Trimeric PEG-Conjugated Exendin-4 for the Treatment of Sepsis

Publication Date (Web): February 23, 2016. Copyright © 2016 American Chemical Society. *(D.H.Na) Tel: 82-53-950-8579; Fax: 82-53-950-8557; E-mail: dh...
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Trimeric PEG-Conjugated Exendin‑4 for the Treatment of Sepsis Wonhwa Lee,†,§ Eun Ji Park,†,§ Soyoung Kwak,† Kang Choon Lee,‡ Dong Hee Na,*,† and Jong-Sup Bae*,† †

College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics based Creative Drug Research Team, Kyungpook National University, Daegu 41566, Republic of Korea ‡ School of Pharmacy, SungKyunKwan University, Suwon 03063, Republic of Korea S Supporting Information *

ABSTRACT: Exendin-4 (EX4), a glucagon-like peptide-1 receptor (GLP-1R) agonist that regulates blood glucose levels, has been used in the management of type-2 diabetes mellitus. EX4 can be PEGylated to improve its antidiabetic effects by enhancing its stability and extending the circulation half-life. Here, to determine whether PEGylated EX4 is effective for the treatment of sepsis, C-terminal thiol-specific PEGylated EX4s with linear maleimide-PEG-2K, -5K, -20K and trimeric maleimide-PEG-50K (hereafter referred to as EX4-2K, EX45K, EX4-20K, and EX4-50K, respectively) were prepared, and their antiseptic responses were investigated. These PEGylated EX4s reduced cecal ligation and puncture (CLP)-induced organ injury by decreasing hyperpermeability, and suppressing interactions between leukocytes and endothelial cells. The binding avidity and stability of EX4-50K toward GLP-1R were superior to that of wild-type EX4, as was the circulation half-life of EX4-50K. In addition, the antiseptic effects of EX4-50K were superior to those of other PEGylated EX4s, which may be attributed to enhanced proteolytic stability, longer circulation half-life, and higher receptor-binding affinity of EX4-50K due to its trimeric PEG structure. Therefore, EX4-50K may decrease CLP-induced septic mortality in vivo. There are currently neither effective preventatives against nor treatment options for sepsis; our results show that EX4-50K has the potential to treat sepsis.

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INTRODUCTION

there are currently no effective preventatives or treatment options for severe sepsis. Exendin-4 (EX4) is a glucagon-like peptide-1 (GLP-1) receptor agonist that was originally isolated from the saliva of the Gila monster lizard. EX4 has a 53% amino acid sequence overlap with GLP-1, a gut incretin hormone secreted from L cells in the intestine in response to food intake.9 Similar to the glucoregulatory action of GLP-1, EX4 also regulates blood glucose levels by inducing glucose-dependent insulin secretion, and reduces apoptotic cell death of pancreatic β-cells.10,11 Although the synthetic version of EX4 (Exenatide, Byetta) is commercially available for the management of type-2 diabetes mellitus, its short plasma half-life of 1.5−4 h limits its therapeutic application, requiring injection twice daily.12−14 Several drug delivery techniques have been developed to improve the pharmacokinetic properties of EX4.15−18 Recently, a once-weekly dosage form of EX4 was commercialized in the form of a biodegradable poly(D,L-lactide-co-glycolide) microsphere (Bydureon);17 however, the complicated preparation process of the microspheres has resulted in irregular drug

Sepsis typically develops as a complication following illnesses such as pneumonia and bacterial infections, and is the most common cause of mortality in intensive care units.1−3 There exists a continuum of clinical manifestations, from systematic inflammatory response syndrome (SIRS), to sepsis, to severe sepsis, to septic shock, to multiple organ dysfunction syndrome (MODS).1−3 Xigris (Eli Lilly) was approved for the treatment of severe sepsis and septic shock in the United States in 2001 by the Food and Drug Administration (FDA), and in 2002 by the European Medicine Agency.4 However, in October 2011, Xigris was withdrawn from the market due to side effects and a lack of beneficial effects on 28-day mortality following the Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and septic shock (PROWESSSHOCK) trials.5 The most common adverse event associated with activated protein C (APC) is bleeding caused by degradation of the procoagulant cofactors Va and VIIIa, which is consistent with the antithrombotic activity of APC.4,6 The average half-life of APC is short (around 15−18 min in whole blood and 31 min in citrated blood),7,8 suggesting that APC is rapidly eliminated from the blood and should be administered in a way that achieves a steady-state dose. Thus, © 2016 American Chemical Society

Received: December 29, 2015 Revised: February 12, 2016 Published: February 23, 2016 1160

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Biomacromolecules

FNI-675 NHS ester fluorescent dyes (Bioacts Inc., Incheon, Korea). Bacterial lipopolysaccharide (LPS, serotype: 0111:B4, L5293) and antibiotics (penicillin G and streptomycin) were purchased from Sigma (St. Louis, MO). Human GLP-1R protein (P43220) and AntiGLP-1 receptor antibody (ab39072) was purchased from abcam and antimouse CD31 (553369) was purchased from BD Falcon. All other reagents, unless indicated, were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Preparation and Characterization of PEGylated EX4s. Sitespecific PEGylation of Ex4 was achieved at free thiol group in Cterminal Cys residue using PEG-MALs or tPEG-50K-MAL. Briefly, 200 μL of PEG-MALs or tPEG-50K-MAL solution in 50 mM phosphate buffer (pH 6) were added to 100 μL of Ex4 (10 mg/mL in deionized water) with a molar ratio of 1:1.5 (Ex4:PEG-MALs or tPEG50K-MAL). The reaction was allowed to continue for 1 h at room temperature. To isolate PEGylated EX4 species, the reaction mixtures were diluted with 10-times volume of equilibrium buffer A (20 mM acetate buffer at pH 4.0) and loaded onto a TSKgel SP-5PW cationexchange column (75 × 7.5 mm id, 10 μm, Tosoh Bioscience, Tokyo, Japan) pre-equilibrated with equilibrium buffer A at a flow rate of 0.8 mL/min. After washing for 8 min, a linear gradient from 0% to 100% of elution buffer (1 M NaCl in buffer A) for 40 min was followed to elute PEGylated EX4. UV absorbance was monitored at 280 nm. The fractions corresponding to PEGylated EX4s were collected and identified by MALDI-TOF MS as previously described.34 The identified PEGylated EX4s were concentrated to approximately 1 mg/mL with Amicon centrifugal filters (MW cutoff 3 or 10 kDa, Millipore, Billerica, MA, USA), filtered with a 0.2 μm membrane filter, and stored at −70 °C until required. The protein concentration was determined by micro-BCA assay (Pierce, Rockford, IL, USA). Molecular masses of PEGylated EX4s were measured by MALDITOF MS analysis using a Bruker Daltonics Microflex MALDI-TOF mass spectrometer (Bremen, Germany) with a 337 nm nitrogen laser. Mass spectra were obtained in the linear and positive-ion mode with an acceleration voltage of 20 kV. As a matrix solution, a saturated solution of sinapinic acid in acetonitrile:water (50:50, v:v) containing 0.1% trifluoroacetic acid (TFA) was used. Each analyte was mixed with a matrix solution at a ratio of 1:1 (analyte:matrix, v:v) and 1 μL of the analyte-matrix solution was applied and air-dried on the sample plate. HPLC analysis of PEGylated EX4s was carried out using a Dionex Ultimate 3000 HPLC system (Sunnyvale, CA, USA) with a Gemini C18 column (250 × 4.6 mm id, 5 μm, Phenomenex, Torrance, CA, USA) at 25 °C. The mobile phase consisted of deionized water containing 0.1% TFA (mobile phase A) and acetonitrile containing 0.1% TFA (mobile phase B). A linear gradient of 30% to 50% (v/v) mobile phase B for 20 min was performed at a flow rate of 1.0 mL/ min. Sample injection volume was 20 μL and UV absorbance was monitored at 280 nm. Stability Study in Plasma of Septic Mice. Stability of WT-EX4 and PEGylated EX4s was evaluated in plasma of CLP-induced septic mice. After the plasma was preincubated in 37 °C for 30 min, WT-EX4 and PEGylated EX4s were added to plasma to be a final concentration of 200 μg/mL and incubated at 37 °C. At predetermined time, samples were taken from the incubation mixture and 100 μL of acetonitrile containing 0.1% TFA was added to terminate the degradation reaction. After centrifugation for 10 min at 4 °C, the remaining amounts in supernatants were analyzed by RP-HPLC method described above. Cell Culture. Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA) and maintained as previously described.35−39 All experiments were performed using HUVECs at passage 3−5. Human neutrophils were freshly isolated from whole blood (15 mL) obtained by venipuncture from five healthy volunteers, and maintained as previously described.35,40 Animals and Husbandry. Male C57BL/6 mice (6−7-weeks-old, weighing 27 g) were purchased from Orient Bio Co. (Sungnam, Kyungki-Do, Republic of Korea) and used after a 12-day acclimatization period. The animals were housed 5 per polycarbonate cage under controlled temperature (20−25 °C) and humidity (40%− 45%) under a 12:12 h light/dark cycle, fed a normal rodent pellet diet,

release, the relatively large particle size causes pain during administration, and its immune reaction potential is a cause of concern for clinical application.19,20 The kidneys are the primary route of elimination and degradation of EX4;21 a bioconjugation approach, such as poly(ethylene glycol) conjugation (PEGylation), may prolong the plasma half-life and improve the stability in vivo.22 PEGylation is an effective approach for improving the therapeutic efficacy of peptide/ protein drugs by extending the plasma half-life, reducing immunogenicity, and increasing their stability in biological fluids.23 Site-specific PEGylation is important for overcoming the inherent problems of random PEGylation, which usually results in a reduction in the binding affinity and bioactivity of peptides, leading to heterogeneous conjugates with different compositions depending on the number and position of the attached PEG molecules.24,25 An EX4 analogue, engineered to have a cysteine residue at the C-terminus, has been used as sitespecific PEGylation, where the PEG molecules attach to the Cterminus using a maleimide-activated PEG reagent.26,27 Because the N-terminal region of EX4 (mainly the amino acid sequence from 1 through 8, i.e., His-Gly-Glu-Gly-Thr-Phe-Thr-Ser) is essential for GLP-1 receptor (GLP-1R) binding and biological activity, the PEGylation site should be located far from the Nterminal region.26,28 GlP-1R agonist has been reported to be neuroprotective and anti-inflammatory CNS (Central Nervous System) effect29 and have the protective effect of ischemic damage in mice.30,31 Furthermore, Cai et al. found that EX-4 inhibited HMGB1 (high mobility group box 1 protein) expression induced by high glucose concentrations in cardiomyocytes.32 Thus, the current study was designed to evaluate the effects of EX-4 on lipopolysaccharide (LPS)-induced vascular inflammatory responses in HUVEC and cecal ligation and puncture (CLP)induced septic mouse model. In this work, a C-terminal-free thiol group was sitespecifically PEGylated using linear PEG-maleimides (PEGMAL), with molecular weights (MWs) of 2, 5, or 20 kDa, as well as trimeric PEG-MAL with a MW of 50 kDa (tPEG-50KMAL). PEGylated EX4s prepared with PEG-2K-MAL, PEG5K-MAL, PEG-20K-MAL, and tPEG-50K-MAL (referred to hereafter as EX4-2K, EX4-5K, EX4-20K, and EX4-50K, respectively) were purified using cation-exchange chromatography. The identities and purities were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and reversed-phase highperformance liquid chromatography (RP-HPLC). It has previously been shown that PEGylated EX4 prepared with tPEG-50K-MAL (i.e., EX4-50K) exhibited considerable therapeutic potential with respect to receptor binding affinity and prolonged therapeutic effects.27,33 We hypothesize that PEGylated EX4 with a high receptor-binding affinity and prolonged half-life in vivo will exhibit enhanced therapeutic effects compared with wild-type EX4 (WT-EX4) for the treatment of sepsis.

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EXPERIMENTAL SECTION

Reagents. Ex4-Cys (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC) with MW of 4290.7 Da was purchased from CS Bio (Menlo Park, CA, USA). Linear-type monomethoxy PEG-maleimides with MW of 2 kDa, 5 kDa, and 20 kDa (PEG-2KMAL, PEG-5K-MAL, and PEG-20K-MAL, respectively) and trimeric PEG-MAL with MW of 50 kDa (tPEG-50K-MAL) were obtained from NOF Corporation (Tokyo, Japan). Peptides were labeled with 1161

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Biomacromolecules and supplied with water ad libitum. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University (IRB No.; KNU 2012−13). Cecal Ligation and Puncture (CLP). For induction of sepsis, male mice were anesthetized with 2% isoflurane (Forane, JW pharmaceutical, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. The CLP-induced sepsis model was prepared as previously described.40,41 In brief, a 2 cm midline incision was made to expose the cecum and adjoining intestine. The cecum was then tightly ligated with a 3.0-silk suture at 5.0 mm from the cecal tip and punctured once using a 22-gauge needle for induction of high grade sepsis.42 It was then gently squeezed to extrude a small amount of feces from the perforation site and returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0-silk. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. This protocol was approved by the Animal Care Committee at Kyungpook National University prior to conduct of the study (IRB No. KNU 2012−13). In Vitro Permeability Assay. For spectrophotometric quantification of endothelial cell permeabilities in response to increasing concentrations of PEGylated EX4 conjugates, the flux of Evans bluebound albumin across functional cell monolayers was measured using a modified 2-compartment chamber model, as previously described.35,40 HUVECs were plated (5 × 104/well) in 12 mm diameter Transwells with a pore size of 3 μm for 3 days. Confluent monolayers of HUVECs were exposed to LPS (100 ng/mL) for 4 h before being subjected to WT-EX4 or EX4-50K (15 or 30 nM). Transwell inserts were then washed with TBS (pH 7.4), followed by the addition of Evans blue (0.5 mL; 0.67 mg/mL) diluted in a growth medium containing 4% BSA. Fresh growth medium was then added to the lower chamber, and the medium in the upper chamber was replaced with Evans blue/BSA. Ten minutes later, the optical density of the sample in the lower chamber was measured at 650 nm. In Vivo Permeability and Total Leukocyte Migration Assays. CLP-operated mice were injected with PEGylated EX4 conjugates intravenously. After 6 h, 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, the mice were killed, and the peritoneal exudates were collected after being washed with normal saline (5 mL) and centrifuged at 200 × g for 10 min. The absorbance of the supernatant was read at 650 nm. The vascular permeability was expressed in terms of dye (μg/mouse), which leaked into the peritoneal cavity according to a standard curve of Evans blue dye, as previously described.35,40 For assessment of total leukocyte migration, CLP operated mice were treated with PEGylated EX4 conjugates after CLP surgery 6 h. The mice were then sacrificed, and the peritoneal cavities were washed with 5 mL of normal saline. Peritoneal fluid (20 μL) was mixed with Turk’s solution (0.38 mL; 0.01% crystal violet in 3% acetic acid) and the number of leukocytes was counted under an optical microscope. The results were expressed as neutrophils ×106 per peritoneal cavity. Expression of CAMs. The expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on HUVECs were determined by a whole-cell ELISA as described.35,40 Briefly, confluent monolayers of HUVECs were treated with PEGylated EX4 conjugates for 6 h followed by LPS (100 ng/mL) for 16 h (VCAM-1 and ICAM-1) or 24 h (E-Selectin). The medium was removed, and cells were washed with PBS and fixed by adding 1% paraformaldehyde (50 μL) for 15 min at room temperature. After washing, mouse antihuman monoclonal VCAM-1 (100 μL; clone; 6C7.1), ICAM-1 (clone; P82A4) and E-selectin (clone; P2H3) antibodies (Millipore Corporation, Billerica, MA, 1:50 each) were added. After 1 h (37 °C, 5% CO2), the cells were washed three times and then 1:2000 peroxidase-conjugated antimouse IgG antibody (100 μL; Sigma, Saint Louis, MO) was added for 1 h. The cells were washed again three times and developed using the o-phenylenediamine substrate (Sigma, Saint Louis, MO). Colorimetric analysis was

performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. Cell−Cell Adhesion Assay. Adherence of neutrophils to endothelial cells was evaluated by fluorescent labeling of monocytes, as previously described.35,40 Briefly, monocytes were labeled with 5 μM Vybrant DiD for 20 min at 37 °C in phenol red-free RPMI containing 5% FBS. After washing, the cells (1.5 × 106 cells/mL, 200 μL/well) were resuspended in adhesion medium (RPMI containing 2% fetal bovine serum and 20 mM HEPES). The cells were then added to confluent monolayers of HUVECs in 96-well plates. Prior to the addition of cells, HUVECs were treated with PEGylated EX4 conjugates for 6 h, followed by treatment with LPS (100 ng/mL, 4 h). Hematoxylin and Eosin (H&E) Staining and Histopathological Examination. Male C57BL/6 mice underwent CLP and were administered PEGylated EX4 conjugates (30 nM) intravenously at 12 and 50 h after CLP (n = 5). Mice were euthanized 96 h after CLP. To analyze the phenotypic change of lung in mice, lung samples were removed from each mouse, washed three times in PBS (pH 7.4) to remove remaining blood, and fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS, pH 7.4 for 20 h at 4 °C. After fixation, the samples were dehydrated through ethanol series, embedded in paraffin, sectioned into 4-μm sections, and placed on a slide. The slides were deparaffinized in a 60 °C oven, rehydrated, and stained with hematoxylin (Sigma). To remove overstaining, the slides were quick dipped three times in 0.3% acid alcohol, and counterstained with eosin (Sigma). They are then washed in ethanol series and xylene, and then coverslipped. Light microscopic analysis of lung specimens was performed by blinded observation to evaluate pulmonary architecture, tissue edema, and infiltration of the inflammatory cells as previously defined.43 The results were classified into four grades where grade 1 represented normal histopathology; grade 2 indicated minimal neutrophil leukocyte infiltration; grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and partial destruction of pulmonary architecture; and grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture. Clinical Chemistry and Cytokine Level in Septic Mice Plasma. Fresh serum was used for assaying aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine, and LDH using biochemical kits (Mybiosource). To determine the concentrations of IL-1β, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), and TNF-α commercially available ELISA kits were used according to the manufacturer’s protocol (R&D Systems). Values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria). Enzyme-Linked Immunosorbent Assays (ELISA) for GLP-1R Binding Affinity. To evaluate the interaction of the wild-type EX4 and EX4−50K with GLP-1R, 96-well flat microtiter plates were coated with soluble GLP-1R in 20 mM carbonate−bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, overnight at 4 °C. After the plates were washed three times in TBS buffer (0.1 M NaCl, 0.02 M Tris-HCl, pH 7.4) containing 0.05% Tween 20, the plates were incubated with wild-type EX4 and EX4-50K (0−3.2 nM) diluted in the buffer for 1 h. After the plates were rinsed again, they were incubated with a rabbit anti-GLP-1 receptor polyclonal antibody (1:1000) for 1 h. Then, the plates were washed and incubated with rabbit antirabbit IgG (Abcam, MA, 1:1000) for 1 h. After washing, the plates were incubated with 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS; KPL, Gaithersburg, MD). Colorimetric analysis was performed by measuring absorbance values at 405 nm. Conjugation of Fluorophores to WT-EX4 or EX4-50K. WTEX4 or EX4-50K was labeled with FNG-675 NHS ester for in vitro assays and in vivo assays at a molar ratio of 1:4. Briefly, each molecule (10 μM) was dissolved in PBS (1.5 mL), and FNG-675 NHS ester (30 μM) was dissolved in DMSO (0.2 mL). Each molecule and fluorescent dye was reacted for 2 h at room temperature (RT). The reaction product was passed through a 0.2-μm filtering unit, and the unreacted dye was separated on a PD midiTrap G-25 (GE Healthcare, UK) that had been pre-equilibrated in PBS with 2 mM sodium azide. This 1162

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Biomacromolecules process yielded more than 3.62 μM of each molecule with more than 1:2.8 ratio of dye per protein. Flow Cytometry. For assessment of the binding of fluorescence dye-labeled WT-EX4 or EX4-50K, HUVECs were incubated with each molecule (30 nM) for 6 h. PBS-washed cells were resuspended in PBS (1 mL), and the fluorescence quantified using a FACS Aria III (BD). Cell-Binding Assay. A direct cell-binding assay was performed on HUVECs and in vivo using fluorescence labeled-WT-EX4 or EX4-50K. The assay was performed that WT-EX4 or EX4-50K treated on HUVECs and injected to mice intravenously and then isolated mouse endothelial cells. To determinate bound WT-EX4 or EX4-50K, the fluorescence value of the HUVECs or murine endothelial cells was measured. The concentrations of WT-EX4 or EX4-50K were measured by using the ratio of fluorescent dye per protein. Isolation of Endothelial Cell from Mouse. The endothelial cells were isolated according to the manufacturer’s (Dynal Biotec, Lake Success, NY) instructions, using Dynabeads coupled to anti-CD31 antibody and the Dynal Magnetic holder. Briefly, for endothelial cell isolation, four to six mice (6−10 weeks old) were anesthetized, followed by exposure of the peritoneal cavity. Excised lungs and hearts put into RPMI media and remove other tissues from heart and lungs, and then rinse once in PBS. The lung and hearts incubate with 1.0 mg/mL of collagenase A in a 50 mL tube for 1 h, rocking at 37 °C. Every 5 min during this incubation, the tube was gently agitated for a few second, and then transfer the suspension into a new 50 mL tube by passing it through the 70 um tissue sieve (BD Falcon). The filtered cell suspension was centrifuged for 10 min at 1000 rpm. After removal of the supernatant, the cell pellet was washed once with cold PBS in a new 15 mL tube. To prepare the Dynabead-coupled antimouse CD31 antibody, Dynabeads (60 μL) were washed with MACS buffer (PBS, 0.5% BSA, 2 mM EDTA) on a magnetic holder (Invitrogen). The Dynabeads were resuspended with MACS buffer (600 μL), antimouse CD31 (5 μg of per 10 μL of beads) was added, and the mixture was incubated for 12 h at 4 °C. Cells were incubated with Dynabeadcoupled antimouse CD31 antibody for 10 min at room temperature and then placed in a magnetic holder. Cell suspension was slowly added to a 15 mL tube by placing the pipet on the wall of the tube. After incubation for 5 min, PBS was carefully removed by aspiration. The Dynabead-coupled antimouse CD31 antibodies were washed three times in cold PBS, the pellet was resuspended in EBM-2 growth medium, and then harvested and lysed in RIPA buffer containing protease inhibitor cocktail (Roche) on ice. Biomolecular Interaction Analysis by Surface Plasmon Resonance. The interaction between both GLP-1R forms (soluble GLP-1R) and WT-EX4 or EX4-50K was studied by surface plasmon resonance in a Biacore T200 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). To compare the binding of WT-EX4 or EX4-50K to soluble GLP-1R (sGLP-1R), sGLP-1R were captured by the antiGLP-1R polyclonal antibody immobilized on a CM5 sensor chip (BIAcore). WT-EX4 or EX4-50K was injected in a Hepes buffer with 3 mM CaCl2, and 0.6 mM MgCl2 (HBS-T). Kinetic and affinity analyses were performed using BIAEVALUATION software 3.2 RC1 (BIAcore). Immunofluorescence Staining. HUVECs were grown to confluence on glass coverslips coated with 0.05% poly-L-lysine in complete media containing 10% FBS and maintained for 48 h. Cells were treated with FNI-675 NHS ester fluorescent labeled WT-EX4 or EX4-50K (30 nM) for 6 h. Cells were washed with PBS, fixed in 4% formaldehyde in TBS (v/v) for 15 min at room temperature, permeabilized in 0.05% Triton X-100 in TBS for 15 min, and blocked in blocking buffer (5% bovine serum albumin (BSA) in TBS) overnight at 4 °C. Then, the cells were incubated with a rabbit antiGLP-1R polyclonal antibody (Abcam, MA). GLP-1R was visualized using an Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, donkey antirabbit IgG), and visualized by confocal microscopy at a magnification of 630× (TCS-Sp5, Leica Microsystems, Germany). Histologic Analysis of the GLP-1R Binding in Vivo. Twentyfour hours post-CLP surgery, fluorescence labeled- WT-EX4 or EX450K (30 nM/mouse) was intravenously injected into the mice. After 24 h, mouse vena cava was enucleated and fixed in visikol for 24 h.

Then, the vena cava was embedded in an optimum cutting temperature (OCT) compound (Tissue Tek) in −80 °C. Consecutive sections (10 mm) were incubated with anti-GLP-1R antibody, antirabbit Alexa 488 (green), anti-CD31 antibody, and antirabbit Alexa 350 (blue), and visualized by confocal microscopy at 200× magnification (TCS-SP5, Leica microsystem, Germany). In Vivo Imaging for Biodistribution. The animal study was conducted under the same protocol as described above. A total of 10 BALB/c-nu/nu mice were randomized and grouped. Two mice were allocated to receive nonlabeled EX4-50K treatment (control). For the WT-EX4 or EX4-50K-treated group, 4 mice were divided into 2 time points (n = 2): 3, 24 h. WT-EX4 or EX4-50K were diluted in saline and respectively administered intravenously once at an equivalent dose of 251 ng/mouse via the tail vein. The animals were imaged by IVIS Spectrum CT (PerkinElmer, Waltham, MA). Statistical Analysis. All experiments were performed independently at least three times. Values are expressed as means ± SEM. The statistical significance of differences between test groups was evaluated using SPSS for Windows, version 16.0 (SPSS, Chicago, IL). Statistical relevance was determined by one-way analysis of variance (ANOVA) and Tukey’s post-test. P values less than 0.05 were considered to indicate significance. Survival analysis of CLP-induced sepsis outcomes was performed using Kaplan−Meier analysis.

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RESULTS AND DISCUSSION The free thiol group in the C-terminal Cys residue of EX4 was site-specifically PEGylated using PEG-2K-MAL, PEG-5K-MAL, PEG-20K-MAL, and tPEG-50K-MAL. It has previously been shown that PEGylated EX4 prepared using tPEG-50K-MAL (i.e., EX4-50K) exhibits considerable therapeutic potential with respect to the receptor binding affinity and prolonged therapeutic effects.27,33 Figure 1 shows a schematic diagram

Figure 1. Schematic diagram showing the structure of trimeric poly(ethylene glycol) (PEG)-conjugated EX4 (EX4-50K).

of the structure of EX4-50K. Each PEGylated EX4 molecule (i.e., EX4-2K, EX4-5K, EX4-20K, or EX4-50K) was successfully isolated via cation-exchange chromatography. MADLI-TOF MS analysis showed that the molecular masses of EX4-2K, EX4-5K, EX4-20K, and EX4-50K were measured to be m/z = 6457, 9200, 25 996, and 57 675, respectively, which corresponds to each of the mono-PEGylated EX4 molecules, based on the known MW of EX4 and those of the PEG-MALs or tPEG-50K-MAL (Figure 2A). The purity of the isolated EX4-2K, EX4-5K, EX4-20K, and EX4-50K was confirmed using RP-HPLC (Figure 2B). 1163

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Figure 2. Characterization of PEGylated EX4 conjugates. (A) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) spectra of EX4-2K, EX4-5K, EX4-20K and EX4-50K. (B) High-performance liquid chromatography (HPLC) chromatograms of EX4-2K, EX4-5K, EX4-20K and EX4-50K.

Figure 3. EX4-50K suppresses LPS- and CLP-mediated vascular inflammatory responses. (A,C,E) Human umbilical vein endothelial cells (HUVECs) were incubated with PEGylated EX4 conjugates (15 or 30 nM, for 4 h) and (B,D) Cecal ligation and puncture (CLP)-operated mice were injected with the indicated concentrations of PEGylated EX4 conjugates for 6 h. (A) The lipopolysaccharide (LPS)-mediated hyperpermeability after 4 h, and (B) the CLP-mediated hyperpermeability after 4 h. (C) The expression levels of vascular cell adhesion molecule-1 (VCAM-1) (16 h, white box), intercellular adhesion molecule-1 (ICAM-1) (16 h, gray box), and E-selectin (24 h, black box). (D) Leukocyte migration into the peritoneal cavities of mice, and (E) adherence of purified human neutrophils to HUVEC monolayers (6 h). *p < 0.05 vs LPS or CLP only. #p < 0.05 vs WT-EX4.

To investigate the effects of PEGylation on stability in biological fluids, we studied the chemical stability of WT-EX4 and PEGylated EX4s in plasma obtained from septic mice. The

degradation rates of WT-EX4 and PEGylated EX4s approximately followed first-order kinetics. Degradation rate constants were obtained from the slope of semilog plots of the 1164

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Figure 4. Antiseptic effects of EX4-50K. (A,B) Male C57BL/6 mice (n = 60, group pooled from three independent experiments) were administered WT-EX4, EX4-20K, or EX4-50K (A, 251 ng/mouse, at 12 and 50 h; B, 418.3 ng/mouse, at 12 h) after CLP. Control CLP mice (●) and shamoperated mice (○) were administered sterile saline. Survival curves of CLP-operated mice (A,B) and H&E staining of lung tissue (C). Images are representative of three independent experiments. (D) Histopathological scores for lung tissue were recorded as described in the Experimental Section. *p < 0.05. #p < 0.05 vs WT-EX4.

PEGylated EX4s inhibited LPS-mediated upregulation of CAMs (Figure 3C), adhesion of leukocytes (Figure 3E), and CLP-induced migration of leukocytes (Figure 3D). These effects demonstrate the potential of WT-EX4 and PEGylated EX4s for managing severe vascular inflammatory diseases. Among its derivatives, the barrier protective effects of EX4-50K were superior to those of other PEGylated EX4s, indicating that the large, trimeric structure of PEG-50K is more effective than the small, linear structure of PEGs in increasing the barrier protective effects of EX4. Therefore, among the PEGylated EX4s, hereafter we compare EX4-50K with WT-EX4 and EX420K. Based on the above data, we hypothesized that treatment with EX4-50K may ameliorate the deleterious effects of CLP on sepsis survival in mice. We also expected that EX4-50K would exhibit a greater protective effect than WT-EX4 or EX4-20K. To test these hypotheses, WT-EX4, EX4-20K, or EX4-50K was administered to mice following CLP surgery (251 ng of EX4 per mouse, administered 12 h and then again 50 h after CLP). As shown in Figure 4A, all three reduced CLP-induced mortality (30% for WT-EX4 or EX4-20K, and 60% for EX450K, at 6 days following CLP). Furthermore, an additional injection of EX4−20K and EX4-50K (418.3 ng of EX4 per mouse) 12 h after CLP, resulted in enhanced survival, as shown in Figure 4B. In the CLP group, interstitial edema with massive infiltration of inflammatory cells into the interstitium and alveolar spaces was observed, and the pulmonary architecture was severely damaged (Figure 4C). In the CLP + EX4-50K groups, these changes were suppressed, the pulmonary architecture was preserved, and lung injury scores were significantly lower (Figure 4C,D). Systemic inflammation during sepsis frequently causes multiple organ failure (MOF),

concentration as a function of time using regression analysis. WT-EX4 degraded rapidly, with a half-life of 16.9 h; however, the PEGylated EX4s exhibited greater stability: the degradation half-life of EX4-2K was 69.2 h, the half-life of EX4-5K was 62.8 h, the half-life of EX4-20K was 69.7 h, and the half-life of EX450K was 234.2 h. EX4-50K was considerably more stable than the other conjugates, which suggests that the prolonged circulation half-life of EX4-50K reported previously27 can be attributed to the enhanced stability in blood, as well as reduced renal filtration. During severe vascular inflammatory responses, overproduction of inflammatory cytokines/chemokines may irreversibly damage the vascular integrity and cause excessive circulatory fluid loss.44 This pathophysiological process leads to prolonged tissue hypoperfusion, organ dysfunction, and may ultimately lead to death;44 thus, vascular permeability is pivotal in sepsis. In this work, we describe the effects of WT-EX4 and PEGylated EX4s on their protective barrier responses. Figure 3A,B shows the permeability and dye leakage; these data indicate that lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) surgery significantly increases the vascular barrier permeability, both in human umbilical vein endothelial cells (HUVECs) and in mice. Furthermore, these phenomena were suppressed by WT-EX4 and PEGylated EX4s. Vascular inflammatory responses are known to be mediated by increased expression of cell adhesion molecules (CAMs) at the surfaces of endothelial cells, such as ICAM-1, VCAM-1, and E-selectin, thereby promoting adhesion and migration of leukocytes across the endothelium to sites of inflammation.45 The transendothelial migration of circulating leukocytes to the vascular endothelium is a fundamental step during the pathogenesis of vascular inflammatory diseases.46 We found that WT-EX4 and 1165

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Figure 5. Binding properties of EX4-50K. (A) The binding affinity of WT-EX4 and of EX4-50K to GLP-1R measured using enzyme-linked immunosorbent assay (ELISA). (B,C) Surface plasmon resonance (SPR) analysis of the binding of WT-EX4 (B) and EX4-50K (C) to GLP-1R. Isolated N-terminal domain of GLP-1R was captured to a level of 70 RU on a CM5 chip using GLP-1R antibody. The binding of 3.125, 6.25, 12.5, 25, and 50 nM WT-EX4 or EX4-50K to GLP-1R was recorded. A representative experiment of three independent repeats is shown. Black lines represent experimental data, red lines represent fittings. (D) Cellular binding of WT-EX4 and EX4-50K to HUVECs, assessed using fluorescenceactivated cell sorting (FACS). (E−H) The binding affinities of WT-EX4 and EX4-50K were determined by calculating the concentrations of WTEX4 and EX4-50K using the ratio of the number of fluorescent dye molecules per protein molecule determined via fluorescence ELISA in HUVECs (E: PBS treated; F, LPS treated) or purified murine endothelial cells (ECs) (G: Sham operated; H: CLP operated). *p < 0.05 vs WT-EX4.

involving the liver and kidneys.47 CLP resulted in a significant increase in plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST), which are markers of hepatic injury (Figure S1A), as well as creatinine and blood urea nitrogen (BUN), which are markers of renal injury (Figures S1B−S1C). The magnitudes of these increases were lower following treatment with EX4-50K. Furthermore, levels of another important marker of tissue injury, lactate dehydrogenase (LDH), were also reduced by EX4-50K in CLP-operated mice (Figure S1D). We explored the role of EX4-50K in regulating the production of IL-1β, -6, -10, and tumor necrosis factor alpha (TNF-α during sepsis). IL-10 and TNF-α appear to be essential mediators in sepsis-induced vascular inflammation,48 and IL-6 blockade with neutralizing antibodies has been shown to protect against CLP-induced sepsis mortality.49 Following CLP, the levels of IL-1β, -6, -10 (Figure S1E), TNF-α (Figure S1F), and MCP-1 (Figure S1G) were significantly lower in the EX450K groups than in the control group. To determine the underlying cause of the enhanced antiseptic efficacy of EX4-50K, it is essential to investigate whether WT-EX4 and EX4-50K have different binding properties toward the GLP-1 receptor (GLP-1R) in vitro and in vivo, as well as whether the stability-enhancing effect of the trimeric PEG conjugation can help to overcome the limited

therapeutic effects of WT-EX4 due to its short half-life.27 We aimed to determine whether site-specific conjugation of trimeric PEG-50K affects the binding dynamics in such a way as to improve the in vitro and in vivo activities. First, we used solid-phase enzyme-linked immunosorbent assay (ELISA) to measure the binding affinities of WT-EX4 and EX4-50K toward the GLP-1R. In this assay, we compared the efficiency of these bindings to the isolated N-terminal domain of GLP-1R.50 As shown in Figure 5A, WT-EX4 bound to GLP-1R, with a dissociation constant of Kd(app) ≈ 0.142 nM, which is similar to the previously reported binding affinity of WT-EX4 to GLP-1R in solution (i.e., 0.136 nM).51 A dissociation constant of Kd(app) ≈ 0.159 nM was obtained for the interaction between GLP-1R and EX4-50K. Surface plasmon resonance (SPR) was used to investigate the binding affinities of WT-EX4 and EX4-50K to GLP-1R (Figure 5B,C). These data suggest that EX4-50K binds to GLP-1R as strongly as does wild-type EX4. In vitro and in vivo bindings of EX4-50K to endothelial cells were compared with those of WT-EX4 following dye-labeled peptide incubation with HUVECs or via intravenous injection into mice. We found that EX4-50K bound more strongly than WTEX4 to HUVECs via fluorescence-activated cell sorting (FACS) analysis (Figure 5D) and confocal imaging (Figure S2). The in vivo binding activity of EX4-50K was investigated using immunohistochemistry, and the results show that injected 1166

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Figure 6. In vivo images of EX4-50K. (A-B) Representative in vivo imaging system (IVIS) images of athymic mice injected with FNI-675 NHS esterconjugated EX4-50K (251 ng/mouse) from the left side, (A) after 3 h and (B) after 24 h. In both cases, nonlabeled EX4-50K (251 ng/mouse) was used as a control. (C) Representative IVIS images of isolated organs from mice injected with FNI-675 NHS ester-conjugated EX4-50K (251 ng/ mouse) after 24 h.

EX4-50K colocalized with GLP-1R in the vena cava. In addition, this binding appeared to dominate over that of WTEX4 (Figure S3). HUVECs were activated with and without LPS, followed by treatment with WT-EX4 and EX4-50K. In addition, control and CLP-operated mice were intravenously injected with WT-EX4 and EX4-50K and assessed for concentrations of dye-labeled peptides bound on GLP-1R in HUVECs, as well as in purified mouse endothelial cells (ECs). The quantity of protein bound to HUVECs and purified mouse ECs indicated that EX4-50K bound more strongly to (and remained longer on) endothelial cells than did WT-EX4 in both settings (Figure 5E−H). There results were corroborated by in vivo body imaging analyses (Figure 6A,B; Figure S4), and also by ex vivo fluorescence staining, which showed that EX4-50K exhibited prolonged duration (Figure 6C). Strong binding of EX4-50K to GLP-1R was elucidated by immunofluorescence staining on HUVEC surface and immunohistochemistry imaging, as shown in Figures S2 and S3. As shown in Figure 6, EX4-50K was observed mostly in the liver, lungs, and kidneys, which are the major organs responsible for end-organ dysfunction during sepsis.52 As shown in Figure S4, EX4-50K exhibited greater in vivo stability than WT-EX4. These in vivo data show that EX4-50K is a more effective antiseptic than WT-EX4, which may be attributed to the higher receptor-binding affinity and enhanced in vivo stability of EX-50K. These results suggest that EX4-50K has a protective effect against polymicrobial septic death, and has potential as a therapeutic strategy for sepsis and septic shock. PEGylation is known to be an effective approach for increasing the circulation half-life of peptides and proteins, as well as decreasing the immunogenicity; however, it often leads to a significant reduction in the receptor-binding affinity, either by directly masking the binding site or due to steric hindrance.

To avoid these problems, site-specific PEGylation is a useful strategy, whereby the PEG molecules are conjugated away from the active site. With EX4, the C-terminus is recommended as a target site for site-specific PEGylation, because the N-terminal domain of EX4 is essential for binding to GLP-1R.15,28 It has been reported that C-terminated PEGylated EX4 using trimeric PEG exhibits a binding affinity to GLP-1 receptors that is comparable to unmodified EX4, as well as a superior circulation half-life and prolonged antidiabetic effects in animals. Here, we have shown that EX4-50K, which was prepared by conjugating trimeric PEG-50K on the C-terminal thiol group of EX4, has a similar GLP-1R-binding affinity to WT-EX4. Furthermore, we demonstrated that EX4-50K has a higher binding affinity to endothelial cells than WT-EX4, both in vitro and in vivo, which suggests that C-terminal PEGylation using trimeric PEG is a useful strategy for increasing the therapeutic efficacy of EX4 for the treatment of sepsis.

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CONCLUSIONS

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ASSOCIATED CONTENT

In summary, EX4-50K is a novel long-acting EX4 analogue prepared via C-terminal thiol PEGylation using tPEG-50KMAL. We have shown that EX4-50K exhibits antiseptic responses in HUVECs and in mice, and we believe that EX450K is a promising potential treatment for severe vascular inflammatory diseases, including severe sepsis and septic shock.

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01756. 1167

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Additional data of antiseptic effects, binding affinity by SPR or IF, and in vivo biodistribution of tPEG-Ex4 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(D.H.Na) Tel: 82-53-950-8579; Fax: 82-53-950-8557; E-mail: [email protected]. *(J.-S.B.) Tel: 82-53-950-8570; Fax: 82-53-950-8557; E-mail: [email protected]. Author Contributions §

These authors contributed equally. W.L., D.H.N., and J.S.B. designed the study and analyzed data. J.S.B. wrote the manuscript. W.L. and E.J.P. conducted experiments with assistance from S.K. K.C.L discussed data analysis. Preliminary results were generated by D.H.N. and J.S.B. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2013R1A2A2A01068858 & 2014R1A2A1A11049526), a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2202), and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI15C0001).

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