Introduction

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The blood-brain barrier (BBB) is well known to restrict the penetration of xenobiotics from the circulating blood into the brain parenchyma (Suzuki et al., 1997; Pardridge, 1999). The tight junction, a specific feature of brain capillary endothelial cells, connects endothelial cells to each other and minimizes nonspecific transport via the paracellular route. Cumulative evidence from many studies suggests that efflux transport systems also provide a barrier function at the BBB. For example, active efflux mediated by P-glycoprotein (P-gp) has been shown to restrict the penetration of its substrates from the circulating blood into the brain in vitro and in a series of in vivo studies using P-gp inhibitors and P-gp knock-out mice (Kusuhara et al., 1997; Tsuji and Tamai, 1997; Schinkel, 1999).

We have demonstrated that the efflux transporters for organic anions are involved in the efflux of taurocholate (TCA), p-aminohippurate (PAH), an endothelin antagonist, BQ-123, and nucleoside analogs such as 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine through the BBB in vivo using the Brain Efflux Index (BEI) method (Kakee et al., 1997; Takasawa et al., 1997b; Kitazawa et al., 1998). Looking at a pharmacokinetic analysis of the disposition of AZT (Takasawa et al., 1997a) and new quinolone antibiotics (Ooie et al., 1997) in the brain using a model that reflects the anatomy of the brain, it appears that the transport directed from the brain to the blood is significantly greater than that in the opposite direction. Therefore, it is hypothesized that the efflux transporters for organic anions prevent their penetration from the circulating blood into the brain, resulting in their low distribution to the brain. The efflux transporters in the BBB are also considered to play important roles in the detoxification systems by removing xenobiotics and their conjugates (glutathione conjugates and glucuronides) from the brain, since: 1) 1-naphthyl-17-glucuronide exhibits nonlinearity in its elimination from the brain following microinjection (Leininger et al., 1991); and 2) there is an abundance of glutathione in the brain (Ghersi-Egea et al., 1994). The efflux transport systems for organic anions are also located in the choroid plexus (Angeletti et al., 1997; Gao et al., 1999; Nishino et al., 1999). It is suggested that the efflux transport systems are responsible for the efflux of glucuronide conjugates formed by UDP-glucuronosyl transferase in the choroid plexus (Nishino et al., 1999; Strazielle and Ghersi-Egea, 1999). Rapid elimination of E₂17G from the cerebrospinal fluid (CSF) was demonstrated in vivo and was inhibited by probenecid (Nishino et al., 1999). Organic anion transporting polypeptide 1 [Oatp1 (Slc21a1)] and multidrug resistance associated protein 1 (MRP1), multispecific transporters for structurally unrelated organic anions including glutathione conjugates and glucuronides, are considered to be responsible for this transepithelial transport of E₂17G from the CSF (Angeletti et al., 1997; Nishino et al., 1999). However, the efflux transport mechanism of glucuronides across the BBB remains to be clarified.

Recently, Gao et al. (1999, 2000) have demonstrated that rat Oatp2 (Slc21a5), an isoform of Oatp1, and human OATP-A (SLC21A3) are localized to the plasma membrane of brain capillary endothelial cells. The substrate specificity of Oatp2 is similar to that of Oatp1, except for digoxin and glutathione conjugates, and accepts organic anions such as TCA, estrone sulfate, and E₂17G as substrates (Reichel et al., 1999). Oatp2 is believed to be responsible for the efflux transport of TCA, BQ-123, and glucuronides across the BBB.

In this study, we examined the involvement of Oatp2 and other organic anion transporters in the efflux transport of E₂17G from the brain using the BEI method by investigating the effect of inhibitors of Oatp1, Oatp2, and organic anion transporters, such as Oat1 (Slc22a6) and Oat3 (Slc22a8), which are also multispecific organic anion transporters expressed in the brain (Sekine et al., 1997; Kusuhara et al., 1999). The selectivity of inhibitors of Oatp1, Oatp2, Oat1, and Oat3 was investigated by examining their inhibitory effects using their transfectants.

	Materials and Methods

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Chemicals. [³H]E₂17G (44 Ci/mmol), [³H]PAH (4.08 Ci/mmol), and [¹⁴C]carboxyl-inulin (2.5 mCi/g) were purchased from PerkinElmer Life Science Products (Boston, MA). [³H]E₂17G and [¹⁴C]carboxyl-inulin were stored at 20°C until use. Unlabeled probenecid and TCA were purchased from Sigma (St. Louis, MO). Unlabeled PAH was purchased from Wako Pure Chemical Industries (Osaka, Japan). Unlabeled digoxin was purchased from Aldrich Chemical (Milwaukee, WI). Ketamine hydrochloride was purchased from Sankyo Co. (Tokyo, Japan). Xylazine and ketamine hydrochloride were used as anesthetics. All other chemicals were commercially available, of reagent grade, and used without any purification.

Animals. Male Wistar rats (Nihon Ikagaku, Tokyo, Japan) weighing 240 to 270 g were used throughout this study and had free access to food and water.

Efflux of [³H]E₂17G from the Rat Brain after Microinjection into the Cerebral Cortex. The in vivo brain efflux experiments were conducted using the BEI method as described previously (Kakee et al., 1996). Rats were anesthetized with intramuscular doses of ketamine (125 mg/kg) and xylazine (1.22 mg/kg). After exposure of the skull, a 1.0-mm hole was made in the skull, 0.20 mm anterior and 5.5 mm lateral to the bregma, using a dental drill. A stereotaxic frame (Narishige, Tokyo, Japan) was used to determine the coordinates of the rat brain coinciding with Par2. The microinjection needle (330-µm diameter; Seiseido Medical Industry, Tokyo, Japan) was inserted into the hole to a depth of 4.5 mm. [³H]E₂17G (0.05 µCi/rat) and [¹⁴C]carboxyl-inulin (0.005 µCi/rat) dissolved in 0.5 µl of buffer containing 122 mM NaCl, 25 mM NaHCO₃, 10 mM D-glucose, 3 mM KCl, 1.4 mM CaCl₂, 1.2 mM MgSO₄, 0.4 mM K₂HPO₄, and 10 mM HEPES, pH 7.4, were injected into the Par2 region. After microinjection of drug into the cerebral cortex, an aliquot of CSF was taken from the cisterna magna. Immediately after CSF sampling, rats were decapitated, and the left and right cerebrum and cerebellum were removed. The excised cerebrum was dissolved in 2.5 ml of 2 N NaOH at 55°C for 1 h after measurement of the wet weight. The radioactivity associated with the brain specimens was determined in a liquid scintillation counter (LS 6000SE; Beckman Instruments, Fullerton, CA) after adding 14 ml of scintillation fluid (Hionic-fluor; Packard Instruments, Meriden, CT) to the scintillation vials. The 100  BEI (%) that represents the remaining percentage of drug in the ipsilateral cerebrum is described by the following:

Effects of Probenecid, TCA, PAH, and Digoxin on the Efflux of [³H]E₂17G from the Brain. To examine the effects of several compounds on the efflux of [³H]E₂17G from the brain, a mixture (0.5 µl) of [³H]E₂17G, [¹⁴C]carboxyl-inulin, and inhibitors was microinjected. The residual percentage of [³H]E₂17G was determined at 20 min after injection. Inhibitory effects were evaluated by comparing the elimination rate constant with respect to the control value. The kinetic parameters for the inhibitory effects of several compounds on the elimination of [³H]E₂17G from the brain were obtained by fitting, assuming competitive inhibition. Because of the limited solubility of digoxin, a higher concentration could not be achieved.

Stable Expression of Oatp1, Oatp2, Oat1, and Oat3 cDNA in LLC-PK1 Cells. The full length of Oatp1 was cut from the original plasmid pSPORT (Life Technologies, Gaithersburg, MD) using MluI, blunt-ended, and then subcloned into the blunt-ended pCXN2 vector [this pCXN2 supplied by Dr. J. Miyazaki, Osaka University, School of Medicine (Niwa et al., 1991)]. The full length of Oatp2 was cut from the original plasmid pBluescript (Life Technologies) using EcoRV and HincII, blunt ended, and then subcloned into the blunt-ended pCXN2 vector. The full length of Oat1 was cut from the original plasmid pSPORT using EcoRI and NotI, blunt-ended, ligated to the EcoRI arm (Life Technologies), and then subcloned into the EcoRI-digested pCXN2 vector. The full length of Oat3 was cut from the original plasmid pBluescript using EcoRI and then subcloned into the EcoRI-digested pCXN2 vector. These constructs were introduced into LLC-PK1 cells by lipofection with LipofectAMINE (Life Technologies) according to the manufacturer's protocols, and stably transfected cells were selected by adding G418 (Life Technologies) to the culture medium. Two weeks after the transfection, positive clones were selected by Northern blot analysis. The uptake from the basal side was comparable with that from apical side in all transfectants, suggesting that a similar amount of transporter transfected to LLC-PK1 cells is expressed on the apical and basal membrane of the cells.

Cell Culture. LLC-PK1 cells were grown in M199 (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and G418 sulfate (400 µg/ml) at 37°C with 5% CO₂ and 95% humidity on the bottom of a dish. Cells were cultured for 48 h with culture medium without sodium butyrate on the bottom of a dish and for an additional 24 h with culture medium supplemented with sodium butyrate (5 mM) before transport studies. In this study, LLC-PK1 cells between the 3rd and 25th passages were used.

Transport Study. Uptake was initiated by adding the radiolabeled ligands to the medium in the presence and absence of inhibitors after cells had been washed three times and preincubated with Krebs-Henseleit buffer at 37°C for 15 min. The Krebs-Henseleit buffer consists of 142 mM NaCl, 23.8 mM NaHCO₃, 4.83 mM KCl, 0.96 mM KH₂PO₄, 1.20 mM MgSO₄, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl₂ adjusted to pH 7.4. The uptake was terminated at designated times by adding ice-cold Krebs-Henseleit buffer. Then, cells were washed twice with 1 ml of ice-cold Krebs-Henseleit buffer, dissolved in 500 µl of 0.2 N NaOH, and kept overnight. The aliquots (350 µl) were transferred to scintillation vials after adding 50 µl of 2 N HCl. The radioactivity associated with the cells and medium was determined in a liquid scintillation counter after adding 2 ml of scintillation fluid (NACALAI TESQUE, Kyoto, Japan) to the scintillation vials. The remaining 50-µl aliquots of cell lysate were used to determine protein concentrations by the method of Lowry (1951), with bovine serum albumin as a standard. Ligand uptake is given as the cell-to-medium concentration ratio determined as the amount of ligand associated with the cells divided by the medium concentration.

	Results

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Time Profile of the Efflux of [³H]E₂17G from the Brain across the BBB. Figure 1 shows the time profile of the remaining fraction of [³H]E₂17G corrected by the recovery of [¹⁴C]carboxyl-inulin in the ipsilateral cerebrum after microinjection. Approximately 60% of the administrated dose of [³H]E₂17G was eliminated from the ipsilateral cerebrum within 20 min (Fig. 1). The apparent elimination rate constant (k_el) was determined as 0.037 ± 0.001 min¹. No significant amount of [³H]E₂17G or [¹⁴C]carboxyl-inulin was found in the contralateral cerebrum, cerebellum, or CSF compartment.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time profile of [3H]E217G in the ipsilateral cerebrum after intracerebral microinjection in the presence of [14C]carboxyl-inulin as an internal reference. A mixture of [3H]E217G (0.05 µCi/rat) and [14C]carboxyl-inulin (0.005 µCi/rat) dissolved in 0.5 µl extracellular fluid buffer was injected into Par2 of the rat cerebrum, and then the animals were decapitated at 2, 5, 10, and 20 min after microinjection. The solid line represents the fitted line obtained by nonlinear regression analysis. Each point represents the mean ± S.E. (n = 4).

Inhibitory Effects of Organic Anions and Digoxin on the Efflux of [³H]E₂17G from the Brain across the BBB. The efflux of [³H]E₂17G from the brain was completely inhibited by TCA and was reduced to about 20% of the total efflux by probenecid at the highest concentration examined (Fig. 2). According to the kinetic analyses, the IC₅₀ values of probenecid and TCA were 33.8 ± 13.0 and 1.75 ± 0.59 nmol/0.5 µl of injectate, respectively. The efflux of [³H]E₂17G from the brain was partially inhibited by PAH and digoxin (Fig. 2). The maximum inhibitory effects of PAH and digoxin at the maximum concentration examined were about 20 and 40%, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of unlabeled probenecid, TCA, PAH, and digoxin on the efflux of [3H]E217G from the brain after microinjection into the rat cerebrum. Extracellular fluid buffer containing [3H]E217G, [14C]carboxyl-inulin, and unlabeled inhibitors (0.5 µl/rat) was injected into the rat cerebrum. A through D show the effect of unlabeled probenecid, TCA, PAH, and digoxin on the efflux of [3H]E217G from the brain across the BBB. Each value of expected concentration is estimated by the concentration in the injectate divided by the dilution factor (Kakee et al., 1996). Results are given as a ratio with respect to the elimination rate constant determined in the absence of unlabeled inhibitors. Each point represents the mean ± S.E. (n = 3). *Significantly different from the control (P < 0.05).

Time Profiles for the Uptake of [³H]E₂17G into Transfectants. Transfection of Oatp1, Oatp2, and Oat3 to LLC-PK1 cells results in an increase in the uptake of E₂17G (Fig. 3). There was no significant accumulation of E₂17G into Oat1-transfected LLC-PK1 cells, compared with that into vector-transfected cells, whereas the accumulation of PAH into Oat1-transfected LLC-PK1 cells was significantly higher than that into vector-transfected cells (Fig. 3). Oatp1- and Oat3-mediated E₂17G uptake increased linearly over 2 min, whereas Oatp2-mediated E₂17G uptake and Oat1-mediated PAH uptake took place over 5 min. Eadie-Hofstee plots for the uptake via Oatp1, Oatp2, Oat1, and Oat3 are shown in Fig. 4. K_m values of E₂17G for Oatp1, Oatp2, and Oat3 were determined as 2.58, 17.0, and 8.43 µM, respectively, and the K_m value of PAH for Oat1 was 85.1 µM (Fig. 4 and Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Time profiles of the uptake of [3H]E217G and [3H]PAH into gene-transfected LLC-PK1 cells. The uptake of [3H]E217G into Oatp1- (A), Oatp2- (B), and Oat3-transfected LLC-PK1 cells (D) and the uptake of [3H]PAH into Oat1-transfected LLC-PK1 cells (C) were examined at 37°C. Open () and closed () squares represent the uptake into cDNA-transfected cells and vector-transfected cells, respectively. Each point represents the mean ± S.E. (n = 3).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of the uptake of [3H]E217G and [3H]PAH into gene-transfected cells. The uptake of [3H]E217G into Oatp1- (A), Oatp2- (B), and Oat3-transfected LLC-PK1 cells (D) in the presence of unlabeled E217G and the uptake of [3H]PAH into Oat1-transfected LLC-PK1 cells in the presence of unlabeled PAH (C) were examined at 37°C. Specific uptake was obtained by subtracting the uptake into vector-transfected cells from that into cDNA-transfected cells. Each point represents the mean ± S.E. (n = 3).

View this table: [in this window] [in a new window]   TABLE 1 Ki and Km values for Oatp1, Oatp2, Oat1, and Oat3 Effects of unlabeled probenecid, TCA, PAH, digoxin, and E217G on the uptake of [3H]E217G into Oatp1-, Oatp2-, and Oat3-transfected LLC-PK1 cells and on the uptake of [3H]PAH into Oat1-transfected LLC-PK1 cells were examined at 37°C. These Ki and Km values were determined by nonlinear regression analysis using data shown in Fig. 5. Each point represents the mean ± S.E. (n = 3).

Inhibitory Effects of Probenecid, TCA, PAH, and Digoxin on the Uptake via Oatp1, Oatp2, Oat1, and Oat3. The inhibitory effects of probenecid, TCA, PAH, and digoxin, on the uptake via Oatp1, Oatp2, Oat1, and Oat3 are shown in Fig. 5. Since no significant uptake of E₂17G was observed in Oat1-tranfected cells, the K_i values for Oat1 were obtained for the uptake of PAH. K_i values obtained assuming competitive inhibition are summarized in Table 1. Probenecid inhibited the transport via Oatp1, Oatp2, Oat1, and Oat3 with similar affinity for these transporters (Fig. 5 and Table 1). TCA inhibited the transport via Oatp1, Oatp2, Oat1, and Oat3, but the affinity of TCA for Oatp1 and Oatp2 was higher than that for Oat1 and Oat3 (Fig. 5 and Table 1). PAH inhibited the transport of E₂17G via Oat3, but did not inhibit the uptake of E₂17G via Oatp1 or Oatp2 (Fig. 5 and Table 1). Digoxin inhibited the transport of E₂17G via Oatp2, but did not inhibit either the transport of E₂17G via Oatp1, Oat3, or the transport of PAH via Oat1 (Fig. 5 and Table 1).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of unlabeled probenecid, TCA, PAH, and digoxin on the uptake of [3H]E217G and [3H]PAH into gene-transfected LLC-PK1 cells. The effects of unlabeled probenecid (A), TCA (B), PAH (C), and digoxin (D) on the uptake of [3H]E217G into Oatp1-, Oatp2-, and Oat3-transfected LLC-PK1 cells, and on the uptake of [3H]PAH into Oat1-transfected LLC-PK1 cells were examined at 37°C. The specific uptake was obtained by subtracting the uptake into vector-transfected cells from that into gene-transfected cells. Results are given as a ratio with respect to the control values determined in the absence of unlabeled compounds. Open () and closed () squares and open () and closed () circles represent the relative uptake via Oatp1, Oatp2, Oat1, and Oat3, respectively. Each point represents the mean ± S.E. (n = 3). The curves were fitted by nonlinear regression analysis, and the solid line represents the fitted curve.

	Discussion

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The efflux transport mechanism of E₂17G from the brain across the BBB was investigated using inhibitors, and examining their selectivity for transfected Oatp1, Oatp2, Oat1, and Oat3. E₂17G disappeared from the brain after the microinjection into the cerebral cortex exhibiting an elimination rate constant of 0.037 min¹ (Fig. 1). This elimination rate constant is higher than that of TCA and BQ-123 and the same as that of PAH (Kakee et al., 1997; Kitazawa et al., 1998). Since the radioactivity associated with E₂17G and inulin was not detected in the CSF at all, it appears that E₂17G undergoes the elimination from the brain through the BBB. Although it was not possible to examine the concentration dependence of the efflux of E₂17G from the brain because of its limited solubility, probenecid and TCA inhibited the efflux of E₂17G from the brain in a concentration-dependent manner, suggesting that an efflux transport system(s) is involved in the efflux of E₂17G from the brain (Fig. 2). Since the IC₅₀ value of TCA for the efflux of E₂17G (1.75 nmol/0.5 µl of injectate) was comparable with the K_m value of the efflux transport of TCA from the brain (0.396 nmol/0.2 µl of injectate) (Kitazawa et al., 1998), it is possible that the same efflux transport system(s) is shared by TCA and E₂17G. Since PAH did not affect the efflux of TCA from the brain at all (Kitazawa et al., 1998), there are, at least, two separate efflux transport systems for TCA and PAH on the BBB. PAH partially inhibited the efflux of E₂17G from the brain. Since the apparent K_m value of the efflux transport of PAH from the brain was found to be 6.0 nmol/0.5 µl of injectate as the injectate concentration (Kakee et al., 1997), the specific transport system(s) for PAH should be saturated at the maximum concentration examined (150 nmol/0.5 µl of injectate), and the maximum inhibitory effects of PAH (about 20%) indicates a contribution from the PAH-sensitive efflux transport system(s) on the BBB (Fig. 2). Digoxin also partially inhibited the efflux of E₂17G from the brain, and the maximum inhibitory effects of digoxin was about 40% (Fig. 2). Therefore, the contribution of the transport system (s) sensitive for PAH and digoxin accounts for, at most, 60% of the total efflux of E₂17G from the brain. Almost complete inhibition by probenecid and TCA suggests that an efflux transport system (s) accounts for the total efflux of E₂17G through the BBB. The total efflux of E₂17G from the brain to the blood consists of two steps: uptake on the abluminal membrane, followed by the excretion through the luminal membrane of the brain capillary endothelial cells. There are two possibilities for the residual fraction of the efflux of E₂17G (about 40%): 1) probenecid- and TCA-sensitive transporters are located on the abluminal membrane; or 2) the remaining fraction is accounted for by passive diffusion on the abluminal membrane, and probenecid and TCA also inhibit the excretion process on the luminal membrane.

Gao et al. (1999) demonstrated that Oatp2 is localized to the plasma membrane of the brain capillary endothelial cells. Northern blot analyses indicated that Oatp1, Oat1, and Oat3 are expressed in the brain (Jacquemin et al., 1994; Sekine et al., 1997; Kusuhara et al., 1999). E₂17G is known to be a substrate for Oatp1 and Oatp2 (Kanai et al., 1996; Noe et al., 1997). In this study, it was demonstrated that E₂17G was also a substrate for Oat3 (Figs. 3 and 4). The K_m value for the transport of E₂17G via Oat3 was 8.4 µM, which is comparable with those for Oatp1 and Oatp2 (Table 1) (Kanai et al., 1996; Noe et al., 1997). Since 1) no significant uptake of E₂17G was observed in Oat1-transfected LLC-PK1 cells and 2) E₂17G did not inhibit the uptake of PAH via Oat1 even at 300 µM (Table 1), E₂17G does not appear to be a substrate for Oat1. According to the kinetic studies using gene-transfected LLC-PK1 cells, probenecid inhibited both the Oatp and the Oat families with exhibiting similar inhibitory constants (K_i) (Fig. 5 and Table 1). TCA had a 20-fold higher affinity for the Oatp family than for the Oat family (Fig. 5 and Table 1). PAH and digoxin selectively inhibited the Oat family and Oatp2, respectively (Fig. 5 and Table 1). This suggests that probenecid is a nonselective inhibitor and TCA (at an optimal concentration), PAH, and digoxin are selective inhibitors for the Oatp family, the Oat family, and Oatp2, respectively.

Since drugs undergo about 40-fold dilution after microinjection into the cerebral cortex (Kakee et al., 1996), the apparent IC₅₀ value of TCA for the efflux of E₂17G via the BBB determined in vivo using the concentration in the injectate divided by this dilution factor indicated as expected concentration in Fig. 2 was estimated to be about 70 µM, and this value is comparable with the K_i values of TCA for the transport of E₂17G via the Oatp family (Table 1). On the other hand, the apparent IC₅₀ value of digoxin for the efflux of E₂17G via the BBB transport system(s) is estimated to be about 2 µM using the expected concentration, assuming that the maximum inhibitory effect of digoxin on the efflux of E₂17G from the brain is about 40%. This value is 50-fold greater than the K_i value of digoxin for Oatp2 (0.037 µM) (Table 1). In addition, the apparent IC₅₀ value of probenecid for the efflux of E₂17G via the BBB is also 20-fold higher than those for Oatp1, Oatp2, Oat1, and Oat3 (Table 1). This may be due to the tissue binding of digoxin and probenecid, and/or accumulation into neurons and/or astrocytes. Taking into consideration the results obtained from in vitro studies using transfectants, it appears that the contribution of Oatp2 and the Oat family, mainly Oat3, to the efflux of E₂17G is about 40 and 20%, respectively.

In the choroid plexus, Mrp1 is considered to be responsible for the excretion on the blood-side membrane (Nishino et al., 1999; Wijnholds et al., 2000a). In the BBB, Oatp2 is also expressed on the luminal membrane (blood-side membrane) (Gao et al., 1999), and it has been demonstrated that Oatp2 mediates bidirectional transport (Li et al., 2000). It is possible that Oatp2 is involved in the total efflux of E₂17G through the BBB, i.e., uptake and excretion across the brain capillary endothelial cells. In addition, Zhang et al. (2000) and Kusuhara et al. (1998) have demonstrated that Mrp1 and Mrp4-6 are expressed on the BBB. Since Mrp1 and Mrp5 accept conjugated metabolites (Wijnholds et al., 2000b), they might also be involved in the excretion process.

In conclusion, it appears from our in vivo and in vitro experiments that Oatp2 and the Oat family, mainly Oat3, account for about 40 and 20% of the total efflux of E₂17G from the brain across the BBB.