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Involvement of the Pyrilamine Transporter, a Putative Organic Cation Transporter, in Blood-Brain Barrier Transport of Oxycodone

Department of Drug Disposition and Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, Sagamihara, Japan (T.O., A.H., Y.T., T.S., Y.D.); Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden (M.H.-U.); and Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (T.T.)

(Received April 25, 2008; Accepted July 3, 2008)

	Abstract

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The purpose of this study was to characterize blood-brain barrier (BBB) transport of oxycodone, a cationic opioid agonist, via the pyrilamine transporter, a putative organic cation transporter, using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13). Oxycodone and [³H]pyrilamine were both transported into TR-BBB13 cells in a temperature- and concentration-dependent manner with K_m values of 89 and 28 µM, respectively. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa. Furthermore, mutual uptake inhibition by oxycodone and pyrilamine suggests that a common mechanism is involved in their transport. Transport of both substrates was inhibited by type II cations (quinidine, verapamil, and amantadine), but not by classic organic cation transporter (OCT) substrates and/or inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridinium, and corticosterone), substrates of OCTN1 (ergothioneine) and OCTN2 (L-carnitine), or organic anions. The transport was inhibited by metabolic inhibitors (rotenone and sodium azide) but was insensitive to extracellular sodium and membrane potential for both substrates. Furthermore, the transport of both substrates was increased at alkaline extracellular pH and decreased in the presence of a protonophore (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). Intracellular acidification induced with ammonium chloride enhanced the uptakes, suggesting that the transport is driven by an oppositely directed proton gradient. The brain uptake of oxycodone measured by in situ rat brain perfusion was increased in alkaline perfusate and was significantly inhibited by pyrilamine. These results suggest that blood-brain barrier transport of oxycodone is at least partly mediated by a common transporter with pyrilamine, and this transporter is an energy-dependent, proton-coupled antiporter.

In relation to the membrane transport of cationic drugs, at least six distinct transporters, OCT1 (SLC22A1), OCT2 (SLC22A2), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), and MATE1 (SLC47A1), for organic cations are involved in the influx and efflux transport of various cations in the kidney and liver (Nezu et al., 1999; Inui et al., 2000; Koepsell et al., 2003; Otsuka et al., 2005). However, relatively little is known about which transporters are involved in the BBB transport of cationic drugs. Yamazaki et al. (1994a,b,c) demonstrated that pyrilamine, a cationic H₁-antagonist, is transferred from the circulating blood into the brain by an organic cation-sensitive transport system. Recently, we reported that pramipexole, a potent dopamine receptor agonist with high efficacy for Parkinson's disease, is also transported across the BBB via the organic cation-sensitive transporter (Okura et al., 2007). Therefore, the putative pyrilamine transporter has been suggested to be an organic cation transporter at the BBB, although its molecular species remains to be identified.

Oxycodone is widely used as an opioid agonist for treatment of moderate to severe cancer pain. Boström et al. (2006) have recently reported that the steady-state unbound concentration of oxycodone in the brain interstitial fluid is 3-fold higher than that in the plasma, suggesting the existence of an active influx transporter(s) for oxycodone at the BBB. Oxycodone is a weak cationic drug with a partial positive charge at physiological pH, so the pyrilamine transporter may mediate the transport of oxycodone across the BBB.

The purpose of this study, therefore, was to clarify the BBB transport properties of oxycodone across the BBB and the role of the pyrilamine transporter. The transports of oxycodone and pyrilamine were characterized using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13 cells) as an in vitro model of the BBB. Furthermore, in vivo BBB transport of oxycodone was examined by the in situ rat brain perfusion technique. The results obtained in this study should provide useful information for the development and proper use of CNS-acting cationic drugs, as well as for pain therapy with opioids.

	Materials and Methods

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Chemicals. Oxycodone was kindly provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). [³H]Pyrilamine (23–30 Ci/mmol) and [³H]inulin (0.59 Ci/mmol) were purchased from GE Healthcare (Little Chalfont, Buckingshamshire, UK). All other chemicals and reagents were commercial products of reagent grade.

Animals. Adult male Wistar rats weighing approximately 350 g were purchased from Japan SLC (Shizuoka, Japan); they were housed, three or four per cage, in a laboratory with free access to food and water and were maintained on a 12-h dark/12-h light cycle in a room with controlled temperature (24 ± 2°C) and humidity (55 ± 5%). This study was conducted according to guidelines approved by the Experimental Animal Ethical Committee of Teikyo University.

Transport Studies in TR-BBB13 Cells. TR-BBB13 cells were seeded on collagen-coated multiwell dishes at a density of 0.1 x 10⁵ cells/cm². At 3 days after seeding, the cells were washed twice with 1 ml of phosphate-buffered saline and preincubated with 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) for 20 min at 37°C. After preincubation, the buffer (0.25 ml) containing oxycodone (30 µM) or [³H]pyrilamine (74 kBq/µl, 90 nM) 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 collected and homogenized by sonication in 300 µl of water, and the homogenate was stored at –20°C until determination of oxycodone as described below. For the determination of [³H]pyrilamine radioactivity, the cells were solubilized with 1 N NaOH for 60 min. The radioactivity was measured using a liquid scintillation counter after the addition of scintillation cocktail Hionic Fluor (PerkinElmer Life and Analytical Sciences, Boston, MA). Cellular protein content was determined with a BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Uptake was expressed as the cell-to-medium ratio (microliters per milligram of protein) obtained by dividing the uptake amount by the concentration of substrate in the incubation buffer. To estimate the kinetic parameters for the uptake by TR-BBB13 cells, the initial uptake rates for oxycodone (1–500 µM for 15 s) and [³H]pyrilamine (6–500 µM for 10 s) were determined by subtracting the uptakes at the concentration of 5 mM oxycodone and pyrilamine, respectively. The initial uptake rates were fitted to the following equation by means of nonlinear least-squares regression analysis with Prism software (GraphPad Software Inc., San Diego, CA):

where v is the initial uptake rate of substrate (nanomoles per minute per milligram of protein), s is the substrate concentration in the medium (micromolar), K_m is the Michaelis-Menten constant (micromolar concentration), and V_max is the maximum uptake rate (nanomoles per minute per milligram of protein). In the trans-stimulation studies, the cells were preloaded with pyrilamine (250 µM) or oxycodone (1 mM) for 20 min. Cells were then rinsed with 1 ml of incubation medium warmed at 37°C. Subsequently, uptake of oxycodone or [³H]pyrilamine by the cells was measured. In the inhibition study, the uptake was measured after incubation with oxycodone (30 µM) for 15 s or [³H]pyrilamine (74 kBq/µl, 90 nM) for 10 s, respectively, in the presence of several inhibitors. After treatment with 25 µM rotenone or 0.1% NaN₃ for 20 min to reduce metabolic energy, the uptakes of oxycodone and [³H]pyrilamine were measured as described above. Uptake was also measured at medium pH values of 7.4 and 8.4 in the presence or absence of 10 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore. When the influence of intracellular pH (pHi) was examined, the uptake was measured in the presence of 30 mM NH₄Cl to elevate pHi (Ohta et al., 2006; Terada et al., 2006). To measure the uptake at acidic pHi, extracellular NH₄Cl was removed after the preincubation with 30 mM NH₄Cl, because intracellular NH₃ rapidly diffuses out of the cells, resulting in the accumulation of protons released from NH ⁺₄ during NH₃ generation in the cells.

In Situ Brain Perfusion Study. Brain perfusion was performed by the method reported previously (Takasato et al., 1984). In brief, each rat was anesthetized, and the right carotid artery was catheterized with polyethylene tubing (SP-10) filled with sodium heparin (100 IU/ml). The perfusate (Krebs-Henseleit buffer: 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgSO₄, and 10 mM D-glucose, pH 7.4) containing oxycodone (30 µM) and [³H]inulin (0.15 µM), a brain intravascular marker, was passed through the catheter at the rate of 4.9 ml/min with an infusion pump (Harvard Apparatus Inc., South Natick, MA, USA). After the infusion pump is started, 5.0 s is required to fill the external carotid artery cannula (Takasato et al., 1984). Therefore 5.0 s was subtracted routinely from the gross perfusion time in each experiment to obtain uptake time for which perfusate was actually within the brain capillary. At the end of uptake for 0 to 30 s, rats were decapitated, and the right cerebral hemisphere was dissected from the perfused brain and weighed. The brain samples were stored at –20°C until determination of oxycodone. The value of the permeability-surface area product (PS_{BBB, inf}), which represents in vivo BBB permeability, was calculated from the following equation after correcting for remaining intravascular oxycodone, estimated from the apparent brain uptake of [³H]inulin:

where F_pf is the flow rate of the perfusate (7.54 ml/min/g of brain) (Takasato et al., 1984), and K_in is a transfer constant. The flow rate substantially exceeded the K_in value, indicating that the flow rate is not the limiting factor for the brain transport of oxycodone. The transfer constant (K_in) for unidirectional uptake was calculated by fitting the equation:

where q_br is the amount of oxycodone in the brain, C_pf is the concentration of oxycodone in the perfusate, T is uptake time (minutes), and V₀ is the extrapolated oxycodone intercept (T = 0).

Determination of Oxycodone. Oxycodone was determined by HPLC with electrochemical detection (Kokubun et al., 2005). Twenty microliters of codeine solution (250 ng/ml), an internal standard, 100 µl of 4 N NaOH, and 800 µl of butyl chloride were added to 200 µl of cell homogenate. The samples were mixed, centrifuged for 10 min at 3000 rpm at 4°C, and then the butyl chloride layer was transferred. The remaining aqueous layer was extracted again with 800 µl of butyl chloride. The combined butyl chloride extract was evaporated to dryness. The residue was reconstituted in 200 µl of mobile phase, and a 40 µl aliquot was injected into the high-performance liquid chromatograph. The cerebral hemisphere was homogenized in 5 volumes of perchloric acid, and the homogenate was centrifuged for 10 min at 3000 rpm at 4°C. Naltrexone solution (250 ng/ml) was added as an internal standard to the supernatant, and the solution was neutralized and subjected to solid-phase extraction using an Oasis HLB cartridge (Waters, Milford, MA). The Oasis HLB cartridge was first prewetted with methanol (1 ml) followed by water (1 ml). The sample was applied to the cartridge, the cartridge was washed with 1 ml of 5% methanol, and oxycodone and naltrexone were then eluted with methanol. The eluate was dried under a nitrogen stream, and the residues were reconstituted in the HPLC mobile phase. The HPLC system consisted of a pump (301E; Eicom, Kyoto, Japan), an electrochemical detector (ECD-300; Eicom). The HPLC analytical column used was an XTerra RP18 (4.6 mm x 50 mm, 5 µm particle size; Waters, Milford, MA). The HPLC separation for oxycodone, codeine, and naltrexone was carried out at a flow rate of 0.5 ml/min with a mobile phase containing 10% acetonitrile, 20% methanol, and 5 mM phosphate buffer (pH 8.0) at 40°C. The retention times of oxycodone, codeine, and naltrexone were 9.1, 6.4, and 23.3 min, respectively. The voltage of the working electrode of the electrochemical detector was set at 800 mV. Standard curves of oxycodone in the amount range of 0.15 to 50 pmol injected onto the column showed good linearity (coefficient of determination >0.99). The detection limit for quantification of oxycodone was 3 nM. Runs were accepted if the precision and accuracy of the quality control samples at low (3 nM), middle (10 nM), and high (100 nM) concentrations had a coefficient of variation less than 15%.

Expression Profiling of Organic Cation Transporters by Real-Time PCR. The mRNA levels of organic cation transporters (rOCT1–3, rOCTN1–2, and rMATE1) in TR-BBB13 cells were measured by quantitative real-time PCR analysis. Total RNA was isolated from TR-BBB13 cells and rat brain and kidney using an RNeasy mini kit (QIAGEN, Valencia, CA). Single-strand cDNA was prepared from 1.0 µg of total RNA by RT (Superscript III; Invitrogen, Carlsbad, CA) using oligo(dT) primer. Quantitative real-time PCR analysis was performed using a 7500 sequence detection system (PE Applied Biosystems, Foster City, CA) with 2x SYBR Green PCR Master Mix (PE Applied Biosystems) according to the manufacturer's protocols. Rat genomic DNA was used as a standard in quantitative PCR. The sense and antisense primers were 5'-TTC ACC CCT GGA CAT TAT TGC-3' and 5'-TCA TGC ACT GGC TGA GGA AG-3' for rOCT1, 5'-CCC CAA ACC CAC ACA AAC C-3' and 5'-CAA CAG ACC GTG CAA GCT ACA-3' for rOCT2, 5'-CCC TGT GTG TTT CAT GGC TGT-3' and 5'-CTT GAA ATG CAA TCC AAA GGC-3' for rOCT3, 5'-GGG ACA GGA GGA TCG AGA AGT-3' and 5'-GGC TGG TCT CAC ACT CCT GAC-3' for rOCTN1, 5'-CGC AAA AAG ATG GTG GAG AAA-3' and 5'-CAG GGT GTT AGA AGG CTG TGC-3' for rOCTN2, 5'-TGC CAT CGG CTA TTA TGT CAT C-3' and 5'-AGC TTG GCA ACG AAC ATC AGT-3' for rMATE1, and 5'-GGT GGA CCT CAT GGC CTA CAT-3' and 5'-TGG GTG GTC CAG GGT TTC T-3' for rGAPDH. All primers were designed on the basis of the published full sequence of each transporter. The thermal protocol was set to 2 min at 50°C, followed by 10 min at 95°C and then 40 cycles of 15 s at 95°C and 1 min at 60°C. To confirm specificity of amplification, the PCR products were subjected to a melting curve analysis. The control lacking the RT enzyme was assayed in parallel to monitor any possible genomic contamination.

Statistical Analysis. Statistical analysis of the data were performed by Student's t test and by one-way analysis of variance followed by Dunnett's test for single and multiple comparisons, respectively. Differences were considered statistically significant at P < 0.05.

	Results

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Uptake Kinetics of Oxycodone and [³H]Pyrilamine by TR-BBB13 Cells. The uptake of oxycodone increased with time at 37°C, and the cell/medium ratios were 40.5 to 40.9 at 30 to 60 s (Fig. 1A). The uptake at 4°C was decreased by 41 to 68% compared with that at 37°C. [³H]Pyrilamine uptake by TR-BBB13 cells also increased with time, and the cell/medium ratio was 68.5 at 60 s. The uptake was decreased by 53 to 62% at 4°C (Fig. 1B). The initial uptake was assessed as the uptake at 15 s for oxycodone and 10 s for [³H]pyrilamine, respectively, in the subsequent kinetic and inhibition studies. The initial uptakes of oxycodone and [³H]pyrilamine were concentration-dependent (Fig. 2). Each Eadie-Hofstee plot gave a single straight line, indicating a single saturable process. Kinetic analysis provided a K_m value of 89 µM and a V_max of 3.5 nmol/mg of protein/15 s for oxycodone and a K_m value of 28 µM and a V_max of 0.7 nmol/mg of protein/10 s for [³H]pyrilamine.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Time course of uptake of oxycodone (A) and [3H]pyrilamine (B) into TR-BBB13 cells. Uptake of oxycodone (10 µM) or [3H]pyrilamine (90 nM) was measured at 37°C () and 4°C (). Each point represents the mean ± S.E. of three or four determinations. Significant difference: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus 37°C.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Concentration dependence of uptake of oxycodone (A) and [3H]pyrilamine (B) into TR-BBB13 cells. Inset, Eadie-Hofstee plots. v, uptake in nanomoles per milligram of protein per 15 s. Substrate concentration in micromolar. Uptake of oxycodone or [3H]pyrilamine was measured at 37°C for 15 or 10 s, respectively. Each point represents the mean ± S.E. from three or four determinations.

Trans-Stimulation Study on the Uptakes of Oxycodone and [³H]Pyrilamine by TR-BBB13 Cells. To examine whether the initial uptake of oxycodone might be attributable to membrane transport or binding to the membrane surface and intracellular components, a trans-stimulation study on the initial uptake of oxycodone into TR-BBB13 cells was performed (Fig. 3). The oxycodone uptake was significantly enhanced by preloading with 250 µM pyrilamine for 20 min. [³H]Pyrilamine uptake was also increased by pretreatment with 1 mM oxycodone.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Trans-stimulation effects on oxycodone (A) and [3H]pyrilamine (B) uptake into TR-BBB13 cells. The cells were preincubated for 20 min in the absence (control) and presence of pyrilamine (250 µM) or oxycodone (1 mM). Then the uptake of oxycodone (30 µM) or [3H]pyrilamine (90 nM) was measured at 37°C for 15 or 10 s, respectively. Each column represents the mean ± S.E. of three or four determinations. Significant difference: **, P < 0.01 versus control.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Lineweaver-Burk plot of mutual inhibitory effects on uptakes of oxycodone (A) and [3H]pyrilamine (B) into TR-BBB13 cells. Oxycodone uptake was measured at 37°C for 15 s in the presence () or absence () of unlabeled pyrilamine (30 µM). [3H]Pyrilamine uptake was also measured at 37°C for 10 s in the presence () or absence () of oxycodone (500 µM). Each point represents the mean ± S.E. of three or four determinations.

Metabolic Energy and Ion Dependence of the Uptakes of Oxycodone and [³H]Pyrilamine by TR-BBB13 Cells. The uptakes of oxycodone and [³H]pyrilamine were significantly inhibited by pretreatment with rotenone and sodium azide in TR-BBB13 cells (Fig. 5). The uptakes of oxycodone and [³H]pyrilamine were not affected by the replacement of extracellular Na⁺ with N-methylglucamine⁺ (Fig. 6). Treatment with valinomycin, a potassium ionophore, did not significantly change the uptake of these drugs. The pH dependence of the uptake is shown in Fig. 7. The uptakes of oxycodone and [³H]pyrilamine were increased at higher extracellular pH (8.4) and decreased by treatment with FCCP, a protonophore (Fig. 7, A and B). To examine the effect of intracellular pH, the cells were treated with NH₄Cl, because intracellular pH rises in the presence of NH₄Cl (acute treatment), and subsequent removal of NH₄Cl (pretreatment) causes a decrease in intracellular pH (Ohta et al., 2006; Terada et al., 2006). Acute treatment with NH₄Cl (intracellular alkalization) markedly reduced the uptakes of oxycodone (Fig. 7C) and [³H]pyrilamine (Fig. 7D), whereas pretreatment with NH₄Cl (intracellular acidification) resulted in stimulation of oxycodone uptake but not [³H]pyrilamine uptake at extracellular pH 8.4.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effect of ATP depletion on uptakes of oxycodone (A) and [3H]pyrilamine (B) into TR-BBB13 cells. Uptake of oxycodone (30 µM) was measured at 37°C for 15 s in the absence and presence of 25 µM rotenone or 0.1% sodium azide. Rotenone and sodium azide were preincubated for 20 min. Each column represents the mean ± S.E. of three or four determinations. Significant difference: **, P < 0.01 versus control.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Effects of sodium-replacement and membrane potential on uptakes of oxycodone (A and B) and [3H]pyrilamine (C and D) into TR-BBB13 cells. Uptakes of oxycodone (30 µM) and [3H]pyrilamine (80 nM) were measured at 37°C for 15 and 10 s, respectively, in sodium-containing (control) or sodium-free (replaced with N-methylglucamine) buffer. The mixtures were preincubated with valinomycin (10 µM) for 10 min. Each column represents the mean ± S.E. of three or four determinations.

View larger version (32K): [in this window] [in a new window]   FIG. 7. Effects of extracellular pH and protonophore treatment on uptakes of oxycodone (A) and [3H]pyrilamine (B) and effect of intracellular pH on uptakes of oxycodone (C) and [3H]pyrilamine (D) into TR-BBB13 cells. Uptakes of oxycodone (30 µM) and [3H]pyrilamine (90 nM) were measured at 37°C for 15 and 10 s, respectively, in the absence and presence of 10 µM FCCP. The mixtures were preincubated with FCCP for 10 min. The cells were preincubated with incubation medium (pH 7.4) in the absence (Control and Acute) or presence (pre) of 30 mM ammonium chloride for 20 min. Then the preincubation medium was removed, and the cells were incubated with 30 µM oxycodone (pH 7.4 and 8.4) in the absence (Control and pre) or presence (Acute) of 30 mM ammonium chloride for 15 s at 37°C. Each column represents the mean ± S.E. of three or four determinations.

In Vivo Blood-to-Brain Transport of Oxycodone. The brain uptake of oxycodone was measured by the in situ brain perfusion technique. The brain/perfusate (B/P) ratio of oxycodone increased linearly with increasing perfusion time up to 30 s (Fig. 8A). The PS_{BBB, inf} value for oxycodone was 224 µl/min/g of brain, which is 20 times greater than the value for brain uptake clearance of morphine (11.4 µl/min/g of brain) (Tunblad et al., 2003) and one-seventh of the value for [³H]pyrilamine (1618 µl/min/g of brain) (Yamazaki et al., 1994a). The B/P ratio at time 0 s was 0.044 ± 0.008 ml/g of brain. This value was 4-fold greater than the measured vascular [³H]inulin space of 0.011 ± 0.001 ml/g of brain. This value of vascular [³H]inulin space is consistent with reported values (0.007–0.017 ml/g of brain) (Takasato et al., 1984; Allen and Smith, 2001), which suggests that the B/P ratio at time 0 s may include a contribution due to extremely rapid binding to the capillary surface. The observation of this rapidly equilibrating component of brain oxycodone uptake emphasizes the importance of a thorough time course analysis to estimate the BBB permeability of cationic compounds, taking account of simple binding or association with the luminal membrane of the brain capillary endothelium (Allen and Smith, 2001). The brain uptake at pH 7.4 for a 15-s perfusion, corrected for the rapidly equilibrating component of oxycodone, was significantly inhibited by 54% by coperfusion with 1 mM pyrilamine (Fig. 8B). The brain uptake of oxycodone was increased by 4.4-fold in alkaline perfusate (pH 8.4), compared with that at pH 7.4. The brain uptake at pH 8.4 was also decreased by 45% by coperfusion of 1 mM pyrilamine.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Brain uptake of oxycodone in rats determined by the in situ brain perfusion method. A, perfusate containing oxycodone (30 µM) with or without pyrilamine (1 mM) was perfused into the brain hemisphere through the carotid arterial catheter at a rate of 4.9 ml/min for 0 to 30 s. B, brain perfusion of oxycodone was measured in perfusate of pH 7.4 or 8.4 in the absence and presence of 1 mM pyrilamine. The brain uptake was correlated with the rapidly equilibrating space of oxycodone. Each value represents the mean ± S.E. of three or four determinations.

	Discussion

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The BBB transport of CNS-acting drugs is an important determinant of their pharmacological action in the CNS. Here, the role of the pyrilamine transporter, a putative cation transporter, in the BBB transport of oxycodone, a cationic opioid, was characterized, using TR-BBB13 cells. The results indicate that transport of both oxycodone and pyrilamine is mediated by an energy-dependent, proton-coupled antiporter. Furthermore, in situ brain perfusion indicated that the pyrilamine transporter is involved in oxycodone transport into the brain across the BBB.

TR-BBB13 cells are stable, immortalized rat brain capillary endothelial cells derived from transgenic rats harboring temperature-sensitive simian virus 40 large-T antigen (Terasaki et al., 2003). They express several transporters, including GLUT1, MCT1, LAT1 with 4F2hc (Hosoya et al., 2000), and OAT3 (Ohtsuki et al., 2002), and these transporters are functional. TR-BBB13 cells retain glucose transport function with 10-fold higher activity than that of primary-cultured bovine brain endothelial cells (Terasaki et al., 2003). Furthermore, the BBB permeability clearances of several compounds estimated in TR-BBB13 cells were quite similar to those measured in vivo (Terasaki et al., 2003). Therefore, this cell line appears to be suitable as an in vitro BBB model for investigating BBB transport mechanisms.

The uptakes of oxycodone and [³H]pyrilamine were time- and temperature-dependent. The values of the cell-to-medium ratio for oxycodone and [³H]pyrilamine reached 40 and 70, respectively, at 60 s, suggesting that both cationic drugs are concentrated into the cells and/or bound to the intracellular constituents of TR-BBB13 cells after having passed through the cell membrane. The uptakes of oxycodone and [³H]pyrilamine by TR-BBB13 cells were saturable (Fig. 2). Saturation of uptake could be explained not only by saturation of the membrane transport but also by saturation of membrane surface binding and/or intracellular binding. Therefore, trans-stimulation studies were carried out to confirm that the initial uptake obtained here is attributable to transport through the plasma membrane. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa (Fig. 3). These results suggest that saturation of oxycodone and pyrilamine uptake is caused by the saturation of a specific transport system at the membrane of TR-BBB13 cells. In addition, kinetic analyses revealed that the calculated uptake clearance (V_max/K_m) for oxycodone (157 µl/min/mg of protein) was in good agreement with that of [³H]pyrilamine (150 µl/min/mg of protein), suggesting participation of a transport system with similar affinity and transport rate for the two substrates. The K_m values for the serotonin transporter and the norepinephrine transporter are 0.1 to 0.5 µM. Therefore, oxycodone transport in TR-BBB13 cells may be mediated by a relatively low-affinity transporter different from the Na⁺-dependent transporters for endogenous cationic neurotransmitters.

To examine commonality in the transport of oxycodone and pyrilamine, mutual inhibitory kinetic studies were carried out. Oxycodone and pyrilamine indeed mutually inhibited the uptake of each other (Fig. 4). The K_i values estimated for oxycodone (135 µM) and for pyrilamine (30 µM) were similar to respective K_m values for oxycodone (89 µM) and pyrilamine (28 µM), suggesting the occurrence of competition between oxycodone and pyrilamine for binding to the transporter.

Bulky hydrophobic cations with widely differing molecular structures, such as pyrilamine, quinidine, verapamil, and amantadine, markedly inhibited the transports of oxycodone and [³H]pyrilamine into TR-BBB13 cells (Table 1). These data suggest that the putative pyrilamine transporter may be polyspecific. In contrast, organic cations, which are classic substrates of OCTs, such as tetraethylammonium and 1-methyl-4-phenylpyridinium, had no significant effect. This finding suggests that oxycodone and pyrilamine transports are mediated by an organic cation-sensitive transporter, but the interaction does not involve electrostatic interaction as in the case of cationic peptides (Deguchi et al., 2003). Ergothioneine (a specific substrate of OCTN1), L-carnitine (an endogenous substrate of OCTN2), choline and hemicholinium-3 (representative substrate and inhibitor, respectively, of the choline transport system), or organic anions showed no or only slight inhibition, suggesting that rOCTN1, rOCTN2, choline transporter, and organic anion transporter may not play major roles in oxycodone and pyrilamine transport.

To identify the driving force for oxycodone and pyrilamine transport, the effects of metabolic inhibitors and extracellular ions were examined. The transports of both drugs were significantly inhibited by pretreatment with metabolic inhibitors but were insensitive to extracellular sodium and membrane potential in TR-BBB13 cells. The uptakes of oxycodone and [³H]pyrilamine were increased at higher extracellular pH (8.4) and decreased in the presence of FCCP. Intracellular acidification induced with NH₄Cl resulted in stimulation of the uptakes. These results suggest that the transport is dependent on an oppositely directed proton gradient. The intracellular pH in rat brain capillary endothelial cells has been estimated to be 6.9 to 7.15 (Sipos et al., 2005; Taylor et al., 2006). Lower intracellular pH than extracellular pH can drive organic cation/H⁺ antiport. Thus, an energy- and proton-dependent transporter seems to be involved in oxycodone and pyrilamine uptakes in TR-BBB13 cells. This organic cation/H⁺ antiporter at the BBB could play a role in establishing the sustained unbound concentration gradient of oxycodone between the brain interstitial fluid and the blood, observed by in vivo microdialysis in rats (Boström et al., 2006).

[³H]Pyrilamine transport in primary-cultured bovine brain capillary endothelial cells has been reported to be insensitive to metabolic inhibitors (Yamazaki et al., 1994b). The difference in metabolic energy dependence between primary and immortalized cells may be explained partially by taking differences in expression of transporters into account, because ATP-dependent efflux transporters such as P-glycoprotein and multidrug resistance-associated protein 4 are down-regulated in TR-BBB13 cells. Therefore, the net uptake of [³H]pyrilamine in primary-cultured bovine brain capillary endothelial cells may be offset by decreases in the efflux rate by metabolic inhibitors.

The in situ brain perfusion technique was used to examine whether the pyrilamine transporter plays a significant role in the BBB transport of oxycodone in vivo. The influx BBB permeability per capillary surface area is estimated to be 2.24 µl/min/cm², assuming that the rat brain capillary surface area is 100 cm²/g of brain (Terasaki et al., 2003). This value approximates to the uptake clearance (7.14 µl/min/cm²) extrapolated from the in vitro uptake study, despite the down-regulation of the P-glycoprotein level in TR-BBB13 cells, indicating that the contribution of the efflux transporter to the brain uptake of oxycodone is not large. Indeed, Boström et al. (2005) reported that the pharmacokinetics of oxycodone in rats was unaffected by coadministration of PSC833, an inhibitor of several efflux transporters including P-glycoprotein and breast cancer resistance protein. The influx BBB permeability of oxycodone in this study was 20 times greater than the reported value for morphine (Tunblad et al., 2003). The difference in the influx BBB permeability between oxycodone and morphine may be partially explained by the P-glycoprotein-mediated active efflux of morphine. However, this does not account for the efficient influx BBB permeability of oxycodone, because the steady-state unbound concentration of oxycodone in the brain interstitial fluid is 3-fold higher than that in the plasma (Boström et al., 2006). An alternative explanation is participation of an active influx transporter(s) for oxycodone at the BBB. The brain uptake of oxycodone was increased in alkaline perfusate (pH 8.4), and it was significantly inhibited by 54% at pH 7.4 and by 45% at pH 8.4 by pyrilamine. The inhibition by pyrilamine is thought to be specific to oxycodone transport, because the coperfusion with pyrilamine did not change the brain uptake of antipyrine, a BBB transfer marker (our unpublished data). These results strongly suggest that the pH- and pyrilamine-sensitive transporter contributes substantially to the efficient transport of oxycodone into the brain across the BBB. This influx transport system at the BBB may play an important role in determining the onset of the analgesic effect and clinical therapeutic outcome of oxycodone.

Quantitative RT-PCR analysis showed that the expression level of rOCTN2 was the highest among the organic cation transporters examined. On the other hand, little or no expression of rOCT1–3 and rMATE1 was observed. Expression levels of these mRNAs in TR-BBB13 cells were similar to those reported in rat brain capillary endothelial cells (Okura et al., 2007). Lack of mRNA expression of these transporters suggests that OCTs and MATE1 do not play major roles in the transport of cationic drugs in this in vitro BBB model. rOCTN2 has been reported to be involved in L-carnitine transport across the BBB (Kido et al., 2001). The lack of any inhibitory effect of L-carnitine on transports of oxycodone and [³H]pyrilamine suggests that rOCTN2 is not the putative pyrilamine transporter. An unidentified organic cation transport system different from previously identified organic cation transporters (OCTs, OCTNs, and MATE1) seems to be a candidate for the pyrilamine transporter.

In conclusion, our results indicate that oxycodone is transported into TR-BBB13 cells by the pyrilamine transporter, previously proposed as a putative cation transporter. The transporter was energy-dependent and oppositely directed proton gradient-dependent but sodium- or membrane potential-independent. It was found that the pyrilamine transporter is also involved in the transport of oxycodone into the brain across the BBB in vivo, although the transport molecule remains unidentified. These findings should be relevant to the treatment of cancer pain with oxycodone, and, more generally, to the BBB transport of CNS-acting cationic drugs.

	Acknowledgments

 
We gratefully acknowledge the technical support of Drs. Hikaru Yabuuchi and Masaaki Takemura (GenoMembrane Inc., Yokohama, Japan).

	Footnotes

 
This work was supported in part by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Young Scientists provided by the Ministry of Education, Culture, Sports, Science and Technology.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.108.022087.

ABBREVIATIONS: BBB, blood-brain barrier; GLUT, glucose transporter; MCT, monocarboxylate transporter; LAT, large neutral amino acid transporter; OAT/Oat, organic anion transporter 3; OATP/Oatp, organic anion transport polypeptide; OCT, organic cation transporter; MATE, multidrug and toxin extrusion; TR-BBB13 cells, conditionally immortalized rat brain capillary endothelial cells; CNS, central nervous system; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; pHi, intracellular pH; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; r, rat; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; B/P, blood/perfusion.

Address correspondence to: Dr. Yoshiharu Deguchi, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195. E-mail: deguchi{at}pharm.teikyo-u.ac.jp

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