Abstract
Transport characteristics of diphenhydramine, an antihistamine, were studied in cultured human intestinal Caco-2 cell monolayers to elucidate the mechanisms of its intestinal absorption. Diphenhydramine accumulation in the monolayers increased rapidly and was influenced by extracellular pH (pH 7.4 > 6.5 > 5.5). Diphenhydramine uptake was temperature dependent, saturable, and not potential sensitive. Kinetic analysis revealed that the apparentKm values were constant (0.8–1.0 mM) in all pH conditions tested, whereas Vmax values decreased at the lower pH. The initial uptake of diphenhydramine was competitively inhibited by another antihistamine, chlorpheniramine, with a Ki value of 1.3 mM. On the other hand, cimetidine and tetraethylammonium, typical substrates for the renal organic cation transport system, had no effect. Moreover, biological amines and neurotransmitters, such as histamine, dopamine, serotonin, and choline, also had no effect on the diphenhydramine accumulation. Finally, diphenhydramine uptake was stimulated by preloading monolayers with chlorpheniramine (trans-stimulation effect). These findings indicate that diphenhydramine transport in Caco-2 cells is mediated by a specific transport system. This pH-dependent transport system may contribute to the intestinal absorption of diphenhydramine.
Diphenhydramine, a tertiary amine compound with one site of ionization with a pKa value of pH 9.0 (de Roos et al., 1970), is widely used as an antihistamine for the symptomatic relief of hypersensitivity reaction. Diphenhydramine is relatively well absorbed, and its plasma concentration is rapidly elevated after oral administration (Paton and Webster, 1985), although it is mostly ionized over the pH range in the gastrointestinal tract.
The intestinal absorption mechanisms of lipophilic organic cations, such as diphenhydramine, have been explained by the passive diffusion of nonionized compounds according to the pH-partition theory. However, recent studies have suggested that carrier-mediated transport systems contribute to the intestinal absorption of various organic cations (Tan et al., 1989; Kuo et al., 1994). With intestinal brush-border membrane vesicles, it was reported that the uptake of organic cations consisted of two steps: binding to the membrane and entrance into intravesicular space stimulated by the inside-negative electrical potential (Saitoh et al., 1988a, 1989; Sugawara et al., 1995). However, these mechanisms have not been well investigated.
Recently, the human colon carcinoma cell line Caco-2 was used to study transport mechanisms of drugs (Artursson, 1990; Hu and Borchardt, 1990;Artursson and Karlsson, 1991). This cell line forms confluent monolayers of well differentiated enterocyte-like cells with functional properties of transporting epithelia (Hidalgo et al., 1989) and has been used to study the transport of nutrients and drugs. Using this cell line, we have demonstrated the absorption mechanisms of some oral cephalosporins and a dipeptide-like anticancer agent, bestatin (Inui et al., 1992; Saito and Inui, 1993; Matsumoto et al., 1994, 1995). In this report, we examined the uptake characteristics of diphenhydramine in Caco-2 cells to elucidate the intestinal absorption mechanisms of this lipophilic organic cation.
Experimental Procedures
Materials.
Diphenhydramine hydrochloride was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). (±)-Chlorpheniramine maleate, cimetidine, histamine, l-histidine monohydrochloride monohydrate, hydroxyzine dihydrochloride, imipramine hydrochloride, ketotifen fumarate, and tetraethylammonium bromide were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals were of the highest purity available.
Cell Culture.
Caco-2 cells at passage 18 obtained from the American Type Culture Collection (ATCC HTB37; Rockville, MD) were maintained by serial passage in plastic culture dishes (Falcon; Becton 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 × 105 cells in 5 ml of the complete culture medium. The medium consisted of Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 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 antihistamines 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)ethanesulfonic 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 extraction solution (0.01 N HCl/methanol, 1:1) and were maintained for 1 h at room temperature. The extraction solution was centrifuged at 13,000 rpm (model 3533; Abbott Laboratories, Abbott Park, IL) for 15 min. The supernatant was filtered through a Millipore filter (SJGVL, 0.22 μm), and the drug was analyzed by HPLC as described 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) under the following conditions: column, TSK-gel ODS 80TM 4.6 mm i.d. × 150 mm (Tohso Co., Tokyo, Japan) for diphenhydramine, l-column ODS 4.6 mm i.d. × 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 diphenhydramine, 20 mM KH2PO4 buffer (pH 5.4)/methanol 6:4 for chlorpheniramine; flow rate, 0.8 ml/min; wavelength, 225 nm; injection volume, 50 μl; temperature, 40°C. The detection limits were approximately 10 pmol for both compounds. The protein content of the cell monolayers 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 γ-globulin as a standard.
Statistical Analysis.
Data were analyzed statistically by nonpaired t test or one-way ANOVA followed by Scheffé’s test when multiple comparisons were needed. Probability values less than 5% were considered significant.
Results
Uptake of Diphenhydramine by Caco-2 Monolayers.
To characterize the diphenhydramine uptake by Caco-2 cells, diphenhydramine accumulation was investigated at three extracellular pH values (7.4, 6.5, 5.5). As shown in Fig.1, the accumulation of diphenhydramine increased rapidly and reached almost steady state at 15 min after starting incubation under all pH conditions. The accumulation was influenced by the extracellular pH, the order of uptake being pH 7.4 > 6.5 > 5.5. Moreover, when the monolayers were incubated with 1 mM diphenhydramine (pH 7.4) for 1 min at 4°C, the amount accumulated decreased to 41% of the amount at 37°C (37°C, 15.5 ± 0.5; 4°C, 6.4 ± 0.2 nmol · mg−1protein · min−1, mean ± S.E. of three monolayers).
Kinetic Analysis of Diphenhydramine Uptake.
The concentration-dependence of diphenhydramine accumulation was examined, and the kinetic parameters were calculated. Figure2 shows the accumulation of diphenhydramine at 1 min as a function of the substrate concentration ranging from 0.1 to 10 mM. Table 1summarizes the kinetic parameters evaluated by nonlinear least-squares regression analysis (Yamaoka et al., 1981) from the following Michaelis-Menten equation:
Effect of Various Organic Cations on Diphenhydramine Uptake.
The effect of various organic cations on diphenhydramine accumulation was investigated. As shown in Fig. 3, chlorpheniramine and imipramine inhibited the diphenhydramine uptake, but other antihistaminic agents (hydroxyzine and ketotifen) had almost no effect. Then the effect of histamine and its precursor histidine were examined. In addition, we studied whether cimetidine and tetraethylammonium, typical substrates for the organic cation transporters in the kidney, affected the diphenhydramine accumulation. These compounds had no effect on diphenhydramine accumulation (control, 20.3 ± 0.9; with histidine, 20.3 ± 0.2; with histamine, 20.6 ± 0.4; with tetraethylammonium, 19.8 ± 0.7; with cimetidine, 20.0 ± 0.4 nmol · mg−1protein · 5 min−1; each value represents the mean ± 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).
Because chlorpheniramine is an antihistamine and its chemical structure is similar to diphenhydramine, the effect of chlorpheniramine on diphenhydramine accumulation was further investigated. As shown in Fig.4, chlorpheniramine inhibited diphenhydramine accumulation in a concentration-dependent manner. Dixon plot analysis demonstrated that chlorpheniramine competitively inhibited diphenhydramine uptake with an apparentKi value of 1.3 mM (Fig.5). We also studied the initial chlorpheniramine uptake by Caco-2 cells and calculated the kinetic parameters. The Km value of chlorpheniramine (0.9 mM) at pH 7.4 was similar to itsKi value against the diphenhydramine uptake (data not shown).
Effect of Membrane Potential on Diphenhydramine Uptake.
To examine whether diphenhydramine uptake by Caco-2 cells is potential dependent, the effect of ion composition of the incubation medium on diphenhydramine uptake was examined. To decrease a transmembrane electrical potential, extracellular Na+ was replaced with K+ (K+medium: 3 mM NaCl, 145 mM KCl). Under this condition, diphenhydramine accumulation at 1 min did not change (control, 12.8 ± 0.3; K+ medium, 13.8 ± 0.4, nmol · mg−1protein · min−1, mean ± S.E. of three monolayers), indicating the potential-insensitive uptake of diphenhydramine by Caco-2 cells.
trans-Stimulation Effect on Diphenhydramine Uptake.
To elucidate whether the diphenhydramine uptake is mediated by a specific transport system, thetrans-stimulation effect on the initial uptake of diphenhydramine was examined. As shown in Table2, the initial uptake was stimulated by preloading the monolayers with chlorpheniramine, which had acis-inhibitory effect on diphenhydramine uptake. In addition, chlorpheniramine uptake was trans-stimulated by diphenhydramine preloading.
Discussion
To characterize the intestinal absorption mechanism of diphenhydramine, we investigated the cellular uptake of diphenhydramine by Caco-2 cells. Intestinal absorption of ionized drugs has been explained by passive diffusion of nonionized compounds. In this study, the amount of diphenhydramine accumulated in Caco-2 cells was decreased at lower pH. This pH dependence might be partly explained by passive diffusion according to the pH-partition theory. However, diphenhydramine (pKa 9.0) was mostly ionized even at the highest pH level tested (de Roos et al., 1970). Moreover, the accumulation of diphenhydramine was temperature dependent, saturable, and competitively inhibited by another organic cation, chlorpheniramine. Therefore, the cellular uptake characteristics of diphenhydramine were not explained only by the pH-partition theory.
We investigated the effect of histamine and other antihistamines on diphenhydramine uptake. The results showed that histamine and all other antihistamines, except chlorpheniramine, had no effect on diphenhydramine accumulation. Thus, it is unlikely that diphenhydramine accumulation implies binding to the histamine receptor, which was reported to exist in the intestine (Morini et al., 1993). Dixon plot analysis demonstrated that chlorpheniramine competitively inhibited the uptake of diphenhydramine with a Kivalue of 1.3 mM, which was comparable with theKm value for chlorpheniramine (0.9 mM). These findings suggest that diphenhydramine and chlorpheniramine are accumulated in Caco-2 cells via a common transport system. On the other hand, cimetidine and tetraethylammonium, which are transported by the organic cation-H+ antiport system in renal brush-border membrane (Takano et al., 1984; McKinney and Kunnemann, 1987; Wright and Wunz, 1987; Katsura et al., 1991), had no effect on diphenhydramine accumulation. It seems likely that diphenhydramine and chlorpheniramine have a common chemical structure recognized by some specific transport system that has not been reported in either Caco-2 cells or the renal brush-border membrane.
Lipophilic organic cations, such as diphenhydramine, imipramine, and chlorpromazine, were reported to have a specific binding site on biological membrane (Saitoh et al., 1988b). Therefore, the binding of diphenhydramine to the cellular membrane surface might partly account for its accumulation in Caco-2 cells. It was also reported that diphenhydramine inhibited the small-intestinal sodium-dependent uptake of α-d-glucoside in rats (Elsenhans et al., 1985). The uptake inhibition occurred by the binding of diphenhydramine to the sodium binding site in intestinal mucosa. Furthermore, it was reported that organic cations entered into the intravesicular space, stimulated by the inside-negative transmembrane electrical potential, after their binding to the membrane surface (Saitoh et al., 1988a, 1989; Sugawara et al., 1995). However, we found that diphenhydramine accumulation was insensitive to the membrane potential. In addition, diphenhydramine accumulation was enhanced under various ATP-depleted conditions (H. Mizuuchi, T. Katsura, Y. Hashimoto and K. Inui, unpublished observations). It is likely that the amount of diphenhydramine accumulated in the cells or associated with the membrane is not increased because the transmembrane potential difference was weakened under such ATP-depleted conditions. Thus, our results exclude the possibility that the cellular accumulation is indicative of the potential-sensitive binding and/or uptake of diphenhydramine.
The diphenhydramine accumulation was stimulated by preloading Caco-2 monolayers with chlorpheniramine (trans-stimulation effect). This finding indicates the existence of a specific transport system for diphenhydramine and chlorpheniramine. Carrier-mediated transport of diphenhydramine was also reported in the central nervous system (Goldberg et al., 1987). However, the driving force for diphenhydramine transport was not determined. Because the amount of diphenhydramine accumulated in Caco-2 cells was decreased at lower extracellular pH, it is speculated that a pH-dependent transport system contributes to the uptake of diphenhydramine in Caco-2 cells. Further examination is necessary to clarify the driving force and/or substrate specificity of this transport system.
In conclusion, diphenhydramine is rapidly accumulated in Caco-2 cells with substrate saturability and pH dependence. The cellular accumulation was influenced by cis- andtrans-interaction with another organic cation, chlorpheniramine. These findings suggest the contribution of a specific transport system to the intestinal absorption of diphenhydramine.
Footnotes
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Send reprint requests to: Professor Ken-ichi Inui, Ph.D., Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. E-mail:inui{at}kuhp.kyoto-u.ac.jp
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↵1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
- Received August 21, 1998.
- Accepted March 17, 1999.
- The American Society for Pharmacology and Experimental Therapeutics