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Identification of Blood−Brain Barrier-Permeable Proteins Derived from a Peripheral Organ: In Vivo and in Vitro Evidence of Blood-toBrain Transport of Creatine Kinase Kazuki Sato, Masanori Tachikawa, Michitoshi Watanabe, Eisuke Miyauchi, Yasuo Uchida, and Tetsuya Terasaki* Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8577, Japan

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ABSTRACT: Certain proteins, such as inflammatory cytokines, that are released from injured or diseased organs are transported from the circulating blood through the blood−brain barrier (BBB) into the brain and contribute to the pathogenesis of related central nervous system dysfunctions. However, little is known about the protein transport mechanisms involved in the central nervous system dysfunctions. The aims of the present study were to identify BBB-permeable protein(s) derived from liver and to clarify their transport characteristics at the BBB. After administration of biotin-labeled liver cytosolic protein fraction to mice in vivo, we identified 9 biotin-labeled proteins in the brain. Among them, we focused here on creatine kinase (CK). In vitro uptake studies with human brain microvessel endothelial cells (hCMEC/D3 cells) showed preferential uptake of muscle-type CK (CK-MM) compared with brain-type CK (CK-BB) at the BBB. Integration plot analysis revealed that CK-MM readily penetrated into brain parenchyma from the circulating blood across the BBB. The uptake of CK-MM by hCMEC/D3 cells was decreased at 4 °C and in the presence of clathrin- and caveolin-dependent endocytosis inhibitors. These results indicate that entry of CK into the brain is mediated by a transport system(s) at the BBB. KEYWORDS: blood−brain barrier, blood−brain barrier-permeable protein, creatine kinase, comprehensive quantitative proteomics, peripheral-to-CNS communication

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INTRODUCTION

cules, such as peptides and proteins, under both physiological and pathological conditions. Previous studies have identified BBB transport systems for proteins,14 such as leptin, insulin, insulin-like growth factor-1 (IGF-1), transferrin, and lipoproteins, and inflammatory cytokines,8 such as interleukin-1α, -1β, -6 (IL-1α, IL-1β, and IL-6), and tumor necrosis factor-α (TNF-α). Also, various proteins are released into the circulating blood in disease states, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in liver failure, and creatine kinase (CK) isozymes in muscular and cardiac disorders. Thus, it is conceivable that multiple proteins that have leaked from various organs may be taken up into the brain via transport systems at the BBB. Clarifying the macromolecule transport systems at the BBB might also be helpful in providing tools for delivery of macromolecular drugs to the brain. Therapeutic macromolecules, such as recombinant proteins, monoclonal anti-

Dynamic communication between the central nervous system (CNS) and peripheral organs via signaling macromolecules plays a crucial role in homeostasis.1,2 For example, leptin, which regulates the body weight as a suppressor of food intake and body adiposity accumulation,3 is transported from the adipose tissues to the brain via leptin receptor (ObR)mediated transcytosis at the blood−brain barrier (BBB).4,5 We previously reported that atrial natriuretic peptide (ANP; generated in the brain), which is involved in the central control of cardiovascular and neuroendocrine functions, is transported from brain parenchyma to circulating blood via natriuretic peptide receptor C expressed at the BBB.6 Moreover, several inflammatory cytokines released into the circulating blood are transported into the brain across the BBB.7,8 The significance of peripheral-to-CNS signaling is further supported by the fact that various disorders in the liver (such as hepatitis C and liver cirrhosis),9 muscle (Duchenne muscular dystrophy),10 gut (inflammatory bowel disease),11,12 or adipose tissue (obesity)13 are associated with multiple brain dysfunctions. Thus, it seems plausible that the BBB regulates peripheral-to-CNS crosstalk by transporting various organ-derived macromole© XXXX American Chemical Society

Received: Revised: Accepted: Published: A

September 15, 2018 November 15, 2018 November 29, 2018 November 29, 2018 DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

with phosphate-buffered saline (PBS) (20 mL) and homogenized in hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH7.4) containing protease inhibitor cocktail (Sigma) and 0.125 mM phenylmethylsulfonyl fluoride with a Potter−Elvehjem homogenizer. The homogenates were transferred to a cavitation camber (Parr Instrument) and lysed with N2 gas at 450 psi for 15 min on ice. The lysates were centrifuged (10 000g, 10 min, 4 °C) to remove debris, and the supernatants were collected. They were centrifuged again (100 000g, 1 h, 4 °C), and the resulting supernatants were collected as cytosolic protein fraction. The solvent was replaced with PBS by using an ultrafiltration column (Concentrators, Spin 5K MWCO, Agilent Technologies), and then the cytosolic proteins of liver and cerebrum were reacted with biotin-SS-sulfo-OSu (20 nmol/mg cytosolic proteins) for 30 min at 25 °C. The biotinylation reaction was quenched by adding a 10-fold excess of glycine (incubation for 15 min at 25 °C). The biotin-labeled liver (160 mg/kg) or cerebrum (80 mg/kg) cytosolic protein fractions were intravenously administered to mice. The cerebrums were extracted 1 h later, after PBS perfusion (20 mL), and the cytosolic protein fraction was prepared. Cytosolic protein fraction was also prepared from a cerebrum of normal mice as a control. Cytosolic proteins were collected by acetone precipitation and washed with ice-cold acetone twice to remove endogenous biotin. The precipitated proteins were dissolved in 8 M urea in Tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-HCl, pH7.5) containing 0.1% Tween 20 and 0.1% Triton X-100 and diluted 10-fold with TBS containing 0.1% Tween 20 and 0.1% Triton X-100. A slurry of streptavidin mag sepharose beads (150 μL) was added to 3 mg of cytosolic proteins and incubated for 1 h at 25 °C in an end-over-end mixer to capture the biotin-labeled proteins. The beads were collected magnetically, and the supernatant was removed. The collected beads were washed 5 times with wash buffer (2 M urea, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% Tween 20, 0.1% Triton X-100). The collected proteins were eluted from the beads with 50 mM dithiothreitol (DTT) in 0.1 M Tris-HCl (pH 8.5) for 2 h at 25 °C, which cleaved the S−S bonds in biotin-SS-sulfo-OSu. The eluted proteins were Scarbamoylmethylated with iodoacetamide (IAA) for 1 h at 25 °C. The alkylated proteins were diluted 10-fold with 0.1 M Tris-HCl (pH 8.5) containing 0.05% protease-max surfactant (Promega) and digested with lysyl endopeptidase (Lys-C, Wako Pure Chemical Industries, Osaka, Japan) at 30 °C for 3 h. The Lys-C-digested peptides were treated with TPCKtreated trypsin (Promega, Madison, WI, USA) at 37 °C for 16 h. The resulting peptides were desalted by GC with GL-tip SDB (GL Sciences Inc., Tokyo, Japan), according to the manufacturer’s instructions, and measured by LC−MS/MS SWATH−MS. LC−MS/MS analysis was performed by coupling a nano-LC ultra 2D plus (Eksigent Technologies, Dublin, California) to an electrospray-ionization Triple TOF 5600 mass spectrometer (SCIEX, Framingham, Massachusetts). SWATH−MS was performed as previously described.21 To generate the spectrum library for SWATH−MS analysis, the digests obtained in the screening, whole-tissue lysates (mouse cerebrum, liver, and brain capillaries), cytosolic proteins (mouse cerebrum and liver), and crude and plasma membrane proteins (mouse cerebrum and liver) were measured by information-dependent acquisition (IDA). Data obtained by IDA measurement were analyzed with ProteinPilot (AB Sciex) and Mascot (Matrix Science). In SWATH−MS

bodies, and gene drugs, generally cannot be applied to treat CNS disorders, because their entry into the brain is blocked by the BBB.15 But, in recent decades, the conjugation of macrodrugs to antibodies against BBB-expressed protein transport receptors, such as transferrin receptor or insulin receptor, has provided a way to deliver therapeutic macromolecules into the brain, and this approach has been used to treat CNS disorders in experimental animal models of Parkinson’s disease16,17 and brain manifestations of mucopolysaccharidosis.18−20 Thus, a better understanding of protein transport systems at the BBB would promote the development of CNS-targeting macromolecular drugs. Mass spectrometry (MS)-based proteomics is a powerful tool not only for biomarker discovery21 but also for quantitative investigations of BBB transporters and receptors.22,23 Several candidates for BBB-permeable proteins present in the circulating blood have been found by using proteomics in conjunction with an in vitro Transwell system.24 However, there has been no comprehensive analysis of BBBpermeable proteins derived from peripheral tissues. SWATH− MS, a data-independent acquisition (DIA) technique, is likely to be especially suitable for this purpose, because it is far more comprehensive, quantitative, and reproducible than conventional proteomics.25 Indeed, the application of SWATH−MS has recently led to great progress in the fields of label-free protein quantification26 and biomarker discovery.21,27 Thus, the aims of the present study were to identify in vivo BBBpermeable protein(s) derived from the liver in mice by means of SWATH−MS and to conduct transport studies of a selected protein among them in order to obtain direct evidence of its BBB transport into the brain.

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MATERIALS AND METHODS Animals and Reagents. C57BL/6J JmsSlc (8 wks old, male) mice were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments were approved by the Animal Care Committee, Graduate School of Pharmaceutical Sciences, Tohoku University, and performed in accordance with the guidelines. BL21-CodonPlus(DE3)-RIPL competent cells were purchased from Agilent Technologies. CK from rabbit muscle was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), biotin-SS-sulfo-OSu from Dojindo Lab (Kumamoto, Japan), IPTG (isopropyl-β-D-thiogalactopyranoside) from Takara Bio Inc. (Shiga, Japan), and streptavidin mag sepharose from GE Healthcare Life Sciences. Chlorpromazine (CPZ), holotransferrin (hTf) from human blood, and albumin from bovine serum (BSA, Fraction V, pH 7.0) were purchased from Wako Pure Chemical Industries (Tokyo, Japan). Methyl-β-cyclodextrin (MβCD) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were obtained from Sigma. Peptide probes were synthesized by Scrum Inc. (Tokyo, Japan) (>95% purity). Other reagents were commercial products of analytical grade. Cell Culture. hCMEC/D3 cells were kindly provided by Pierre-Olivier Couraud (Department of Cell Biology, Institut Cochin, Paris, France)28 and routinely cultured in EndoGRO basal medium (Merck Millipore) containing 0.2% EndoGROLS supplement, 5 ng/mL rh EGF, 10 mM L-glutamine, 1 μg/ mL hydrocortisone hemisuccinate, 0.75 U/mL heparin sulfate, 50 μg/mL ascorbic acid, 1 ng/mL bFGF, 5% fetal bovine serum (FBS), and 1% penicillin−streptomycin, in an atmosphere of 95% air and 5% CO2 at 37 °C for 3−4 days. In Vivo Screening of BBB-Permeable Proteins. The liver and cerebrum were extracted from mice after perfusion B

DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. BBB-Permeable Protein Candidates Identified by in Vivo Screeninga accession number

protein name

theoretical isoelectric point (pI)b

P05201 Q04447 P16125 P52480−2 Q99KI0 P06745 P15105 P10649 Q9QZZ4

aspartate aminotransferase, cytoplasmic creatine kinase B-type L-lactate dehydrogenase B chain pyruvate kinase isoform M1 aconitate hydratase, mitochondrial glucose-6-phosphate isomerase glutamine synthetase glutathione S-transferase Mu 1 unconventional myosin-XV

6.68 5.40 5.70 6.69 8.08 8.14 6.64 7.72 9.26

identified group (liver or cerebrum administration) liver, liver, liver, liver, liver liver liver liver liver

cerebrum cerebrum cerebrum cerebrum

a

Mouse liver and cerebrum cytosol proteins were biotinylated with biotin-SS-sulfo-Osu. Biotin-labeled liver or cerebrum cytosol proteins were intravenously administered to mice. Cerebrum cytosolic proteins were extracted at 1 h, after PBS perfusion (20 mL), to wash out proteins in the vascular space. Nontreated normal mouse cerebrum cytosolic proteins were also extracted as a control. Biotin-labeled proteins were collected on streptavidin beads. The collected proteins were eluted from the beads and digested with Lys-C and trypsin. The peptide digests were measured by LC−MS/MS with SWATH−MS. Peak identification and calculation of the peak areas of the product ions of identified peptides were done by PeakView software. Among the detected peptides, reliable peptides were extracted on the basis of the peptide selection criteria, as described in the Materials and Methods section. The peak area ratios of product ions of identified peptides were calculated between biotin-labeled liver or cerebrum cytosol protein-administered mice and the control. The source proteins of peptides showing at least a 2-fold increase in the administered mice are listed in Table 1. Details of SWATH−MS-detected peptides are listedin Tables S1 and S2. bTheoretical isoelectric points of the identified proteins were calculated from their amino acid sequences by using ExPASY.77

CodonPlus(DE3)-RIPL competent cells). For protein expression, E. coli was incubated for 3 h in LB broth medium (Thermo Fisher Scientific, IL, USA) with 1 mM IPTG as an inducer. Harvested E. coli were collected by centrifugation (3000g, 10 min, 4 °C), washed with saline, pelleted, and solubilized in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0) with a tip sonicator (Sonifier 150D, Brason Ultrasonics Corp, USA) and nitrogen cavitation (1000 psi, 15 min, 4 °C). The lysates were centrifuged (15 000g, 10 min, 4 °C), and the supernatants and pellets were collected as the soluble fraction and insoluble fraction, respectively. Protein synthesis was confirmed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue (Figure S1). The soluble fractions were loaded on a HisPur Cobalt Spin column (Thermo Fisher Scientific, IL, USA), and the synthesized CKB and CK-M were purified in 6 M urea according to the manufacturer’s instructions. The purified proteins were diluted 60-fold with refolding buffer (5 mM DTT, 0.5 M NaCl, 50 mM Tris-HCl, pH 8.3) and incubated for 24 h at 4 °C to generate the dimeric form.34,35 The refolded CK-BB and CKMM dimers were loaded on a Strep-Tactin Sepharose column and purified in extracellular fluid (ECF, 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 0.4 mM K2HPO4, 10 mM D-glucose, 1.4 mM CaCl2, 1.2 mM MgSO4, 10 mM HEPES, pH 7.4, 300 ± 20 mOsm) buffer according to the manufacturer’s instructions. To quantify the synthesized proteins, the purified CK-BB and CK-MM were subjected to Lys-C and trypsin digestions. The purified CK-BB and CK-MM were dissolved in 6 M urea in 0.1 M Tris-HCl (pH 8.5) and S-carboxymethylated with DTT (1 h, 25 °C) and IAA (1h, 25 °C). The alkylated proteins were diluted 5-fold with 0.1 M Tris-HCl (pH 8.5) and treated with Lys-C for 3 h at 30 °C. The Lys-C-digested peptides were treated with TPCK-treated trypsin (Promega, Madison, WI, USA) at 37 °C for 16 h. The digests were spiked with stable isotope-labeled reference peptide^VI and ^LF as internal standards, and subjected to LC−MS/MS. LC−MS/MS analysis was performed with an electrospray ionization-triple quadrupole mass spectrometer (QTRAP5500; AB SCIEX, Framingham, MA) equipped with a Turbo V ion source (AB SCIEX) and coupled with a micro LC system (expert microLC 200; Eksigent Technologies, Dublin, CA, USA). The digests

data analysis, spectral alignment and peptide identification from SWATH−MS data were performed with the SWATH− MS Processing Micro App in Peakview (Version 2.0, SCIEX). Among identified peptides, unreliable peptides were removed by using amino acid sequence-based peptide selection29 and the following criteria: peptides with a glutamine residue at the N-terminus were removed because they are instable,30 and peptides, including a continuous sequence of asparagine and alanine (NA) or asparagine and glycine (NG), were removed because they are susceptible to degradation through deamination.31−33 Finally, the amounts of peptides in the liver or cerebrum cytosolic protein administration group were compared with those in the control, and the source proteins of peptides showing over a 2-fold increase in the administered group were identified as BBB-permeable protein candidates. These are listed in Table 1. Details of identified peptides are listed in Tables S1 and S2. Recombinant Creatine Kinase. Total RNAs from mouse cerebrum and muscle were extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNAs were synthesized by reverse transcription (ReverTra Ace, Toyobo Co., Ltd., Osaka, Japan) with Oligo(dT)15 Primer (Takara Bio Inc., Shiga, Japan). The creatine kinase B-type (CK-B) and M-type (CK-M) genes were each amplified from the cDNAs of brain and muscle by PCR with the following primers: for CK-B, 5′- CTTAGGATCCATGCCCTTCTCCAACAGCC-3′ and 5′-TATTGAATTCCTTCTGGGCCGGCATGAGGTC-3′, and for CK-M, 5′-ATTAGGATCCATGCCGTTCGGCAACACCCAC-3′ and 5′-TATTGAATTCCTTCTGCGCGGGGATCATGTCG-3′. To construct the protein expression vectors, the PCR products were inserted into the pET17b vector (Novagen), together with the purification tags of strep-tag (WSHPQFEK) and HAT-tag (KDHLIHNVHKEEHAHAHNK) at the 5′- and 3′terminal sides, respectively. We also inserted reference peptides Reference^VI (VIAPVLGR) and Reference^LF (LFGPSIPLAR) between the strep-tag and the 5′-terminus of CK, and between the 3′-terminus of CK and the HAT-tag, respectively, in order to evaluate the amount of the synthesized protein. The protein expression vectors of pET17b-CK-B and CK-M were transfected into Escherichia coli (E. coli, BL21C

DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics were injected onto a HALO Fused-Core C18 column (2.7 μm, 100 mm × 0.5 mm, 90 Å, Eksigent Technologies) at a flow rate of 10 μL/min. The peptides were eluted from the column using the following gradient sequences: 1% B (0−2 min), 1− 30% B (2−17 min), increased to 30−100% B (17−20 min), maintained at 100% B (20−22 min), reduced from 100 to 1% B (22−24 min), and then maintained at 1% B (24−30 min) at a flow rate of 10 μL/min. Mobile phase A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Reference peptides were monitored with four sets of SRM/MRM transitions (see Table S3). The reference peptide amounts were calculated as the average of three or four quantitative values, as described previously,29 and the amount of synthesized recombinant protein was determined as the average of the amounts of reference peptide^VI and ^LF. Mouse CK-BB and CK-MM Transwell Assays. hCMEC/ D3 cells were cultured on Transwells (6.5 mm, 0.4 μm pore size, polycarbonate, Corning International, Inc.) for 7 days. ECF buffer containing recombinant mouse CK-BB (1.57 ± 0.07 μM) or CK-MM (1.65 ± 0.07 μM) was added to the Tranwell insert and incubated for 3 h at 37 or 4 °C. ECF buffer in the inset and the bottom compartment was aspirated and evaporated in a centrifugal concentrator CC-105 (TOMY; heat off, 1 h) under vacuum. In addition, hCMEC/D3 cells on the Transwell insert were disrupted in hypotonic buffer and collected as whole-cell lysate. The protein concentration after cell disruption was measured by the Lowry method using the DC protein assay reagent (Bio-Rad, Hercules, CA). The evaporated medium and whole-cell lysates were solubilized in 6 M urea in 0.1 M Tris-HCl (pH 8.5), then subjected to Lys-C and trypsin digestions, spiked with internal standard reference peptides, and desalted as described previously. The desalted samples were measured by LC−MS/MS in the SRM/MRM mode. The protein amounts were calculated as the average of the amounts of the two reference peptides. Medium concentrations indicated in the description of the method for Transwell assay were determined by LC−MS/MS. Transflux rate was calculated as follows: Transflux rate (μL/cm2) = total amount (fmol) of CK in the bottom compartment/CK concentration in the inset chamber (fmol/μL)/cell growth area in the inset chamber (0.33 cm2). Influx rate (μL/cm2) was calculated as follows: Influx rate (μL/cm2) = total uptake amount (fmol) of CK in hCMEC/D3 cells/CK concentration in the inset chamber (fmol/μL)/cell growth area in the inset chamber (0.33 cm2). The apparent permeability coefficient (Papp) was calculated as follows: Papp (cm/s) = the permeability rate of CK from the inset chamber to the bottom compartment (fmol/s)/(cell growth area in the inset chamber (0.33 cm2) × *initial CK concentration in the inset chamber (fmol/mL)). *Initial CK concentration in the inset chamber was calculated as follows: (total amount (fmol) of CK left in the inset chamber after Transwell assay incubation + total amount (fmol) of CK internalized in hCMEC/D3 cells + total amount (fmol) of CK in the bottom compartment)/medium volume in the inset chamber (mL). In Vivo Detection of Administered Rabbit CK-MM in Cytosol of Mouse Cerebrum and Integration Plot Analysis. To examine the BBB permeability of rabbit CKMM in mouse, rabbit CK-MM (232 nmol) or PBS was intravenously administered to mice, and the cerebrums were extracted 12 h later, after PBS perfusion (20 mL). The cytosolic proteins were prepared, solubilized in 6 M urea in 0.1 M Tris-HCl (pH 8.5), and subjected to Lys-C and trypsin

digestions. The digest was desalted as described previously. The desalted peptides were subjected to isoelectric focusing and divided into 12 fractions with a 3−10 linear pH range by a 3100 OFFGEL Fractionator (Agilent Technologies, Böblingen, Baden-Württemberg, Germany) according to the manufacturer’s instructions. The peptides distributed in fraction 1 were aspirated and desalted again. The desalted samples were subjected to LC−MS/MS, and rabbit CK-MM specific peptide was measured by LC−MS/MS in the SRM/MRM mode (Q1/ Q3:598.8/559.3). In the integration plot analysis, rabbit CK-MM (589 nmol/ kg) was administered to mice, the plasma was collected after 1, 5, 15, 30, 60, 180, 360, and 720 min, and the cerebrum was extracted after 30, 60, 180, and 360 min. Cerebral parenchyma fraction and cerebral capillary fraction were isolated from whole cerebrum according to the capillary depletion method.36 Briefly, whole cerebrum was homogenized with 20 strokes in 5 volumes of physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 1 mM NaH2PO4, 2.8 mM CaCl2, 1 mM MgSO4, 10 mM D-glucose, pH 7.4). An equal volume of 32% dextran was added, and the mixture was homogenized again with 3 strokes. The homogenate in 16% dextran was centrifuged at 5400g for 15 min at 4 °C, and the pellet and supernatant were collected as cerebral capillary fraction and cerebral parenchyma fraction, respectively. The capillary fraction pellet was suspended in hypotonic buffer and disrupted by a tip sonicator. To remove dextran from the cerebral parenchyma fraction, trichloroacetic acid (TCA) was added (final conc. 20% TCA), and the mixture was incubated for 30 min on ice and centrifuged at 12 000g for 5 min at 4 °C. The resulting pellet was suspended in 20% TCA in physiological buffer and centrifuged again at 12 000g for 5 min at 4 °C. The pellet was washed with ice-cold acetone twice, and solubilized in 8 M urea in TBS containing 0.1% Tween 20/0.1% Triton X-100. The solution was diluted 10fold with TBS containing 0.1% Tween 20/0.1% Triton X-100. The protein concentrations of plasma, whole cerebrum tissue lysate, cerebral capillary fraction, and cerebral parenchyma fractions were measured by the Lowry method. These proteins were subjected to Lys-C and trypsin digestions as described previously. The digests were spiked with stable isotope-labeled peptides of rabbit CK-MM and desalted. Rabbit CK-MMspecific peptide in the desalted samples was measured by LC− MS/MS with four sets of SRM/MRM transitions (listed in Table S3). The CK concentration in cerebrum samples (whole cerebrum, cerebral parenchyma fraction, and cerebral capillary fraction) and plasma were calculated, and plots were made of cerebrum sample/plasma ratios (apparent Kp values) against normalized time, calculated as the area under the plasma concentration−time curve divided by the plasma concentration. The slope of the regression line represents the influx rate from the circulating blood into the whole cerebrum and cerebral parenchyma and the accumulation rate from blood into cerebral capillaries (μL/g brain/min). Immunohistochemical Study of Rabbit CK-MM Uptake by hCMEC/D3 Cells. hCMEC/D3 cells were exposed to 7.65 μM rabbit CK-MM for 3 h at 37 or 4 °C, then the medium was removed, and the cells were washed with PBS 3 times and fixed with 4% paraformaldehyde (Merck) in 0.1 M phosphate buffer (PB) (0.019 M NaH2PO4 and 0.081 M Na2HPO4) for 30 min at room temperature (rt). The fixed cells were permeabilized with 90% methanol/5% acetic acid for 10 min at −20 °C, incubated with 1% Triton X-100 for 30 min D

DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Among them, AST, CK B-type, L-lactate dehydrogenase B chain, and pyruvate kinase isoform M1 were commonly identified in both the liver- and cerebrum-derived proteinadministered groups. Five proteins, aconitate hydratase, glucose-6-phosphate isomerase, glutamine synthetase, glutathione S-transferase Mu1, and unconventional myosin-XV, were only identified in the liver-derived protein-administered group. In Vitro Transcellular Transport of CK-BB and CK-MM in Human Brain Microvessel Endothelial (hCMEC/D3) Cells. Transcellular transport of CK, which was identified as a candidate BBB-permeable protein (Table 1), was validated in a Transwell culture system of human brain microvessel endothelial cells (hCMEC/D3 cells), which are considered to be an in vitro model of human BBB. hCMCE/D3 cells retain not only small-molecule transport systems but also peptide and protein transport systems,40−42 and the expression profiles of transporters and receptors, including transport systems of insulin receptor and transferrin receptor, in hCMEC/D3 cells have much in common with those in in vivo human isolated brain capillaries.43 Indeed, hCMCE/D3 cells can construct a proper functional barrier, including the low density lipoprotein receptor-related protein 1 (LRP-1)mediated protein transport system, similar to the primarycultured human brain microvascular endothelial cells.44 The apical-to-cell uptakes of recombinant mouse CK-BB and CKMM proteins were significantly greater at 37 °C than at 4 °C (Figure 1a). The uptake rate of CK-MM was 10-fold greater

at rt, washed with PBS, and blocked with 10% goat serum for 30 min at rt. The blocked cells were reacted with anti-creatine kinase MM antibody (Abcam, ab151465, 1:500) diluted in 3% BSA-PBS overnight at 4 °C. The cells were washed with PBS and stained with donkey anti-rabbit IgG Alexa546 (Invitrogen) for 1 h at rt. Nuclei were counterstained with 4′,6-diamidino-2phenylindole (DAPI) for 30 min at rt. Photographs were taken with a confocal laser scanning microscope LSM 800 (Zeiss). In Vitro Uptake Study. In the uptake studies of rabbit CKMM, human hTf, and BSA, hCMEC/D3 cells were incubated with a mixture of 5.93 ± 0.11 μM rabbit CK-MM, 9.40 ± 0.22 μM hTf, and 5.66 ± 0.07 μM BSA in ECF buffer for 3 h at 37 or 4 °C. In the uptake studies with endocytosis inhibitors, hCMEC/D3 cells were preincubated with 5 μg/mL CPZ for 30 min, 5 mM MβCD for 1 h, or 100 μM EIPA for 30 min at 37 °C. After the preincubation medium was removed, the cells were exposed to 2.96 ± 0.07 μM rabbit CK-MM in ECF buffer for 1 h at 37 °C in the presence of CPZ or EIPA or in the absence of MβCD. To examine concentration-dependent uptake, hCMEC/D3 cells were exposed to stepwise increases of CK calculated as 0.1 μM × 5 to the X-th power, up to 62.5 μM (actual measured medium concentrations covered the range of 0.156−62.3 μM rabbit CK-MM) in ECF buffer for 3 h at 37 or 4 °C. In all cases, the medium was aspirated after the incubation, and cells were collected and disrupted in hypotonic buffer with a tip sonicator. The medium and cells were solubilized in 6 M urea in 0.1 M Tris-HCl (pH 8.5) and subjected to Lys-C and trypsin digestions as indicated previously. The digests were spiked with stable isotope-labeled peptides of only rabbit CK-MM or rabbit CK-MM, hTf, and BSA and desalted. The desalted samples were measured by LC−MS/MS with four sets of SRM/MRM transitions (listed in Table S3). Medium concentrations indicated in the description of the methods for each uptake study were determined by LC−MS/MS. The cell-to-medium (C/M) ratio was calculated as follows: C/M ratio (μL/mg protein) = intracellular uptake amount (fmol/mg protein)/medium concentration (fmol/μL). The influx rate was calculated as follows: Influx rate (fmol/mg protein/min) = intracellular uptake amount (fmol/mg protein)/exposure time (min). Statistical Analysis. All statistical analysis was performed using an unpaired two-tailed Student’s t test (equal variance) or Welch’s test (unequal variance) according to the result of the F test. p < 0.05 was used as the criterion of a significant difference.

Figure 1. Transwell assay of mouse recombinant CK-BB and CKMM. hCMEC/D3 cells cultured on Transwells were exposed to E. coli-synthesized recombinant mouse CK B-type (CK-BB) and M-type (CK-MM) for 3 h at 37 °C (■) or 4 °C (□). The influx rate from the apical side into hCMEC/D3 cells (a), and the transflux rate from the apical to basolateral side (b) are shown as the mean ± SEM (n = 3). The transflux rates of CK-BB and CK-MM (b) were converted into the following values of apparent permeability coefficient (Papp): 2.50 ± 0.46 (×10−7 cm/s) and 3.28 ± 0.85 (×10−7 cm/s) at 37 °C, and 1.58 ± 0.16 (×10−7 cm/s) and 1.59 ± 0.21 (×10−7 cm/s) at 4 °C, respectively. * (p < 0.05) and * * (p < 0.01) indicate significant differences between two groups. N.S. indicates no significant difference between the two groups (p > 0.05).

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RESULTS Quantitative and Comprehensive Proteomics-Based in Vivo Screening of BBB-Permeable Proteins Derived from Liver and Cerebrum in Mouse. Biotin-labeled cytosolic proteins of liver and cerebrum, which were transported to mouse brain cytosol fraction across the BBB after intravenous administration, were collected on streptavidin beads and identified as candidate BBB-permeable proteins by quantitative and comprehensive proteomics (SWATH−MS). The cytosolic proteins of liver and cerebrum were used because, (i) among the major organs, the liver has the highest protein-producing ability, including BBB-permeable proteins, such as transferrin,37 IGF-1,38 and α-2-macroglobulin,39 and (ii) proteins that have actually been transported across the BBB should exist in the cerebrum. As shown in Table 1, 9 candidate proteins were identified from mouse brain after intravenous administration of the biotin-labeled proteins.

than that of CK-BB at 37 °C (Figure 1a). The apical-to-basal transcellular transport of CK-BB and CK-MM proteins was decreased at 4 °C, compared to 37 °C, although without statistical significance (Figure 1b). The transflux rates of CKBB and CK-MM (Figure 1b) were converted into the following values of the apparent permeability coefficient (Papp): 2.50 ± 0.46 (×10−7 cm/s) and 3.28 ± 0.85 (×10−7 cm/s) at 37 °C, and 1.58 ± 0.16 (×10−7 cm/s) and 1.59 ± 0.21 (×10−7 cm/s) at 4 °C, respectively. These results indicate that transcellular transport of CK-BB and CK-MM occurs across monolayers of hCMEC/D3 cells, with CK-MMpreferential uptake at the apical side. E

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Molecular Pharmaceutics In Vivo Blood-to-Brain Transport of CK-MM across Mouse BBB. To distinguish intravenously administered CKMM from mouse endogenous CK, rabbit CK-MM, which has 98% amino acid sequence similarity with mouse CK-M, was administered to mice, and rabbit CK-MM in the brain was selectively quantified by QTAP. As shown in Figure 2, rabbit

Figure 3. Integration plot analysis of influx rates of CK-MM from blood to whole cerebrum and from blood to cerebral parenchyma, and the accumulation rate from blood into cerebral capillaries in mice. Rabbit CK-MM (589 nmol/kg) was intravenously administered to mice. Apparent Kp values of cerebrum whole-tissue lysates (●), cerebral parenchymal fractions (○), and cerebral capillary fractions (◇) were measured at 30, 60, 180, and 360 min after intravenous administration of rabbit CK-MM. The apparent Kp value was calculated as the concentration ratio of cerebrum whole-tissue lysate, cerebral parenchymal fraction, and cerebral capillary fraction to plasma. Normalized time on the horizontal axis was calculated as the area under the plasma concentration−time curve divided by the plasma concentration.

Figure 2. Rabbit CK-MM penetrated into mouse cerebrum cytosol from the circulating blood. (a) Rabbit CK-MM or (b) PBS were intravenously administered to mice. The cerebrums were extracted at 12 h after PBS perfusion (20 mL), and cytosol fractions were prepared. The proteins were digested with Lys-C and trypsin. The specific target peptide for rabbit CK-MM was separated by isoelectric focusing and measured by LC−MS/MS in the SRM/MRM mode (Q1/Q3:598.8/559.3).

CK-MM was detected in mouse brain cytosolic fraction after intravenous administration, although there was no signal of rabbit CK-MM in the PBS-administered mice. Furthermore, blood-to-brain parenchymal transport across the BBB was confirmed by integration plot analysis. The plasma concentration−time profile and compartmental pharmacokinetic parameters of rabbit CK-MM intravenously administered to mice are shown in Figure S2 and Table S4, respectively. The influx transport rates of rabbit CK-MM from the circulating blood into the whole cerebrum and cerebral parenchyma and the accumulation rate from blood into isolated cerebral capillaries were estimated to be 8.66 ± 0.33 (×10−3 μL/ min/g brain), 4.15 ± 0.95 (×10−3 μL/min/g brain), and 0.403 ± 0.042 (×10−3 μL/min/g brain), respectively (Figure 3). The influx rate of blood-to-cerebral parenchyma transport was over 10-fold greater than the accumulation rate of blood-to-cerebral capillaries. In contrast, the rates of blood-to-whole cerebrum and blood-to-cerebral parenchymal transport showed less than a 2-fold difference. These results indicate that rabbit CK-MM is transported into the brain parenchyma across the BBB. Characteristics of CK-MM Uptake by hCMEC/D3 Cells. The characteristics of rabbit CK-MM transport at the BBB were studied by using hCMEC/D3 cells. Immunofluorescence showed that the intracellular internalization of rabbit CK-MM was diminished at 4 °C, compared to that at 37 °C (Figure 4a,b). No signal was observed in the absence of rabbit CK-MM (Figure 4c). The uptakes of rabbit CK-MM and hTf, which undergoes transferrin receptor-mediated endocytosis, at 37 °C were significantly decreased by 68.6 and 59.7%, respectively, at 4 °C, whereas the uptake of albumin, a poorly BBB-permeable protein, was at the same at both 37 and 4 °C (Figure 5). These results indicate that an energy-dependent process is involved in the uptake of CK-MM in hCMEC/D3 cells; further, the endocytosis activity was comparable to that of transferrin, a well-known substrate of receptor-mediated endocytosis at the BBB. Rabbit CK-MM uptake by hCMEC/D3 cells was significantly inhibited by 59.0%, and 54.5% in the presence of the clathrin-mediated endocytosis inhibitor CPZ and the caveolinmediated endocytosis inhibitor MβCD, respectively, whereas

Figure 4. Temperature-dependent internalization of rabbit CK-MM in hCMEC/D3 cells. hCMEC/D3 cells were incubated with 7.65 μM rabbit CK-MM for 3 h at (a) 37 or (b) 4 °C. (c) Untreated hCMEC/ D3 cells cultured at 37 °C were used as the control. Rabbit CK-MM was removed, and the cells were incubated overnight with anti-CKMM antibody and stained with Alexa546-cojugated secondary antibody(red). Nuclei were stained with DAPI (blue). Scale bar = 20 μm.

the macropinocytosis inhibitor EIPA had no effect (Figure 6). Rabbit CK-MM uptake at 37 °C largely increased concentration-dependently compared to that at 4 °C, but it was not saturated up to 62.3 μM (Figure 7). There results suggest the involvement of clathrin- and caveolin-mediated endocytosis in the uptake of rabbit CK-MM.

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DISCUSSION Up to now, proteins that are released or leak from tissues have been primarily regarded as biomarkers for tissue disorders, and their physiological functions and kinetics in the circulating blood have not been studied much. The present work is the first to clarify the in vivo and in vitro BBB transport characteristics of creatine kinase, as a representative protein released from peripheral tissue under pathological conditions. CK catalyzes the reversible phosphorylation of creatine by adenosine triphosphate (ATP) and regulates ATP homeostasis. There are three dimeric isozymes of CK, brain-type (CK-BB), muscle-type (CK-MM), and hybrid (CK-MB) in cytosol; CK-BB is predominantly found in the brain, CK-MM in sarcomeric muscle, and CK-MB in heart muscle.45 Mouse F

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Figure 7. Temperature and concentration dependence of the uptake of rabbit CK-MM by hCMEC/D3 cells. hCMEC/D3 cells were exposed to rabbit CK-MM at various concentrations at 37 (●) or 4 °C (○) for 3 h. The influx rate was calculated from the uptake amount and exposure time (mean ± S.E.M, n = 3).

Figure 5. Comparative uptake study of rabbit CK-MM, hTf, and BSA in hCMEC/D3. hCMEC/D3 cells were exposed to a mixture of rabbit CK-MM, human hTf, and BSA for 3 h at 37 (■) or 4 °C (□), and the C/M ratio was calculated from the uptake amount and medium concentration (mean ± S.E.M., n = 5). * (p < 0.01) indicates a significant difference between the two groups. N.S. indicates no significant difference between the two groups (p > 0.05).

failure patients with encephalopathy.48 In addition, the blood concentration of CK-MM is increased by around 100-fold in Duchenne muscular dystrophy (DMD) patients,49 who show cognitive dysfunction as a major feature,10,50 compared to that under normal conditions (around 40 ng/mL). Thus, the circulating blood-to-brain transport system of CKs across the BBB is likely to have a functional impact on peripheral organto-brain communications, especially in patients with peripheral organ injuries. While the involvement of CKs in hepatic failureinduced encephalopathy remains to be determined, the abnormality of energy metabolism, including phosphocreatine/creatine system products in the brain, has been suggested to play a role in cognitive dysfunction with DMD.50,51 From this viewpoint, the blood-to-brain transport of CK-MM derived from the muscles of DMD patients may change the balance of the phospho-creatine/creatine system in the brain, which might be associated with brain functional abnormality. On the other hand, it was reported that intraperitoneal administration of cell-penetrating peptideconjugated CK-BB reduced acute seizure susceptibility in rats.52 This implies that BBB transport of CK-BB may rescue brain dysfunction due to abnormality of the CK system. Therefore, it seems plausible that the BBB transport system(s) of CKs would have pathophysiological relevance to the progression of brain pathology. CK is typically released from multiple injured peripheral organs during rhabdomyolysis,53 extreme exercise,54 intestinal infarction,55 and acute myocardiac infarction.56 Thus, clarifying the transport system of CK at the BBB will be helpful for understanding CNS-peripheral organ interactions. However, further study is needed to clarify the pathophysiological role of transport of CKs at the BBB, for example, by using animals with a knockout of CK transporter genes. To exclude the possibility that high-dose administration of liver cytosol proteins (160 mg/kg) might have caused BBB disruption, trypan blue staining of the brain parenchyma was performed. As shown in Figure S3, there was little staining of the brain parenchyma after the intracardial trypan blue perfusion in the mice given either biotinylated liver cytosolic proteins (320 mg/kg) or saline. These results indicate that the administration of liver cytosol proteins at the doses used here did not induce marked BBB disruption. Table 1 summarizes the theoretical pI (isoelectric point) values of identified candidates, ranging from 5.40 to 9.26. It has been reported,

Figure 6. The effect of endocytosis inhibitors on rabbit CK-MM uptake in hCMEC/D3 cells. hCMEC/D3 cells were preincubated with 5 μg/mL CPZ for 30 min, 5 mM MβCD for 1 h, or 100 μM EIPA for 30 min at 37 °C. The preincubation medium was removed, and the cells were exposed to rabbit CK-MM for 1 h at 37 °C in the presence of CPZ or EIPA, or in the absence of MβCD. C/M ratio was calculated from the uptake amount and medium concentration and shown as the mean ± SEM (% of control, n = 3). * (p < 0.05) and * * (p < 0.01) indicate a significant difference between the two groups. N.S. indicates no significant difference between the two groups (p > 0.05).

liver has been reported to express both CK-BB and CK-MM isozymes,46 but human47 or rat liver46 only expresses CK-BB. The present screening only detected CK B-type as a BBBpermeable protein in the liver cytosolic proteins administrated group, even though our findings indicate that the BBB transport system(s) for CK favors CK-MM over CK-BB; the reason for this could be that peptides corresponding to CK Btype, but not CK M-type, were included in the peptide spectrum library used for SWATH−MS analysis, because the identification of peptides/proteins by SWATH−MS analysis depends on the peptide spectrum library used.25 In this study, the library was constructed using IDA data, which includes the brain, which abundantly expresses CK B-type but not CK Mtype-expressing organs, such as muscle. Thus, it is possible that CK M-type was present but was not detected by our screening. Although CK-BB is generally absent in serum under normal conditions, it has been reported to be elevated in hepatic G

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Drug delivery utilizing protein transport systems at the BBB has great potential for the delivery of macromolecular drugs to treat CNS disorders.65 In particular, the transferrin receptor is a good target for drug delivery to the brain because its expression is limited to endothelial cells of the brain capillaries, and it is not present on peripheral endothelial cells.66 However, transferrin itself cannot be utilized as a drug delivery carrier, because the dissociation constant of transferrin receptor (against hTf: 5.6 nM)67 is much lower than the serum concentration of transferrin (35 μM) under physiological conditions.68 The Km values for transport of other proteins, such as the blood-to-brain transport of leptin,69 insulin,70 and the brain-to-blood transport of amyloid-beta,71 are in a range of 0.014 nM−0.25 μM. On the other hand, the present results indicate that CK-MM uptake by hCMEC/D3 cells was not saturated up to at least the concentration of 62.3 μM (Figure 7), suggesting that CK-MM transport is mediated by a highcapacity transport system(s). This would be advantageous for blood-to-CNS drug delivery. In addition to CK, our in vivo screening found eight other proteins as BBB-permeable protein candidates. Some of these candidates are known to be released into the blood from diseased liver, e.g., hepatic carcinoma (glutamine synthetase)72 and acute hepatitis (glutathione S-transferase).73 Notably, elevation of blood levels of AST has been reported in not only hepatic disorders74 but also hemolysis,75 extreme exercise,54 polymyositis,54 and acute myocardiac infraction.76 Thus, it would be intriguing in future studies to examine the BBB transport of other candidates identified as in vivo BBBpermeable proteins in order to uncover their possible pathophysiological relevance to the brain functional changes in patients with peripheral injuries.

that cationized peptides and proteins with pI values of more than 10 are internalized in in vitro brain capillary endothelial cells57 and isolated brain capillaries58 via absorptive-mediated endocytosis and can penetrate into the brain.59 Therefore, unconventional myosin-XV with the pI value of 9.26 might be transported into the brain across the BBB via the absorptivemediated endocytosis pathway. We observed mild temperature dependency of the transcellular transport of CK-BB and CK-MM across the hCMCE/ D3 cell monolayer (Figure 1b). In the Transwell system, the tightness of the tight junctions (TJ) of hCMEC/D3 affects the apparent permeability coefficient (Papp) values, because low tightness is associated with large paracellular leakage. The TJ tightness of hCMEC/D3 cells is affected by multiple factors, such as culture period (days) and cocultivation with other cells of neurovascular unit (e.g., astrocytes and pericytes).60 Indeed, the Papp values of CK-BB (2.50 × 10−7 cm/s) and CK-MM (3.28 × 10−7 cm/s) at 37 °C across an hCMCE/D3 monolayer constructed by culture for 7 days are about 10-fold lower than the reported value of albumin, a poorly BBB-permeable protein (about 30 × 10−7 cm/s), across a monolayer constructed by culture for 3 days.61 Thus, it could be considered, that the higher Papp of albumin reflects large paracellular leakage. However, even though hCMEC/D3 cells were cultured for 7 days in our analysis, this might still be insufficient for complete TJ formation. The Papp values of CK-BB and CK-MM at 4 °C are considered to represent paracellular leakage across the monolayer, and the large background accounts for the modest difference of apparent transflux activity of CK between 37 and 4 °C. To obtain Papp values relevant to the transcellular transport activity in in vivo human BBB, it will be essential to use culture models with characteristics closely resembling those of the in vivo human BBB. The influx rate of CK-MM from blood to whole cerebrum determined by the integration plot analysis seems very low, because it is only 8.7-fold higher than that of albumin (1 × 10−3 μL/min/g cerebrum).62 However, the influx rate obtained by the integration plot could be underestimated if the transport system(s) and/or peripheral clearance mechanism(s) are saturated.63 Therefore, the influx rate of CK might have been underestimated here, because the dosage of CK-MM was quite high (589 nmol/kg). The integration plot analysis also indicated that rabbit CK-MM penetrated into the cerebral parenchyma across the BBB. But, it is possible that, in the capillary depletion process, the plasma-membrane-bound rabbit CK-MM could have been dissociated to some extent into the parenchymal fraction, which would cause overestimation of transcellular transport across the BBB. As shown in Figure 3, the y-intercept of blood-to-whole cerebrum fraction was 7.33 (μL/g brain), which is the same as the luminal space volume of the blood vessels in mouse, i.e., 7.3 (μL/g brain).64 This indicates that rabbit CK-MM does not have strong binding affinity for the plasma membrane of the brain capillary endothelial cells. Therefore, the results shown in Figure 3 suggest that rabbit CK-MM permeates across the BBB and is not trapped in the capillary endothelial cells. However, we cannot exclude the possibility that rabbit CK-MM may bind to a putative receptor located in the luminal membrane of brain capillary endothelial cells with an extremely slow onset time, and that the bound CK-MM dissociates rapidly during the centrifugal separation process in the capillary depletion method.

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CONCLUSION In this work, we identified nine BBB-permeable liver-derived protein candidates in the mouse brain by means of SWATH− MS. Among them, we focused here on CK as a representative protein released from peripheral tissues under pathological conditions, and obtained evidence that it is actively transported at the BBB. These findings may provide insight into pathophysiological CNS-peripheral organ communication.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00975. Synthesis of mouse creatine kinase B-type and M-type recombinant proteins, plasma concentration−time profile of rabbit CK-MM after intravenous administration to mice, BBB integrity in mice treated with biotinylated liver cytosol proteins, details of SWATH-MS-identified peptides-derived from in vivo-BBB permeable cytosolic proteins in cerebrum, details of SWATH-MS-identified peptides-derived from in vivo-BBB permeable cytosolic proteins in liver, target peptide transitions in LC−MS/ MS analysis, and compartmental pharmacokinetic parameters of rabbit CK-MM intravenously administered to mice (PDF) H

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(8) Banks, W. A. Blood-brain barrier transport of cytokines: a mechanism for neuropathology. Curr. Pharm. Des. 2005, 11 (8), 973− 84. (9) D’Mello, C.; Swain, M. G. Liver-brain interactions in inflammatory liver diseases: implications for fatigue and mood disorders. Brain, Behav., Immun. 2014, 35, 9−20. (10) Cyrulnik, S. E.; Hinton, V. J. Duchenne muscular dystrophy: a cerebellar disorder? Neurosci. Biobehav. Rev. 2008, 32 (3), 486−96. (11) Dolapcioglu, C.; Dolapcioglu, H. Structural brain lesions in inflammatory bowel disease. World J. Gastrointest Pathophysiol 2015, 6 (4), 124−30. (12) Bonaz, B. L.; Bernstein, C. N. Brain-gut interactions in inflammatory bowel disease. Gastroenterology 2013, 144 (1), 36−49. (13) Monda, V.; La Marra, M.; Perrella, R.; Caviglia, G.; Iavarone, A.; Chieffi, S.; Messina, G.; Carotenuto, M.; Monda, M.; Messina, A. Obesity and brain illness: from cognitive and psychological evidences to obesity paradox. Diabetes, Metab. Syndr. Obes.: Targets Ther. 2017, 10, 473−479. (14) Preston, J. E.; Joan Abbott, N.; Begley, D. J. Transcytosis of macromolecules at the blood-brain barrier. Adv. Pharmacol. 2014, 71, 147−63. (15) Pardridge, W. M. Blood-brain barrier delivery. Drug Discovery Today 2007, 12 (1−2), 54−61. (16) Zhang, Y.; Schlachetzki, F.; Zhang, Y. F.; Boado, R. J.; Pardridge, W. M. Normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism with intravenous nonviral gene therapy and a brain-specific promoter. Hum. Gene Ther. 2004, 15 (4), 339−50. (17) Zhou, Q. H.; Sumbria, R.; Hui, E. K.; Lu, J. Z.; Boado, R. J.; Pardridge, W. M. Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor. J. Pharmacol. Exp. Ther. 2011, 339 (2), 618−23. (18) Boado, R. J.; Hui, E. K.; Lu, J. Z.; Zhou, Q. H.; Pardridge, W. M. Reversal of lysosomal storage in brain of adult MPS-I mice with intravenous Trojan horse-iduronidase fusion protein. Mol. Pharmaceutics 2011, 8 (4), 1342−50. (19) Boado, R. J.; Zhang, Y.; Xia, C. F.; Wang, Y.; Pardridge, W. M.; Zhang, Y. Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier. Biotechnol. Bioeng. 2008, 99 (2), 475−484. (20) Boado, R. J.; Ka-Wai Hui, E.; Zhiqiang Lu, J.; Pardridge, W. M. Insulin receptor antibody-iduronate 2-sulfatase fusion protein: pharmacokinetics, anti-drug antibody, and safety pharmacology in Rhesus monkeys. Biotechnol. Bioeng. 2014, 111 (11), 2317−25. (21) Miyauchi, E.; Furuta, T.; Ohtsuki, S.; Tachikawa, M.; Uchida, Y.; Sabit, H.; Obuchi, W.; Baba, T.; Watanabe, M.; Terasaki, T.; Nakada, M. Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics. PLoS One 2018, 13 (3), e0193799. (22) Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.; Kamiie, J.; Terasaki, T. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J. Neurochem. 2011, 117 (2), 333−45. (23) Uchida, Y.; Tachikawa, M.; Obuchi, W.; Hoshi, Y.; Tomioka, Y.; Ohtsuki, S.; Terasaki, T. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for interstrain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS 2013, 10 (1), 21. (24) Kim, S. Y.; Choi, E. S.; Lee, H. J.; Moon, C.; Kim, E. Transthyretin as a new transporter of nanoparticles for receptormediated transcytosis in rat brain microvessels. Colloids Surf., B 2015, 136, 989−96. (25) Gillet, L. C.; Navarro, P.; Tate, S.; Rost, H.; Selevsek, N.; Reiter, L.; Bonner, R.; Aebersold, R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol. Cell. Proteomics 2012, 11 (6), O111016717.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-22-795-6831; Fax: +81-22-795-6886; E-mail: [email protected]. ORCID

Kazuki Sato: 0000-0002-7455-2356 Masanori Tachikawa: 0000-0001-5711-5691 Tetsuya Terasaki: 0000-0002-6332-7575 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for JSPS Research Fellows (16J02046, K.S.), a Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan (N150602, K.S.), and a JSPS Bilateral Open Partnership Joint Research Projects Japan-Spain Research Cooperative Program (T.T.). We thank Ms. A. Niitomi and Ms. N. Handa for secretarial assistance.

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ABBREVIATIONS USED BBB, blood−brain barrier; LC−MS/MS, liquid chromatography coupled with tandem mass spectrometry; CK, creatine kinase; CNS, central nervous system; ANP, atrial natriuretic peptide; IGF-1, insulin-like growth factor-1; IL, interleukin; TNF, tumor necrosis factor; DIA, data-independent acquisition; IPTG, isopropyl-β-D-thiogalactopyranoside; SRM/ MRM, selected/multiple reaction monitoring; IDA, information-dependent acquisition; CPZ, chlorpromazine; MβCD, methyl-β-cyclodextrin; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; hTf, holo-transferrin; BSA, bovine serum albumin; C/M ratio, cell-to-medium ratio; LRP-1, low density lipoprotein receptor-related protein 1; DMD, Duchenne muscular dystrophy; pI, isoelectric point; TJ, tight junction; Papp, apparent permeability coefficient

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DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.8b00975 Mol. Pharmaceutics XXXX, XXX, XXX−XXX