceeded 10% of the
donor concentration to ensure that the transport process was
fit to the linear dynamics10. In the transwell cultivation system,
there is an unstirred water layer (UWL) between enteric cavity
and cell monolayers. The previous study showed that UWL is
an important barrier of drug absorption, and it can restrict
some liposoluble drug’s diffusion11. To avoid the influence of
UWL on drug transport, all studies were conducted with
shaking (50 r/min) by a humidified incubator at 37 1C. Prior to
all experiments, each monolayer was washed twice with HBSS
and pre-incubation for 30 min. The transport study was
started by replacing the donor buffer with transport buffer
containing individual concentration of these drugs. The
volume added to the apical (AP) side of the monolayer was
0.5 mL and 1.5 mL at the basolateral (BL) side. Transport
studies were conducted at both the absorptive direction (AP-
BL) and the secretory direction (BL-AP)12. After incubating
at 37 1C for indicated time, 200 mL samples were collected
from the receiving sides of the cell monolayers for HPLC
analysis. Drug concentrations loaded onto the Caco-2 cell
monolayers were shown in Table 1, and the incubation time
were shown in Table 2.
2.4. Enantioselective HPLC analysis
The (S)- and (R)-enantiomers of six drugs in the samples were
separated and quantified by enantioselective RP-HPLC with
GITC as a pre-column derivatizing agent6.
2.5. Calculations and statistics
The transport rate of six enantiomers (V) was calculated using
the following equation:
V ¼ dQ=ðdt  AÞ ð1Þ
The apparent permeability coefficient (Papp) which repre-
sents the permeability of these enantiomers, was estimated by
calculating as follows:
Papp ¼ dQ=ðdt  A  C0 Þ ð2Þ
where dQ/dt is the rate of appearance of drugs on the receiver
side, A is the surface area of the monolayer and C0 is the initial
concentration in the donor compartment.
Data was expressed as the mean7SD (n ¼5). Differences
in Papp of enantiomers were evaluated using paired t-test. A
P valueo0.05 was considered to be statistically significant.

Table 1 Drug concentrations loaded onto the donor side 3. Results

in transport study.
We have investigated the transport across Caco-2 cell mono-
Drug Racemic concentration (mM) layers of six chiral aryloxy aminopropanol compounds. The
experimental results indicated that the transports of racemic
Atenolol 75.0 187.5 375.0 750.0 1500
atenolol, sotalol, metoprolol and propafenone were not enan-
Sotalol 130.0 325.0 650.0 1300 3250
tioselective in the concentration ranges tested. The transport
Celiprolol 96.0 240.0 480.0 960.0 2400
Carvedilol 5.0 12.5 25.0 50.0 100.0 rates of (S)-enantiomers were similar to (R)-enantiomers both
Metoprolol 6.0 15.0 30.0 75.0 150.0 in the absorptive direction and the secretory direction
Propafenone 2.0 5.0 10.0 25.0 50.0 (P40.05). During the transport process of enantiomers, neither
concentration-dependence nor directionality (Papp (AP-BL)

Table 2 Permeability of chiral drug enantiomers across Caco-2 cell monolayers (mean7 SD, n ¼5).

Drug Incubation Racemic Papp (AP-BL) (  106 cm/s) Papp (BL-AP) (  106 cm/s) ER
time (h) conc. (mM)
R S R S R S

Atenolol 4.0 75.0 0.16270.139 0.15070.101 0.17170.078 0.16670.084 1.06 1.11

Sotalol 4.0 130.0 0.35870.006 0.38570.049 0.42270.076 0.40970.058 1.18 1.06
Celiprolol 3.0 96.0 0.78570.309 2.21870.349 7.03470.48 7.57970.68 8.96 3.42
Carvedilol 2.0 5.0 9.93572.631 11.2771.849 23.9571.48 22.3770.98 2.41 1.98
Metoprolol 3.0 6.0 4.31970.556 4.48770.673 3.69370.392 3.92770.327 0.855 0.875
Propafenone 1.0 2.0 12.9071.171 13.0671.106 13.2370.898 12.2070.859 1.03 0.934
Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs 171

Figure 2 Comparison of Papp values of celiprolol enantiomers in AP-BL (A) and BL-AP (B) transport experiments, incubation time
was 3 h. Values were shown as mean 7SD (n ¼5).
 represents very significant difference between S- and R-celiprolol by paired t-test, Po0.01;  represents significantly difference between
S- and R-celiprolol by paired t-test, Po0.05.

Figure 3 Comparision of Papp values of carvedilol enantiomers in AP-BL (A) and BL-AP (B) transport experiments, the incubation time
was 2 h. Values were shown as mean 7SD (n¼ 5).  represents significant difference between S- and R-carvedilol by paired t-test, Po0.05.

and Papp (BL-AP) did not have significant differences) were
observed in the concentration ranges studied, suggesting that the
transmembrane transports of these drugs were mainly by passive
diffusion pathway (Table 2). A series of concentrations of atenolol,
sotalol, metoprolol and propafenone have been tested, but the Papp
values in both two directions and efflux ratios (ER) in higher
concentrations were similar to the lowest concentrations used in
these experiments, so only these parameters determined at the
lowest concentration were shown in Table 2.
These studies also demonstrated that transports of celi-
prolol and carvedilol were significantly stereoselective. In the
absorptive direction, there was significant difference between
the Papp values of (S)-()- and (R)-(þ)-celiprolol (Po0.01).
The values for (R)-(þ)-isomer were almost 3-fold smaller
than (S)-()-isomer which indicated (S)-()-isomer was
easier to penetrate through biomembrane. The transports
of celiprolol enantiomers were concentration-dependent, with
the increasing of the drug’s concentration, the transport rates
raised, though it dropped a little at the highest concentration
of (S)-()-isomer. But the distinction between (S)-()- and
(R)-(þ)- celiprolol was decreased while drugs’ concentration
increased. In secretory direction, the Papp values of (S)-()-
celiprolol were slightly smaller than (R)-(þ)-celiprolol at low
concentrations (96.0–480.0 mM) (Po0.05), but as the concen-
tration increased to 960.0, 2400.0 mM the differences between
celiprolol enantiomers diminished (P40.05). Compared the
bidirectional (AP-BL and BL-AP) transport, we conclude
that celiprolol enantiomers have conspicuous excretion trans-
port, and the ER value were between 9.0–2.2 in the con-
centration range studied (Fig. 2).
As shown in Fig. 3, the transport of carvedilol was
enantioselective and excretive, but no concentration-depen-
dence was observed. In the absorptive direction, the Papp
values of (S)-()-carvedilol were greater than (R)-(þ)-carve-
dilol, while in the secretory direction, Papp values of (S)-()-
carvedilol were smaller than (R)-(þ)-carvedilol (Po0.05). So
the ER values of (S)-()-carvedilol were conspicuously smaller
than (R)-(þ)-carvedilol. It is likely that an excretive transpor-
ter participated in the transport process across Caco-2 cell
monolayers, and the transporter favors (R)-(þ)-enantiomer of
the molecule. The ER values were between 2.4–1.9 in the
concentrations studied (Fig. 3).
4. Discussion
In Caco-2 cell monolayers, the transports of atenolol, sotalol,
metoprolol and propafenone were not stereoselective, suggest-
ing that the differences of pharmacological effects of sotalol
isomers were not caused by the differences of cell permeability
between two isomers. But the enantiomers of celiprolol and
carvedilol displayed different transport behaviors.
172
Our data are in accordance with previous studies roughly.
Bachmakov et al.13 have observed that the BL-AP transport
of carvedilol was greater than the AP- BL transport, and the
polarized transport of celiprolol has been reported by Kuo
et al.14. Karlsson et al.15 reported that the basal-to-apical
transport (secretion) of [14C]-celiprolol (50 mM) was 5 times
higher than apical-to-basal transport (absorption). And as
reported, atenolol with low permeability was used as a
reference compound for low intestinal absorption and was
mainly transported paracellularly16–19. These results are in
agreement with our observation because small Papp values for
atenolol were obtained in our experiments (Table 2). Yang
et al.20 have determined the permeability of seven b-blockers
using Caco-2 cell line, and the Papp values of metoprolol,
atenolol and sotalol were close to our results calculated as
racemate. They also believed that the transmembrane trans-
port of the three drugs were mainly by passive diffusion
pathway. But some investigations indicated atenolol and
sotalol had slight polarized transport13,21. These minor incon-
sistencies may be caused by the varied cultural conditions of
Caco-2 cells and different experimental circumstances from
lab to lab. The study of Bachmakov et al.22 revealed that
although propafenone was not the substrate of P-gp, the
inhibitive activity in P-gp-mediated digoxin transport of
(S)-(þ)- and (R)-()-propafenone were different. (R)-()-pro-
pafenone reduced the digoxin transport more significantly.
Comparing with our result, (R)-propafenone has a little
higher ER value than (S)-(þ)-isomer, indicating (R)-()-pro-
pafenone may have higher affinity to P-gp which makes
(R)-()-isomer interact with digoxin more effectively. However,
they all did not investigate whether the transport characteristics
of the enantiomers were different. Because the enantiomers may
differ in terms of pharmacological properties and disposition,
stereoselective disposition of the enantiomers can arise from
absorption of the enantiomers via intestine; therefore, uncover-
ing the transport characteristics of enantiomers, which have
been evaluated in this study, has significant meaning in predict-
ing the pharmacological effect and pharmacokinetic behaviors
of chiral drugs’ enantiomers.
The transport of celiprolol and carvedilol were stereoselec-
tive and excretive, which were significantly different from the
other four chiral drugs, and the similar transport behavior has
been observed in esmolol23 and propranolol24 enantiomers in
our previous studies. It has been proved that both celiprolol
and carvedilol are the substrates of P-gp.25,26 There are several
articles investigated the interaction between celiprolol and
P-gp. Karlsson et al.15 reported that the secretion of celiprolol
could be inhibited by typical substrates of P-gp (such as
vinblastine, verapamil and nifedipine), furthermore, celiprolol
inhibited the basal-to-apical transport of vinblastine. These
results indicated that celiprolol was transported by P-gp. In
our observation, the Papp values for (S)-()-celiprolol were
almost 3-fold larger than (R)-(þ)-isomer in the absorptive
direction, and in secretory direction, they were slightly smaller
than (R)-(þ)-celiprolol, which indicated that (S)-()-isomer
was easier to penetrate through biomembrane. So, the recog-
nition of P-gp for (R)-(þ)-celiprolol was much stronger than
(S)-()-isomer, and (R)-(þ)-isomer may be a more potent
substrate for P-gp. The transports of celiprolol enantiomers
were concentration-dependent in our experiment. With the
drug’s concentration increased, the transport rates raised. For
(R)-(þ)-isomer, it raised all the way within the concentration
Ye Tian et al.
range tested, while it dropped a little at the highest concentra-
tion for (S)-()-isomer (Fig. 2). The reason for this phenom-
enon may because (S)-()-isomer penetrated more than
(R)-(þ)-isomer, so it reached the steady-stage of penetration
more rapidly. The penetration at 960 mM has reached the
maximum and the decreasing was caused by the experimental
error. On the other hand, another reason could be undefined
transport mechanisms existed to make (S)-()-isomer easier to
penetrate through biomembrane, and at high concentration
these transport systems were inhibited which contributed to
the less permeability of (S)-()-isomer.
The transport behaviors of carvedilol enantiomers were
different but no concentration-dependence was observed. In
the concentration range studied, the Papp values of (S)-()-
isomer were higher than (R)-(þ)-isomer in absorptive direc-
tion, while in secretory direction, it was contrary (Po0.05).
It is likely that an excretive transporter participated in the
transport process across Caco-2 cell monolayers, and the
transporter favors (R)-(þ)-enantiomer (Fig. 3). Several articles
have reported carvedilol-digoxin interaction in children and
adults13,27–29. Carvedilol could increase serum concentrations
and the area under the plasma concentration time-curve of
orally administered digoxin. As digoxin is a typical substrate
of P-gp, so carvedilol is likely to have the ability to inhibit
P-gp. This result has been proved on MDR1-transfected
Madin-Darby canine kidney (MDR1-MDCK) cells; these
MDR1-mediated reversing effects of carvedilol on vinblastine,
paclitaxel, doxorubicin and daunorubicin were similar to those
of verapamil29. Taken together, P-gp is likely to be one
determinant of carvedilol disposition in humans.
Combined the results above and our pre-studies of esmolol
and propranolol, we presume that the chiral recognition of
P-gp may lead to the stereoselective transport. But more
investigations are still needed to confirm that if P-gp indeed
play a role in the enantioselective recognition. Because the
interactions are restricted by the shape of the binding pocket,
the conformation differences between enantiomers may lead to
the chiral recognition and stereoselective transport of P-gp.
Further studies in molecular model are needed to help us learn
more about structure activity relationship between P-gp and
these drugs.
Acknowledgment
This project was supported by National Major Projects of
China (2011CB710800, 2012ZX09506001-004) and NSFC
(30873122).
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