Article pubs.acs.org/molecularpharmaceutics
Involvement of Carrier-Mediated Transport in the Retinal Uptake of Clonidine at the Inner Blood−Retinal Barrier Yoshiyuki Kubo, Ai Tsuchiyama, Yoshimi Shimizu, Shin-ichi Akanuma, and Ken-ichi Hosoya* Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan S Supporting Information *
ABSTRACT: In the present study, the blood-to-retina transport across the inner BRB was investigated for clonidine, a compound which is expected to exhibit a neuroprotective effect for the treatment of severe retinal diseases. In the in vivo study, the integration plot analysis for [3H]clonidine exhibited an apparent influx permeability clearance of 457 μL/(min·g retina) in the retina. The in vivo inhibition study suggests that the blood-to-retina transport of clonidine at the BRB is organic cation-sensitive since clonidine, pyrilamine, and propranolol, at a concentration of 40 mM, significantly reduced the retinal uptake index (RUI) of [3H]clonidine, and an inhibitory effect on the RUI was also exhibited by verapamil, at a concentration of 3 mM. The in vitro study with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests that carrier-mediated transport is involved in the blood-to-retina transport of clonidine at the inner BRB since the results obtained demonstrated time-, temperature-, pH-, and concentration-dependent [3H]clonidine uptake, with a Km of 286 μM. In the in vitro inhibition study, the [3H]clonidine uptake was significantly reduced by several organic cations, such as clonidine, verapamil, pyrilamine, and propranolol, and was competitively inhibited by 200 μM verapamil, in spite of slight or no significant alteration being produced with organic anions. Furthermore, the typical substrates and inhibitors of well-known organic cation transporters had no significant effect on the uptake of [3H]clonidine to suggest the involvement of novel transporter molecules in the transport of clonidine across the inner BRB. These results suggest that the blood-to-retina transport of clonidine across the inner BRB involves a carrier-mediated transport manner, suggesting the contribution of a novel organic cation transporter expressed by the retinal capillary endothelial cells. KEYWORDS: inner blood−retinal barrier, organic cation transport, clonidine, transporter
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INTRODUCTION The progression of retinal diseases, such as diabetic retinopathy and macular degeneration, is known to cause severe neurological dysfunction in the retina, leading to blindness that dramatically affects the quality of life of patients. Studies of neurological dysfunction have shown that a neuroprotective effect is exerted by several cationic drugs, such as desipramine, nipradilol, imipramine, and memantine,1−6 and the neuroprotective effect of several cationic α2 agonists, such as brimonidin, dexmedetomidine and tizanidine, has been reported in cerebral ischemia or optic nerve injury,7−12 suggesting their usefulness in the treatment of the neurological dysfunction. Recently, a neuroprotective effect was also suggested for clonidine, a cationic α2 agonist used to treat essential and renal hypertensions, and it has been reported that the loss of neurons in the rat partially injured optic nerve system is suppressed by the intraperitoneal administration of clonidine,11,12 suggesting the potential usefulness of clonidine for the future treatment of retinal diseases. In the development of drugs to treat retinal diseases, it is essential to improve our understanding of the systems involved in transport from the circulating blood to the retina since their advances will contribute to the efficient and safe delivery of drugs to the retina across the blood−retinal barrier (BRB).1,2 © 2014 American Chemical Society
However, little is known about the transport of clonidine at the BRB, while several reports have suggested the involvement of carrier-mediated transport processes in the uptake of clonidine by placental epithelial cells, brain capillary endothelial cells, and keratinocytes.13−16 In the retina, the retinal capillary endothelial cells and the retinal pigment epithelial cells are responsible for the inner BRB and outer BRB, respectively, and these barrier structures separate the circulating blood and the neural retina. At the BRB, efficient nutrient supply is performed by blood-toretina transport mediated by various membrane transporters during the paracellular transport suppressed by tight junction.1,2,17,18 In particular, it is known that the inner BRB nourishes two-thirds of the retina, and the recent advances in research involving the inner BRB suggest that the transport of nutrients, such as sugar, amino acids, and vitamins, is carried out by glucose transporter (GLUT1/Slc2a1), L (leucinereferring) amino acid transporter 1 (LAT1/Slc7a5), and Na+dependent multivitamin transporter (SMVT/Slc5a6), respectively.19−22 Received: Revised: Accepted: Published: 3747
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Regarding cationic drugs, our previous studies using in vivo and in vitro analyses have demonstrated the blood-to-retina transport of verapamil, pyrilamine, and propranolol across the BRB, and suggested the involvement of novel organic cation transporters at the inner BRB.23−26 In the in vivo inhibition study, these transport systems were significantly inhibited in the presence of several cationic neuroprotectants, showing their possible contribution to the transport of cationic neuroprotectants at the inner BRB.25,26 However, it has been exceptionally implied that the putative transport of clonidine at the inner BRB involves a transport system distinct from those for verapamil, pyrilamine, and propranolol, since the inhibitory potential of clonidine is much greater than that predicted from the lipophilicity−inhibitory effect relationship developed in the in vitro inhibition study of propranolol transport.26 This cumulative evidence suggests that the elucidation of the transport system for clonidine at the BRB will lead to the safe and effective treatment of retinal diseases. In the present study, the blood-to-retinal transport of clonidine was investigated by in vivo vascular injection method and an in vitro uptake study with an in vitro model cell line of retinal capillary endothelial cells, TR-iBRB2 cells, the transport of which is reported to be correlated with in vivo BRB permeability.24,27,28
RUI = ([3H]/[14C] (dpm in the retina)) /([3H]/[14C] (dpm in the injectate solution)) × 100
Cellular Uptake Study. TR-iBRB2 cells, a conditionally immortalized rat capillary endothelial cell line, were used in the cellular uptake study. TR-iBRB2 cells have been used as an in vitro tool in studies of the inner BRB because of their similar cell morphology and gene expression to retinal capillary endothelial cells.24,27,28 The cells (passage number 23−35) were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals, Tokyo, Japan) containing 10% fetal bovine serum, 20 mM NaHCO3, and antibiotics at 33 °C to allow temperature-sensitive large T-antigen expression as described elsewhere.27,28 In the uptake study, as described previously, cells were rinsed with extracellular fluid (ECF)-buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 10 mM D-glucose, and 10 mM HEPES (pH 7.4)) and incubated in 200 μL of ECF-buffer containing 12 nM [3H]clonidine (0.1 μCi) at 37 °C. The Na+ dependence, membrane potential dependence, and pH dependence of [3H]clonidine uptake were studied with special buffers, such as Na+-free ECF buffers, as previously reported.31 Radioactivity measurements were performed using a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical) after the cells were lysed with 1 N NaOH and neutralized with 1 N HCl, respectively. The cell-to-medium ratio was used to express the [3H]clonidine uptake by TR-iBRB2 cells, in the data analysis using eq 3, and the protein content of the cells was measured with a DC protein assay kit (Bio-Rad, Hercules, CA).
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MATERIALS AND METHODS Reagents. Commercially available chemicals of reagent grade were used in the present study. n-[1-14C]Butanol ([14C]n-butanol, 2 mCi/mmol) and [benzene ring-3H]clonidine hydrochloride ([3H]clonidine, 57.8 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer (Boston, MA), respectively. Animals. The animal experiments were designed in accordance with the institutional guidelines established by the Animal Care Committee of the University of Toyama, which approved the protocol for the animal experiments of this study. In addition, the animals were treated in accordance with the statement issued by the Association for Research in Vision and Ophthalmology (ARVO). Male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan). In Vivo Blood-to-Tissue Transport Study. Integration plot was used to estimate the apparent influx permeability clearance (Kin) of [3H]clonidine in the retina, as reported previously.22 In brief, [3H]clonidine (3 μCi/rat) was injected into the femoral vein of anesthetized rats, and blood samples were collected at designated times. The retinas were collected after decapitation of the rats, and lysed and then neutralized with 2 N NaOH and 2 N HCl, respectively. For the radioactivity measurements, a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan) was used, and the Kin of [3H]clonidine was estimated using eq 1; the details have been described previously (Supporting Information).22 Vd(t ) = K in,retina × AUC(t )/Cp(t ) + Vi
(2)
Cell‐to‐medium ratio = ([3H] dpm per cell protein (mg)) /([3H] dpm per μL of medium)
(3)
MULTI, the nonlinear least-squares regression analysis program, was used in the kinetic analysis as described previously,22,31,32 and the data obtained in the uptake study were fitted to eq 4, where C, V, Km, and Vmax are the substrate concentration, the uptake rate, the Michaelis constant, and the maximal uptake rate, respectively. V = Vmax × C /(K m + C)
(4)
MULTI was also used in the kinetic analysis for the inhibition of clonidine uptake involving eq 5, where Ki and I are the inhibitory constant and the concentration of inhibitors, respectively. V = Vmax × C /[K m × (1 + I /K i) + C)]
(5)
Statistical Analysis. Unless otherwise indicated, data represent means ± SEM except for kinetic parameters, and the kinetic parameter data represent means ± SD. In the assessment of statistical differences, a one way analysis of variance (ANOVA) followed by the modified Fisher’s leastsignificant difference method was used for several groups, and an unpaired two-tailed Student’s t-test was used for two groups.
(1)
To evaluate the in vivo effect of inhibitors, the retinal uptake index (RUI) in rats was measured by injecting 200 μL of Ringer-HEPES buffer containing [3H]clonidine (2.5 μCi, 220 nM) and [14C]-n-butanol (0.5 μCi).22,29,30 The RUI was calculated using eq 2, and the values reflect the fractional uptake of [3H]clonidine as a percentage of the fractional uptake of [14C]-n-butanol.
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RESULTS Blood-to-Retina Transport of [3H]Clonidine across the BRB. Integration plot analysis of [3H]clonidine was carried out to investigate the in vivo blood-to-retina transport of clonidine 3748
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across the BRB. Analysis of the [3H]clonidine data using eq 1 showed that the apparent influx permeability clearance, Kin,retina, was 457 ± 130 μL/(min·g retina) (Figure 1), while the Kin,retina
2a). In the study of ion dependence, Na+- and K+-free buffers had no significant effect on the uptake of [3H]clonidine by TR-
Figure 1. Initial uptake of [3H]clonidine by the retina in rats. In the integration plot analysis, [3H]clonidine (3 μCi/rat) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3).
Figure 2. Uptake of [3H]clonidine (0.1 μCi, 12 nM) by TR-iBRB2 cells. (a) Time dependence and temperature dependence of [3H]clonidine uptake by TR-iBRB2 cells were investigated at 37 °C (closed circles) and 4 °C (open circle), respectively. (b) The effects of Na+ and membrane potential on the uptake of [3H]clonidine were examined at 37 °C for 15 s. Each column and point represent the mean ± SEM (n = 3). *p < 0.01, significantly different from the control.
for [3H]-D-mannitol, a marker of paracellular solute transport, was 0.626 μL/(min·g retina).33 In particular, the Kin,retina for clonidine was 730-fold greater than that of [3H]-D-mannitol, suggesting the influx transport of clonidine across the BRB. A carotid artery injection showed that the value of RUI for [3H]clonidine was 359 ± 77% (Table 1), and this was greater
iBRB2 cells (Figure 2b). The study carried out under different extracellular pH conditions revealed a 57% decrease and a 47% increase in [3H]clonidine uptake at pH 6.4 and pH 8.4, respectively (Figure 3a), and the study of the effect of
Table 1. In Vivo Uptake Study of [3H]Clonidine by the Retina in Ratsa inhibitors control clonidine pyrilamine propranolol verapamil nicotine TEA PAH probenecid
concentration (mM) 40 40 40 3 40 40 40 3
RUI (%)
% of control
± ± ± ± ± ± ± ± ±
100 ± 21 54.1 ± 5.9* 37.2 ± 4.4* 41.5 ± 10.2* 64.5 ± 5.9 74.3 ± 2.8 91.3 ± 7.8 100 ± 3 107 ± 9
359 185 134 149 232 267 328 360 386
77 21* 16* 37* 21 10 28 9 33
[ H]Clonidine (2.5 μCi/rat) and [14C]-n-butanol (0.5 μCi/rat) were injected with or without inhibitors. Decapitation was carried out 15 s after injection. 3 mM concentration of verapamil and probenecid were set because of its limitation of solubility. Each value represents means ± SEM (n = 3−5). *p < 0.01, significantly different from the control. TEA, tetraethylammonium. PAH, p-aminohippuric acid. a 3
Figure 3. Effect of pH on the uptake of [3H]clonidine (0.1 μCi, 12 nM) by TR-iBRB2 cells. The effects of extracellular pH (a) and intracellular pH (b) were examined at 37 °C for 15 s. Intracellular pH was increased and decreased by acute treatment and pretreatment (pre) of cells with 30 mM NH4Cl, respectively. Each column represents the mean ± SEM (n = 3). *p < 0.01, significantly different from the control (pH 7.4).
than that for [3H]-D-mannitol reported in a previous study,34 also supporting the influx transport of clonidine across the BRB. The RUI value of [3H]clonidine was significantly reduced by more than 30% in the presence of 40 mM clonidine, 40 mM pyrilamine, and 40 mM propranolol, and the tendency of inhibition (more than 25% reduction) was also exhibited by 3 mM verapamil and 40 mM nicotine, while no significant effect was exhibited in the presence of 40 mM tetraethylammonium (TEA), 40 mM p-aminohippuric acid (PAH), and 3 mM probenecid (Table 1). Uptake of [3H]Clonidine by TR-iBRB2 Cells. The in vitro uptake study was carried out using TR-iBRB2 cells, an in vitro model of the inner BRB, to examine the influx transport of clonidine across the inner BRB. TR-iBRB2 cells showed a timedependent increase of [3H]clonidine for 15 s at least, with an initial uptake rate of 1.99 ± 0.30 μL/(min·mg protein), while the uptake was significantly reduced by 31% at 4 °C (Figure
intracellular pH revealed a 90% decrease and a 70% increase in [3H]clonidine uptake at an alkaline (acute) and acidic (pretreatment) intracellular pH, respectively (Figure 3b). As clonidine is a cationic drug with a pKa of 8.05, the ratios of uncharged clonidine were estimated to be 2%, 18%, and 69% under the extracellular condition of pH 6.4, pH 7.4, and pH 8.4, respectively. This indicated a much smaller change in clonidine uptake than that of the uncharged clonidine (Figure 3), showing that passive diffusion is not sufficient to explain the alteration of clonidine uptake under different pH conditions. The TR-iBRB2 cells exhibited concentration-dependent uptake of [3H]clonidine (Figure 4a), and the Eadie−Scatchard plot showed that a one saturable process model fits the uptake data of clonidine with Km and Vmax values of 286 ± 44 μM and 43.7 ± 4.7 nmol/(min·mg protein), respectively (Figure 4b). These 3749
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(by more than 61%) of [3H]clonidine uptake was found in the presence of amantadine and timolol, although no significant effect was observed in the presence of typical organic cations, such as TEA, MPP+, choline, L-carnitine, cimetidine, serotonin, and putrescine (Table 2). Regarding organic anions, acetylsalicylic acid and diclofenac exhibited no significant effect on [3H]clonidine uptake by TR-iBRB2 cells, and a significant but weak effect was observed in the presence of PAH (Table 2). Kinetic Inhibition Analysis of [3H]Clonidine Uptake by TR-iBRB2 Cells. The inhibitory effect on the clonidine uptake by TR-iBRB2 cells was kinetically investigated. Clonidine uptake, with or without 200 μM verapamil, exhibited very similar y-intercepts corresponding to Vmax values of 48.1 ± 14.1 and 43.7 ± 4.7 nmol/(min·mg protein), respectively (p = 0.417; Figure 5a), suggesting the competitive inhibitory effect of verapamil on clonidine uptake with a Ki of 90.4 ± 46.8 μM. Clonidine uptake with pyrilamine and propranolol at a concentration of 200 μM exhibited Vmax values of 89.2 ± 51.1 and 82.6 ± 23.3 nmol/(min·mg protein), which were
Figure 4. Concentration dependence of [3H]clonidine uptake by TRiBRB2 cells. The uptake was investigated at 37 °C for 15 s, over the concentration range 10−1000 μM. Michaelis−Menten (a) and Eadie− Scatchard (b) plots were used to analyze the data obtained. Each point represents the mean ± SEM (n = 3).
results suggest that a carrier-mediated transport system contributes to the uptake of [3H]clonidine by TR-iBRB2 cells. Inhibition Study of [3H]Clonidine Uptake by TR-iBRB2 Cells. The inhibitory effects on [3H]clonidine uptake by TRiBRB2 cells were investigated using a variety of compounds at a concentration of 1 mM, and the results obtained are summarized in Table 2. Marked inhibition (by more than 81%) of [3H]clonidine uptake was exhibited in the presence of organic cations, such as desipramine, propranolol, nicotine, nipradilol, mecamylamine, verapamil, imipramine, memantine, clonidine, and quinidine (Table 2), while moderate inhibition Table 2. Inhibitory Effects on [3H]Clonidine Uptake by TRiBRB2 Cellsa compound
% of control
control desipramine propranolol nicotine nipradilol mecamylamine pyrilamine verapamil imipramine memantine clonidine quinidine amantadine timolol TEA MPP+ choline L-carnitine cimetidine serotonin PAH putrescine acetylsalicylic acid diclofenac
100 ± 5 3.61 ± 0.42* 7.50 ± 1.20* 8.49 ± 0.41* 10.1 ± 1.1* 10.5 ± 0.5* 12.0 ± 0.2* 14.6 ± 2.3* 16.0 ± 2.3* 17.3 ± 1.8* 17.9 ± 1.3* 19.0 ± 2.5* 33.0 ± 1.6* 38.6 ± 3.2* 86.8 ± 4.9 93.3 ± 2.0 95.1 ± 11.6 95.5 ± 6.5 98.4 ± 4.6 101 ± 8 83.1 ± 1.3* 108 ± 4 109 ± 8 115 ± 3
[ H]Clonidine (12 nM, 0.1 μCi) uptake by TR-iBRB2 cells was performed in the absence or presence of 1 mM compounds at 37 °C for 15 s. Each value represents the means ± SEM (n = 3−9). *p < 0.01, significantly different from the control. TEA, tetraethylammonium. PAH, p-aminohippuric acid. MPP+, 1-methyl-4-phenylpyridinium. a 3
Figure 5. Lineweaver−Burk plot of clonidine uptake by TR-iBRB2 cells. [3H]Clonidine uptake was studied with (closed circles) or without (open circles) 200 μM verapamil (a), pyrilamine (b), and propranolol (c). The uptake was examined at 37 °C for 15 s. Each point represents the mean ± SEM (n = 3). 3750
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The results of the in vitro inhibition study support the inhibitory profile obtained in the in vivo inhibition study of clonidine transport at the BRB, and they suggest the contribution of an organic cation transporter to the blood-toretina transport of clonidine at the inner BRB since organic cations, such as desipramine, propranolol, nicotine, nipradilol, mecamylamine, verapamil, imipramine, memantine, clonidine, quinidine, amantadine, and timolol, markedly inhibited the uptake of [3H]clonidine by TR-iBRB2 cells. This is also supported by weak or insignificant effects exhibited by anions, such as acetylsalicylic acid, diclofenac, and PAH (Table 2). Based on the significant inhibitory effects of desipramine, nipradilol, imipramine, and memantine (Table 2), it appears that the contribution of the clonidine transport system also has a significant effect on the retinal uptake of cationic neuroprotectants.3−6 In addition, the inhibitory profile given in Table 2 suggests that different molecules are involved in clonidine transport at the BBB and inner BRB since the verapamil- and desipramine-sensitive transport of clonidine was found at the inner BRB while the study using the in situ mouse brain perfusion method suggested that the BBB involves the verapamil- and desipramine-insensitive transport of clonidine.15 Furthermore, nicotine showed the significant inhibitory effect on the uptake of [3H]clonidine by TR-iBRB2 cells in spite of its tendency of inhibition in the in vivo study (Table 1 and Table 2), and the different results obtained in the in vivo and in vitro studies implied a clonidine transport system at the outer BRB that is poorly sensitive to nicotine. It is suggested that OCTs (Slc22a1−3), OCTNs (Slc22A4 and -5), MATE1 (Slc47a1), and PMAT (Slc29a4/ENT4) make only a minor contribution to clonidine transport at the inner BRB since no significant effect on the uptake of [3H]clonidine by TR-iBRB2 cells was found for typical substrates and inhibitors, such as TEA, MPP+, choline, L-carnitine, cimetidine, and serotonin,25,26,35,36 suggesting the contribution of a novel organic cation transporter to the blood-to-retina transport of clonidine across the inner BRB. In the kinetic analysis of inhibitory effect on the uptake of [3H]clonidine by TR-iBRB2 cells, a competitive inhibition manner was exhibited in the presence of verapamil (Ki = 90.4 μM) while no competitive inhibition was observed in the presence of pyrilamine and propranolol (Figure 5a). Previously, the influx transport of verapamil was reported at the inner BRB with a Km value of 61.9 μM,25 and comparison of the Ki and Km values implies the possible identity of the molecules responsible for the blood-toretina transport of verapamil and clonidine at the inner BRB. However, while the transport of clonidine is suggested to be Lcarnitine insensitive and pH sensitive, it has been reported that verapamil is transported in an L-carnitine-sensitive and pHinsensitive manner at the inner BRB,25 which suggests that the transport of clonidine and verapamil is different at the inner BRB. To date, we have proposed the transport systems for cationic drugs at the inner BRB,23−26 and their physiological meaning at the BRB remains unclear. The clarification of whole picture for organic cation transports at the BRB can have an impact on the efficient drug delivery to the retina and is expected to be elucidated through the molecular identification of responsible transporters. In conclusion, the present study will improve our understanding of the blood-to-retina transport of cationic drugs across the BRB. The in vivo study suggests the blood-to-retina transport of clonidine at the BRB, and the uptake study with an in vitro model cell line suggests the involvement of a carrier-
significantly different from the Vmax value of the control (p < 0.05; Figures 5b and 5c).
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DISCUSSION Some cationic drugs exert a neuroprotective effect,3−10 and the systemic administration of clonidine has been reported to reduce the loss of neurons in the injured retina,12 suggesting its possible use for the treatment of retinal diseases. Our previous reports have suggested the involvement of carrier-mediated transport systems in the blood-to-retina transport of cationic drugs, such as verapamil, pyrilamine, and propranolol, across the inner BRB, and the in vitro inhibition study implied that the putative transport of clonidine at the inner BRB is different from the transport systems for verapamil, pyrilamine, and propranolol,23−26 suggesting that the clarification of clonidine transport across the BRB will lead to the efficient and safe treatment of retinal diseases. In this study, the blood-to-retina transport of clonidine was investigated using in vivo and in vitro analyses, and the results obtained strongly suggest the carriermediated blood-to-retina transport of clonidine at the inner BRB. In the in vivo study in rats, integration plot analysis revealed the transport of clonidine to the retina from the circulating blood across the BRB since [3H]clonidine transport exhibited a Kin,retina value of 457 μL/(min·g retina) which is greater than that of D-mannitol, a nonpermeable paracellular marker (Figure 1).33 The RUI study of [3H]clonidine revealed an approximately 31-fold greater RUI value (359%) than that of [3H]-Dmannitol (11.6%),34 and suggests the involvement of blood-toretina transport in the retinal uptake of clonidine at the BRB. The in vivo inhibition study suggests that clonidine transport at the BRB is pyrilamine and propranolol sensitive because of the significant inhibitory effects exhibited by pyrilamine and propranolol (Table 1). In addition, it would appear that the transport of clonidine across the BRB is verapamil sensitive since inhibition was exhibited by verapamil as low as 3 mM (Table 1). The in vivo study suggested that blood-to-retina transport of clonidine at the BRB involves two barrier structures, the inner BRB and the outer BRB. In particular, the inner BRB nourishes two-thirds of the retina, and an in vitro uptake study with TRiBRB2 cells was carried out to investigate the function of the blood-to-retina transport of clonidine at the inner BRB. In the cellular uptake study, the involvement of carrier-mediated transport is suggested in the blood-to-retina transport of clonidine at the inner BRB since TR-iBRB2 cells exhibited temperature- and concentration-dependent [3H]clonidine uptake with a Km value of 286 μM (Figure 2a, Figure 4). pH-sensitive clonidine transport at the inner BRB is suggested in the study of ion dependence since TR-iBRB2 cells exhibited a significant decrease and increase in [3H]clonidine uptake at an extracellular condition of pH 6.4 and pH 8.4, respectively (Figure 3a). This is also supported by the result that the uptake of [3H]clonidine was significantly altered by changes in intracellular pH (Figure 3b). Na+ independent and membrane potential independent transport of clonidine at the inner BRB is suggested since choline replacements and K+ replacements had no significant effect on the uptake of [3H]clonidine by TRiBRB2 cells (Figure 2b). These results suggest the involvement of a pH-sensitive carrier-mediated transport system, including H+/organic cation antiporter, in the blood-to-retina transport of clonidine at the inner BRB. 3751
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(10) Maier, C.; Steinberg, G. K.; Sun, G. H.; Zhi, G. T.; Maze, M. Neuroprotection by the alpha 2-adrenoreceptor agonist dexmedetomidine in a focal model of cerebral ischemia. Anesthesiology 1993, 79 (2), 306−312. (11) Gavras, I.; Manolis, A. J.; Gavras, H. The alpha2-adrenergic receptors in hypertension and heart failure: experimental and clinical studies. J. Hypertens. 2001, 19 (12), 2115−2124. (12) Yoles, E.; Wheeler, L. A.; Schwartz, M. Alpha2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration. Invest. Ophthalmol. Visual Sci. 1999, 40 (1), 65−73. (13) Fischer, W.; Metzner, L.; Hoffmann, K.; Neubert, R. H.; Brandsch, M. Substrate specificity and mechanism of the intestinal clonidine uptake by Caco-2 cells. Pharm. Res. 2006, 23 (1), 131−137. (14) Müller, J.; Neubert, R.; Brandsch, M. Transport of clonidine at cultured epithelial cells (JEG-3) of the human placenta. Pharm. Res. 2004, 21 (4), 692−694. (15) André, P.; Debray, M.; Scherrmann, J. M.; Cisternino, S. Clonidine transport at the mouse blood-brain barrier by a new H+ antiporter that interacts with addictive drugs. J. Cereb. Blood Flow Metab. 2009, 29 (7), 1293−1304. (16) Grafe, F.; Wohlrab, W.; Neubert, R.; Brandsch, M. Carriermediated transport of clonidine in human keratinocytes. Eur. J. Pharm. Sci. 2004, 21 (2−3), 309−312. (17) Cunha-Vaz, J. G. The blood−retinal barriers system. Basic concepts and clinical evaluation. Exp. Eye Res. 2004, 78 (3), 715−721. (18) Stewart, P. A.; Tuor, U. I. 1994. Blood−eye barriers in the rat: Correlation of ultrastructure with function. J. Comp. Neurol. 1994, 340 (4), 566−576. (19) Takata, K.; Kasahara, T.; Kasahara, M.; Ezaki, O.; Hirano, H. Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in cells of the blood-retinal barrier in the rat. Invest. Ophthalmol. Visual Sci. 1992, 33 (2), 377−383. (20) Matsuyama, R.; Tomi, M.; Akanuma, S.; Tabuchi, A.; Kubo, Y.; Tachikawa, M.; Hosoya, K. Up-regulation of L-type amino acid transporter 1 (LAT1) in cultured rat retinal capillary endothelial cells in response to glucose deprivation. Drug Metab. Pharmacokinet. 2012, 27 (3), 317−324. (21) Tomi, M.; Mori, M.; Tachikawa, M.; Katayama, K.; Terasaki, T.; Hosoya, K. L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier. Invest. Ophthalmol. Visual Sci. 2005, 46 (7), 2522−2530. (22) Ohkura, Y.; Akanuma, S.; Tachikawa, M.; Hosoya, K. Blood-toretina transport of biotin via Na+-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier. Exp. Eye Res. 2010, 91 (3), 387−392. (23) Hosoya, K.; Yamamoto, A.; Akanuma, S.; Tachikawa, M. Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability. Pharm. Res. 2010, 27 (12), 2715−2724. (24) Kubo, Y.; Fukui, E.; Akanuma, S.; Tachikawa, M.; Hosoya, K. Application of membrane permeability evaluated in in vitro analyses to estimate blood-retinal barrier permeability. J. Pharm. Sci. 2012, 101 (7), 2596−2605. (25) Kubo, Y.; Kusagawa, Y.; Tachikawa, M.; Akanuma, S.; Hosoya, K. Involvement of a novel organic cation transporter in verapamil transport across the inner blood-retinal barrier. Pharm. Res. 2013, 30 (3), 847−856. (26) Kubo, Y.; Shimizu, Y.; Kusagawa, Y.; Akanuma, S.; Hosoya, K. Propranolol transport across the inner blood-retinal barrier: potential involvement of a novel organic cation transporter. J. Pharm. Sci. 2013, 102 (9), 3332−3342. (27) Hosoya, K.; Tomi, M.; Ohtsuki, S.; Takanaga, H.; Ueda, M.; Yanai, N.; Obinata, M.; Terasaki, T. Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. Exp. Eye Res. 2001, 72 (2), 163−172. (28) Hosoya, K.; Tomi, M. Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions. Biol. Pharm. Bull. 2005, 28 (1), 1−8.
mediated transport system in the blood-to-retina transport of clonidine at the inner BRB. In addition, the in vitro inhibition study suggests the contribution of a novel organic cation transporter, showing its possible usefulness for the systemic delivery of cationic neuroprotectants to the retina.
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ASSOCIATED CONTENT
S Supporting Information *
Definition of the terms of eq 1 and related references. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama, 930-0194, Japan. Tel: +81-76-434-7505. Fax: +81-76-434-5172. E-mail: [email protected]. Author Contributions
Y.K. and A.T. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported in part by Grants-in Aid for Scientific Research (B) [KAKENHI: 25293036] and Scientific Research (C) [KAKENHI: 26460193] from Japan Society for the Promotion of Science (JSPS). We are grateful to Kowa Co., Ltd. (Nagoya, Japan), for providing nipradilol.
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REFERENCES
(1) Hosoya, K.; Tomi, M.; Tachikawa, M. Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier. Expert Opin. Drug Delivery 2011, 8 (12), 1571−1587. (2) Tomi, M.; Hosoya, K. The role of blood-ocular barrier transporters in retinal drug disposition: an overview. Expert Opin. Drug Metab. Toxicol. 2010, 6 (9), 1111−1124. (3) Mizuno, K.; Koide, T.; Yoshimura, M.; Araie, M. Neuroprotective effect and intraocular penetration of nipradilol, a betablocker with nitric oxide donative action. Invest. Ophthalmol. Visual Sci. 2001, 42 (3), 688−694. (4) Rojas, J. C.; Saavedra, J. A.; Gonzalez-Lima, F. Neuroprotective effects of memantine in a mouse model of retinal degeneration induced by rotenone. Brain Res. 2008, 1215, 208−217. (5) Albino Teixeira, A.; Azevedo, I.; Branco, D.; Rodrigues-Pereira, E.; Osswald, W. Sympathetic denervation caused by long-term noradrenaline infusions; prevention by desipramine and superoxide dismutase. Br. J. Pharmacol. 1989, 97 (1), 95−102. (6) Ono, Y.; Shimazawa, M.; Ishisaka, M.; Oyagi, A.; Tsuruma, K.; Hara, H. Imipramine protects mouse hippocampus against tunicamycin-induced cell death. Eur. J. Pharmacol. 2012, 696 (1−3), 83−88. (7) Wheeler, L. A.; Lai, R.; Woldemussie, E. From the lab to the clinic: activation of an alpha-2 agonist pathway is neuroprotective in models of retinal and optic nerve injury. Eur. J. Ophthalmol. 1999, No. Suppl. 1, 17−21. (8) Berkman, M. Z.; Zirh, T. A.; Berkman, K.; Pamir, M. N. Tizanidine is an effective agent in the prevention of focal cerebral ischemia in rats: an experimental study. Surg. Neurol. 1998, 50 (3), 264−270. (9) Kuhmonen, J.; Pokorný, J.; Miettinen, R.; Haapalinna, A.; Jolkkonen, J.; Riekkinen, P., Sr.; Sivenius, J. Neuroprotective effects of dexmedetomidine in the gerbil hippocampus after transient global ischemia. Anesthesiology 1997, 87 (2), 371−377. 3752
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(29) Alm, A.; Törnquist, P. The uptake index method applied to studies on the blood-retinal barrier. I. A methodological study. Acta Physiol. Scand. 1981, 113 (1), 73−79. (30) Pardridge, W. M.; Fierer, G. Blood-brain barrier transport of butanol and water relative to N-isopropyl-p-iodoamphetamine as the internal reference. J. Cereb. Blood Flow Metab. 1985, 5 (2), 275−281. (31) Kubo, Y.; Tomise, A.; Tsuchiyama, A.; Akanuma, S. I.; Hosoya, K. I. Involvement of the carrier-mediated process in the retina-toblood transport of spermine at the inner blood-retinal barrier. Exp. Eye Res. 2014, 124, 17−23. (32) Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. A pharmacokinetic analysis program (multi) for microcomputer. J. Pharmacobiodyn. 1981, 4 (11), 879−885. (33) Tachikawa, M.; Takeda, Y.; Tomi, M.; Hosoya, K. Involvement of OCTN2 in the transport of acetyl-L-carnitine across the inner blood-retinal barrier. Invest. Ophthalmol. Visual Sci. 2010, 51 (1), 430− 436. (34) Nagase, K.; Tomi, M.; Tachikawa, M.; Hosoya, K. Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier. Biochim. Biophys. Acta 2006, 1758 (1), 13−19. (35) Okura, T.; Higuchi, K.; Kitamura, A.; Deguchi, Y. Protoncoupled organic cation antiporter-mediated uptake of apomorphine enantiomers in human brain capillary endothelial cell line hCMEC/ D3. Biol. Pharm. Bull. 2014, 37 (2), 286−291. (36) Okura, T.; Hattori, A.; Takano, Y.; Sato, T.; HammarlundUdenaes, M.; Terasaki, T.; Deguchi, Y. Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone. Drug Metab. Dispos. 2008, 36 (10), 2005−13.
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Involvement of Carrier-Mediated Transport in the Retinal Uptake of Clonidine at the Inner Blood−Retinal Barrier Yoshiyuki Kubo, Ai Tsuchiyama, Yoshimi Shimizu, Shin-ichi Akanuma, and Ken-ichi Hosoya* Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan S Supporting Information *
ABSTRACT: In the present study, the blood-to-retina transport across the inner BRB was investigated for clonidine, a compound which is expected to exhibit a neuroprotective effect for the treatment of severe retinal diseases. In the in vivo study, the integration plot analysis for [3H]clonidine exhibited an apparent influx permeability clearance of 457 μL/(min·g retina) in the retina. The in vivo inhibition study suggests that the blood-to-retina transport of clonidine at the BRB is organic cation-sensitive since clonidine, pyrilamine, and propranolol, at a concentration of 40 mM, significantly reduced the retinal uptake index (RUI) of [3H]clonidine, and an inhibitory effect on the RUI was also exhibited by verapamil, at a concentration of 3 mM. The in vitro study with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests that carrier-mediated transport is involved in the blood-to-retina transport of clonidine at the inner BRB since the results obtained demonstrated time-, temperature-, pH-, and concentration-dependent [3H]clonidine uptake, with a Km of 286 μM. In the in vitro inhibition study, the [3H]clonidine uptake was significantly reduced by several organic cations, such as clonidine, verapamil, pyrilamine, and propranolol, and was competitively inhibited by 200 μM verapamil, in spite of slight or no significant alteration being produced with organic anions. Furthermore, the typical substrates and inhibitors of well-known organic cation transporters had no significant effect on the uptake of [3H]clonidine to suggest the involvement of novel transporter molecules in the transport of clonidine across the inner BRB. These results suggest that the blood-to-retina transport of clonidine across the inner BRB involves a carrier-mediated transport manner, suggesting the contribution of a novel organic cation transporter expressed by the retinal capillary endothelial cells. KEYWORDS: inner blood−retinal barrier, organic cation transport, clonidine, transporter
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INTRODUCTION The progression of retinal diseases, such as diabetic retinopathy and macular degeneration, is known to cause severe neurological dysfunction in the retina, leading to blindness that dramatically affects the quality of life of patients. Studies of neurological dysfunction have shown that a neuroprotective effect is exerted by several cationic drugs, such as desipramine, nipradilol, imipramine, and memantine,1−6 and the neuroprotective effect of several cationic α2 agonists, such as brimonidin, dexmedetomidine and tizanidine, has been reported in cerebral ischemia or optic nerve injury,7−12 suggesting their usefulness in the treatment of the neurological dysfunction. Recently, a neuroprotective effect was also suggested for clonidine, a cationic α2 agonist used to treat essential and renal hypertensions, and it has been reported that the loss of neurons in the rat partially injured optic nerve system is suppressed by the intraperitoneal administration of clonidine,11,12 suggesting the potential usefulness of clonidine for the future treatment of retinal diseases. In the development of drugs to treat retinal diseases, it is essential to improve our understanding of the systems involved in transport from the circulating blood to the retina since their advances will contribute to the efficient and safe delivery of drugs to the retina across the blood−retinal barrier (BRB).1,2 © 2014 American Chemical Society
However, little is known about the transport of clonidine at the BRB, while several reports have suggested the involvement of carrier-mediated transport processes in the uptake of clonidine by placental epithelial cells, brain capillary endothelial cells, and keratinocytes.13−16 In the retina, the retinal capillary endothelial cells and the retinal pigment epithelial cells are responsible for the inner BRB and outer BRB, respectively, and these barrier structures separate the circulating blood and the neural retina. At the BRB, efficient nutrient supply is performed by blood-toretina transport mediated by various membrane transporters during the paracellular transport suppressed by tight junction.1,2,17,18 In particular, it is known that the inner BRB nourishes two-thirds of the retina, and the recent advances in research involving the inner BRB suggest that the transport of nutrients, such as sugar, amino acids, and vitamins, is carried out by glucose transporter (GLUT1/Slc2a1), L (leucinereferring) amino acid transporter 1 (LAT1/Slc7a5), and Na+dependent multivitamin transporter (SMVT/Slc5a6), respectively.19−22 Received: Revised: Accepted: Published: 3747
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Regarding cationic drugs, our previous studies using in vivo and in vitro analyses have demonstrated the blood-to-retina transport of verapamil, pyrilamine, and propranolol across the BRB, and suggested the involvement of novel organic cation transporters at the inner BRB.23−26 In the in vivo inhibition study, these transport systems were significantly inhibited in the presence of several cationic neuroprotectants, showing their possible contribution to the transport of cationic neuroprotectants at the inner BRB.25,26 However, it has been exceptionally implied that the putative transport of clonidine at the inner BRB involves a transport system distinct from those for verapamil, pyrilamine, and propranolol, since the inhibitory potential of clonidine is much greater than that predicted from the lipophilicity−inhibitory effect relationship developed in the in vitro inhibition study of propranolol transport.26 This cumulative evidence suggests that the elucidation of the transport system for clonidine at the BRB will lead to the safe and effective treatment of retinal diseases. In the present study, the blood-to-retinal transport of clonidine was investigated by in vivo vascular injection method and an in vitro uptake study with an in vitro model cell line of retinal capillary endothelial cells, TR-iBRB2 cells, the transport of which is reported to be correlated with in vivo BRB permeability.24,27,28
RUI = ([3H]/[14C] (dpm in the retina)) /([3H]/[14C] (dpm in the injectate solution)) × 100
Cellular Uptake Study. TR-iBRB2 cells, a conditionally immortalized rat capillary endothelial cell line, were used in the cellular uptake study. TR-iBRB2 cells have been used as an in vitro tool in studies of the inner BRB because of their similar cell morphology and gene expression to retinal capillary endothelial cells.24,27,28 The cells (passage number 23−35) were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals, Tokyo, Japan) containing 10% fetal bovine serum, 20 mM NaHCO3, and antibiotics at 33 °C to allow temperature-sensitive large T-antigen expression as described elsewhere.27,28 In the uptake study, as described previously, cells were rinsed with extracellular fluid (ECF)-buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 10 mM D-glucose, and 10 mM HEPES (pH 7.4)) and incubated in 200 μL of ECF-buffer containing 12 nM [3H]clonidine (0.1 μCi) at 37 °C. The Na+ dependence, membrane potential dependence, and pH dependence of [3H]clonidine uptake were studied with special buffers, such as Na+-free ECF buffers, as previously reported.31 Radioactivity measurements were performed using a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical) after the cells were lysed with 1 N NaOH and neutralized with 1 N HCl, respectively. The cell-to-medium ratio was used to express the [3H]clonidine uptake by TR-iBRB2 cells, in the data analysis using eq 3, and the protein content of the cells was measured with a DC protein assay kit (Bio-Rad, Hercules, CA).
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MATERIALS AND METHODS Reagents. Commercially available chemicals of reagent grade were used in the present study. n-[1-14C]Butanol ([14C]n-butanol, 2 mCi/mmol) and [benzene ring-3H]clonidine hydrochloride ([3H]clonidine, 57.8 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO) and PerkinElmer (Boston, MA), respectively. Animals. The animal experiments were designed in accordance with the institutional guidelines established by the Animal Care Committee of the University of Toyama, which approved the protocol for the animal experiments of this study. In addition, the animals were treated in accordance with the statement issued by the Association for Research in Vision and Ophthalmology (ARVO). Male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan). In Vivo Blood-to-Tissue Transport Study. Integration plot was used to estimate the apparent influx permeability clearance (Kin) of [3H]clonidine in the retina, as reported previously.22 In brief, [3H]clonidine (3 μCi/rat) was injected into the femoral vein of anesthetized rats, and blood samples were collected at designated times. The retinas were collected after decapitation of the rats, and lysed and then neutralized with 2 N NaOH and 2 N HCl, respectively. For the radioactivity measurements, a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan) was used, and the Kin of [3H]clonidine was estimated using eq 1; the details have been described previously (Supporting Information).22 Vd(t ) = K in,retina × AUC(t )/Cp(t ) + Vi
(2)
Cell‐to‐medium ratio = ([3H] dpm per cell protein (mg)) /([3H] dpm per μL of medium)
(3)
MULTI, the nonlinear least-squares regression analysis program, was used in the kinetic analysis as described previously,22,31,32 and the data obtained in the uptake study were fitted to eq 4, where C, V, Km, and Vmax are the substrate concentration, the uptake rate, the Michaelis constant, and the maximal uptake rate, respectively. V = Vmax × C /(K m + C)
(4)
MULTI was also used in the kinetic analysis for the inhibition of clonidine uptake involving eq 5, where Ki and I are the inhibitory constant and the concentration of inhibitors, respectively. V = Vmax × C /[K m × (1 + I /K i) + C)]
(5)
Statistical Analysis. Unless otherwise indicated, data represent means ± SEM except for kinetic parameters, and the kinetic parameter data represent means ± SD. In the assessment of statistical differences, a one way analysis of variance (ANOVA) followed by the modified Fisher’s leastsignificant difference method was used for several groups, and an unpaired two-tailed Student’s t-test was used for two groups.
(1)
To evaluate the in vivo effect of inhibitors, the retinal uptake index (RUI) in rats was measured by injecting 200 μL of Ringer-HEPES buffer containing [3H]clonidine (2.5 μCi, 220 nM) and [14C]-n-butanol (0.5 μCi).22,29,30 The RUI was calculated using eq 2, and the values reflect the fractional uptake of [3H]clonidine as a percentage of the fractional uptake of [14C]-n-butanol.
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RESULTS Blood-to-Retina Transport of [3H]Clonidine across the BRB. Integration plot analysis of [3H]clonidine was carried out to investigate the in vivo blood-to-retina transport of clonidine 3748
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across the BRB. Analysis of the [3H]clonidine data using eq 1 showed that the apparent influx permeability clearance, Kin,retina, was 457 ± 130 μL/(min·g retina) (Figure 1), while the Kin,retina
2a). In the study of ion dependence, Na+- and K+-free buffers had no significant effect on the uptake of [3H]clonidine by TR-
Figure 1. Initial uptake of [3H]clonidine by the retina in rats. In the integration plot analysis, [3H]clonidine (3 μCi/rat) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3).
Figure 2. Uptake of [3H]clonidine (0.1 μCi, 12 nM) by TR-iBRB2 cells. (a) Time dependence and temperature dependence of [3H]clonidine uptake by TR-iBRB2 cells were investigated at 37 °C (closed circles) and 4 °C (open circle), respectively. (b) The effects of Na+ and membrane potential on the uptake of [3H]clonidine were examined at 37 °C for 15 s. Each column and point represent the mean ± SEM (n = 3). *p < 0.01, significantly different from the control.
for [3H]-D-mannitol, a marker of paracellular solute transport, was 0.626 μL/(min·g retina).33 In particular, the Kin,retina for clonidine was 730-fold greater than that of [3H]-D-mannitol, suggesting the influx transport of clonidine across the BRB. A carotid artery injection showed that the value of RUI for [3H]clonidine was 359 ± 77% (Table 1), and this was greater
iBRB2 cells (Figure 2b). The study carried out under different extracellular pH conditions revealed a 57% decrease and a 47% increase in [3H]clonidine uptake at pH 6.4 and pH 8.4, respectively (Figure 3a), and the study of the effect of
Table 1. In Vivo Uptake Study of [3H]Clonidine by the Retina in Ratsa inhibitors control clonidine pyrilamine propranolol verapamil nicotine TEA PAH probenecid
concentration (mM) 40 40 40 3 40 40 40 3
RUI (%)
% of control
± ± ± ± ± ± ± ± ±
100 ± 21 54.1 ± 5.9* 37.2 ± 4.4* 41.5 ± 10.2* 64.5 ± 5.9 74.3 ± 2.8 91.3 ± 7.8 100 ± 3 107 ± 9
359 185 134 149 232 267 328 360 386
77 21* 16* 37* 21 10 28 9 33
[ H]Clonidine (2.5 μCi/rat) and [14C]-n-butanol (0.5 μCi/rat) were injected with or without inhibitors. Decapitation was carried out 15 s after injection. 3 mM concentration of verapamil and probenecid were set because of its limitation of solubility. Each value represents means ± SEM (n = 3−5). *p < 0.01, significantly different from the control. TEA, tetraethylammonium. PAH, p-aminohippuric acid. a 3
Figure 3. Effect of pH on the uptake of [3H]clonidine (0.1 μCi, 12 nM) by TR-iBRB2 cells. The effects of extracellular pH (a) and intracellular pH (b) were examined at 37 °C for 15 s. Intracellular pH was increased and decreased by acute treatment and pretreatment (pre) of cells with 30 mM NH4Cl, respectively. Each column represents the mean ± SEM (n = 3). *p < 0.01, significantly different from the control (pH 7.4).
than that for [3H]-D-mannitol reported in a previous study,34 also supporting the influx transport of clonidine across the BRB. The RUI value of [3H]clonidine was significantly reduced by more than 30% in the presence of 40 mM clonidine, 40 mM pyrilamine, and 40 mM propranolol, and the tendency of inhibition (more than 25% reduction) was also exhibited by 3 mM verapamil and 40 mM nicotine, while no significant effect was exhibited in the presence of 40 mM tetraethylammonium (TEA), 40 mM p-aminohippuric acid (PAH), and 3 mM probenecid (Table 1). Uptake of [3H]Clonidine by TR-iBRB2 Cells. The in vitro uptake study was carried out using TR-iBRB2 cells, an in vitro model of the inner BRB, to examine the influx transport of clonidine across the inner BRB. TR-iBRB2 cells showed a timedependent increase of [3H]clonidine for 15 s at least, with an initial uptake rate of 1.99 ± 0.30 μL/(min·mg protein), while the uptake was significantly reduced by 31% at 4 °C (Figure
intracellular pH revealed a 90% decrease and a 70% increase in [3H]clonidine uptake at an alkaline (acute) and acidic (pretreatment) intracellular pH, respectively (Figure 3b). As clonidine is a cationic drug with a pKa of 8.05, the ratios of uncharged clonidine were estimated to be 2%, 18%, and 69% under the extracellular condition of pH 6.4, pH 7.4, and pH 8.4, respectively. This indicated a much smaller change in clonidine uptake than that of the uncharged clonidine (Figure 3), showing that passive diffusion is not sufficient to explain the alteration of clonidine uptake under different pH conditions. The TR-iBRB2 cells exhibited concentration-dependent uptake of [3H]clonidine (Figure 4a), and the Eadie−Scatchard plot showed that a one saturable process model fits the uptake data of clonidine with Km and Vmax values of 286 ± 44 μM and 43.7 ± 4.7 nmol/(min·mg protein), respectively (Figure 4b). These 3749
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(by more than 61%) of [3H]clonidine uptake was found in the presence of amantadine and timolol, although no significant effect was observed in the presence of typical organic cations, such as TEA, MPP+, choline, L-carnitine, cimetidine, serotonin, and putrescine (Table 2). Regarding organic anions, acetylsalicylic acid and diclofenac exhibited no significant effect on [3H]clonidine uptake by TR-iBRB2 cells, and a significant but weak effect was observed in the presence of PAH (Table 2). Kinetic Inhibition Analysis of [3H]Clonidine Uptake by TR-iBRB2 Cells. The inhibitory effect on the clonidine uptake by TR-iBRB2 cells was kinetically investigated. Clonidine uptake, with or without 200 μM verapamil, exhibited very similar y-intercepts corresponding to Vmax values of 48.1 ± 14.1 and 43.7 ± 4.7 nmol/(min·mg protein), respectively (p = 0.417; Figure 5a), suggesting the competitive inhibitory effect of verapamil on clonidine uptake with a Ki of 90.4 ± 46.8 μM. Clonidine uptake with pyrilamine and propranolol at a concentration of 200 μM exhibited Vmax values of 89.2 ± 51.1 and 82.6 ± 23.3 nmol/(min·mg protein), which were
Figure 4. Concentration dependence of [3H]clonidine uptake by TRiBRB2 cells. The uptake was investigated at 37 °C for 15 s, over the concentration range 10−1000 μM. Michaelis−Menten (a) and Eadie− Scatchard (b) plots were used to analyze the data obtained. Each point represents the mean ± SEM (n = 3).
results suggest that a carrier-mediated transport system contributes to the uptake of [3H]clonidine by TR-iBRB2 cells. Inhibition Study of [3H]Clonidine Uptake by TR-iBRB2 Cells. The inhibitory effects on [3H]clonidine uptake by TRiBRB2 cells were investigated using a variety of compounds at a concentration of 1 mM, and the results obtained are summarized in Table 2. Marked inhibition (by more than 81%) of [3H]clonidine uptake was exhibited in the presence of organic cations, such as desipramine, propranolol, nicotine, nipradilol, mecamylamine, verapamil, imipramine, memantine, clonidine, and quinidine (Table 2), while moderate inhibition Table 2. Inhibitory Effects on [3H]Clonidine Uptake by TRiBRB2 Cellsa compound
% of control
control desipramine propranolol nicotine nipradilol mecamylamine pyrilamine verapamil imipramine memantine clonidine quinidine amantadine timolol TEA MPP+ choline L-carnitine cimetidine serotonin PAH putrescine acetylsalicylic acid diclofenac
100 ± 5 3.61 ± 0.42* 7.50 ± 1.20* 8.49 ± 0.41* 10.1 ± 1.1* 10.5 ± 0.5* 12.0 ± 0.2* 14.6 ± 2.3* 16.0 ± 2.3* 17.3 ± 1.8* 17.9 ± 1.3* 19.0 ± 2.5* 33.0 ± 1.6* 38.6 ± 3.2* 86.8 ± 4.9 93.3 ± 2.0 95.1 ± 11.6 95.5 ± 6.5 98.4 ± 4.6 101 ± 8 83.1 ± 1.3* 108 ± 4 109 ± 8 115 ± 3
[ H]Clonidine (12 nM, 0.1 μCi) uptake by TR-iBRB2 cells was performed in the absence or presence of 1 mM compounds at 37 °C for 15 s. Each value represents the means ± SEM (n = 3−9). *p < 0.01, significantly different from the control. TEA, tetraethylammonium. PAH, p-aminohippuric acid. MPP+, 1-methyl-4-phenylpyridinium. a 3
Figure 5. Lineweaver−Burk plot of clonidine uptake by TR-iBRB2 cells. [3H]Clonidine uptake was studied with (closed circles) or without (open circles) 200 μM verapamil (a), pyrilamine (b), and propranolol (c). The uptake was examined at 37 °C for 15 s. Each point represents the mean ± SEM (n = 3). 3750
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The results of the in vitro inhibition study support the inhibitory profile obtained in the in vivo inhibition study of clonidine transport at the BRB, and they suggest the contribution of an organic cation transporter to the blood-toretina transport of clonidine at the inner BRB since organic cations, such as desipramine, propranolol, nicotine, nipradilol, mecamylamine, verapamil, imipramine, memantine, clonidine, quinidine, amantadine, and timolol, markedly inhibited the uptake of [3H]clonidine by TR-iBRB2 cells. This is also supported by weak or insignificant effects exhibited by anions, such as acetylsalicylic acid, diclofenac, and PAH (Table 2). Based on the significant inhibitory effects of desipramine, nipradilol, imipramine, and memantine (Table 2), it appears that the contribution of the clonidine transport system also has a significant effect on the retinal uptake of cationic neuroprotectants.3−6 In addition, the inhibitory profile given in Table 2 suggests that different molecules are involved in clonidine transport at the BBB and inner BRB since the verapamil- and desipramine-sensitive transport of clonidine was found at the inner BRB while the study using the in situ mouse brain perfusion method suggested that the BBB involves the verapamil- and desipramine-insensitive transport of clonidine.15 Furthermore, nicotine showed the significant inhibitory effect on the uptake of [3H]clonidine by TR-iBRB2 cells in spite of its tendency of inhibition in the in vivo study (Table 1 and Table 2), and the different results obtained in the in vivo and in vitro studies implied a clonidine transport system at the outer BRB that is poorly sensitive to nicotine. It is suggested that OCTs (Slc22a1−3), OCTNs (Slc22A4 and -5), MATE1 (Slc47a1), and PMAT (Slc29a4/ENT4) make only a minor contribution to clonidine transport at the inner BRB since no significant effect on the uptake of [3H]clonidine by TR-iBRB2 cells was found for typical substrates and inhibitors, such as TEA, MPP+, choline, L-carnitine, cimetidine, and serotonin,25,26,35,36 suggesting the contribution of a novel organic cation transporter to the blood-to-retina transport of clonidine across the inner BRB. In the kinetic analysis of inhibitory effect on the uptake of [3H]clonidine by TR-iBRB2 cells, a competitive inhibition manner was exhibited in the presence of verapamil (Ki = 90.4 μM) while no competitive inhibition was observed in the presence of pyrilamine and propranolol (Figure 5a). Previously, the influx transport of verapamil was reported at the inner BRB with a Km value of 61.9 μM,25 and comparison of the Ki and Km values implies the possible identity of the molecules responsible for the blood-toretina transport of verapamil and clonidine at the inner BRB. However, while the transport of clonidine is suggested to be Lcarnitine insensitive and pH sensitive, it has been reported that verapamil is transported in an L-carnitine-sensitive and pHinsensitive manner at the inner BRB,25 which suggests that the transport of clonidine and verapamil is different at the inner BRB. To date, we have proposed the transport systems for cationic drugs at the inner BRB,23−26 and their physiological meaning at the BRB remains unclear. The clarification of whole picture for organic cation transports at the BRB can have an impact on the efficient drug delivery to the retina and is expected to be elucidated through the molecular identification of responsible transporters. In conclusion, the present study will improve our understanding of the blood-to-retina transport of cationic drugs across the BRB. The in vivo study suggests the blood-to-retina transport of clonidine at the BRB, and the uptake study with an in vitro model cell line suggests the involvement of a carrier-
significantly different from the Vmax value of the control (p < 0.05; Figures 5b and 5c).
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DISCUSSION Some cationic drugs exert a neuroprotective effect,3−10 and the systemic administration of clonidine has been reported to reduce the loss of neurons in the injured retina,12 suggesting its possible use for the treatment of retinal diseases. Our previous reports have suggested the involvement of carrier-mediated transport systems in the blood-to-retina transport of cationic drugs, such as verapamil, pyrilamine, and propranolol, across the inner BRB, and the in vitro inhibition study implied that the putative transport of clonidine at the inner BRB is different from the transport systems for verapamil, pyrilamine, and propranolol,23−26 suggesting that the clarification of clonidine transport across the BRB will lead to the efficient and safe treatment of retinal diseases. In this study, the blood-to-retina transport of clonidine was investigated using in vivo and in vitro analyses, and the results obtained strongly suggest the carriermediated blood-to-retina transport of clonidine at the inner BRB. In the in vivo study in rats, integration plot analysis revealed the transport of clonidine to the retina from the circulating blood across the BRB since [3H]clonidine transport exhibited a Kin,retina value of 457 μL/(min·g retina) which is greater than that of D-mannitol, a nonpermeable paracellular marker (Figure 1).33 The RUI study of [3H]clonidine revealed an approximately 31-fold greater RUI value (359%) than that of [3H]-Dmannitol (11.6%),34 and suggests the involvement of blood-toretina transport in the retinal uptake of clonidine at the BRB. The in vivo inhibition study suggests that clonidine transport at the BRB is pyrilamine and propranolol sensitive because of the significant inhibitory effects exhibited by pyrilamine and propranolol (Table 1). In addition, it would appear that the transport of clonidine across the BRB is verapamil sensitive since inhibition was exhibited by verapamil as low as 3 mM (Table 1). The in vivo study suggested that blood-to-retina transport of clonidine at the BRB involves two barrier structures, the inner BRB and the outer BRB. In particular, the inner BRB nourishes two-thirds of the retina, and an in vitro uptake study with TRiBRB2 cells was carried out to investigate the function of the blood-to-retina transport of clonidine at the inner BRB. In the cellular uptake study, the involvement of carrier-mediated transport is suggested in the blood-to-retina transport of clonidine at the inner BRB since TR-iBRB2 cells exhibited temperature- and concentration-dependent [3H]clonidine uptake with a Km value of 286 μM (Figure 2a, Figure 4). pH-sensitive clonidine transport at the inner BRB is suggested in the study of ion dependence since TR-iBRB2 cells exhibited a significant decrease and increase in [3H]clonidine uptake at an extracellular condition of pH 6.4 and pH 8.4, respectively (Figure 3a). This is also supported by the result that the uptake of [3H]clonidine was significantly altered by changes in intracellular pH (Figure 3b). Na+ independent and membrane potential independent transport of clonidine at the inner BRB is suggested since choline replacements and K+ replacements had no significant effect on the uptake of [3H]clonidine by TRiBRB2 cells (Figure 2b). These results suggest the involvement of a pH-sensitive carrier-mediated transport system, including H+/organic cation antiporter, in the blood-to-retina transport of clonidine at the inner BRB. 3751
dx.doi.org/10.1021/mp500516j | Mol. Pharmaceutics 2014, 11, 3747−3753
Molecular Pharmaceutics
Article
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mediated transport system in the blood-to-retina transport of clonidine at the inner BRB. In addition, the in vitro inhibition study suggests the contribution of a novel organic cation transporter, showing its possible usefulness for the systemic delivery of cationic neuroprotectants to the retina.
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ASSOCIATED CONTENT
S Supporting Information *
Definition of the terms of eq 1 and related references. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama, 930-0194, Japan. Tel: +81-76-434-7505. Fax: +81-76-434-5172. E-mail: [email protected]. Author Contributions
Y.K. and A.T. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported in part by Grants-in Aid for Scientific Research (B) [KAKENHI: 25293036] and Scientific Research (C) [KAKENHI: 26460193] from Japan Society for the Promotion of Science (JSPS). We are grateful to Kowa Co., Ltd. (Nagoya, Japan), for providing nipradilol.
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