Transport Characteristics of Diphenhydramine in Human Intestinal Epithelial Caco-2 Cells: Contribution of pH-Dependent Transport System1
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0022-3565/99/2901-0388$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 1
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 290:388 –392, 1999
Transport Characteristics of Diphenhydramine in Human
Intestinal Epithelial Caco-2 Cells: Contribution of
pH-Dependent Transport System1
HIROSHI MIZUUCHI, TOSHIYA KATSURA, HIDEYUKI SAITO, YUKIYA HASHIMOTO, and KEN-ICHI INUI
Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan
Accepted for publication March 17, 1999 This paper is available online at http://www.jpet.org
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 29, 2015
ABSTRACT
Transport characteristics of diphenhydramine, an antihista- niramine, with a Ki value of 1.3 mM. On the other hand, cime-
mine, were studied in cultured human intestinal Caco-2 cell tidine and tetraethylammonium, typical substrates for the renal
monolayers to elucidate the mechanisms of its intestinal ab- organic cation transport system, had no effect. Moreover, bio-
sorption. Diphenhydramine accumulation in the monolayers in- logical amines and neurotransmitters, such as histamine, do-
creased rapidly and was influenced by extracellular pH (pH pamine, serotonin, and choline, also had no effect on the di-
7.4 . 6.5 . 5.5). Diphenhydramine uptake was temperature phenhydramine accumulation. Finally, diphenhydramine uptake
dependent, saturable, and not potential sensitive. Kinetic anal- was stimulated by preloading monolayers with chlorphenira-
ysis revealed that the apparent Km values were constant (0.8 – mine (trans-stimulation effect). These findings indicate that di-
1.0 mM) in all pH conditions tested, whereas Vmax values de- phenhydramine transport in Caco-2 cells is mediated by a
creased at the lower pH. The initial uptake of diphenhydramine specific transport system. This pH-dependent transport system
was competitively inhibited by another antihistamine, chlorphe- may contribute to the intestinal absorption of diphenhydramine.
Diphenhydramine, a tertiary amine compound with one 1991). This cell line forms confluent monolayers of well dif-
site of ionization with a pKa value of pH 9.0 (de Roos et al., ferentiated enterocyte-like cells with functional properties of
1970), is widely used as an antihistamine for the symptom- transporting epithelia (Hidalgo et al., 1989) and has been
atic relief of hypersensitivity reaction. Diphenhydramine is used to study the transport of nutrients and drugs. Using this
relatively well absorbed, and its plasma concentration is cell line, we have demonstrated the absorption mechanisms
rapidly elevated after oral administration (Paton and Web- of some oral cephalosporins and a dipeptide-like anticancer
ster, 1985), although it is mostly ionized over the pH range in agent, bestatin (Inui et al., 1992; Saito and Inui, 1993;
the gastrointestinal tract. Matsumoto et al., 1994, 1995). In this report, we examined
The intestinal absorption mechanisms of lipophilic organic the uptake characteristics of diphenhydramine in Caco-2
cations, such as diphenhydramine, have been explained by cells to elucidate the intestinal absorption mechanisms of
the passive diffusion of nonionized compounds according to this lipophilic organic cation.
the pH-partition theory. However, recent studies have sug-
gested that carrier-mediated transport systems contribute to
the intestinal absorption of various organic cations (Tan et Experimental Procedures
al., 1989; Kuo et al., 1994). With intestinal brush-border Materials. Diphenhydramine hydrochloride was purchased from
membrane vesicles, it was reported that the uptake of or- Tokyo Kasei Kogyo Co. (Tokyo, Japan). (6)-Chlorpheniramine mal-
ganic cations consisted of two steps: binding to the membrane eate, cimetidine, histamine, L-histidine monohydrochloride monohy-
and entrance into intravesicular space stimulated by the drate, hydroxyzine dihydrochloride, imipramine hydrochloride, ke-
inside-negative electrical potential (Saitoh et al., 1988a, totifen fumarate, and tetraethylammonium bromide were obtained
1989; Sugawara et al., 1995). However, these mechanisms from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals were of
have not been well investigated. the highest purity available.
Cell Culture. Caco-2 cells at passage 18 obtained from the Amer-
Recently, the human colon carcinoma cell line Caco-2 was
ican Type Culture Collection (ATCC HTB37; Rockville, MD) were
used to study transport mechanisms of drugs (Artursson, maintained by serial passage in plastic culture dishes (Falcon; Bec-
1990; Hu and Borchardt, 1990; Artursson and Karlsson, ton Dickinson & Co., Lincoln Park, NJ) as described previously (Inui
et al., 1992; Matsumoto et al., 1994). For uptake studies, 60-mm
plastic dishes were inoculated with 5 3 105 cells in 5 ml of the
Received for publication August 21, 1998.
1
This work was supported in part by a Grant-in-Aid for Scientific Research complete culture medium. The medium consisted of Dulbecco’s mod-
from the Ministry of Education, Science, Sports, and Culture of Japan. ified Eagle’s medium (Gibco, Grand Island, NY) supplemented with
3881999 Transport of Diphenhydramine in Caco-2 Cells 389
10% fetal calf serum (Microbiological Associates, Bethesda, MD) and
1% nonessential amino acids (Gibco) without antibiotics. The cells
were grown in an atmosphere of 5% CO2/95% air at 37°C and given
fresh medium every 3 or 4 days. The cell monolayers were used at the
12 to 14 days in culture for the uptake experiments. In this study,
cells between the 33rd and 47th passages were used.
Measurement of Antihistamine Uptake. The uptake of anti-
histamines was measured in Caco-2 monolayer cultures grown in
60-mm plastic culture dishes. The composition of the incubation
medium was as follows: 145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5
mM MgCl2, 5 mM D-glucose, and 5 mM 2-(N-morpholino)ethanesul-
fonic acid (pH 5.5) or HEPES (pH 6.5, 7.4). After removal of the
culture medium, each dish was washed once with 5 ml of incubation
medium (pH 7.4) and further incubated with 2 ml of the same
medium for 10 min at 37°C. The cells were then incubated with 2 ml
of incubation medium containing a test drug for specific periods at
37°C. Thereafter, the medium was aspirated off, and the dishes were
rapidly rinsed twice with 5 ml of ice-cold incubation medium (pH
7.4). The cells were scraped off with a rubber policeman into 1 ml of
Fig. 1. Time course of diphenhydramine accumulation by Caco-2 cells.
extraction solution (0.01 N HCl/methanol, 1:1) and were maintained
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Caco-2 cells were incubated for specified periods at 37°C with incubation
for 1 h at room temperature. The extraction solution was centrifuged medium (pH 7.4, E; pH 6.5, F; pH 5.5, ‚) containing 1 mM diphenhydra-
at 13,000 rpm (model 3533; Abbott Laboratories, Abbott Park, IL) for mine. At the end of incubation, the accumulation of diphenhydramine
15 min. The supernatant was filtered through a Millipore filter was measured. Each point represents the mean 6 S.E. of four monolayers
(SJGVL, 0.22 mm), and the drug was analyzed by HPLC as described from two separate experiments.
below.
Analytical Methods. Antihistamines were assayed with a high-
performance liquid chromatograph LC-10A (Shimadzu Co., Kyoto,
Japan) equipped with a UV spectrophotometric detector SPD-6A
(Shimadzu) and an integrator (Chromatopac C-R1A; Shimadzu) un-
der the following conditions: column, TSK-gel ODS 80TM 4.6 mm i.d.
3 150 mm (Tohso Co., Tokyo, Japan) for diphenhydramine, L-column
ODS 4.6 mm i.d. 3 150 mm (Chemicals Inspection and Testing
Institute, Tokyo, Japan) for chlorpheniramine; mobile phase, 20 mM
KH2PO4 buffer (pH 5.4)/methanol/2-propanol 6:3:1 for diphenhydra-
mine, 20 mM KH2PO4 buffer (pH 5.4)/methanol 6:4 for chlorphenira-
mine; flow rate, 0.8 ml/min; wavelength, 225 nm; injection volume,
50 ml; temperature, 40°C. The detection limits were approximately
10 pmol for both compounds. The protein content of the cell mono-
layers solubilized in 1.0 ml of 1 N NaOH was determined by the
method of Bradford (1976) with a Bio-Rad protein assay kit (Bio-Rad
Laboratories, Richmond, CA) with bovine g-globulin as a standard. Fig. 2. Concentration dependence of diphenhydramine uptake by Caco-2
Statistical Analysis. Data were analyzed statistically by non- cells. A, Caco-2 cells were incubated for 1 min at 37°C with incubation
medium (pH 7.4, E; pH 6.5, F; pH 5.5, ‚) containing various concentra-
paired t test or one-way ANOVA followed by Scheffé’s test when tions of diphenhydramine. The uptake of diphenhydramine was then
multiple comparisons were needed. Probability values less than 5% measured. B, Eadie-Hofstee plots of diphenhydramine uptake after cor-
were considered significant. rection for the nonsaturable component. V, uptake rate (nmol z mg21
protein z min21); S, diphenhydramine concentration (mM). Each point
represents the mean of two monolayers from a typical experiment.
Results
0.1 to 10 mM. Table 1 summarizes the kinetic parameters
Uptake of Diphenhydramine by Caco-2 Monolayers. evaluated by nonlinear least-squares regression analysis
To characterize the diphenhydramine uptake by Caco-2 cells, (Yamaoka et al., 1981) from the following Michaelis-Menten
diphenhydramine accumulation was investigated at three equation:
extracellular pH values (7.4, 6.5, 5.5). As shown in Fig. 1, the
accumulation of diphenhydramine increased rapidly and V max@ S#
reached almost steady state at 15 min after starting incuba- V5 1 K d@ S #
K m 1 @ S#
tion under all pH conditions. The accumulation was influ-
enced by the extracellular pH, the order of uptake being pH where V is the initial uptake rate, [S] is the initial concen-
7.4 . 6.5 . 5.5. Moreover, when the monolayers were incu- tration, Vmax is the maximum uptake rate, Km is the Michae-
bated with 1 mM diphenhydramine (pH 7.4) for 1 min at 4°C, lis constant, and Kd is the coefficient of simple diffusion
the amount accumulated decreased to 41% of the amount at (nonsaturable process). Whereas apparent Km and Kd values
37°C (37°C, 15.5 6 0.5; 4°C, 6.4 6 0.2 nmol z mg21 were almost unchanged for all pH conditions, the Vmax values
protein z min21, mean 6 S.E. of three monolayers). decreased markedly at lower pH levels.
Kinetic Analysis of Diphenhydramine Uptake. The Effect of Various Organic Cations on Diphenhydra-
concentration-dependence of diphenhydramine accumulation mine Uptake. The effect of various organic cations on di-
was examined, and the kinetic parameters were calculated. phenhydramine accumulation was investigated. As shown in
Figure 2 shows the accumulation of diphenhydramine at 1 Fig. 3, chlorpheniramine and imipramine inhibited the di-
min as a function of the substrate concentration ranging from phenhydramine uptake, but other antihistaminic agents (hy-390 Mizuuchi et al. Vol. 290
TABLE 1
Kinetic parameters for diphenhydramine uptake by Caco-2 cells under various pH conditions
Caco-2 cells were incubated at 37°C for 1 min with incubation medium containing various concentrations of diphenhydramine. Diphenhydramine uptake was then assayed,
and kinetic parameters were calculated. Each value represents the mean of two independent experiments. Individual data are shown in the parentheses.
pH Km Vmax Kd
mM nmol z mg21 protein z min21 nmol z mg21 protein z min21 z mM21
7.4 0.9 (0.9, 0.9) 21.9 (21.6, 22.2) 2.8 (3.1, 2.6)
6.5 0.8 (0.8, 0.7) 9.7 (9.6, 9.7) 2.0 (2.1, 1.8)
5.5 1.0 (1.0, 1.0) 2.9 (2.8, 3.0) 1.3 (1.5, 1.2)
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Fig. 4. Effect of chlorpheniramine on diphenhydramine uptake by Caco-2
Fig. 3. Effect of various organic cations on diphenhydramine accumula- cells. Caco-2 cells were incubated for 1 min at 37°C with incubation
tion in Caco-2 cells. Caco-2 cells were incubated for 5 min at 37°C with medium (pH 7.4) containing 1 mM diphenhydramine in the absence or
incubation medium (pH 7.4) containing diphenhydramine (1 mM) in the presence of various concentrations of chlorpheniramine. Thereafter, the
absence (control) or presence of various organic cations. Thereafter, the accumulation of diphenhydramine was measured. Each point represents
accumulation of diphenhydramine was measured. The concentrations of the mean 6 S.E. of three monolayers.
ketotifen and hydroxyzine were 2.5 and 1 mM, respectively. Those of
chlorpheniramine and imipramine were 5 mM. Each column represents
the mean 6 S.E. of three monolayers. *P , .01, significant difference from
the control value.
droxyzine and ketotifen) had almost no effect. Then the effect
of histamine and its precursor histidine were examined. In
addition, we studied whether cimetidine and tetraethylam-
monium, typical substrates for the organic cation transport-
ers in the kidney, affected the diphenhydramine accumula-
tion. These compounds had no effect on diphenhydramine
accumulation (control, 20.3 6 0.9; with histidine, 20.3 6 0.2;
with histamine, 20.6 6 0.4; with tetraethylammonium,
19.8 6 0.7; with cimetidine, 20.0 6 0.4 nmol z mg21 pro-
tein z 5 min21; each value represents the mean 6 S.E. of
three monolayers). Moreover, some neurotransmitters and/or
biological amines such as choline, dopamine, and serotonin
also had no effect on diphenhydramine accumulation (data
not shown). Fig. 5. Inhibition of diphenhydramine uptake by varying concentrations
Because chlorpheniramine is an antihistamine and its of chlorpheniramine. Caco-2 cells were incubated for 1 min at 37°C with
chemical structure is similar to diphenhydramine, the effect incubation medium (pH 7.4) containing 0.5 (E), 1 (F), and 5 (‚) mM
diphenhydramine in the absence or presence of chlorpheniramine, shown
of chlorpheniramine on diphenhydramine accumulation was in the abscissa. Thereafter, the accumulation of diphenhydramine was
further investigated. As shown in Fig. 4, chlorpheniramine measured. The data are presented as a Dixon plot. Each point represents
inhibited diphenhydramine accumulation in a concentration- the mean of three determinations.
dependent manner. Dixon plot analysis demonstrated that
chlorpheniramine competitively inhibited diphenhydramine Effect of Membrane Potential on Diphenhydramine
uptake with an apparent Ki value of 1.3 mM (Fig. 5). We also Uptake. To examine whether diphenhydramine uptake by
studied the initial chlorpheniramine uptake by Caco-2 cells Caco-2 cells is potential dependent, the effect of ion compo-
and calculated the kinetic parameters. The Km value of chlor- sition of the incubation medium on diphenhydramine uptake
pheniramine (0.9 mM) at pH 7.4 was similar to its Ki value was examined. To decrease a transmembrane electrical po-
against the diphenhydramine uptake (data not shown). tential, extracellular Na1 was replaced with K1 (K1 me-1999 Transport of Diphenhydramine in Caco-2 Cells 391
dium: 3 mM NaCl, 145 mM KCl). Under this condition, ported by the organic cation-H1 antiport system in renal
diphenhydramine accumulation at 1 min did not change (con- brush-border membrane (Takano et al., 1984; McKinney and
trol, 12.8 6 0.3; K1 medium, 13.8 6 0.4, nmol z mg21 Kunnemann, 1987; Wright and Wunz, 1987; Katsura et al.,
protein z min21, mean 6 S.E. of three monolayers), indicating 1991), had no effect on diphenhydramine accumulation. It
the potential-insensitive uptake of diphenhydramine by seems likely that diphenhydramine and chlorpheniramine
Caco-2 cells. have a common chemical structure recognized by some spe-
trans-Stimulation Effect on Diphenhydramine Up- cific transport system that has not been reported in either
take. To elucidate whether the diphenhydramine uptake is Caco-2 cells or the renal brush-border membrane.
mediated by a specific transport system, the trans-stimula- Lipophilic organic cations, such as diphenhydramine, imip-
tion effect on the initial uptake of diphenhydramine was ramine, and chlorpromazine, were reported to have a specific
examined. As shown in Table 2, the initial uptake was stim- binding site on biological membrane (Saitoh et al., 1988b).
ulated by preloading the monolayers with chlorpheniramine, Therefore, the binding of diphenhydramine to the cellular
which had a cis-inhibitory effect on diphenhydramine up- membrane surface might partly account for its accumulation
take. In addition, chlorpheniramine uptake was trans-stim- in Caco-2 cells. It was also reported that diphenhydramine
ulated by diphenhydramine preloading. inhibited the small-intestinal sodium-dependent uptake of
a-D-glucoside in rats (Elsenhans et al., 1985). The uptake
inhibition occurred by the binding of diphenhydramine to the
Discussion sodium binding site in intestinal mucosa. Furthermore, it
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 29, 2015
To characterize the intestinal absorption mechanism of was reported that organic cations entered into the intrave-
diphenhydramine, we investigated the cellular uptake of sicular space, stimulated by the inside-negative transmem-
diphenhydramine by Caco-2 cells. Intestinal absorption brane electrical potential, after their binding to the mem-
of ionized drugs has been explained by passive diffusion of brane surface (Saitoh et al., 1988a, 1989; Sugawara et al.,
nonionized compounds. In this study, the amount of diphen- 1995). However, we found that diphenhydramine accumula-
hydramine accumulated in Caco-2 cells was decreased at tion was insensitive to the membrane potential. In addition,
lower pH. This pH dependence might be partly explained by diphenhydramine accumulation was enhanced under various
passive diffusion according to the pH-partition theory. How- ATP-depleted conditions (H. Mizuuchi, T. Katsura, Y. Hashi-
ever, diphenhydramine (pKa 9.0) was mostly ionized even at moto and K. Inui, unpublished observations). It is likely that
the highest pH level tested (de Roos et al., 1970). Moreover, the amount of diphenhydramine accumulated in the cells or
the accumulation of diphenhydramine was temperature de- associated with the membrane is not increased because the
pendent, saturable, and competitively inhibited by another transmembrane potential difference was weakened under
organic cation, chlorpheniramine. Therefore, the cellular up- such ATP-depleted conditions. Thus, our results exclude the
take characteristics of diphenhydramine were not explained possibility that the cellular accumulation is indicative of the
only by the pH-partition theory. potential-sensitive binding and/or uptake of diphenhydra-
We investigated the effect of histamine and other antihis- mine.
tamines on diphenhydramine uptake. The results showed The diphenhydramine accumulation was stimulated by
that histamine and all other antihistamines, except chlor- preloading Caco-2 monolayers with chlorpheniramine (trans-
pheniramine, had no effect on diphenhydramine accumula- stimulation effect). This finding indicates the existence of a
tion. Thus, it is unlikely that diphenhydramine accumulation specific transport system for diphenhydramine and chlorphe-
implies binding to the histamine receptor, which was re- niramine. Carrier-mediated transport of diphenhydramine
ported to exist in the intestine (Morini et al., 1993). Dixon was also reported in the central nervous system (Goldberg et
plot analysis demonstrated that chlorpheniramine competi- al., 1987). However, the driving force for diphenhydramine
tively inhibited the uptake of diphenhydramine with a Ki transport was not determined. Because the amount of di-
value of 1.3 mM, which was comparable with the Km value for phenhydramine accumulated in Caco-2 cells was decreased
chlorpheniramine (0.9 mM). These findings suggest that di- at lower extracellular pH, it is speculated that a pH-depen-
phenhydramine and chlorpheniramine are accumulated in dent transport system contributes to the uptake of diphen-
Caco-2 cells via a common transport system. On the other hydramine in Caco-2 cells. Further examination is necessary
hand, cimetidine and tetraethylammonium, which are trans- to clarify the driving force and/or substrate specificity of this
transport system.
TABLE 2 In conclusion, diphenhydramine is rapidly accumulated in
trans-Stimulation effect on antihistamine uptake by Caco-2 cells Caco-2 cells with substrate saturability and pH dependence.
Caco-2 cells were preloaded for 30 min at 37°C with 1 mM chlorpheniramine or The cellular accumulation was influenced by cis- and trans-
diphenhydramine (pH 7.4). After removing this medium, cell monolayers were
washed once with ice-cold incubation medium and then incubated for 1 min at 37°C interaction with another organic cation, chlorpheniramine.
with 5 mM diphenhydramine or chlorpheniramine (pH 7.4). Thereafter, intracellular These findings suggest the contribution of a specific trans-
accumulation of drugs was measured. Each value represents the mean 6 S.E. of
three monolayers. port system to the intestinal absorption of diphenhydramine.
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