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Organic Anion Transporting Polypeptide 1a4 (Oatp1a4/ Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats Yuka Yaguchi, Masanori Tachikawa, Zhengyu Zhang, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00005 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Molecular Pharmaceutics

Organic Anion Transporting Polypeptide 1a4 (Oatp1a4/Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats

AUTHOR NAMES Yuka Yaguchi1, Masanori Tachikawa1,2, Zhengyu Zhang1, Tetsuya Terasaki1*

AUTHOR AFFILIATIONS 1Division

of Membrane Transport and Drug Targeting, Graduate School of

Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan 2Graduate

School of Biomedical Sciences, Tokushima University, Tokushima, 770-8505,

Japan

*Corresponding

author: Professor Tetsuya Terasaki, Ph.D.

Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki, Aoba, Sendai 980-8578, Japan. Voice: +81-22-795-6831, FAX: +81-22-795-6886, Email: [email protected]

1 ACS Paragon Plus Environment

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

ABSTRACT GRAPHIC For Table of Contents Use Only

Manuscript Title Organic Anion Transporting Polypeptide 1a4 (Oatp1a4/Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats

Author Names Yuka Yaguchi1, Masanori Tachikawa1,2, Zhengyu Zhang1, Tetsuya Terasaki1*


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

ABSTRACT (250 words)

The blood-arachnoid barrier (BAB), which is formed by arachnoid epithelial cells linked by tight junctions, has generally been considered impermeable to water-soluble substances. However, we recently demonstrated that organic anion transporters 1 and 3 (oat1, oat3) play roles in drug clearance at the BAB.1 Here, we examined whether organic anion-transporting polypeptide (Oatp) also plays a role, using the fluorescent organic anion sulforhodamine-101 (SR-101) as a model substrate. SR-101 was injected into the cisterna magna of rats in order to minimize the contribution of choroid plexus transport. The in vivo CSF elimination clearance of SR-101 after intracisternal administration was 9-fold greater than that of fluorescein-labeled inulin, a bulk flow marker. In the case of pre-administration of taurocholate, a broad-spectrum inhibitor of Oatps, or digoxin, a strong substrate/inhibitor for Oatp1a4 but not for Oatp1a1, Oat1, and Oat3, the CSF elimination of SR-101 was significantly reduced, becoming similar to that of inulin, and thus indicating complete inhibition of SR-101 clearance from the CSF. The distribution of SR-101 fluorescence was restricted to the arachnoid mater in the absence of inhibitor, whereas the fluorescence was increased in the parenchyma of the spinal cord after coinjection of taurocholate or digoxin. Immunostaining confirmed the localization of



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Oatp1a4 in the arachnoid mater. These results indicate that Oatp1a4 at the BAB acts as an avid clearance pathway of SR-101 in the CSF to the blood. Thus, Oatp1a4 appears to play a major role in CSF detoxification by limiting the distribution of organic anions to the brain and spinal cord.

KEYWORDS organic

anion-transporting

polypeptide

1a4,

blood-arachnoid

barrier,

pharmacoproteomics, CSF clearance, arachnoid epithelial cells, blood-cerebrospinal fluid barrier, drug delivery to the brain,


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ABBREVIATIONS

BAB, blood−arachnoid barrier; Oatp, organic anion transporting polypeptide; Oat, organic anion transporter; CSF, cerebrospinal fluid; BCSFB, blood−cerebrospinal fluid barrier; SR-101, sulforhodamine-101; Slco, Solute carrier organic anion transporter family member; P-gp, P-glycoprotein; i.c.v., intracerebroventricular



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INTRODUCTION

Organic anion transporter polypeptide 1a4 (Oatp1a4/Slco1a4), the functional ortholog of human OATP1A2 at the blood-brain barrier (BBB), has attracted considerable attention as a possible pathway of drug delivery to the brain.2 Although rat Oatp1a4 transports many anionic drugs,3, 4 the apparent blood-to-brain concentration ratio (Kp,app) of substrates such as taurocholate, pitavastatin, and rosuvastatin is only equivalent to, or up to 5-fold greater than, the volume of brain capillaries in mice.5 The relatively low BBB permeability of Oatp1a4 substrates would be explained by the facts that Oatp1a4 is localized both on the luminal and the abluminal membranes of rat brain capillary endothelial cells,6 and it mediates brain-to-blood efflux transport as well as blood-to-brain influx transport across the BBB.5 On the other hand, Oatp1a4 is selectively localized on the basolateral membrane of rat choroid plexus epithelial cells, which form the bloodcerebrospinal fluid barrier (BCSFB),6 suggesting a role of Oatp1a4 in blood-to-CSF transport. The potential value of Oatp1a4 at the BBB as a route of drug delivery to the brain at the BBB and BCSFB remains uncertain. Recent studies have proposed that exchange of endogenous substances between the brain parenchyma and CSF occurs mainly in the subarachnoid space via the glymphatic


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system.7 Indeed, in the development of central nervous system (CNS)-acting drugs, the unbound drug concentration in the CSF of the subarachnoid space is generally used as a surrogate for the unbound concentration in the brain parenchyma, due to the difficulty of measuring the brain parenchymal concentration directly.8 However, some cases of inequivalence between the unbound drug concentrations in the CSF of the subarachnoid space and the brain parenchyma have been reported in rats.9 This raises the possibility that distinct regulatory mechanisms could be involved in controlling the transport kinetics in the two locations. We previously demonstrated that Oatp1a4 exhibits the highest expression level of 8.86 fmol/µg protein among the transporters tested in the plasma membrane fraction of rat arachnoid-containing leptomeninges, and its expression level is 3.2-fold and 1.3-fold greater than those of Oat1 (2.73 fmol/µg protein) and Oat3 (6.65 fmol/µg protein), which mediate CSF elimination of organic anion drugs at the blood-arachnoid barrier (BAB).1 Most of the CSF faces the BAB, because the CSF volume in the subarachnoid space accounts for approximately 80% of the total CSF volume in human and rat. 10 The surface area of arachnoid mater can be estimated to be about three-fold greater than that of choroid plexus.1 Therefore, the BAB could play a more substantial role than the BCSFB in regulating the kinetics of various substances in the CSF. Assuming that Oatp1a4, as



well as Oat1 and Oat3, is involved in the CSF elimination of drugs at the BAB, the low brain distribution of Oatp1a4 substrates may be due to Oatp1a4-mediated CSF-to-blood efflux transport after blood-to-brain influx transport across the BBB. Therefore, clarifying the localization of Oatp1a4 at the BAB and its contribution to influx and/or efflux transport between the blood and the CSF is important for a better understanding of its role in the CSF distribution of drugs. Thus, the purpose of the present study was to clarify the localization of Oatp1a4 and to examine its functional role at the BAB in rats.


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MATERIALS AND METHODS

Animals. Male adult Wistar rats (10 weeks age, 220-280 g body weight) were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were maintained on a 12-h light/dark cycle in a temperature-controlled environment with free access to food and water. All experiments were approved by the Institutional Animal Care and Use Committee in Tohoku University, and were performed in accordance with Tohoku University guidelines. The rats were randomly divided into 8 groups. They were anesthetized by intramuscular injection of ketamine and xylazine (50 mg/kg body weight, respectively), and their heads were fixed in a stereotaxic apparatus (Narishige, Tokyo, Japan). In vivo SR-101 and Alexa Fluor 594 hydrazide (A594) elimination from the CSF after intracisternal administration. Since SR-101 is a fluorescent substrate of rat Oatp1a4, 11 we considered that it would be a useful tool to elucidate the transport kinetics of CSF elimination and to visualize the distribution in the arachnoid mater. Under anesthesia, a 30-gage needle was inserted into the cisterna magna of the rats. A mixture of SR-101 or A594 (250 pmol) and FITC-inulin (10 µg) dissolved in 10 µL artificial CSF (122 mM NaCl, 15 mM NaHCO3, 10 mM glucose, 0.4 mM K2HPO4, 1.2 mM



MgSO4·7H2O, 3 mM KCl, 1.4 mM CaCl2, 10 mM HEPES)12 was injected at the speed of 20 µL/min via the cisterna magna. To exclude a major contribution of the choroid plexus to elimination from the CSF, intracisternal administration was conducted as previously reported1. In inhibition studies, a mixture of SR-101 (250 pmol) and FITC-inulin (10 µg) was injected with or without pre-administration of inhibitors such as taurocholate, digoxin, para-aminohippuric acid (PAH) or bromosulfophthalein (BSP) dissolved in 10 µL artificial CSF at the concentrations of 10 mM, 1.25 mM (containing 2.5% dimethyl sulfoxide; DMSO), 25 mM or 2.5 mM, respectively. CSF samples (approximately 80~120 µL) were withdrawn via cisterna magna puncture using a 1 mL syringe at the designated time after administration (2, 10, 20, or 40 min), or 20 min for inhibition studies. The fluorescence in CSF specimens was measured using a Fluoroskan Ascent FL (Labsystems, Helsinki, Finland). The kinetic parameters for the elimination of FITCinulin, A594 (Eq. 1) and SR-101 (Eq. 2) were determined using the time points of 2, 10, 20, and 40 min for FITC-inulin and SR-101, and the time points of 2, and 20 min for A594 by using the nonlinear least-squares regression analysis program MULTI, as previously reported.13, 14 The Y-intercept of SR101 was assumed to be the same as that of FITC-inulin. CCSF(t) = C0 × exp(-kel × t) 　　　　　　　　　　　　　　　(1)


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CCSF(t) = A × exp(-α × t) + B × exp(-β×t)　　　　　　　　　

(2)

The elimination clearance were calculated by dividing the administered dose by the area under the curve (AUC), which was calculated as (C0/kel) or (A/α＋B/β). Distribution of SR-101 in brain and spinal cord tissues. 　The rats were randomly divided into 3 groups. To examine the distribution of SR-101 in the brain and spinal cord, a solution of SR-101 (2.5 nmol) in 10 µL artificial CSF was injected at the speed of 20 µL/min via cisterna magna puncture with or without pre-administration of inhibitor taurocholate, digoxin or PAH dissolved in 10 µL artificial CSF at the concentrations described in the previous section. Brain and spinal cord were isolated at 20 min after the intracisternal administration and frozen on dry ice. Sections (20 µm in thickness) were prepared with a cryostat (Leica CM 1900; Leica Microsystems, Bensheim, Germany), and SR-101 fluorescence in the tissues was detected with a fluorescence microscope (BX51WI, Olympus, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera (ORCA-Flash4.0, Hamamatsu Photonics, Hamamatsu, Japan). Immunohistochemistry.　　The anesthetized rats were decapitated and the brain, and cervical, thoracic, and lumbar spinal cords were excised and frozen on dry ice. Frozen tissue sections (20 µm in thickness) were prepared and fixed with 4 % paraformaldehyde in phosphate buffer for 30 minutes at room temperature. The sections were immersed in



0.2 % Triton-X in PBS, followed by blocking with 10% goat serum or donkey for 30 min. They were immune-reacted overnight with guinea pig anti-rat Oatp1c1 antibody (1 µg/mL)15, or with guinea pig anti-rat Oatp1a4 antibody (1 µg/mL)15 singly or in combination with mouse monoclonal anti-human P-gp antibody (C219, GeneTex, California, USA) (1:12 dilution) at 4℃. Subsequently, they were incubated with goat anti guinea pig IgG Alexa Fluor 488-conjugated and goat anti mouse IgG Alexa Fluor 546conjugated (Life Technologies Inc., Maryland) secondary antibodies for 2 h. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL) for 10 min. Photographs were taken with a confocal laser scanning microscopes at the resolution of 96 dpi (AXIO Observer.Z1, ZEISS, Jena, Germany; TCS SP8, Leica Microsystems, Wetzlar, Germany). Statistical Analysis.

All data are presented as the mean±S.D. An unpaired, two-

tailed Student's t test was used to determine the significance of differences between means of two groups. One-way analysis of variance followed by the modified Fisher's leastsquares difference method was used to assess the statistical significance of differences among means of more than two groups.


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RESULTS

In vivo elimination of SR-101 from the CSF after intracisternal administration. The apparent elimination clearance of SR-101 (13.4±2.5 µL/min) was 6.5-fold greater than that of FITC-inulin (2.07±0.47 µL/min), a CSF bulk flow marker (Fig. 1A). The elimination clearance of inulin was close to the CSF bulk flow rate (2.9 µL/min) obtained in a previous study, in which the bulk flow marker was injected into the lateral ventricle and CSF was obtained from the cisternal magna.16 SR-101 fluorescence was detected in the leptomeninges along the cerebellum at 2 min after intracisternal administration (Fig. 1B). As shown in Table 1, taurocholate, a broad-spectrum inhibitor of Oatps, and digoxin, a strong substrate/inhibitor of Oatp1a4, but not Oatp1a1, Oat1, or Oat3,17, 18 significantly inhibited the elimination of SR-101 from the CSF at 20 min after its administration, whereas they had no effect on inulin elimination. Bromosulfophthalein (BSP), which is reported to inhibit multiple Oatp isoforms except for Oatp1a4,17,

19, 20

exhibited 20%

inhibition. PAH, a typical substrate of oat1 and oat3, did not inhibit the elimination of SR-101.

Distribution of SR-101 in the brain and spinal cord tissues.



In the absence of inhibitors, SR-101 fluorescence was predominantly detected in the leptomeninges at the surface of the cerebral cortex (Fig. 2A and B) and cerebellum (Fig. 2A and C) and in the choroid plexus located in the fourth ventricle (Fig. 2 C) at 20 min after intracisternal administration. In contrast, there was no signal in the choroid plexus located in the lateral (Fig. 2D) and third ventricles (data not shown). In the absence of inhibitors, the fluorescence intensity was greatest in the cervical spinal cord near the SR101 injection site (Fig. 2E) and was weaker in the thoracic and lumbar spinal cords, which are far from the injection site (Fig. 2F-G). As shown in Fig. 3, when taurocholate (Fig. 3C-D) and digoxin (Fig. 3E-F) were pre-administered into the cisterna magna, the SR101 fluorescence was diminished in the leptomeninges of control rats (Fig. 3A-B) and instead was increased in the parenchyma of the cervical spinal cord.

Localization of Oatp1a4 and Oatp1c1 in arachnoid mater. Immunohistochemistry showed that the fluorescence of Oatp1a4 was localized in the arachnoid mater as well as the capillaries in the cervical spinal cord (Fig. 4A) and brain (Fig. 4B). P-gp was previously reported to be localized on the dura-facing membrane of arachnoid mater epithelial cells,21 and double immunofluorescence showed that the signals of Oatp1a4 and P-gp were not overlapped (Fig. 4C). This suggests distinct


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localizations of P-gp and Oatp1a4 on the dura-facing and CSF-facing membranes, respectively, across the nucleus of the arachnoid mater in the cervical spinal cord. No staining was detected in the arachnoid mater when the sections were incubated without a primary antibody (negative control) (Fig. 4D). In contrast, signals of Oatp1c1 were not detected in the arachnoid mater, but were present in the blood vessels in the cervical spinal cord (Fig. 4E) and cerebral cortex (Fig. 4F). These expression patterns are identical with those in the thoracic and lumbar spinal cords (data not shown).

In vivo elimination of A594, a membrane-impermeable fluorescent tracer, from the CSF after intracisternal administration. It has been reported that fluorescent tracer and horseradish peroxidase are distributed into the perivascular spaces after intracisternal injection.22,

23

FITC-inulin used in the

present study is suitable to calculate the CSF bulk flow rate, since it has a large molecular weight of 2000-5000 Da and is membrane-impermeable. On the other hand, we cannot rule out a contribution of diffusion into the spinal cord, followed by parenchymal endothelial cell-mediated efflux transport, to the CSF elimination of SR-101, because SR101 has the small molecular weight of 606.71 Da. Since Oatp1a4 is localized in the capillaries of the brain and spinal cord (Fig. 4), it is possible that SR-101 in the CSF flows



to the perivascular spaces and is eliminated via these capillaries. To estimate the elimination clearance via the perivascular route, Alexa Fluor 594 hydrazide (A594), which has the low molecular weight of 758.79 Da and is membrane-impermeable, was injected from the cisterna magna. The apparent elimination clearance of A594 was estimated to be 4.31±2.43 µL/min, and this value is close to that of FITC-inulin (3.05±1.49 µL/min). This result suggests that the spinal cord endothelial cell-mediated efflux transport of SR-101 could be negligible at least up to 20 min.


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

DISCUSSION

The present study is the first to demonstrate the functional significance of Oatp1a4mediated organic anion transport at the BAB in the elimination of a substance from the CSF. We found (i) a 6.5-fold greater elimination rate of SR-101 from the CSF after intracisternal administration compared to a CSF bulk flow marker, (ii) significant inhibition of SR-101 elimination by taurocholate and digoxin, a broad-spectrum inhibitor of Oatps24 and a strong substrate/inhibitor for Oatp1a4 but not for Oatp1a1, Oat1, and Oat3,17,

18

respectively, and (iii) localization of Oatp1a4 protein on the CSF-facing

membrane of BAB epithelial cells. These results suggest that Oatp1a4 most likely serves as the elimination pathway at the BAB for SR-101 present in the CSF. We also found that the fluorescence of SR-101 after intracisternal administration is distributed in the arachnoid mater. Administration of taurocholate and digoxin diminishes this fluorescence, while the parenchymal distribution of SR-101 is significantly increased in the spinal cord. It is thus likely that the Oatp1a4-mediated clearance system for SR-101 serves to prevent SR-101 distribution into parenchymal cells in the spinal cord. Our previous quantitative targeted absolute proteomics study demonstrated that Oatps besides Oatp1a4 are expressed in rat leptomeninges,1 suggesting that other Oatp isoforms



might be expressed in arachnoid mater epithelial cells. In the experiment with BSP, which is reported to inhibit multiple Oatp isoforms except for Oatp1a4, 17, 19, 20 we observed 20% inhibition (p

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