Visualization and Quantitative Assessment of the Brain Distribution of

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Visualization and quantitative assessment of the brain distribution of insulin through nose-to-brain delivery based on the cell-penetrating peptide noncovalent strategy Noriyasu Kamei, Tomotaka Shingaki, Yousuke Kanayama, Misa Tanaka, Riyo Zochi, Koki Hasegawa, Yasuyoshi Watanabe, and Mariko Takeda-Morishita Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00854 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Molecular Pharmaceutics

Visualization and quantitative assessment of the brain distribution of insulin through nose-to-brain delivery based on the cell-penetrating peptide noncovalent strategy Authors: Noriyasu Kamei †, Tomotaka Shingaki ‡, Yousuke Kanayama ‡, Misa Tanaka †, Riyo Zochi ‡, Koki Hasegawa §,‖, Yasuyoshi Watanabe ‡, Mariko Takeda-Morishita *,†

Affiliations: †

Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University,

1-1-3 Minatojima, Chuo-ku, Kobe, Hyogo 650-8586, Japan ‡

RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe,

Hyogo 650-0047, Japan §

Department of Pathology and Experimental Medicine, Graduate School of Medical Sciences,

Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan ‖

Present Address: Center for Instrumental Analysis, Kyoto Pharmaceutical University,

Misasagi-Shichonocho, Yamashina-ku, Kyoto 607-8412, Japan

*Corresponding author: Tel: +81-78-974-4816 Fax: +81-78-974-4820 E-mail address: [email protected]

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ABSTRACT: Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive

64

Cu-NODAGA–

insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of 64Cu-NODAGA–insulin with L- or D-penetratin.

The brain accumulation of 64Cu-NODAGA–insulin with penetratin was confirmed by

ELISA using unlabeled insulin, in which intact insulin was delivered to the brain after intranasal coadministration with L- or D-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with L-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

KEYWORDS: Brain delivery, intranasal administration, cell-penetrating peptides, insulin, biopharmaceuticals

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■ Table of Contents/Abstract Graphic

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■ INTRODUCTION

The delivery of biopharmaceuticals to the brain is not feasible at present because such a strategy cannot overcome the biological function of the blood–brain barrier (BBB) effectively and safely 1-3. Intranasal administration is thought to be an ideal route for drug delivery to the brain 4-6. Many studies have reported that various peptides and proteins can be delivered from the nasal cavity to the brain parenchyma, but most studies have not provided quantitative data to prove the effectiveness of brain transport 7-13. Intranasal administration of drugs has an advantage of reducing the risk of systemic drug exposure because the distribution to the circulation is lower for intranasal compared with intravenous administration. However, our recent work has shown that the brain transport of the model peptide drug insulin is inefficient after intranasal administration 14. In the same work, we also demonstrated that penetratin, a typical cell-penetrating peptide (CPP), could significantly boost the efficiency of insulin delivery to the brain after intranasal administration 14. In that study, insulin was distributed rapidly to the brain parenchyma, as shown by its appearance 15 min after intranasal administration with penetratin, and that insulin eventually penetrated into deeper brain regions such as the hippocampus. This suggested that our novel strategy using CPP administered via the intranasal route might be feasible for the delivery of biopharmaceuticals such as insulin and other neurotrophic agents to the brain. However, further verification study, including highly sensitive visualization and quantitative methods, are required to document the safety and effectiveness of our strategy. In addition, the mechanism responsible for the penetratin-induced increase in the delivery of insulin from the nasal cavity to the brain parenchyma should be clarified. There are two possible pathways: (1) a direct route from the nose to the brain via the olfactory bulb and/or cerebrospinal fluid (CSF), or (2) an indirect route via permeation of the BBB after systemic absorption. Therefore, in the present study, we used autoradiography and gamma counting to examine the distribution of insulin tagged with the 4


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radioactive tracer

64

Cu delivered with a proper chelator, 1,4,7-triazacyclononane,1-glutaric

acid-4,7-acetic acid (NODAGA), into the brain. The data obtained by measuring the 64Cu-NODAGA– insulin distribution was confirmed in experiments using unlabeled insulin and an enzyme-linked immunosorbent assay (ELISA). This is the first trial to demonstrate insulin brain distribution through nose-to-brain delivery based on CPP noncovalent strategy using autoradiography techniques. The evidences and findings obtained in this study are significantly beneficial to establish the CPPs for delivering biopharmaceuticals from the nose to the brain.

■ MATERIALS AND METHODS

Materials. Recombinant human insulin (27.5 IU/mg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

L-Penetratin

(RQIKIWFQNRRMKWKK, capital letters indicate the L-form of

amino acids) and D-penetratin (rqikiwfqnrrmkwkk, lowercase letters indicate the D-form of amino acids) were synthesized by Sigma-Genosys, Life Science Division of Sigma-Aldrich Japan Co. (Hokkaido, Japan).

NODAGA-N-hydroxysuccinimide (NHS) was purchased from CheMatech

(Dijon, France). N,N-dimethylformamide (DMF), acetonitrile, and trifluoroacetic acid (TFA) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). cOmplete tablet protease inhibitor cocktail was purchased from Roche Diagnostics GmbH (Mannheim, Germany). 2-Morpholinoethanesulfonic acid (MES) monohydrate was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals were of analytical grade and are commercially available.

Preparation of insulin–penetratin mixed solution. To prepare the insulin solution, specific amounts of recombinant insulin were dissolved in 100 5



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µL of 0.1 M HCl. The insulin solution was diluted with 0.8 mL of phosphate-buffered saline (PBS, pH 6.0) containing 0.001% methylcellulose, which prevents the adsorption of insulin and penetratin to the plastic tube surface, and was then normalized with 100 µL of 0.1 M NaOH. Specific amounts of L- or D-penetratin

were dissolved in PBS (pH 6.0) with 0.001% methylcellulose. In the study without

64

Cu-NODAGA–insulin, equal volumes of insulin and L- or D-penetratin solution were mixed gently

and adjusted to concentrations of 20 IU/mL and 0.5 mM, respectively. Each insulin–L- or insulin– D-penetratin

solution was clear after mixing. In the study using

insulin solution containing

64

64

Cu-NODAGA–insulin, 25 µL of

Cu-NODAGA–insulin was added to 25 µL of L- or D-penetratin and

adjusted to 10 IU/mL and 0.5 mM, respectively.

Labeling of NODAGA with insulin. NODAGA–insulin was synthesized according to Lee’s method modified by replacement of deoxycholic acid with NODAGA

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. Briefly, insulin (20 mg, 3.5 µmol) was dissolved in 0.1 M

carbonate–bicarbonate buffer (2 mL, pH 10). NODAGA-NHS (5.1 mg, 7 µmol) was dissolved in DMF (200 µL) and added dropwise to the insulin solution. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was passed through a Sephadex G-25 column (16 mm × 250 mm) equilibrated with 20% acetonitrile in water and the obtained crude sample fraction was lyophilized. The crude sample was purified by preparative reversed-phase high-performance liquid chromatography (HPLC) (Gilson, Middleton, WI) with a Cosmosil 5C18-ARII column (4.6 mm × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan). The mobile phase comprised 0.1% TFA in water (eluent A) and acetonitrile containing 0.1% TFA (eluent B). The mobile phase was eluted with a linear gradient of 20% to 30% eluent B for 20 min at a flow rate of 1 mL/min, and the UV absorbance of the eluent was monitored at 220 nm.

Labeling of 64Cu with NODAGA–insulin. 6


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Copper-64 solution was prepared from 64Ni as described previously 16, 17. Briefly, enriched 64Ni (99%) electroplated on a gold disk was irradiated with a 12 MeV proton beam at 20 µA current for 3 h using a small cyclotron (CYPRIS HM-12S, Sumitomo Heavy Industries, Ltd., Tokyo, Japan). Copper-64 produced in 64Ni plating was chemically separated using anion-exchange resin (Muromac 1×8, Cl– form, 100–200 mesh, Muromachi Chemicals Inc., Fukuoka, Japan) column.

For the

radiolabeling, about 700 MBq of 64Cu solution was added to NODAGA–insulin (2 nmol) dissolved in 0.1 M of sodium acetate buffer and incubated at 40 °C for 30 min. The final pH of the reaction solution was 6.5. The reaction mixture was passed over a solid-phase extraction cartridge (Sep-Pak C18 1cc Vac, Waters Corp., Milford, MA, USA), washed with ultrapure water, and eluted with 400 µL of ethanol. Finally, the purified 64Cu-NODAGA–insulin in ethanol was evaporated by heating at 40 °C with nitrogen blowdown and then dissolved in 120 µL of PBS containing unlabeled insulin (20 IU/mL). The 64Cu-NODAGA–insulin solution containing unlabeled insulin was added to PBS or the penetratin solution just before the animal study, and the results were analyzed using an HPLC radioanalyzer (RLC-700, Aloka Co., Ltd., Tokyo, Japan). The radioactive purity of synthesized 64

Cu-NODAGA–insulin was calculated based on the peak areas. The average yield of decay-corrected

radioactivity was 87%. The overall scheme of 64Cu-NODAGA–insulin preparation is shown in Fig. 1.

In vivo intranasal administration. Male Sprague-Dawley rats weighing 200–220 g (or 250–270 g for the CSF collection study) were purchased from Japan SLC Inc. (Shizuoka, Japan). The experiments using animals were performed at Kobe Gakuin University and RIKEN Center for Life Science Technologies, and complied with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals. The animals were housed in rooms maintained at 23 ± 1 °C and 55 ± 5% relative humidity, and were allowed free access to water and food during acclimatization. Rats were anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg, Somnopentyl, Kyoritsu Seiyaku 7



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Corp., Tokyo, Japan), and they were restrained in a supine position on a thermostatically controlled board at 37 °C. The in vivo operation was performed in rats in accordance with the method originally described by Hirai et al 18. After an incision was made in the neck, the trachea and esophagus were cannulated with polyethylene tubing (Hibiki No. 6) to maintain respiration and to keep the solution in the nasal cavity, respectively. The nasopalatine ducts were closed with medical Aron Alpha A Super Glue (Daiichi Sankyo Co., Tokyo, Japan) to prevent drainage of the solution from the nasal cavity to the oral cavity via the nasopalatine duct. The rats were then given 50 µL of the test solution with a micropipette (Pipetman P-100, Gilson) inserted directly into the left nostril.

Brain distribution study with 64Cu-NODAGA–insulin. Following the surgical method of Hirai et al, the animal was placed under anesthesia with sodium pentobarbital as described above, 50 µL of 64Cu-NODAGA–insulin (20–35 MBq/50 µL) and unlabeled insulin (2.5 IU/kg) with or without penetratin (0.5 mM) was administered intranasally to the left nostril of the rat (200–220 g). At the designated time point after administration (15 min), the abdominal cavity was opened, and 0.3 mL of blood was collected from the postcaval vein. The rat was quickly decapitated, the administered solution was flushed by perfusion of saline, and the whole brain was carefully isolated and washed with ice-cold saline. The brain sample was placed onto a brain matrix (Muromachi Kikai Co., Ltd., Tokyo, Japan), and sliced into 12 fractions. The sliced brain samples were weighed, placed on film, and then placed in contact with an imaging plate (BAS IP SR 2040 E, GE Healthcare Japan Corp., Tokyo, Japan) for 120 min. Following the exposure, the distribution characteristics of radioactivity to the brain were analyzed by autoradiography (FLA-7000, Fujifilm Corp., Tokyo, Japan). The radioactivity of the blood and sliced brain samples was measured by using an automatic gamma counter (Wallac Wizard 3” 1480, Perkin Elmer Japan Co., Ltd., Kanagawa, Japan).

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Brain distribution study based on ELISA measurement. Following the surgical method of Hirai et al, with the animal under anesthesia with sodium pentobarbital as described above, 50 µL of insulin (5 IU/kg) with or without penetratin (0.5 mM) was administered intranasally to the left nostril of the rat (200–220 g). At the designated time point after administration (15 min), 0.3 mL of blood was collected from the left jugular vein. After removal of the administered solution from the nasal cavity by flushing with PBS, the abdominal cavity was opened, and the intravascular content in the brain was flushed by perfusion of ice-cold PBS (pH 7.4) into the left ventricle of the heart at 15 mL/min for 5 min using a peristaltic pump (ATTO Corp., Tokyo, Japan). The rat was decapitated, and the whole brain was collected in the same manner as described above. The isolated brain sample was separated on ice into the olfactory bulbs, hypothalamus, hippocampus, cerebral cortex, cerebellum, and brainstem. These samples were weighed and homogenized with a 2× volume of ice-cold 0.1 M MES solution (pH 3.5) with protease inhibitor cocktail using a glass or microtube tissue grinder. The blood samples and homogenized samples were centrifuged at 4 °C and 5,400 ×g for 15 min, and the insulin concentration in the resultant plasma or homogenate supernatant was measured using an ultrasensitive insulin ELISA kit (Mercodia AB, Uppsala, Sweden).

CSF transport study. Following the surgical method of Hirai et al, with the animal under anesthesia with sodium pentobarbital as described above, 50 µL of insulin (5 IU/kg) with or without penetratin (0.5 mM) was administered intranasally to the left nostril of the rat (250–270 g). At the designated time point after administration (15 min), 0.3 mL of blood was collected from the left jugular vein, and 0.2 mL of CSF was collected by cisternal puncture and divided into the initial and latter halves to measure the concentrations of the posterior and anterior portions of CSF, respectively. The concentrations of insulin in both portions of CSF were determined using an ultrasensitive insulin ELISA kit (Mercodia AB). 9



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Statistical analysis. Each value is expressed as the mean and standard error of the mean (SEM) of multiple determinations. The significance of the differences in the mean values of multiple groups was evaluated using analysis of variance (ANOVA) with Dunnett’s test. The IBM SPSS Statistics Version 22 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Differences were considered significant when the p value was

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