BIOPHARMACEUTICS & DRUG DISPOSITION
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`Published online 8 April 2010 in Wiley InterScience
`(www.interscience.wiley.com) DOI: 10.1002/bdd.707
`
`Involvement of an Influx Transporter in the Blood–Brain
`Barrier Transport of Naloxone
`
`Toyofumi Suzukia, , Aya Ohmuroa, Mariko Miyataa, Takayuki Furuishia, Shinji Hidakaa, Fumihiko Kugawab,
`Toshiro Fukamia, and Kazuo Tomonoa
`aDepartment of Pharmaceutics, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
`bDepartment of Biopharmaceutics, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-Ku, Kobe,
`Hyogo 650-8530, Japan
`
`ABSTRACT: Naloxone, a potent and specific opioid antagonist, has been shown in previous
`studies to have an influx clearance across the rat blood–brain barrier (BBB) two times greater than
`the efflux clearance. The purpose of the present study was to characterize the influx transport of
`naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of
`[3H]naloxone exhibited saturability in a concentration-dependent manner (concentration range
`0.5 mM to 15 mM) in the presence of unlabeled naloxone. These results indicate that both passive
`diffusion and a carrier-mediated transport mechanism are operating. The in vivo kinetic parameters
`were estimated as follows: the Michaelis constant, Kt, was 2.9970.71 mM; the maximum uptake rate,
`Jmax, was 0.47770.083 mmol/min/g brain; and the nonsaturable first-order rate constant, Kd, was
`0.16070.044 ml/min/g brain. The uptake of [3H]naloxone by the rat brain increased as the pH of
`the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic
`H1-antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and
`propranolol. In contrast, the BBB transport of [3H]naloxone was not affected by any typical
`substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or
`L-carnitine or substrates for organic anion transport systems such as p-aminohippuric acid,
`benzylpenicillin or pravastatin. The present results suggest that a pH-dependent and saturable
`influx transport system that is a selective transporter for cationic H1-antagonists is involved in the
`BBB transport of naloxone in the rat. Copyright r 2010 John Wiley & Sons, Ltd.
`
`Key words: blood–brain barrier; influx transport; brain uptake index; naloxone; opioid antagonist
`
`Introduction
`
`The transport of naloxone across the blood–brain
`barrier (BBB) may be an important characteristic
`affecting its ability to inhibit opioid-related side
`effects in the central nervous system. Naloxone, a
`potent opioid antagonist, is transported across
`
`*Correspondence to: Department of Pharmaceutics, School of
`Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi,
`Chiba 274-8555, Japan.
`E-mail: suzuki.toyofumi@nihon-u.ac.jp
`
`the (BBB) about 8–10 times more effectively than
`morphine, an opioid agonist [1]. Furthermore,
`the initial brain concentration of naloxone is 4.6
`times higher than that in serum at 5 min follow-
`ing intravenous administration in rats [2]. It was
`recently demonstrated quantitatively using
`in vivo approaches that the influx clearance of
`naloxone across the BBB, which is not affected by
`efflux transporters such as P-glycoprotein, is two
`times greater than the efflux clearance [3]. This
`result suggests that a certain predominant influx
`mechanism is involved in the transport of
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`Received 30 November 2009
`Revised 16 February 2010
`Accepted 9 March 2010
`
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`244
`
`T. SUZUKI ET AL.
`
`naloxone from the blood to the brain. It was
`hypothesized that the uptake of naloxone by the
`brain may be determined not only by lipophili-
`city and molecular size, but also by some carrier-
`mediated transport system.
`The BBB is composed of brain capillary
`endothelial cells connected by tight junctions.
`Several
`influx transport
`systems which are
`expressed at the luminal and abluminal mem-
`branes of the endothelial cells control the entry of
`many drugs from systemic circulation into the
`brain across the BBB [4,5]. The uptake of fentanyl,
`a cationic opioid agonist, into the bovine brain-
`microvascular endothelial cells has been shown to
`exhibit energy dependence, suggesting the invol-
`vement of an active influx-transport system [6]. In
`the rat brain, Elkiweri et al. recently reported that
`fentanyl uptake, which was mediated by an
`organic anion-transporting polypeptide (rodents:
`Oatps, human: OATPs)
`influx
`transporter,
`decreased to one-third when naloxone was used
`as a competitive substrate for rat Oatps [7].
`Naloxone also strongly inhibits deltorphine II
`transport in Xenopus oocytes expressing human
`OATP-A (OATP1A2/SLC21A3) [8]. Since the rat
`Oatp2 (Oatp1a4/Slc21a5) and human OATP-A
`transport systems are highly expressed at the BBB
`[8,9], it is possible that Oatp2 may be involved in
`the influx transport of naloxone at the rat BBB.
`Because of the cationic nature of naloxone [3], a
`second possible mechanism can be speculated to
`involve
`organic
`cation
`transport
`systems.
`Although polyspecific organic cation transpor-
`ters are known to be expressed in the brains of
`rodents and/or humans [10], it has been shown
`through in vivo and in vitro studies [11–17] that a
`specific carrier-mediated transport system for
`H1-antagonists such as pyrilamine and diphen-
`hydramine that
`is different
`from previously
`reported organic cation transporters is present
`at the BBB. Okura et al. have reported that an as
`yet unidentified organic cation-sensitive trans-
`port system mediates the uptake of the cationic
`opioid agonist oxycodone by conditionally
`immortalized rat brain endothelial cells as an
`in vitro BBB model [18]. Furthermore, the rat
`brain perfusion study demonstrated that
`the
`transporter for oxycodone is also partly respon-
`sible for the influx transport of
`the cationic
`H1-antagonist pyrilamine [18]. It was reported
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`previously that pentazocine, a cationic drug and
`a mixed opioid agonist/antagonist,
`is trans-
`ported predominantly into the rat brain by an
`organic cation-sensitive transport system, rather
`than via passive diffusion [19,20]. Pentazocine
`transport across the BBB was strongly inhibited
`by pyrilamine and diphenhydramine [19]. More
`recently, using an in vitro BBB model, it was
`shown that pentazocine strikingly inhibits 97% of
`pyrilamine uptake [21]. Given these results, it is
`possible to conclude that the opioid antagonist
`naloxone could be a substrate for the unidenti-
`fied organic-cation sensitive transporter or the
`polyspecific organic cation transporters partici-
`pating in the brain uptake of H1-antagonists.
`Treatment of cancer-related pain with opioids
`can result in adverse effects including respiratory
`depression, sedation and hypotension. Evalua-
`tion of
`the transporter-mediated uptake of
`naloxone by the brain could provide information
`useful for developing methods to prevent or
`reverse such effects. The primary objective of
`the present study was to clarify the involvement
`of the influx transport system(s) of naloxone
`at the rat BBB using the brain uptake index
`(BUI) method.
`
`Materials and Methods
`
`Radioisotopes and reagents
`[N-allyl-2,3-3H]naloxone ([3H]naloxone, 67.0 Ci/
`mmol; 497% purity) was purchased from Perkin-
`Elmer Life and Analytical Sciences, Inc. (Boston,
`([14C]butanol,
`MA, USA). N-[1-14C]butanol
`2 mCi/mmol; 99% purity) and 3-O-[methyl-3H]-
`methyl-D-glucose ([3H]3OMG, 60 Ci/mmol; 99%
`purity) were purchased from American Radiola-
`beled Chemicals Inc.
`(St Louis, MO, USA).
`Unlabeled naloxone hydrochloride was pur-
`chased from Sigma-Aldrich Co. (St Louis, MO,
`USA). Digoxin (Digosins injection) was pur-
`chased from Chugai Pharmaceutical Co. Ltd
`(Tokyo, Japan) and was composed of digoxin
`(0.25 mg), propylene glycol
`(208 mg), ethanol
`(86 mg) and benzyl alcohol (21 mg) in 1 ml of
`distilled water for injection. Ketamine hydro-
`chloride (Ketarals 50, Sankyo Co., Ltd, Tokyo,
`Japan) was used as an anesthetic. All other
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
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`NALOXONE UPTAKE BY THE RAT BRAIN
`
`245
`
`reagents were commercial products of reagent
`grade and were used without further purification.
`
`Animals
`
`All experiments were conducted according to
`guidelines approved by Nihon University Ani-
`mal Care and Use Committee (Nihon University,
`Japan). Adult Male Wistar/ST rats (7 weeks old)
`were obtained from Japan SLC, Inc. (Shizuoka,
`Japan). The rats were housed in stainless steel
`cages with a 12-h light/dark cycle (light on 8:00
`am–8:00 pm) under conditions of controlled
`temperature maintained at 23711C with a
`humidity of 55710% for at least 1 week before
`use. The rats were fed and given water ad libitum
`prior to the experiments.
`
`BUI measurements by carotid injections
`
`The in vivo brain uptake study was performed
`using the carotid artery injection technique as
`
`cerebral hemisphere (approximately 0.2 g) to the
`injected side was isolated and placed in a
`scintillation vial. Samples were solubilized in
`1 ml of Soluenes-350 (PerkinElmer Life and
`Analytical Sciences, Inc., Boston, MA, USA) at
`501C for 4 h and neutralized. The liquid scintilla-
`tion cocktail (Hionic-fluorTM, PerkinElmer Life
`and Analytical Sciences, 10 ml) was then added
`to the brain sample. A 50 ml aliquot of
`the
`injection solution was transferred to a scintilla-
`tion vial and dissolved in 3 ml of Pico-fluorTM 40
`(PerkinElmer Life and Analytical Sciences). The
`[3H] and [14C]
`disintegrations per minute of
`radioactivity in the brain sample and the applied
`solution were measured in a liquid scintillation
`counter equipped with an appropriate crossover
`correction for [3H] and [14C] (Tri-carb 2800TR,
`PerkinElmer Life and Analytical Sciences).
`
`Data analysis
`
`The percentage of BUI was calculated as follows:
`
`BUI ð%Þ ¼
`
`amount of ½3HŠtest substrate in the brain=amount of ½14CŠreference in the brain
`amount of ½3HŠtest substrate in the injectate=amount of ½14CŠreference in the injectate
`
` 100
`
`ð1Þ
`
`reported previously [19]. Rats (250–350 g) were
`anesthetized with an intraperitoneal injection of
`ketamine (100 mg/kg) and xylazine (2 mg/kg).
`An aliquot of 200 ml Ringer’s/HEPES buffer
`(141 mM NaCl, 4.0 mM KCl, 2.8 mM CaCl2 and
`10 mM HEPES; pH 7.4) containing [3H]naloxone
`(5 mCi/ml, 0.1 mM) and the reference compound,
`[14C]butanol (0.5 mCi/ml), was injected rapidly
`into the right common carotid artery. In the study
`of concentration-dependency, several concentra-
`tions of naloxone in the injection solution were
`adjusted by adding unlabeled naloxone. For the
`investigation of the effect of pH, the injection
`solution was adjusted to pH 5.5–8.5 with HCl or
`NaOH, respectively. In the inhibition study, the
`selected organic cation or anion was dissolved in
`the injection solution to yield the final concentra-
`tion. The pH of the injection solution containing
`the unlabeled naloxone or inhibitor was adjusted
`by buffering the solution using HCl or NaOH
`before injection. Fifteen seconds after injection,
`the rat was decapitated and the ipsilateral
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`The BUI values
`follows [22,23]:
`BUI ð%Þ ¼ Etest=Eref  100
`
`can also be defined as
`
`ð2Þ
`
`where Etest and Eref represent the extraction of the
`test substrate ([3H]naloxone) and reference com-
`pound in the brain, respectively. In this study,
`[14C]butanol, with a known extraction in the
`brain of 0.64, was used as the reference com-
`pound [23].
`
`Estimation of kinetic parameters
`
`To estimate the kinetic parameters, the initial
`uptake rate (J) and mean capillary concentration
`(Ccap) were calculated using the following
`Equations (3) and (4), respectively:
`J ¼ ðEtest=100Þ  F  Cin
`
`ð3Þ
`
`Ccap ¼ Cin  ðEtest=100Þ=lnð1 Etest=100Þ
`
`ð4Þ
`
`where F and Cin are the cerebral blood flow rate
`reported previously as 0.93 ml/min/g brain [23]
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
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`246
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`T. SUZUKI ET AL.
`
`and the concentration of substrate (mM) in the
`carotid injection solution, respectively. The initial
`uptake rates of substrate (J) with various Ccap
`were fitted to the following Equation (5), which
`contains both saturable and nonsaturable linear
`terms, using the nonlinear least-squares regres-
`sion program (WinNONLIN):
`J ¼ Jmax  Ccap =ðKt1Ccap Þ1Kd  Ccap
`
`ð5Þ
`
`where Jmax represents the maximal uptake rate
`(mmol/min/g
`for
`the
`saturable
`component
`brain), Kt
`represents the Michaelis constant
`(mM), Kd represents the first-order rate constant
`for
`the nonsaturable component
`(ml/min/g
`brain) and Ccap represents the mean capillary
`concentration of naloxone (mM).
`
`Statistical analysis
`
`An unpaired, two-tailed Student’s t-test was used
`to assess the significance of differences between
`the means of two groups. The significance of the
`difference among means of more than two groups
`was determined by one-way analysis of variance
`(ANOVA) followed by the modified Fisher’s least-
`squares difference method. Differences were
`considered statistically significant at po0.01.
`
`Results
`
`dependence
`Concentration
`uptake by the brain
`
`of
`
`[3H]naloxone
`
`The BBB permeability of naloxone in the rat was
`examined using the BUI method. The BUI value
`of [3H]naloxone (0.1 mM) was determined to be
`43.770.6% (n 5 3; mean7SE) at 15 s after carotid
`injection, corresponding to a BBB influx clearance
`of 0.30570.005 ml/min/g brain [3]. The concen-
`tration dependence of [3H]naloxone uptake by
`the rat brain is shown in Figure 1. The brain
`uptake of [3H]naloxone was determined in the
`presence of unlabeled naloxone in the concentra-
`tion range between 0.5 mM and 15 mM, and
`the initial uptake rates were related to the
`substrate concentration using Equation (5). The
`initial uptake rate of [3H]naloxone was saturated
`as Ccap increased. The values for Ccap were lower
`than Cin by approximately 10%. An Eadie-
`Hofstee plot showed a single straight
`line,
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`Figure 1. Concentration dependence of [3H]naloxone uptake
`by the rat brain. The BUI values of [3H]naloxone at various
`concentrations of unlabeled naloxone (0.5 mM15 mM) were
`measured 15 s after carotid artery injection. Initial uptake rate
`and mean capillary concentration were calculated from
`Equations (3) and (4), respectively. The solid line represents
`the total uptake rate generated from Equation (5), using
`WinNonlin fitted parameters (mean7SE): Jmax 5 0.47770.083 m
`mol/min/g brain; Kt 5 2.9970.71 mM; Kd 5 0.16070.044 ml/
`min/g brain. The broken line and dotted line represent
`saturable and nonsaturable uptake rates, respectively. Each
`point with a vertical bar represents the mean7SE (n 5 3–10).
`Data points without vertical bars include the SE within the
`points. Inset: Eadie-Fofstee plot of [3H]naloxone uptake by the
`rat brain. V, initial uptake rate in mmol/min/g brain. S, mean
`capillary concentration of substrate (mM)
`
`(see
`saturable process
`single
`indicating a
`Figure 1). Kinetic analysis provided a Jmax of
`0.47770.083 mmol/min/g
`Kt
`brain,
`a
`of
`2.9970.71 mM and a Kd of 0.16070.044 ml/
`min/g brain (mean7SE). Since no inhibitory
`effect at
`the high concentration of naloxone
`(15 mM) was observed on the BUI of [3H]3OMG
`(data not shown), the concentration-dependent
`uptake of naloxone by the brain was not attributed
`to a toxic effect of naloxone on the BBB.
`
`pH dependence of [3H]naloxone uptake by the brain
`The effect of pH in the range 5.5–8.5 on
`[3H]naloxone uptake by the brain is shown in
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
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`NALOXONE UPTAKE BY THE RAT BRAIN
`
`247
`
`Table 1. Inhibitory effect of selected cationic compounds on
`[3H]naloxone uptake by the rat brain
`
`Compound
`
`No.
`studied
`
`Concentration
`(mM)
`
`Relative uptake
`(% of control)
`
`Control
`Lidocaine
`Propranolol
`Pethidine
`Eptazocine
`Naloxone
`methiodide
`Morphine
`Pentazocine
`Tramadol
`Naltrexone
`Clonidine
`Nicotine
`TEA
`MPP
`Procainamide
`Amantadine
`NMN
`Guanidine
`Choline
`Ergothioneine
`L-Carnitine
`
`3
`3
`3
`3
`3
`3
`
`3
`3
`3
`3
`4
`5
`3
`4
`3
`4
`4
`3
`3
`3
`3
`
`40
`40
`40
`40
`40
`
`40
`40
`40
`40
`40
`20
`40
`40
`40
`40
`40
`40
`40
`40
`40
`
`100
`51.878.3a
`41.571.5a
`69.376.5a
`68.276.5a
`73.972.8a
`
`89.675.3
`99.979.1
`87.378.7
`79.878.3
`11271
`96.375.7
`10377
`12178
`95.978.7
`11178
`11271
`10576
`88.174.5
`97.674.0
`82.677.3
`
`1-methyl-4-phenylpyridinium;
`tetraethylammonium; MPP,
`TEA,
`NMN, N-methylnicotinamide. A mixture of 5 mCi/ml [3H]naloxone
`(0.1 mM) and 0.5 mCi/ml [14C]butanol dissolved in 200 ml RHB buffer in
`the absence or presence of a cationic compound was injected to the
`common carotid artery. Rats were decapitated at 15 s after the injection.
`Each value represents the mean7SE.
`apo0.01, significantly different from the control uptake.
`
`Table 2. Inhibitory effect of H1-antagonists on [3H]naloxone
`uptake by the rat brain
`
`H1-antagonist
`
`No.
`studied
`
`Concentration
`(mM)
`
`Relative uptake
`(% of control)
`
`Control
`Pyrilamine
`
`3
`3
`3
`Diphenhydramine 6
`3
`4
`3
`5
`
`Ketotifen
`Cyproheptadine
`Fexofenadine
`
`20
`40
`20
`40
`5
`1
`1
`
`100
`10671
`25.373.2a
`71.874.2a
`29.771.0a
`59.875.4a
`98.678.0
`95.273.3
`
`A mixture of 5 mCi/ml [3H]naloxone (0.1 mM) and 0.5 mCi/ml [14C]bu-
`tanol dissolved in 200 ml RHB buffer in the absence or presence of a
`H1-antagonist was injected to the common carotid artery. Rats were
`decapitated at 15 s after the injection. Each value represents the
`mean7SE.
`apo0.01, significant difference from the control uptake.
`
`(Table 1). Morphine, pentazocine, tramadol, nal-
`trexone, clonidine, nicotine, tetraethylammonium
`(TEA), 1-methyl-4-phenylpyridinium (MPP), pro-
`cainamide, amantadine, N-methylnicotinamide
`(NMN), guanidine, choline, ergothioneine and
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
`
`Figure 2. Effect of the pH of injection solution on [3H]nalox-
`one uptake by the rat brain. The BUI value of [3H]naloxone
`(0.1 mM) was determined in the absence (open circles) or
`presence (closed circles) of unlabeled naloxone (10 mM). Each
`point with a vertical bar represents the mean7SE (n 5 3–9).
` po0.01, significantly different from the absence of unlabeled
`naloxone at pH 7.4
`
`Figure 2. In the absence of unlabeled naloxone,
`the uptake was elevated from 28.771.1 (n 5 3;
`mean7SE)
`to 52.171.3 (n 5 3; mean7SE) by
`raising the injection solution pH from 5.5 to 8.5.
`This apparent pH-dependence of [3H]naloxone
`uptake was affected by the presence of 10 mM
`unlabeled naloxone in the pH range 5.5–7.4. The
`BUI values in the presence of unlabeled naloxone
`were also 1.4-fold lower than that in the absence
`of the unlabeled naloxone, although this differ-
`ence was not statistically significant, except for
`pH 7.4. It was not possible to prepare an alkaline
`solution (pH 8.0 and 8.5) for solutions containing
`unlabeled naloxone (10 mM).
`
`Effects of various compounds on [3H]naloxone
`uptake by the brain
`
`To characterize the carrier responsible for the
`uptake of naloxone by the rat brain, the effect of
`various compounds on the uptake of [3H]nalox-
`one was investigated. Effects of selected cationic
`compounds and H1-antagonists on the uptake of
`[3H]naloxone are summarized in Tables 1 and 2,
`respectively. The uptake of [3H]naloxone by the
`brain was strongly diminished by lidocaine and
`propranolol, and moderately reduced by pethi-
`dine, eptazocine and naloxone methiodide,
`the quaternary ammonium analog of naloxone
`
`Copyright r 2010 John Wiley & Sons, Ltd.
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`248
`
`T. SUZUKI ET AL.
`
`Table 3. Effect of selected anionic compounds on [3H]naloxone
`uptake by the rat brain
`
`Compound
`
`No.
`studied
`
`Concentration
`(mM)
`
`Relative uptake
`(% of control)
`
`Control
`p-Aminohippuric
`acid
`Benzylpenicillin
`Pravastatin
`Taurocholate
`Digoxin
`
`3
`3
`
`3
`5
`3
`4
`
`40
`
`40
`40
`40
`0.2
`
`100
`92.174.1
`
`90.876.9
`11376
`13674a
`13174a
`
`[3H]naloxone (0.1 mM) and 0.5 mCi/ml
`A mixture of 5 mCi/ml
`[14C]butanol dissolved in 200 ml RHB buffer in the absence or presence
`of an anionic compound was injected to the common carotid artery.
`Rats were decapitated at 15 s after the injection. Each value represents
`the mean7SE.
`apo0.01, significant difference from the control uptake.
`
`L-carnitine had no effect. H1-antagonists such as
`pyrilamine, diphenhydramine and ketotifen sig-
`nificantly diminished the uptake of [3H]naloxone,
`whereas cyproheptadine and fexofenadine did
`not (Table 2). As shown in Table 3, there was no
`significant change in the [3H]naloxone uptake in
`the presence of selected anionic compounds such
`as p-aminohippuric acid, benzylpenicillin and
`pravastatin. The uptake was significantly in-
`creased by taurocholate and digoxin.
`
`Discussion
`
`It was reported previously that P-gp-efflux
`transport is not involved in naloxone transport
`across the BBB, but a nonsaturable mechanism is
`likely [3]. Because transport of naloxone across
`the BBB is not mediated by a specific efflux
`transporter,
`the BUI method is particularly
`applicable for investigating influx upon initial
`brain uptake of this highly lipophilic drug. The
`advantages of
`this method include ease and
`flexibility of application. Surgical preparation of
`the animal is simple, and the short sampling time
`of 15 s results in relatively efficient experimental
`throughput under approximate sink conditions
`[24]. By modulating the composition of
`the
`injection solution, it is possible to probe specific
`mechanisms of BBB translocation [24]. The
`present study attempted to clarify whether a
`specific influx transport system at the BBB may
`be responsible for
`the high distribution of
`naloxone to the brain.
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`Initially it was determined that a carrier-
`mediated system acts in the uptake of naloxone
`by the brain. Concentration dependence of the
`brain uptake rate of [3H]naloxone indicated the
`participation of a saturable system (Figure 1).
`The analysis of uptake kinetics provided one
`saturable component with a Kt value of 2.997
`0.71 mM, a Jmax value of 0.47770.083 mmol/min/g
`brain and a Kd value of 0.16070.044 ml/min/g
`brain. The ratio of Jmax/Kt
`is estimated to be
`0.159 ml/min/g brain, which is equivalent to the
`value of Kd and constitutes approximately
`50% of
`the
`total
`brain influx
`clearance
`(0.30570.005 ml/min/g
`brain).
`This
`result
`suggests that a carrier-mediated mechanism con-
`stitutes half of the naloxone uptake by the rat
`brain.
`Evidence of a saturable transport mechanism
`with a low-affinity system at the rat BBB has been
`found for several lipophilic and cationic drugs
`using the BUI method [11,19,25,26]. It is impor-
`tant to note, though, that while the BUI method
`can prove transporter saturability and specificity,
`methodological
`limitations exist
`that prevent
`calculation of more accurate kinetic parameters.
`Estimation of the true concentration of a test drug
`in the brain capillary is almost
`impossible.
`Mixing of endogenous plasma with the injection
`solution has been estimated at 5% [27]. This level
`of mixing is relevant
`in cases of saturable
`transport with a low Kt and for drugs with high
`plasma protein binding.
`Next the study determined whether carrier-
`mediated uptake of naloxone depends on the pH
`of the injection solution. The uptake of [3H]na-
`loxone was markedly increased (20%) with an
`alkaline injection solution of pH 8.5 and sig-
`nificantly decreased (30%) with an acidic
`injection solution of pH 5.5 when compared with
`a solution of neutral pH 7.4 (Figure 2). Since
`naloxone is an organic cation (pKa 9.12), the
`concentration of the ionized form of naloxone
`increases slightly to 99.9% at pH 5.5 compared
`with a concentration of 98.1% at pH 7.4. The
`pH-related decrease in naloxone uptake may
`have resulted from this increase in the concentra-
`tion of ionized naloxone in accordance with the
`pH-partition theory.
`The inhibitory effect of [3H]naloxone uptake
`at pH 7.4 in the presence of 10 mM naloxone,
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
`
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`NALOXONE UPTAKE BY THE RAT BRAIN
`
`249
`
`however, was similar to the effect observed at
`acidic pH, indicating the presence of a saturable
`process. This result suggests that a pH-sensitive
`carrier-mediated system is involved in naloxone
`transport. A decrease in transport efficiency
`(Jmax/Kt) in vivo may occur in a saturable uptake
`system for naloxone when the pH is lowered
`from neutral to acidic, as has been suggested
`with pyrilamine [11] and pentazocine [19,20]. It
`has also been reported that the BBB transport of
`organic cations such as clonidine [28,29], oxyco-
`done [18], pyrilamine [11] and pramipexole [30]
`are
`saturable
`and exhibit
`an accelerated
`pH-dependency at elevated pH in both in vivo
`and in vitro studies. Both transport systems utilize
`an oppositely directed H1 gradient as a driving
`force. These reports lead us to speculate that
`naloxone uptake by the BBB can be explained by
`an H1/organic cation antiport mechanism in
`which the pH-dependence of naloxone transport
`is regulated by both H1 and naloxone gradients.
`Physiological and biopharmaceutical roles of
`polyspecific organic cation transporters consist-
`ing of the solute carrier 22 (SLC22) family, the
`multidrug and toxin extrusion (MATE/SLC47A)
`transporter family and the plasma membrane
`(PMAT/SLC29A4)
`monoamine
`transporter
`family have been reported [10,31,32]. The SLC22
`family contains the cation transporters (OCTs:
`OCT1/SLC22A1, OCT2/SLC22A2 and OCT3/
`SLC22A3) and the cation/carnitine transporters
`(OCTNs: OCTN1/SLC22A4
`and OCTN2/
`SLC22A5).
`To determine whether any OCTs, OCTNs,
`MATE1 and PMAT transporters for organic
`cations are involved in naloxone influx at the
`BBB, the effect of substrates for these transporters
`on the uptake of [3H]naloxone was investigated.
`TEA (a substrate of OCTs, OCTNs, MATEs and
`PMAT), MPP (a substrate of OCTs, MATEs and
`PMAT) and procainamide (a substrate of OCTs
`and the MATEs family) did not have a significant
`[3H]naloxone
`inhibitory
`effect
`on
`uptake
`(Table 1). Moreover, naloxone transport was not
`affected by amantadine (a substrate of OCTs),
`NMN, guanidine and choline (endogenous sub-
`strates of OCTs), ergothioneine (a specific sub-
`strate of OCTN1) and L-carnitine (a specific
`substrate of OCTN2) (Table 1). Notably, rOCTN1
`and rMATE1 are known among the polyspecific
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`organic cation transporters to be H1/organic
`cation antiporters [10,31]. The present observa-
`tions
`suggest
`that no previously identified
`members of
`the OCTs, OCTNs, MATE1 and
`PMAT organic cation transporter families are
`responsible for naloxone transport at
`the rat
`BBB. It was concluded, therefore, that unidenti-
`fied pH-dependent transport systems are candi-
`date transporters for naloxone uptake across the
`rat BBB.
`In contrast, the organic cationic drugs lidocaine
`and propranolol and cationic H1-antagonists
`pyrilamine, diphenhydramine
`and ketotifen
`strongly inhibited [3H]naloxone uptake by the
`rat brain (Tables 1 and 2). These results are in
`accord with previous findings regarding pyrila-
`mine transport at the BBB. In both in vivo and
`in vitro systems, pyrilamine uptake is inhibited
`significantly by lidocaine [12,21], propranolol
`[11,12], diphenhydramine [11] and ketotifen
`[12,14]. Earlier studies also demonstrated that a
`specific transport mechanism for cationic drugs
`such as lidocaine and propranolol exists in the rat
`brain and isolated bovine brain microvessels and
`it has been suggested that this mechanism is
`active for H1-antagonists [25,33]. Saturable trans-
`port systems for diphenhydramine were also
`found in the BBB through in vivo microdialysis
`[15,16] and in situ brain perfusion [17] studies. In
`addition, diphenhydramine accumulation in
`Caco-2 cells was suggested to occur with an
`H1/diphenhydramine antiport mechanism and
`was not affected by any typical substrate (MPP,
`NMN, cimetidine) for the renal organic cation
`transport system [34]. From the available evi-
`dence, it can be interpreted that the substrate
`specificity and pH-dependency of the transport
`system for naloxone are very similar to those for
`pyrilamine and diphenhydramine.
`To complete our study, the effect of substrates
`for organic anion transporters on the uptake of
`naloxone was evaluated. Rat organic anion
`transporter 3 (rOat3/Slc22a8) and organic anion
`transporting polypeptide (rodents: Oatp, human;
`OATP) 2 (Oatp2/Slc1a5) have been identified on
`both the luminal and abluminal membranes of
`rat brain capillary endothelial cells [9,35]. rOat3
`can mediate not only efflux but also influx
`transport across the BBB depending on the
`concentration gradient of the substrate and its
`
`Biopharm. Drug Dispos. 31: 243–252 (2010)
`DOI: 10.1002/bdd
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`250
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`T. SUZUKI ET AL.
`
`driving force, as speculated by Kikuchi et al. [35].
`Rat Oatp2 was demonstrated in vivo to be
`responsible for influx transport of substrates
`such as prostaglandin E1 [36] and [D-penicilla-
`mine2,5]-enkephalin (an opioid peptide)
`[37].
`Human OATP1A2 (SLC21A3) exhibits a particu-
`larly strong expression in the brain [38], and
`could mediate the uptake of opioid peptides [8].
`Naloxone strongly inhibits human OATP1A2-
`mediated uptake of deltorphine II, an anionic
`opioid peptide, into the oocyte expression system
`[8]. Rat Oatp2 shares a high amino acid sequence
`identity (73%) with human OATP-A [39]. There-
`fore, it is possible that multispecific organic anion
`transporters (rOat3 and Oatp2) may be involved
`in the influx transport of naloxone across the BBB
`in rats.
`Involvement of rOat3 can be excluded in
`naloxone uptake across the BBB (Table 3), how-
`ever, because the uptake of [3H]naloxone was not
`affected by substrates of rOat3 such as p-amino-
`hippuric acid, benzylpenicillin and pravastatin
`[34,40]. Pravastatin has also been reported to be a
`good substrate of rOatp2 [39,41]. Interestingly,
`taurocholate, a substrate of the Oatp family [42],
`and digoxin, a specific substrate of
`rOatp2
`[43], stimulated [3H]naloxone uptake rather than
`having an inhibitory effect (Table 3). The reason
`for this increase in [3H]naloxone uptake has not
`been clarified yet. The results do confirm, though,
`that rOatp2, one of several members of the Oatp
`family [44], does not serve as a transporter for the
`uptake of naloxone by the brain.
`
`Conclusion
`
`The present study provides in vivo evidence that
`a pH-dependent and saturable transport system
`is involved in the influx of naloxone at the BBB in
`parallel with simple diffusion.
`Inhibition by
`cationic H1-antagonists such as pyrilamine and
`diphenhydramine suggests that naloxone trans-
`port
`is mediated by an as yet unidentified
`organic cation-sensitive transport system at the
`BBB. We cannot exclude, however, the possibility
`that in vivo saturation of naloxone uptake by the
`brain can be explained not only by saturation of
`the membrane transport system, but also by
`saturation of membrane surface binding and/or
`
`Copyright r 2010 John Wiley & Sons, Ltd.
`
`by trapping in an acidic intracellular component.
`While this in vivo study helps to confirm the
`involvement of an influx transport system in
`naloxone uptake, further investigation is needed
`fully to characterize the influx transport mechan-
`ism for naloxone through cellular uptake experi-
`ments using rat-brain capillary-endothelial cells.
`The present information obtained in vivo, how-
`ever, will be valuable for understanding the high
`distribution of opioid antagonists in the brain
`and for
`the development of cationic drugs
`affecting the central nervous system.
`
`Acknowledgements
`
`This work was supported in part by a grant from
`the High-Tech Research Center (to T.S. and T.F.)
`and the Academic Frontier (to T.F. and K.T.)
`Projects for Private Universities: matching fund
`subsidy from the Ministry of Education, Culture,
`Sports, Science and Technology (MEXT) for 2007-
`2009 in Japan, and Nihon University Individual
`Research Grant (No. 08-136) for 2008 (to T.S.).
`
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