Self-Assembly PEGylation Retaining Activity (SPRA) Technology via a

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Self-assembly PEGylation Retaining Activity (SPRA) Technology via a Host– guest Interaction Surpassing Conventional PEGylation Methods of Proteins Tatsunori Hirotsu, Taishi Higashi, Irhan Ibrahim Abu Hashim, Shogo Misumi, Koki Wada, Keiichi Motoyama, and Hidetoshi Arima Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00678 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on December 30, 2016

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Molecular Pharmaceutics

Article

Revised

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Self-assembly PEGylation Retaining Activity (SPRA) Technology via a Host–

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guest Interaction Surpassing Conventional PEGylation Methods of Proteins

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Tatsunori Hirotsu †, ‡, #, Taishi Higashi †, #, Irhan Ibrahim Abu Hashim †, §, Shogo Misumi †, Koki Wada ǁ,

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Keiichi Motoyama †, Hidetoshi Arima †, ‡, *

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†

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Kumamoto 862-0973, Japan

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‡

Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku,

Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and Glocal

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Oriented) Program”, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan

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§

Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

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ǁ

Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji, Shizuoka 417-8539, Japan

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#

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* Corresponding author.

These authors contributed equally to this work.

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Hidetoshi Arima

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Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku,

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Kumamoto 862-0973, Japan.

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E-mail: [email protected], TEL: +81 96 371 4160, FAX: +81 96 371 4420

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ACS Paragon Plus Environment


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ABSTRACT

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Polyethylene glycol (PEG) modification (PEGylation) is one of the best approaches to improve the

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stabilities and blood half-lives of protein drugs; however, PEGylation dramatically reduces the

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bioactivities of protein drugs. Here, we present “self-assembly PEGylation retaining activity” (SPRA)

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technology via a host–guest interaction between PEGylated β-cyclodextrin (PEG-β-CyD) and

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adamantane-appended (Ad)-proteins. PEG-β-CyD formed stable complexes with Ad-insulin and Ad-

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lysozyme to yield SPRA-insulin and SPRA-lysozyme, respectively. Both SPRA-proteins showed high

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stability against heat and trypsin digest, comparable with that of covalently PEGylated protein equivalents.

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Importantly, the SPRA-lysozyme possessed ca. 100% lytic activity, whereas the activity of the covalently

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PEGylated lysozyme was ca. 23%. Additionally, SPRA-insulin provided a prolonged and peakless blood

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glucose profile when compared with insulin glargine. It also showed no loss of activity. In contrast, the

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covalently PEGylated insulin showed a negligible hypoglycemic effect. These findings indicate that

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SPRA technology has the potential as a generic method, surpassing conventional PEGylation methods for

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proteins.

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KEYWORDS : cyclodextrin, adamantine, polyethylene glycol, protein, supramolecular host–guest

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interaction

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Table of contents graphic

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1. INTRODUCTION

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Protein drugs have been used widely as valuable therapeutic agents, with worldwide sales in 2014

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showing that protein drugs represent 7 out of the top 10 pharmaceuticals used.1 However, protein drugs

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often show low physicochemical stability, low enzymatic stability, immunogenicity and short circulating

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half-lives.2 Therefore, pharmaceutical techniques are needed to develop protein drug formulations that

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show superior stability and activity.

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Covalent polyethylene glycol (PEG) modification (PEGylation) of proteins is one of the best

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approaches to improve pharmaceutical properties of protein drugs, and a number of PEGylated proteins

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have been used in the clinical field.3, 4 However, bioactivities of proteins are dramatically reduced by

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PEGylation, because PEGylated proteins bind weakly to their cognate receptors or substrates due to steric

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hindrance of PEG or structural changes to the PEGylated proteins.3, 5 For example, PEGylated interferon

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α-2a (branched PEG, M.W. 40 kDa) and PEGylated erythropoietin (linear PEG, M.W. 30 kDa), both of

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which are commercially available products, show only 7% and 1–2% bioactivities, respectively,

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compared with those of native interferon and erythropoietin.6, 7

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Steric hindrance by the PEG chain is due to the bulky hydrated PEG, which forms a layer on the

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surface of the protein. Here, PEG entangles around the protein surface through hydrophobic interactions

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and concurrently forms hydrogen bonds with the surrounding water molecules.8 The hydrodynamic radius

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of PEG (M.W. 2–30 kDa) is ca. 1.36–5.98 nm.9 Although these features of PEGylated proteins provide

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crucial stability and avoid glomerular filtration (slit-pores are 3.5 nm), they reduce the bioactivity of the

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protein drug considerably.

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To develop PEGylated proteins, the benefits and disadvantages of PEGylation must be considered, and

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an acceptable M.W. of PEG should be determined.5 For example, mono- and multi-substituted PEGylated

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lysozyme (PEG 20 kDa) showed high stabilities; however, their lytic activities were only 1.4% and 1.1%,

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respectively.10 In addition, PEGylation of insulin with PEG 2 kDa improved its physicochemical stability,

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but it did not prolong its hypoglycemic effect (the activity was 85%).11, 12 In our previous report, the 3



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enzymatic stability of insulin was not improved by PEGylation with PEG 2 kDa.13, 14 Torosantucci et al.

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also reported that PEGylation with PEG 5 kDa does not protect insulin against forced aggregation.15 In

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addition, a PEGylated insulin analog (PEG-lispro, linear PEG, M.W. 20 kDa) was in Phase III of clinical

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trials as a once-daily treatment for type-1 and type-2 diabetes, although it was recently stopped.16-18 PEG-

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lispro has a >10-fold longer blood half-life than insulin lispro, leading to a prolonged hypoglycemic effect,

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compared with that of insulin glargine. However, PEG-lispro possesses only 6% of the bioactivity of

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intact insulin lispro.16,

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protein drugs’ pharmaceutical properties and bioactivity. Hence, PEGylation that can improve the

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pharmaceutical properties of proteins without loss of activity will lead to development of a large number

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of PEGylated proteins.

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Thus, PEGylation of proteins represents a serious dilemma with respect to

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Recently, site-specific PEGylation19-21 and reversible PEGylation22-24 technologies have been developed.

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These approaches improve the pharmaceutical properties of protein drugs without significant loss of

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bioactivity. Nonetheless, these PEGylated proteins showed only 10–40% bioactivity, compared with

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unmodified proteins. Thus, improving the pharmaceutical properties of proteins by PEGylation without

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significant loss of activity is challenging.

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Cyclodextrins (CyDs), cyclic oligosaccharides, are acknowledged to form inclusion complexes with

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hydrophobic compounds spontaneously through a host–guest interaction.25, 26 Recently, a large number of

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CyD-based supramolecular drug carriers have been developed for low-molecular weight drugs, protein

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drugs, genes and oligonucleotides.27-29 However, little is known about the fabrication of reversible

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PEGylated proteins utilizing CyD-based supramolecular properties.

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Based on these studies, we propose “Self-assembly PEGylation Retaining Activity (SPRA)”

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technology via a host–guest interaction between β-CyD and adamantane (Ad). We prepared PEGylated

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proteins through supramolecular inclusion complexation rather than covalent bond linkage and evaluated

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their pharmaceutical properties. These SuPRAmolecular PEGylated proteins (SPRA-proteins) are based

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on a reversible host–guest interaction, and are expected to show prolonged and high bioactivities. We 4


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employed β-CyD as a host molecule and Ad as a guest molecule because they form a stable complex,30

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and they are also used as a part of siRNA carrier in human.31 Additionally, we used insulin as a model

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protein because PEG-lispro shows only 6% bioactivity of unmodified insulin.16 Also, lysozyme as a

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model protein was used to demonstrate the utility of SPRA technology for other protein drugs.

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2. MATERIAL AND METHODS

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2.1. Materials. Bovine Zn-insulin (27.5 IU/mg, approximately 0.5% Zn) and hen egg lysozyme (58,100

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IU/mg) were obtained from Sigma Chemicals (St. Louis, MO, USA). β-CyD and 6-O-α-(4-O-α-D-

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glucuronyl)-D-glucosyl-β-CyD (GUG-β-CyD) were donated by Nihon Shokuhin Kako (Tokyo, Japan)

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and Ensuiko Sugar Refining (Tokyo, Japan), respectively. Both mono- and per-NH2-β-CyDs were

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prepared according to the previous method.32, 33 SUNBRIGHT® ME-200 CS (mPEG-NHS) was obtained

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from NOF (Tokyo, Japan). All other chemicals and solvents were of analytical reagent grade and

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deionized double-distilled water was used throughout the study.

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2.2. Preparation of Ad-proteins. To obtain Ad conjugate with protein (Ad-protein), adamantane acetic

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acid (190 mg), N-hydroxysuccinimide (NHS) (230 mg) and N,N'-dicyclohexylcarbodiimide (DCC) (820

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mg) were dissolved in 6 mL of DMSO. The solution was stirred at room temperature for 1 h and

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centrifuged at 12,890 G for 15 min. The supernatant was added to 300 mL of water to precipitate

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activated Ad and resulting precipitates were dried under reduced pressure. Insulin (28.5 mg) and activated

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Ad (20 mg) were dissolved in 5 mL of dimethylformamide (DMF)/water (3:2 v/v, pH 10) and stirred at

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room temperature for 10 min. Water (40 mL) was added, and the pH adjusted to 2 with 1.0 M

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hydrochloric acid to terminate the reaction. The reaction solution was dialyzed using a membrane filter

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(Spectra/Por® membrane MWCO: 3,500) and lyophilized. Finally, crude product was purified by HPLC

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with the following conditions: GL Pack NUCLEOSIL 100–10C18 (4.6 mm i.d. × 150 mm), a mobile

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phase of acetonitrile/water/trifluoroacetic acid (30:69.9:0.1) and acetonitrile/water/trifluoroacetic acid 5



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(95:4.9:0.1), a gradient flow increasing the ratio of the latter solution (0–100%/60 min), a flow rate of 1.0

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mL/min and detection at 280 nm. Likewise, Ad-lysozyme was prepared by mixing lysozyme (58.8 mg)

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and activated Ad (7.2 mg) in 5 mL of DMF/PBS (2:3 v/v, pH 8) solution at 4 °C for 24 h. After dialysis

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using a membrane filter (Spectra/Por® membrane MWCO: 8,000), the sample was lyophilized and

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purified by HPLC.

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2.3. Preparation of PEG-β β-CyDs. To obtain mono-substituted PEGylated β-CyD (mono-PEG-β-CyD),

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SUNBRIGHT® ME-200 CS (mPEG-NHS, 1,000 mg) and mono-NH2-β-CyD (590 mg) were reacted in 20

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mL of DMF/water (3:2 v/v) at room temperature for 24 h. The reaction solution was dialyzed using a

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membrane filter (Spectra/Por® membrane MWCO: 8,000) until free NH2-β-CyD was completely removed.

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The resulting sample was lyophilized. Likewise, multi-substituted PEGylated-β-CyD (multi-PEG-β-CyD)

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was prepared by mixing mPEG-NHS (1,000 mg) and per-NH2-β-CyD (5.64 mg) in 20 mL of DMF/water

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(3:2 v/v) at room temperature for 24 h. After dialysis using a membrane filter (Spectra/Por® membrane

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MWCO: 50,000), the sample was lyophilized.

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2.4. Formation of Supramolecular Complexes. Isothermal titration calorimetry (ITC) was measured at

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a titration rate of 2.5 µL/2 min and stirring speed of 350 rpm at 25 °C using a Nano ITC LV (Tokyo,

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Japan). Ad-proteins and PEG-β-CyDs were dissolved in PBS. The mono-PEG-β-CyD (0.1 or 0.5 mM)

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and multi-PEG-β-CyD (0.1 or 0.5 mM) solutions were loaded into the sample cell. After the titrations of

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the Ad-insulin solution (1 or 2 mM) or Ad-lysozyme solution (1 or 2 mM) into the sample cell, the

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stability constants (Kc) were determined from the titration curve using an independent model. Particle

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sizes of the Ad-proteins/mono-PEG-β-CyD (mono-SPRA-proteins) and Ad-proteins/multi-PEG-β-CyD

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(multi-SPRA-proteins) were determined by dynamic light scattering using a Zetasizer Nano (Malvern

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Instruments, Worcestershire, UK). The solvent was PBS, and the concentration of Ad-insulin or Ad6


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lysozyme was 0.025 mM. The molar ratios of Ad-proteins and PEG-β-CyDs were 1:0.1, 1:0.5, 1:1, 1:2,

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1:5 and 1:10.

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2.5. Thermal Stability.34 Ad-proteins (0.05 mM) and unmodified proteins (0.05 mM) were mixed with

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PEG-β-CyDs in PBS (pH 7.4) at a molar ratio of 1:10. Proteins alone, Ad-proteins alone and covalently

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PEGylated proteins (PEG-proteins) (0.05 mM) were also dissolved in PBS. These solutions were

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incubated at 37 °C (the insulin system) or 70 °C (the lysozyme system). At appropriate intervals, 0.7 mL

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of the samples were collected and centrifuged at 8,952 G for 10 min. The absorbance of the supernatant

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was measured by a spectrophotometer (V-630, JASCO, Tokyo, Japan) at 280 nm.

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2.6. Enzymatic Stability.34 Ad-proteins (5 mg) and unmodified proteins (5 mg) were mixed with PEG-β-

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CyDs in 5 mL of PBS (pH 7.4) at a molar ratio of 1:10. Proteins alone, Ad-proteins alone and PEG-

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proteins (5 mg) were also dissolved in 5 mL of PBS. PBS (100 µL) including 1.0 mg/mL trypsin was

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added to 300 µL of protein solutions. These solutions were incubated at 37 °C and 50 rpm for 6 h, and the

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non-degraded insulin or lysozyme was measured by HPLC.

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2.7. In Vitro Lytic Activity.35 Ad-lysozyme (0.4 µM) or lysozyme (0.4 µM) was mixed with PEG-β-

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CyDs in 5 mL of PBS (pH 7.4) at a molar ratio of 1:5. Lysozyme, Ad-lysozyme and PEG-lysozyme (0.4

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µM) were also dissolved in 5 mL of PBS. The suspension of M. lysodeikticus cells (1 mg/mL, 50 µL) was

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added to the lysozyme solutions (900 µL), and then incubated at 25 °C for 2 min. The turbidity of the

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suspension was measured at 540 nm using a spectrophotometer. The lytic activity was estimated as a

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relative activity (% activity) based on a decrease in turbidity relative to the lysozyme alone system.

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2.8. In Vivo Serum Insulin Level, Hypoglycemic Effect and Blood Chemistry Values.13 All animal

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procedures were carried out in accordance with the approved guidelines and with the approval of the

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Ethics Committee for Animal Care and Use of Kumamoto University (Approval ID: A27-139). Insulin,

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Ad-insulin, PEG-insulin, multi-SPRA-insulin (molar ratio = 1:5), insulin/multi PEG-β-CyD mixture

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(molar ratio = 1:5) and insulin glargine were dissolved in PBS (pH 7.4). The samples (insulin 2 U/kg)

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were subcutaneously injected into male Wistar rats (200–250 g), and at appropriate intervals, blood

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samples were taken from the jugular veins. The serum insulin and glucose levels of rats were determined

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by the enzyme immunoassay using a Rat Insulin ELISA Kit (Shibayagi, Gunma, Japan) and mutarotase-

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glucose oxidase method using the Glucose-CII-Test Wako (Wako Pure Chemical Industries, Osaka,

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Japan), respectively. Blood chemistry values were measured by a clinical chemistry analyzer, JCA-

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BM2250 (JEOL, Tokyo, Japan) after injection of the samples.

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3. RESULTS

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3.1. Design and Preparation of Components of SPRA-insulin and SPRA-lysozyme. We previously

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prepared the insulin conjugate with GUG-β-CyD.34 Therefore, we initially employed this conjugate as a

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component of SPRA-proteins. However, 89% of the hypoglycemic effect of insulin was lost by

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conjugation with GUG-β-CyD, when assuming that the upper area of the blood glucose level–time curve

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(AUCG) corresponds with in vivo bioactivity of insulin (Figure S1). Thus, we prepared an insulin

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conjugate with Ad; a guest molecule (Figure 1). The Ad-conjugation reaction was performed at pH 10,

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because an electrophilic moiety selectively reacts with the amino group of LysB29 of insulin at pH 10 and

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LysB29 is also employed to prepare commercially available bioconjugated insulins, such as insulin

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detemir, insulin degludec and PEG-lispro.18, 36, 37 According to the results of HPLC of the reaction mixture

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(Figure S2), 47.2% of mono-substituted Ad-insulin was included in the reaction mixture. After the

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purification of crude products with HPLC, the purity of Ad-insulin was determined as >99% by HPLC

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and single peak derived from mono-substituted Ad-insulin was observed in the MALDI-TOF MS 8


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spectrum (Figure 2A, B). Importantly, Ad-insulin retained ca. 100% of the hypoglycemic effect of insulin

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as described later, suggesting negligible adverse effects of Ad-conjugation on the bioactivity of insulin.

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Likewise, mono-substituted lysozyme conjugate with Ad was successfully prepared with 15.2% of crude

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yield (Figure S2) and purified with >99% of purity (Figures 1 and 2C, D). Hereafter, the exact position of

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Ad moiety in the Ad-proteins should be determined by the measurement of MALDI-TOF MS after the

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trypsin digestion.

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Next, we prepared mono-PEG-β-CyD (Figure 3A) and multi-PEG-β-CyD (Figure 3B); the PEGylated

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host molecules. The results of the MALDI-TOF MS (Figure 3C-E), FT-IR (Figure S3) and 1H-NMR

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(Figure S4) spectra indicated successful preparation of these PEG-β-CyDs, and average degrees of

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substitution of the PEG chain per a β-CyD molecule were 1.0 and 2.8 in mono-PEG-β-CyD and multi-

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PEG-β-CyD, respectively. Hereafter, gel permeation chromatography of multi-PEG-β-CyD should be

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measured to determine its distribution, heterogeneity and accurate degree of substitution of PEG.

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Figure 1. Preparation pathways of SPRA-insulin and SPRA-lysozyme. Kc represents the stability constant

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of SPRA-proteins, and was determined by ITC.

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Figure 2. MALDI-TOF MS spectra of (A) insulin, (B) Ad-insulin, (C) lysozyme and (D) Ad-lysozyme. 10


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Figure 3. (A, B) Preparation pathways and (C, D, E) MALDI-TOF MS spectra of (C) PEG (20 kD), (A,

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D) mono-PEG-β-CyD and (B, E) multi-PEG-β-CyD. 11



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3.2. Preparation of SPRA-insulin and SPRA-lysozyme. As shown in Figure 1, four kinds of SPRA-

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proteins, consisting of Ad-insulin/mono-PEG-β-CyD (mono-SPRA-insulin), Ad-insulin/multi-PEG-β-

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CyD

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lysozyme/multi-PEG-β-CyD (multi-SPRA-lysozyme), were prepared by mixing both components in

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aqueous solution. According to the results of ITC, exothermic peaks were observed by the addition of

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mono-PEG-β-CyD or multi-PEG-β-CyD solutions (Figure S5), indicating complexation between the Ad

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moiety of Ad-proteins and the β-CyD moiety of PEG-β-CyDs. Additionally, Kc values of SPRA-proteins

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were determined as 2.7 × 104–4.8 × 105 M−1 by ITC (Figure 1). Meanwhile, the Kc value of adamantane

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acetic acid and multi-PEG-β-CyD was determined as 1.1 × 105 M−1 (Figure S6), and was higher than that

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of multi-SPRA-insulin (2.7 × 104 M−1) or multi-SPRA-lysozyme (9.3 × 104 M−1), indicating that protein-

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modification somewhat reduces the interaction between Ad and multi-PEG-β-CyD.

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proteins and PEG-β-CyDs are highly likely to form SPRA-proteins with a certain level of Kc values.

(multi-SPRA-insulin),

Ad-lysozyme/mono-PEG-β-CyD

(mono-SPRA-lysozyme)

and

Ad-

Anyhow, Ad-

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In addition, particle sizes of Ad-insulin and Ad-lysozyme were found to increase by the addition of

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mono-PEG-β-CyD and multi-PEG-β-CyD in a concentration-dependent manner due to complexation of

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both components (Figure 4). Hence, these results indicate the formation of SPRA-proteins. Covalently

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PEGylated insulin (PEG-insulin) and PEGylated lysozyme (PEG-lysozyme) were prepared as controls

228

with activated PEG (M.W. 20 kDa) (Figure 1).

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12


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Figure 4. Particle sizes of SPRA-insulin and SPRA-lysozyme determined by dynamic light scattering.

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3.3. Physicochemical and Enzymatic Stabilities of SPRA-insulin and SPRA-lysozyme. Protein drugs

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often form aggregates during long-term storage, resulting in loss of bioactivity, immunogenicity and

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alteration of pharmacokinetics. In addition, enzyme degradation of protein drugs also causes loss of

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bioactivity. In this context, PEGylation is a powerful approach to improve the physicochemical and

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enzymatic stabilities of protein drugs. Thus, thermal and enzymatic stabilities of SPRA-proteins were

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examined (Figures 5 and 6). Here, to evaluate the thermal stabilities of SPRA-insulin and SPRA-

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lysozyme, the temperature was set at 37 °C and 70 °C, respectively, according to results of preliminary

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experiment, which showed that insulin or lysozyme were certainly degraded. As shown in Figure 5,

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insulin and lysozyme gradually formed aggregates at 37 °C and 70 °C, respectively. Also, the addition of

242

multi-PEG-β-CyD to insulin or lysozyme did not improve their thermal stability, suggesting the

243

negligible stabilizing effects of multi-PEG-β-CyD (Figure S7). In contrast, mono- and multi-SPRA-

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proteins showed high thermal stability compared with the results of protein-only samples. In addition, the

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stability of SPRA-insulin was equivalent to that of PEG-insulin and SPRA-lysozyme showed higher

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stability than PEG-lysozyme after 6.5 h of incubation. 13



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The stability of SPRA-proteins against trypsin digest, especially multi-SPRA-proteins, was also

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superior to protein-only samples or mixtures of proteins/multi-PEG-β-CyD and equivalent to covalent

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PEG-proteins (Figure 6, Figure S8). These results show that SPRA technology dramatically improves the

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thermal and enzymatic stabilities of protein drugs.

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252 253

Figure 5. Thermal stability of SPRA-insulin at 37 °C and SPRA-lysozyme at 70 °C. Each point

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represents the mean ± S.E. of 4–7 experiments. *p < 0.05 versus insulin or lysozyme.

255 256

Figure 6. Enzymatic stability of SPRA-insulin and SPRA-lysozyme against trypsin treatment for 6 h.

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Each value represents the mean ± S.E. of 3–6 experiments. *p < 0.05 versus insulin or lysozyme.

258 259

3.4. In Vitro Enzymatic Activities of SPRA-lysozyme. To evaluate the in vitro enzymatic activity of

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SPRA-lysozyme, we measured the lytic activity of lysozyme against M. lysodeikticus cells (Figure 7).

261

PEG-lysozyme showed only 23% lytic activity, compared with the lytic activity of native unmodified 14


Page 15 of 34

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262

lysozyme. Moreover, the mixture of lysozyme with multi-PEG-β-CyD showed ca. 100% of the lytic

263

activity (Figure S9), suggesting that the addition of multi-PEG-β-CyD to lysozyme does not affect the

264

activity. Importantly, the lytic activities of mono- and multi-SPRA-lysozymes were 90% and 100%,

265

respectively, compared with those of unmodified lysozyme, indicating near complete preservation of the

266

in vitro activity of SPRA-lysozyme.

267

268 269

Figure 7. Lytic activities of SPRA-lysozyme to M. Lysodeikticus cells. Each value represents the mean ±

270

S.E. of 5–13 experiments. *p < 0.05 versus lysozyme.

271 272

3.5. In Vivo Serum Insulin Level and Hypoglycemic Effect of SPRA-insulin. To achieve optimum

273

treatment of diabetes, rapid- and long-acting insulins have been utilized in the clinical field. In the case of

274

long-acting insulin, a flat blood glucose profile resulting from the prolonged hypoglycemic effect of

275

insulin is strongly required. Thus, the serum insulin and glucose levels were evaluated after subcutaneous

276

administration of SPRA-insulin in rats (Figure 8). Here, we chose multi-SPRA-insulin because of its

277

higher physicochemical and enzymatic stabilities and larger molecular weight than mono-SPRA-insulin.

278

High serum insulin levels were retained after subcutaneous administration of multi-SPRA-insulin

279

compared to that of insulin alone (Figure 8A). In addition, area under the blood concentration-time curve

280

(AUC) of SPRA-insulin (81.3 ng/mL·h) was higher than that of intravenously administrated insulin alone 15



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Page 16 of 34

281

(Table S1). As a result, absolute bioavailability of multi-SPRA-insulin after subcutaneous administration

282

was 195%, and was higher than that of insulin. This is probably due to the high enzymatic stability and

283

high retention in the blood of multi-SPRA-insulin. As shown in Figure 8B, duration of the hypoglycemic

284

effect of Ad-insulin was equivalent to that of insulin alone. On the other hand, multi-SPRA-insulin

285

provided the prolonged and flat blood glucose level for 24 h (Figure 8B, left). In fact, the mean residence

286

time of serum glucose levels (MRTG) of multi-SPRA-insulin was significantly higher than that of insulin

287

(Figure 8C). Besides, the cumulative percentage of change in plasma glucose levels up to 24 h post-

288

administration (AUCG) of multi-SPRA-insulin was also superior to that of insulin alone, indicating higher

289

in vivo activity of multi-SPRA-insulin (Figure 8D). In sharp contrast, PEG-insulin showed the negligible

290

hypoglycemic effect, and its AUCG was only 10% versus insulin alone. Importantly, a mixture of insulin

291

and multi-PEG-β-CyD did not show the prolonged the hypoglycemic effect (data not shown). Next, to

292

evaluate the utility of multi-SPRA-insulin as long-acting insulin preparation, the hypoglycemic effect of

293

multi-SPRA-insulin was compared with insulin glargine, a commercially available long-acting insulin

294

analog and the best-selling diabetes drug in 2014. Multi-SPRA-insulin showed higher AUCG and MRTG

295

than insulin glargine (Figure 8C, D). Most importantly, the blood glucose profile after administration of

296

multi-SPRA-insulin was flat when compared with that of insulin glargine (Figure 8B, right). Thus, multi-

297

SPRA-insulin is a promising long-acting insulin.

298 299

16


Page 17 of 34

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300 301

Figure 8. (A) Serum insulin levels after subcutaneous or intravenous administration of insulin (2 U/kg)

302

and multi-SPRA-insulin (2 U/kg) to rats. (B) Serum glucose levels after subcutaneous administration of

303

insulin (2 U/kg), Ad-insulin (2 U/kg), PEG-insulin (2 U/kg) and multi-SPRA-insulin (2 U/kg) to rats. To

304

compare the effects of multi-SPRA-insulin (2 U/kg) and insulin glargine (2 U/kg), the same results of

305

multi-SPRA-insulin are described in the right figure. (C) MRTG and (D) AUCG of multi-SPRA-insulin.

306

Each point represents the mean ± S.E. of 5–13 experiments. *p < 0.05 versus insulin. † p < 0.05 versus

307

insulin glargine. 17



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Page 18 of 34

308 To evaluate a safety profile of multi-SPRA-insulin, blood

309

3.6. Safety Profile of SPRA-insulin.

310

chemistry values, such as creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (AST)

311

and alanine aminotransferase (ALT) were measured 24 h after subcutaneous administration of multi-

312

SPRA-insulin to rats (Table 1). A negligible change in blood chemistry values was observed after

313

subcutaneous administration of multi-SPRA-insulin, suggesting a high safety profile. Additionally, lower

314

hemolytic activity of multi-PEG-β-CyD than β-CyD (Figure S10), and negligible hemolytic activity of

315

multi-SPRA-insulin (Figure S11) supported that high safety of multi-SPRA-insulin after subcutaneous

316

administration.

317 318

319

Table 1. Blood Chemistry Values 24 h after Subcutaneous Administration of Multi-SPRA-insulin to Rats. System

CRE (mg/dL)

BUN (mg/dL)

AST (U/L)

ALT (U/L)

Saline

0.37 ± 0.01

23.1 ± 0.9

73 ± 11

44 ± 3

Insulin

0.35 ± 0.01

19.5 ± 0.8

77 ± 21

62 ± 5

Ad-insulin

0.31 ± 0.02

15.5 ± 1.3

69 ± 12

42 ± 3

multi-SPRA-insulin

0.33 ± 0.01

20.3 ± 1.1

81 ± 23

36 ± 6

Each value represents the mean ± S.E. of three experiments.

320 321

4. DISCUSSION

322

The development of a long-acting insulin drug without loss of the activity has proven to be challenging.

323

Currently, commercially available long-acting insulin products such as insulin/myristic acid (insulin

324

detemir) and insulin/hexadecanedioic acid (insulin degludec) conjugates are used in the clinical field, but

325

these drugs possess only 46% and 13–15% of the bioactivity versus native, unmodified insulin,

326

respectively.36, 37 In addition, PEG-lispro which was in Phase III of clinical trials only possesses 6% 18


Page 19 of 34

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327

activity when compared with that of insulin lispro.16, 18 To achieve advanced PEGylation of insulin that

328

does not lead to significant reduction in activity, a number of reversible PEGylation techniques have been

329

developed. Shechter et al. prepared reversible PEGylated insulin (PEG 40 kDa) through the 9-

330

hydroxymethyl-2-aminofluorene moiety and the resulting PEGylated insulin showed a prolonged

331

hypoglycemic effect.23 However, its in vitro bioactivity was 11% compared with the activity of insulin,

332

and a dose > 100-fold was required for in vivo study. Reversible PEGylation using complexation between

333

insulin and PEGylated basic oligopeptides through an electrostatic interaction38 and using lysine

334

modification through boronic acid39 have also been reported. However, both in vitro and in vivo

335

bioactivities of these PEGylated insulins were not examined.

336

In this context, SPRA technology dramatically improved the stabilities and prolonged the bioactivities

337

of insulin and lysozyme without their loss, highly probably owing to the reversible interaction between

338

Ad-proteins and PEG-β-CyD. Importantly, the addition of multi-PEG-β-CyD negligibly affected thermal

339

stability, enzymatic stability and hypoglycemic effect of insulin or lysozyme, suggesting that the

340

interaction of PEG-β-CyDs with Ad moiety of Ad-proteins is important for improvement of the

341

pharmaceutical properties of the proteins.

342

To achieve long and high bioactivity in vivo, the SPRA-insulin and SPRA-lysozyme should maintain

343

the supramolecular structure in blood and dissociate near the insulin receptor on cells or the rigid layer of

344

bacterial cell walls as its substrate, respectively. Herein, Stella et al. reported that a Kc > 104 M−1 for a

345

CyD/guest molecule complex is required to exist as an inclusion complex in vivo40. In addition, Leong et

346

al.41 demonstrated that sulfobutylether β-CyD is able to alter pharmacokinetics of Ad derivatives when

347

intravenously administered, resulting from complexation with a Kc value equal to ca. 2 × 104 M−1.

348

Moreover, Davis and coworkers developed the CyD nanoparticle containing siRNA in which transferrin

349

is modified through the host-guest interaction between β-CyD and Ad. This particle is stable in the blood,

350

and can accumulate in tumor after intravenous administration.31 Therefore, SPRA-proteins (Kc ~105 M−1)

351

should retain supramolecular structures following subcutaneous administration. 19



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Page 20 of 34

352

Meanwhile, the binding constants of insulin/insulin receptor and lysozyme/substrate are ca. ~109 M−1

353

and ~106 M−1, respectively,42, 43 evoking a competitive dissociation of SPRA-proteins in the presence of

354

their receptors or substrates, and subsequently leading to targeted activities. Thus, SPRA technology is

355

based on a reversible host–guest interaction and the Kc values were moderate (~105 M−1). This moderate

356

association could be suitable for complexation both in vitro and in vivo and for dissociation in the

357

presence of the receptor and substrate.

358

As the other possible mechanism for the in vivo prolonged effects of multi-SPRA-insulin, its absorption

359

via lymph could be attributed. In general, the absorption of high-molecular-weight drug into the blood is

360

restricted after subcutaneous administration by their limited permeability across the vascular endothelia.

361

In this case, the lymphatics provide an alternative absorption pathway from the interstitial space. Lymph

362

flows more slowly than blood, resulting in prolonged high blood drug levels.44 Therefore, multi-SPRA-

363

insulin could be absorbed through the lymph. Hereafter, we should investigate the pharmacokinetics of

364

multi-SPRA-insulin after subcutaneous administration.

365

In the present study, we fabricated reversible PEGylated proteins through the host-guest interaction

366

between PEG-β-CyDs and Ad-proteins. Future efforts should examine the complexation between Ad-

367

proteins and PEG-β-CyD under in vivo conditions or in the presence of receptor and substrate. In

368

addition, to exhibit whether SPRA-insulins retain the supramolecular structure in the blood, after this we

369

should investigate the pharmacokinetics of both PEG-β-CyDs and Ad-insulin.

370

covalently PEGylated insulin prepared by ourselves as a control. Hereafter, we should compare stability

371

and hypoglycemic effects of SPRA-insulins with PEG-lispro produced under GMP.

However, we used

372 373

5. CONCLUSION

374

In this study, we proposed a novel PEGylation technology, SPRA technology, via a host–guest interaction

375

between β-CyD and Ad. We believe that SPRA technology is one of the most successful PEGylation

376

approaches to improve the pharmaceutical properties of proteins without loss of bioactivity. In addition, 20


Page 21 of 34

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377

SPRA technology could be theoretically applied to a large number of peptides and proteins. Currently, we

378

are examining the utility of SPRA technology using various protein drugs. Moreover, SPRA technology

379

should be useful for the PEGylation of low molecular weight drugs, biomaterials and drug carriers.

380

Hereafter, detailed safety profiles, including immunogenicity of SPRA-proteins should be clarified. We

381

believe that SPRA technology has the potential as a generic method, surpassing conventional PEGylation

382

methods of proteins.

383 384

ASSOCIATED CONTENT

385

Supporting Information

386

The Supporting Information is available free of charge on the ACS Publications website at DOI:

387 388

AUTHOR INFORMATION

389

T.H. and T.H. contributed equally to this work.

390

Corresponding Author

391

*Address: Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-

392

ku, Kumamoto 862-0973, Japan. TEL: +81 96 371 4160, FAX: +81 96 371 4420, E mail:

393

[email protected].

394

Notes

395

This study was funded by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). K. Wada is researcher with

396

Nihon Shokuhin Kako Co., Ltd.

397 398

ACKNOWLEDGMENTS

399

The authors thank Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) for providing β-CyD. This work was

400

partially supported by a Program for Leading Graduate Schools “HIGO (Health life science:

401

Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, a Grant-in-Aid for Young 21



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Page 22 of 34

402

Scientists (Start-up) from the Ministry of Education, Science and Culture of Japan (23890161), Adaptable

403

and Seamless Technology Transfer Program through target-driven R&D, JST (AS262Z02650P) and

404

Kumayaku Research Support from the KUMAYAKU Alumni Research Foundation.

405 406

REFERENCES

407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445

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(35) Higashi, T.; Hirayama, F.; Yamashita, S.; Misumi, S.; Arima, H.; Uekama, K. Slow-release system of pegylated lysozyme utilizing formation of polypseudorotaxanes with cyclodextrins. Int. J. Pharm. 2009, 374, 26-32. (36) Markussen, J.; Havelund, S.; Kurtzhals, P.; Andersen, A. S.; Halstrom, J.; Hasselager, E.; Larsen, U. D.; Ribel, U.; Schaffer, L.; Vad, K.; Jonassen, I. Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs. Diabetologia 1996, 39, 281-288. (37) Gough, S. C.; Harris, S.; Woo, V.; Davies, M. Insulin degludec: overview of a novel ultra longacting basal insulin. Diabetes Obes. Metab. 2013, 15, 301-309. (38) Tsiourvas, D.; Sideratou, Z.; Sterioti, N.; Papadopoulos, A.; Nounesis, G.; Paleos, C. M. Insulin complexes with PEGylated basic oligopeptides. J. Colloid. Interface Sci. 2012, 384, 61-72. (39) Cal, P. M.; Frade, R. F.; Cordeiro, C.; Gois, P. M. Reversible lysine modification on proteins by using functionalized boronic acids. Chemistry 2015, 21, 8182-8187. (40) Stella, V. J.; Rao, V. M.; Zannou, E. A.; Zia, V. V. Mechanisms of drug release from cyclodextrin complexes. Adv. Drug Deliv. Rev. 1999, 36, 3-16. (41) Leong, N. J.; Prankerd, R. J.; Shackleford, D. M.; McIntosh, M. P. The effect of intravenous sulfobutylether7-β-cyclodextrin on the pharmacokinetics of a series of adamantane-containing compounds. J. Pharm. Sci. 2015, 104, 1492-1498. (42) Gammeltoft, S.; Gliemann, J. Binding and degradation of 125I-labelled insulin by isolated rat fat cells. Biochim. Biophys. Acta. 1973, 320, 16-32. (43) Malcolm, B. A.; Rosenberg, S.; Corey, M. J.; Allen, J. S.; de Baetselier, A.; Kirsch, J. F. Sitedirected mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc. Natl. Acad. Sci. U S A 1989, 86, 133-137. (44) McLennan, D. N.; Porter, C. J.; Charman, S. A. Subcutaneous drug delivery and the role of the lymphatics. Drug Discov. Today Technol. 2005, 2, 89-96.

519 520

24


Page 25 of 34

1 2 3 4 5A. SPRA-insulin 6 7 8 O COON 9 O 10 11 12 13 14 Insulin 15 16 17 O 18 COON O 19 20 21 22 23 24 25 B. SPRA-lysozyme 26 27 O 28 COON O 29 30 31 32 33 34 Lysozyme 35 36 37 O COON 38 O 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


mono-PEG-b-CyD

mono-SPRA-insulin (Kc = 4.8 x 105 M-1)

Ad-insulin

multi-PEG-b-CyD

multi-SPRA-insulin (PEG : ave. 2.8)

(Kc = 2.7 x 104 M-1)

PEG-insulin

mono-PEG-b-CyD

mono-SPRA-lysozyme (Kc = 6.4 x 104 M-1)

Ad-lysozyme

multi-PEG-b-CyD

(PEG : ave. 2.8)

multi-SPRA-lysozyme (Kc = 9.3 x 104 M-1)

PEG-lysozyme


Figure 1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A. Insulin

Page 26 of 34

C. Lysozyme

14,308

5,734

B. Ad-insulin

D. Ad-lysozyme 5,910

5000

6000

14,483

7000

m/z

14000

16000

m/z


Figure 2

Page 27 of 34


A. mono-PEG-b-CyD (OH)6

p-Toluenesulfonyl chloride

= =

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

O O-SO

-CH3

(OH)6

NaN3

N3

H2O NaOH a.q.

Azido b-CyD

Tosylated b-CyD

(OH)7

Activated PEG (20 kD)

b-CyD

Ph3P (OH)6

O COON O

NH3 a.q. NH2

DMF/H2O

Amino b-CyD

mono-PEG-b-CyD

B. multi-PEG-b-CyD (Cl)7

(N3)7

NaN3

CH3SO2Cl DMAc/H2O

DMF

(OH)7

per-Chloro b-CyD

per-Azido b-CyD Activated PEG (20 kD)

b-CyD

Ph3P

NH3 a.q. (NH2)7

O COON O

DMF/H2O

per-Amino b-CyD

multi-PEG-b-CyD

C. PEG (20 kD)

E. multi-PEG-b-CyD

D. mono-PEG-b-CyD

18

20

x

103

22

24

20

40

m/z

60

x

103

80

100

120

m/z


Figure 3


Number (%)

A. mono-SPRA-insulin

PEG-b-CyD/Ad-Protein 0.1 0.5

30

1 2 10

20 10

Ad-insulin

0 1

10

100

10

100

10

100

10

100

Number (%)

B. multi-SPRA-insulin 30 20 10

Ad-insulin

0 1

Number (%)

C. mono-SPRA-lysozyme 30 20 10

Ad-lysozyme

0 1

D. multi-SPRA-lysozyme Number (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Page 28 of 34

30 20 10

Ad-lysozyme

0 1

Size (nm)


Figure 4

Page 29 of 34

A. SPRA-insulin

B. SPRA-lysozyme

: multi-SPRA-insulin : PEG-insulin : mono-SPRA-insulin : Ad-insulin : Insulin

* * * *

120 100

: multi-SPRA-lysozyme : PEG-lysozyme : mono-SPRA-lysozyme : Ad-lysozyme : Lysozyme

* * *

* * * *

80

*

60 40 20 0

2

4

6

Lysozyme remaining (%)

Insulin remaining (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


120

*

*

100

*

80

* * *

60 40

*

* * *

20 0

2

4

6

Time (h)

Time (day)


Figure 5


B. SPRA-lysozyme

100 80 60 40 20 0

*

*

Lysozyme undegraded (%)

A. SPRA-insulin Insulin undegraded (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Page 30 of 34

100

*

80 60

*

*

40 20 0


Figure 6

Page 31 of 34

120

Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


100 80 60 40

*

20 0


Figure 7


Serum insulin level (ng/mL)

A. Serum insulin level

30 : Insulin (i.v., 2 U/kg) : Insulin (s.c., 2 U/kg) : multi-SPRA-insulin (s.c., 2 U/kg)

* 20 10

*

0

2

*

4

6

8

Time (h)

Serum glucose level (%)

B. Serum glucose level 120

120

100

100

: PEG-insulin (2 U/kg) 40 : Ad-insulin (2 U/kg) : Insulin (2 U/kg) 20 : multi-SPRA-insulin (2 U/kg)

40 20 2

4

6

8 10 Time (h)

12

: Insulin glargine (2 U/kg) : multi-SPRA-insulin (2 U/kg)

0

24

C. MRTG

2

4

6

8 10 Time (h)

12

24

D. AUCG *

*† * AUCG (%・h)

6

† †

60

60

0

80

*

80

MRTG (h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Page 32 of 34

4

2

*

600

400

200

* 0

0


Figure 8

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


Adamantane-protein conjugate

Dissociation

PEGylated b-cyclodextrin

SPRA-protein



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Adamantane-protein conjugate Dissociation

PEGylated b-cyclodextrin

SPRA-protein


Page 34 of 34

Self-Assembly PEGylation Retaining Activity (SPRA) Technology via a

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