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INFLUX AND EFFLUX TRANSPORT OF H1-ANTAGONIST EPINASTINE ACROSS THE BLOOD-BRAIN BARRIER

Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan (N.I., T.N., T.Y., I.T.); Department of Drug Metabolism and Pharmacokinetics, Kawanishi Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., Hyogo, Japan (N.I., A.T., A.S., W.K., K.Y., K.S., T.I.); CREST, Japan Science and Technology Cooperation, Kawaguchi, Japan (I.T.)

(Received September 8, 2003; accepted February 12, 2004)

	Abstract

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We investigated influx and efflux transporters involved in blood-brain barrier transport of the nonsedative H1-antagonist epinastine. The basal-to-apical transport of [¹⁴C]epinastine was markedly higher than that in the opposite direction in LLC-GA5-COL150 cells stably transfected with human multidrug resistance (MDR)1 gene. The brain-to-plasma concentration ratio of [¹⁴C]epinastine in mdr1a/b(-/-) mice was 3.2 times higher than that in wild-type mice. The uptake of both [³H]mepyramine and [¹⁴C]epinastine into immortalized rat brain capillary endothelial cells (RBEC)1 showed temperature and concentration dependence. The kinetic parameters, K_m, V_max, and uptake clearance (V_max/K_m), of the initial uptake of [³H]mepyramine and [¹⁴C]epinastine by RBEC1 were 150 µM, 41.8 nmol/min/mg protein, and 279 µl/min/mg protein for mepyramine and 10.0 mM, 339 nmol/min/mg protein, and 33.9 µl/min/mg protein for epinastine, respectively. The uptake of [³H]mepyramine and [¹⁴C]epinastine by RBEC1 was inhibited by organic cations such as quinidine, amantadine, and verapamil, but not by other organic cations, tetraethyl ammonium, guanidine, and carnitine. Organic anions such as benzoic acid, estrone-3-sulfate, taurocholate, and neutral digoxin were not inhibitory. Furthermore, some cationic H1 antagonists (chlorpheniramine, cyproheptadine, ketotifen, and desloratadine) inhibited the [³H]mepyramine and [¹⁴C]epinastine uptake into RBEC1. In conclusion, the present study demonstrated that the combination of efficient efflux transport by P-glycoprotein and poor uptake by the influx transporter, which is identical with that responsible for the uptake of mepyramine, account for the low brain distribution of epinastine.

Epinastine is an antiallergic agent having histamine H1-receptor antagonistic activity, and it does not exhibit sedative properties in the clinical dose range (Adamus et al., 1987). In rats, the brain concentration of epinastine is about 10 times lower than the plasma concentration (Kato et al., 1997). It has also been reported that only about 10% of the CNS-H1 receptors are occupied in humans after a single dose of 20 mg of epinastine, as determined with positron emission tomography (Yanai et al., 1995).

The aim of the present study was to elucidate the mechanisms of the limited brain distribution of epinastine by focusing on both influx and efflux transport processes at the BBB using various in vitro and in vivo methods. The structures of radiolabeled epinastine and mepyramine used in this study are shown in Fig. 1.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Chemical structures and the positions of the radiolabel () of mepyramine (A) and epinastine (B).

	Materials and Methods

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Chemicals. [¹⁴C]Epinastine (46.76 mCi/mmol), unlabeled epinastine, and LY335979 were synthesized by Boehringer Ingelheim Co. (Biberach, Germany). Loratadine, decarboethoxyloratadine (desloratadine), cetirizine, and ebastine were extracted from tablets and purified to >98%. [³H]Mepyramine and [¹⁴C]mannitol were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckingshamshire, UK). All other chemicals and reagents were commercial products of reagent grade.

Immortalized rat brain capillary endothelial cells (RBEC)1. Immortalized rat brain capillary endothelial cells (RBEC)1 were established by transfecting recombinant plasmids containing origin-defective simian virus 40 gene, SVori-8-16, into primary cultured rat BCECs as described previously (Kido et al., 2000). RBEC1 cells were cultured in Dulbecco's modified Eagle's medium (high glucose) with 5% fetal bovine serum and 5% donor horse serum (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO₂, 95% air atmosphere, and were maintained by serial passages in plastic culture dishes.

LLC-PK1 and LLC-GA5-COL150. LLC-PK1 cells were obtained from Japan Health Science Research Resources Bank (Osaka, Japan). LLC-GA5-COL150 cells were purchased from Riken Gene Bank (Tsukuba, Japan). LLC-GA5-COL150 cells were established by transfection of human MDR1 cDNA into LLC-PK1 cells (Tanigawara et al., 1992; Ueda et al., 1992) and were maintained by serial passage in plastic culture dishes. LLC-PK1 cells were incubated in complete medium consisting of Medium 199 (Nissui Pharmaceutical, Tokyo, Japan) with 3% fetal bovine serum (Invitrogen). For LLC-GA5-COL150 cells, 150 ng/ml colchicine was added to the complete medium consisting of Medium 199 with 10% fetal bovine serum. LLC-PK1 and LLC-GA5-COL150 cells were seeded in plastic dishes containing complete medium. Monolayer cultures were grown at 37°C in a 5% CO₂, 95% air atmosphere.

Transport Study in RBEC1. RBEC1 cells were seeded on collagen-coated multiwell dishes (Nalge Nunc International, Naperville, IL) at a cell density of 7.5 x 10⁴ cells/well. At 3 days after seeding, the cells were washed with 1 ml of incubation buffer (122 mM NaCl, 3 mM KCl, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.4 mM CaCl₂, 10 mM D-glucose, and 10 mM HEPES, pH 7.4) and preincubated for 20 min. After preincubation, the buffer (0.25 ml) containing [³H]mepyramine and [¹⁴C]epinastine was added to initiate uptake. The cells were incubated at 37°C for a designated time, and then washed three times with 1 ml of ice-cold incubation buffer to terminate the uptake. The cells were solubilized with 1 N NaOH for 60 min, and then neutralized with HCl. The radioactivity was measured using a liquid scintillation counter (TRI-CARB 2500TR; PerkinElmer Life and Analytical Sciences, Boston, MA) after the addition of 3 ml of scintillation cocktail, Hionic fluor (PerkinElmer Life and Analytical Sciences). Cellular protein content was measured by the Lowry method with bovine serum albumin as a standard (Lowry et al., 1951). Uptake was expressed as the cell-to-medium ratio (µl/mg protein) obtained by dividing the uptake amount by the concentration of substrate in the incubation buffer. To estimate the kinetic parameters for the uptake of mepyramine and epinastine by RBEC1, the initial uptake rate after subtracting the uptake rate at 4°C was fitted to the following equation by means of nonlinear least-squares regression analysis using Multi (Yamaoka et al., 1981): v = V_max x s/K_m + s, where v is the initial uptake rate of substrate (nmol/min/mg protein), s is the substrate concentration in the medium (µM), K_m is the Michaelis-Menten constant (µM), and V_max is the maximum uptake rate (nmol/min/mg protein).

Transcellular Transport Study in LLC-GA5-COL150 and LLC-PK1 Cells. LLC-PK1 and LLC-GA5-COL150 cells were seeded on microporous polycarbonate membrane Transwells (3-µm pore size, 1-cm diameter; Costar, Cambridge, MA) at a cell density of 2.0 x 10⁵ and 2.5 x 10⁵ cells/cm², respectively. Cells were cultured on the membrane with 1.5 and 0.5 ml of complete medium without colchicine in the donor and receiver compartments, respectively. Cells were supplemented with fresh medium on the second day and used for the transport studies on the fifth day after seeding. Transepithelial electrical resistance was measured in each well using a Millicell ohmmeter (model ERS; Millipore Corporation, Bedford, MA). Cells exhibiting a resistance of 200 or greater, after correcting for the resistance obtained in control blank wells, were used in the transport experiments. About 1 h before the initiation of the transport experiments, the medium in both the donor and receiver compartments was replaced with transport medium. The transport experiments were initiated by replacing the medium with medium containing or not containing a test compound. After 0.5, 1, 1.5, and 2 h, 100 µl were taken from the receiver compartment and replaced with the same amount of fresh transport medium. The radioactivity was measured in the same way as described above. To examine the inhibitory effect of LY335979 on the P-gp-mediated transport in LLC-GA5-COL150, LY335979 was added to the medium on both sides of the cell monolayers 1 h before adding the substrate. The paracellular leakage was monitored in terms of the appearance of [¹⁴C]mannitol in the opposite compartment and was <5 µl/cm² of total radioactivity per hour.

In Vivo Brain Distribution Study. Male FVB and mdr1a/b(-/-) mice, 5 to 8 weeks of age, were purchased from Taconic Farms, Inc. (Germantown, NY). [¹⁴C]Epinastine (2 mg/0.1 mCi/2.5 ml/kg) dissolved in distilled water was intravenously injected via the tail vein. At 1 h after injection, the mice were sacrificed under ether anesthesia, and blood, liver, brain, and heart were immediately collected for analysis. Plasma samples were immediately separated by centrifugation. Brain and other tissues were homogenized with 1 or 2 ml of water, respectively. Samples were kept frozen at -20°C until analysis.

Quantification of Epinastine in Plasma and Tissues. Plasma and tissue homogenates were subjected to liquid-liquid extraction. Briefly, 50 µl of 2 µg/ml diphenidol as an internal standard was added to 200 µl of plasma or 100, 700, or 800 µl of liver, heart, and brain homogenate, respectively. Then, 1.5 ml of 0.5 N NaOH and 10 ml of iso-octane with 2% iso-butanol were added to each sample, and the mixture was shaken mechanically for 10 min. After centrifugation at 2800g for 10 min at room temperature, the upper organic phase was transferred into another tube and 120 µl of 0.05 N hydrochloric acid was added for back-extraction. The mixture was shaken and centrifuged as above; then, the upper organic phase was discharged by aspiration. Finally, 30 µl of the hydrochloric acid phase was injected into a high-performance liquid chromatography system, model 2690 with a 996 photodiode array (Waters, Milford, MA). Quantification of epinastine was performed under isocratic conditions using an Inertsil ODS-2 (5-µm, 2.1 x 150 mm) column (GL Science, Tokyo, Japan). The mobile phase, 0.1% sodium octane sulfonate containing 0.05% phosphoric acid/acetonitrile (67.5:32.5, v/v) was delivered at a flow rate of 0.28 ml/min. The eluate was monitored at 210 nm.

	Results

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Transport of Epinastine across Cultured Cell Monolayers of LLC-PK1 and LLC-GA5-COL150 Cells. The role of P-gp in epinastine efflux transport was assessed using LLC-PK1 and LLC-GA5-COL150 cells stably transfected with human MDR1 gene. Figure 2 shows the time profiles of transcellular transport of [¹⁴C]epinastine across LLC-PK1 and LLC-GA5-COL150 monolayers. For epinastine, the basal-to-apical transport markedly exceeded transport in the opposite direction in LLC-GA5-COL150 cells, whereas in LLC-PK1 cells, such polarized transport was absent. Furthermore, the basal-to-apical and apical-to-basal transports in the presence of LY335979, a specific P-gp inhibitor, were 42.2 and 46.7 µl/cm² for LLC-GA5-COL150 and 50.0 and 52.3 µl/cm² for LLC-PK1 at 60 min, respectively. Namely, the greater basal-to-apical transport as compared with apical-to-basal transport of epinastine across the monolayer of LLC-GA5-COL150 was completely abolished by 0.1 µM LY335979, which had no significant effect on the epinastine transport across a monolayer of LLC-PK1 (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Transepithelial transport of [14C]epinastine (5 µM) across LLC-GA5-COL150 (A) and LLC-PK1 (B) cell monolayers. Open and closed circles indicate the basal-to-apical and apical-to-basal transports, respectively. Each point represents the mean ± S.D. from three experiments. , significantly different from the data with the apical-to-basal transport as determined with a two-sided Student's t test (p < 0.05).

Brain Distribution of Epinastine in mdr1a/b(+/+) and mdr1a/b(-/-) Mice. To evaluate the importance of P-gp in determining the in vivo brain distribution of epinastine, the brain concentration of unchanged epinastine was determined at 1 h after intravenous injection of epinastine into wild-type [mdr1a/b(+/+)] mice and mice that lack the mdr1a/b gene [mdr1a/b(-/-)] (Table 1). The tissue-to-plasma concentration ratio (K_p) in brain in mdr1a/b(-/-) mice (0.70) was 3.2 times higher than that in mdr1a/b(+/+) mice (0.22). In heart, in which the mdr1a/b gene products are not expressed, no significant difference in K_p value was observed between these two types of mice. These results demonstrate that P-gp limits the in vivo brain distribution of epinastine.

Uptake of [³H]Mepyramine and [¹⁴C]Epinastine by Immortalized Rat Brain Capillary Endothelial Cells (RBEC1). The time courses of [³H]mepyramine and [¹⁴C]epinastine uptake at 37°C and 4°C by RBEC1 are shown in Fig. 3. The uptakes of [³H]mepyramine and [¹⁴C]epinastine at 37°C and 4°C increased with time, and marked temperature dependence was observed. Based on the results of time courses, 10 and 30 s for mepyramine and epinastine, respectively, were chosen for kinetic and inhibition studies as the shortest time we can evaluate the initial uptake in a quantitatively reliable manner. The concentration dependence of the initial uptake of [³H]mepyramine and [¹⁴C]epinastine was studied from 1 µM to 1 mM and 0.1 µM to 5 mM, respectively. The results are shown in Fig. 4. The initial uptakes of [³H]mepyramine and [¹⁴C]epinastine at 37°C and 4°C were used to evaluate the total and nonsaturable uptakes, respectively, since the uptakes of [³H]mepyramine and [¹⁴C]epinastine at 4°C increased linearly with increasing substrate concentration. Therefore, it is considered that the difference between the uptake rates at 37°C and 4°C represents the saturable process. The Eadie-Hofstee plots of the saturable uptake component of [³H]mepyramine and [¹⁴C]epinastine each exhibited a single straight line (Fig. 4). The K_m and V_max values estimated using the equation v = V_max x s/K_m + s were 150 ± 53.3 µM and 41.8 ± 4.51 nmol/min/mg protein for mepyramine and 10.0 ± 0.547 mM and 339 ± 12.7 nmol/min/mg protein for epinastine, respectively. The uptake efficiencies of mepyramine and epinastine in terms of uptake clearance were 279 µl/min/mg protein and 33.9 µl/ml/mg protein, respectively, with about 8 times lower efficiency for epinastine compared with mepyramine. Accordingly, both mepyramine and epinastine are taken up by the endothelial cells that form the blood-brain barrier through a carrier-mediated process, but the uptake of epinastine was less efficient than that of the sedative mepyramine.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Time courses for the uptake of [3H]mepyramine (A) and [14C]epinastine (B) by cultured monolayer of RBEC1 at 37°C (closed circles) and 4°C (open circles). The substrate concentrations used were 5 µM for mepyramine and epinastine. Each point represents the mean ± S.D. from three experiments. Inset represents the uptake over 10 and 60 s for mepyramine and epinastine, respectively.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Concentration-dependence of uptake of mepyramine and epinastine by RBEC1. Inset, Eadie-Hofstee plots of initial uptake rates of mepyramine (10 s) and epinastine (30 s) after subtracting the initial uptake rate at 4°C. v, uptake in nmol/mg protein/10 s or 30 s; v/[S], pmol/mg protein/10 s or 30 s/µM. The substrate concentrations used were 1 to 1000 µM and 0.1 to 5000 µM for mepyramine and epinastine, respectively. Each point represents the mean ± S.D. from three experiments. Closed circles, open circles, and closed triangles indicate the uptake at 37°C, 4°C, and the difference between 37°C and 4°C, respectively. The dashed lines were generated from the equation v = Vmax x s/Km + s, using Multi-fitted parameters.

Inhibitory Effect of Several Compounds on the Uptake of [³H]Mepyramine and [¹⁴C]Epinastine in RBEC1. To identify the transporter involved in [³H]mepyramine and [¹⁴C]epinastine uptake by RBEC1, the inhibitory effects of various compounds were examined (Table 2). We used tetraethyl ammonium (TEA), guanidine, carnitine, quinidine, amantadine, and verapamil, which are the inhibitors of organic cation transporters (OCTs and OCTNs), p-aminohip-purate as an inhibitor of organic anion transporters (OATs), estrone-3-sulfate, taurocholate and digoxin as inhibitors of organic anion transporting polypeptides (OATPs), and benzoic acid as an inhibitor of MCT1 (Ohashi et al., 1999; Yabuuchi et al., 1999; Asaba et al., 2000; Kido et al., 2000; Han et al., 2001; Sugiyama et al., 2001; Goralski et al., 2002). Among them, quinidine, amantadine, and verapamil significantly inhibited the uptake of [³H]mepyramine and [¹⁴C]epinastine, whereas other cationic compounds or anionic compounds did not. These results indicate that a transporter(s) that is sensitive to organic cations is involved in the uptake of mepyramine and epinastine by RBEC1 cells.

Inhibitory Effect of Several H1-Antagonists on the Uptake of [³H]Mepyramine and [¹⁴C]Epinastine in RBEC1. To clarify whether other classical and second-generation H1-antagonists have affinity for the transporter(s) that is responsible for the uptake of mepyramine and epinastine in RBEC1, we examined the inhibitory effects of H1-antagonists (Table 3). Chlorpheniramine, cyproheptadine, ketotifen, and desloratadine significantly reduced the initial uptakes of both [³H]mepyramine and [¹⁴C]epinastine by RBEC1, whereas cetirizine, loratadine, ebastine, and fexofenadine were not inhibitory.

	Discussion

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Epinastine is a second-generation H1-antagonist and occupies the central H1 receptor to the extent of about 10% after oral dosing (Yanai et al., 1995), whereas over 50% of central H1 receptors are occupied by some classical H1-antagonists, such as chlorpheniramine, administered orally (Yanai et al., 1992; Tagawa et al., 2001). To enter the brain, it is necessary for drugs to cross the BCECs, which form the BBB, due to the presence of tight junctions between adjacent BCECs. Transporters such as P-gp, MRPs, MCT, OATPs, OCTNs, and OCTs are expressed in the BCECs (Tamai and Tsuji 2000b), and they may control the brain distribution of H1-antagonists, resulting in the observed differential sedative effects among the H1-antagonists. Furthermore, previous studies suggested that there are both influx and efflux transporters for the BBB transport of H1-antagonists. As regards influx transporters, the presence of a mepyramine transporter was suggested, and the cationic derivatives, rather than the zwitterionic derivatives, of H1-antagonists exhibited higher affinity, resulting in greater accumulation of cationic derivatives in the brain. Since the cationic derivatives also exhibit stronger sedative effects, the influx transporter was supposed to be a determinant of the sedative effect of H1-antagonists (Yamazaki et al., 1994a,b,c). Then, involvement of the efflux transporter P-gp as a determinant was proposed, and several studies found a positive correlation between the sedative effect and transport by P-gp (Cvetkovic et al., 1999; Tamai et al., 2000a; Chishty et al., 2001; Chen et al., 2003). Accordingly, both influx and efflux transporters should be important to determine the apparent brain distribution of H1-antagonists. In the present study, we have examined the mechanisms of the limited brain distribution of epinastine, which shows a low sedative effect, by focusing on the influx and efflux transporters at the BBB.

First of all, we evaluated the involvement of the efflux transporter P-gp by means of in vivo and in vitro cultured-cell studies. The transport of epinastine from brain to bloodstream was evaluated by using P-gp-overexpressing LLC-GA5-COL150 cells compared with parental LLC-PK1 cells, and mdr1a/b(-/-) mice compared with parental FVB mice. In the LLC-GA5-COL150 cells, which express P-gp on the apical membrane, the basal-to-apical transport was greater than the oppositely directed transport, which was not the case in LLC-PK1 cells (Fig. 2). Furthermore, the brain-to-plasma concentration ratio in mdr1a/b(-/-) was 3.2 times higher than that in wild-type mice (Table 1). These results clearly indicated that P-gp plays a substantial role in the limited brain distribution of epinastine. In mdr1a/b(-/-) mice, significant differences in the brain-to-plasma concentration or AUC ratios were observed among various H1-antagonists (Table 4). For example, a more than 100 times higher brain-to-plasma AUC ratio was observed for desloratadine as compared with cetirizine. Similarly, the K_p,brain of ebastine was 10 times higher than that of carebastine. Such large differences in brain penetration among H1-antagonists in the absence of P-gp strongly suggested that transporters other than P-gp could affect the apparent BBB perme-ability of H1-antagonists.

There are several transporters functioning for drug influx at the BBB, and H1-antagonists may be taken up by the BCECs through such transporter(s) (Yamazaki et al., 1994a,b,c; Tamai et al., 2000a). In the present study, RBEC1 cells were used to elucidate the involvement of influx transporters in epinastine transport. The immortalized RBEC1 cells established in our laboratory meet various morphological and enzymatic criteria for the BBB (Kido et al., 2000), and expression of several transporters, including monocarboxylic acid transporter MCT1 (Kido et al., 2000), large neutral amino acid transporters LAT1 and LAT2 with 4F2hc (Kido et al., 2001b), carnitine transporter OCTN2 (Kido et al., 2001a), and MRP1, has been confirmed, whereas the cell line showed negligible expression of P-gp (Tamai et al., 2000c). Thus, we used immortalized RBEC1 cells as the model for in vitro BBB transport studies and evaluated the uptake of epinastine into the cells in comparison with that of mepyramine, a classical sedative H1-antagonist. The uptake of mepyramine into RBEC1 showed temperature and concentration dependence (Figs. 3 and 4), as has already been demonstrated in primary cultured bovine BCECs (Yamazaki et al., 1994a,b,c). Epinastine was also taken up by the RBEC1 cells in both a temperature- and concentration-dependent manner. The Eadie-Hofstee plots of the uptakes of mepyramine and epinastine were linear, indicating that mepyramine and epinastine were apparently transported into RBEC1 by a single transporter (Figs. 3 and 4). The K_m and V_max/K_m values of epinastine uptake were about 67 times higher and 8 times lower than those of mepyramine uptake, respectively. The difference in the estimated K_m and V_max/K_m values between mepyramine and epinastine demonstrated that epinastine was taken up less efficiently than mepyramine by the RBEC1 cells. To clarify the transport mechanisms of mepyramine and epinastine into RBEC1, we examined the inhibitory effects of various compounds. The uptakes of [³H]mepyramine and [¹⁴C]epinastine were not affected by substrates of organic anion transporters such as MCT1, OATs, and OATPs. Similarly, several organic cations, including TEA, carnitine, and guanidine, had no effect. However, the uptakes of mepyramine and epinastine were inhibited by organic cations, including quinidine, amantadine, and verapamil (Table 2). The inhibitory effects of several organic cations were very similar to those on amantadine uptake into isolated rat renal proximal tubules (Goralski et al., 2002) and verapamil uptake into human retinal pigment epithelial cells (Han et al., 2001). It was reported that an unidentified carrier, which is different from rOCT1, rOCT2, rOCT3, and OCTN2, transfers amantadine into isolated rat renal tubules, and a novel cation transporter, which is not OCT1, OCT2, OCT3, OCTN1, or OCTN2, transfers verapamil into retinal pigment epithelial cells (Han et al., 2001; Goralski et al., 2002). Amantadine and verapamil significantly inhibited the uptake of [³H]mepyramine and [¹⁴C]epinastine, indicating that a transporter, which is similar to that involved in amantadine and verapamil uptake, is responsible for the uptake of epinastine and mepyramine at the BBB. Furthermore, cationic H1-antagonists such as chlorpheniramine, ketotifen, cyproheptadine, and desloratadine significantly inhibited the uptake of both mepyramine and epinastine, whereas zwitterionic derivatives, cetirizine and fexofenadine, were not inhibitory (Table 3). Epinastine exhibited a negligible inhibitory effect on mepyramine uptake by RBEC1 cells, and the extent of inhibition was comparable with that of zwitterionic derivatives, suggesting that epinastine is not efficiently transported by the transporter responsible for the uptake of mepyramine. Desloratadine, a nonsedative H1-antagonist and metabolite of loratadine, showed a stronger inhibitory effect than epinastine on the mepyramine uptake. The absence of a sedative effect of desloratadine may be explained by efficient efflux via P-gp, as demonstrated previously (Chen et al., 2003), that negates the uptake by the BCECs. Nonsedative fexofenadine was reported to show relatively low transport via P-gp, as evaluated in terms of the flux ratio of basal-to-apical/apical-to-basal transport in a monolayer of MDR1-expressing cells, although it is a substrate of P-gp (Cvetkovic et al., 1999). The lack of a sedative effect of fexofenadine may be explained largely by insufficient uptake by the BCECs, because it exhibited less efficient efflux via P-gp. In the case of epinastine, an insufficient uptake by RBEC1 cells and sufficiently high efflux via P-gp were demonstrated in the present study. Accordingly, if the influx transporter responsible for H1-antagonists at the BBB is shared by these derivatives, the affinity of H1-antagonists for both influx and efflux transporters would affect the apparent BBB permeability for these drugs.

In conclusion, the present study demonstrated that the limited brain distribution of epinastine is partly a consequence of active efflux transport via P-gp. In addition to the efflux transport, 8-fold lower uptake clearance by the influx transporter, as compared with mepyramine, also contributes to the low brain distribution of epinastine. Our results suggest that the brain distribution of H1-antagonists is affected by both influx and efflux transporters, and the relative contributions are variable among different antagonists, resulting in the differential sedative effects. Further studies to identify the influx transporter(s) will be important to understand in detail the brain distribution of H1-antagonists.

	Footnotes

 
¹ Abbreviations used are: CNS, central nervous system; AUC, area under the curve; BBB, blood-brain barrier; BCEC, brain capillary endothelial cell; LY335979, (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo suber-5-yl]-2-hydroxypropoxy}-quinilone trihydrochloride; MCT, monocarboxylic acid transporter; MDR, multi-drug resistance; MRP, multidrug resistance-associated protein; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; (r)OCT, (rat) organic cation transporter; OCTN, novel organic cation transporter; P-gp, P-glycoprotein; RBEC, rat brain capillary endothelial cells; TEA, tetraethyl ammonium.

Address correspondence to: Ikumi Tamai, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 Japan. E-mail address: tamai{at}rs.noda.tus.ac.jp

	References

Top Abstract Materials and Methods Results Discussion References

 


Adamus WS, Oldigs-Kerber J, and Lohmann HF (1987) Antihistamine activity and central effects of WAL 801 CL in man. Eur J Clin Pharmacol 33: 381-385.[CrossRef][Medline]

Asaba H, Hosoya K, Takanaga H, Ohtsuki S, Tamura E, Takizawa T, and Terasaki T (2000) Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2. J Neurochem 75: 1907-1916.[CrossRef][Medline]

Chen C, Hanson E, Watson JW, and Lee JS (2003) P-Glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab Dispos 31: 312-318.[Abstract/Free Full Text]

Chishty M, Reichel A, Siva J, Abbott NJ, and Begley DJ (2001) Affinity for the P-glycoprotein efflux pump at the blood-brain barrier may explain the lack of CNS side-effects of modern antihistamines. J Drug Target 9: 223-228.[Medline]

Cvetkovic M, Leake B, Fromm MF, Wilkinson GR, and Kim RB (1999) OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27: 866-871.[Abstract/Free Full Text]

Goralski KB, Lou G, Prowse MT, Gorboulev V, Volk C, Koepsell H, and Sitar DS (2002) The cation transporters rOCT1 and rOCT2 interact with bicarbonate but play only a minor role for amantadine uptake into rat renal proximal tubules. J Pharmacol Exp Ther 303: 959-968.[Abstract/Free Full Text]

Han YH, Sweet DH, Hu DN, and Pritchard JB (2001) Characterization of a novel cationic drug transporter in human retinal pigment epithelial cells. J Pharmacol Exp Ther 296: 450-457.[Abstract/Free Full Text]

Kato M, Nishida A, Aga Y, Kita J, Kudo Y, Narita H, and Endo T (1997) Pharmacokinetic and pharmacodynamic evaluation of central effect of the novel antiallergic agent betotastine besilate. Arzneim-Forsch 47: 1116-1124.[Medline]

Kido Y, Tamai I, Ohnari A, Sai Y, Kagami T, Nezu J, Nikaido H, Hashimoto N, Asano M, and Tsuji A (2001a) Functional relevance of carnitine transporter OCTN2 to brain distribution of L-carnitine and acetyl-L-carnitine across the blood-brain barrier. J Neurochem 79: 959-969.[CrossRef][Medline]

Kido Y, Tamai I, Okamoto M, Suzuki F, and Tsuji A (2000) Functional classification of MCT1-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies. Pharm Res (NY) 17: 55-62.

Kido Y, Tamai I, Uchino H, Suzuki F, Sai Y, and Tsuji A (2001b) Molecular and functional identification of large neutral amino acid transporters LAT1 and LAT2 and their pharmacological relevance at the blood-brain barrier. J Pharm Pharmacol 53: 497-503.[CrossRef][Medline]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-267.[Free Full Text]

Mattila MJ and Paakkari I (1999) Variations among non-sedating antihistamines: are there real differences? Eur J Clin Pharmacol 55: 85-93.[CrossRef][Medline]

Nicholson AN (1987) New antihistamines free of sedative side effects. Trends Pharmacol Sci 8: 247-249.

Ohashi R, Tamai I, Yabuuchi H, Nezu JI, Oku A, Sai Y, Shimane M, and Tsuji A (1999) Na(+)-dependent carnitine transport by organic cation transporter (OCTN2): its pharmacological and toxicological relevance. J Pharmacol Exp Ther 291: 778-784.[Abstract/Free Full Text]

Sugiyama D, Kusuhara H, Shitara Y, Abe T, Meier PJ, Sekine T, Endou H, Suzuki H, and Sugiyama Y (2001) Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from brain across the blood-brain barrier. J Pharmacol Exp Ther 298: 316-322.[Abstract/Free Full Text]

Tagawa M, Kano M, Okamura N, Higuchi M, Matsuda M, Mizuki Y, Arai H, Iwata R, Fujii T, Komemushi S, et al. (2001) Neuroimaging of histamine H1-receptor occupancy in human brain by positron emission tomography (PET): a comparative study of ebastine, a second-generation antihistamine and (+)-chlorpheniramine, a classical antihistamine. Br J Clin Pharmacol 52: 501-509.[CrossRef][Medline]

Tamai I, Kido Y, Yamashita J, Sai Y, and Tsuji A (2000a) Blood-brain barrier transport of H1-antagonist ebastine and its metabolite carebastine. J Drug Target 8: 383-393.[Medline]

Tamai I and Tsuji A (2000b) Transporter-mediated permeation of drugs across the blood-brain barrier. J Pharm Sci 89: 1371-1388.[CrossRef][Medline]

Tamai I, Yamashita J, Kido Y, Ohnari A, Sai Y, Shima Y, Naruhashi K, Koizumi S, and Tsuji A (2000c) Limited distribution of new quinolone antibacterial agents into brain caused by multiple efflux transporters at the blood-brain barrier. J Pharmacol Exp Ther 295: 146-152.[Abstract/Free Full Text]

Tanigawara Y, Okamura N, Hirai M, Yasuhara M, Ueda K, Kioka N, Komano T, and Hori R (1992) Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). J Pharmacol Exp Ther 263: 840-845.[Abstract/Free Full Text]

Ueda K, Okamura N, Hirai M, Tanigawara Y Saeki T, Kioka N, Komano T, and Hori R (1992) Human P-glycoprotein transports cortisol, aldosterone and dexamethasone, but not progesterone. J Biol Chem 267: 24248-24252.[Abstract/Free Full Text]

Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, Sai Y, and Tsuji A (1999) Novel membrane transporter OCTN1 mediates multispecific, bidirectional and pH-dependent transport of organic cations. J Pharmacol Exp Ther 289: 768-773.[Abstract/Free Full Text]

Yamaoka K, Tanigawara Y, Nakagawa T, and Uno T (1981) A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn 4: 879-885.[Medline]

Yamazaki M, Fukuoka H, Nagata O, Kato H, Ito Y, Terasaki T, and Tsuji A (1994a) Transport mechanism of an H1-antagonist at the blood-brain barrier: transport mechanism of mepyramine using the carotid injection technique. Biol Pharm Bull 17: 676-679.[Medline]

Yamazaki M, Terasaki T, Yoshioka K, Nagata O, Kato H, Ito Y, and Tsuji A (1994b) Carrier-mediated transport of H1-antagonist at the blood-brain barrier: mepyramine uptake into bovine brain capillary endothelial cells in primary monolayer cultures. Pharm Res (NY) 11: 975-978.

Yamazaki M, Terasaki T, Yoshioka K, Nagata O, Kato H, Ito Y, and Tsuji A (1994c) Carrier-mediated transport of H1-antagonist at the blood-brain barrier: a common transport system of H1-antagonists and lipophilic basic drugs. Pharm Res (NY) 11: 1516-1518.

Yanai K, Ryu JH, Watanabe T, Iwata R, Ido T, Asakura M, Matsumura R, and Itoh M (1995) Positron emission tomographic study of central histamine H1-receptor occupancy in human subjects treated with epinastine, a second-generation antihistamine. Methods Find Exp Clin Pharmacol 17: 64-69.

Yanai K, Watanabe T, Yokoyama H, Hatazawa J, Iwata R, Ishiwata K, Meguro K, Itoh M, Takahashi T, Ido T, and Matsuzawa T (1992) Mapping of histamine H1 receptors in the human brain using [¹¹C]pyrilamine and positron emission tomography. J Neurochem 59: 128-136.[Medline]

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