BdA

CH,

Ta

b

c

d

h

•

•

•

a

ARRAA

KAAAmDISt

T

f

h0

Neuroscience Letters 581 (2014) 103–108

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me page: www.elsev ier .com/ locate /neule t

iological evaluation of the radioiodinated imidazo[1,2-a]pyridineerivative DRK092 for amyloid-� imaging in mouse model oflzheimer’s disease

hun-Jen Chena,b,c, Kazunori Bandoa, Hiroki Ashinoa, Kazumi Taguchia,ideaki Shiraishia, Keiji Shimaa, Osuke Fujimotoa, Chiemi Kitamuraa, Yasuaki Morimotoa

Hiroyuki Kasaharaa, Takao Minamizawaa, Cheng Jiangd, Ming-Rong Zhangb,etsuya Suharab, Makoto Higuchib, Kazutaka Yamadac,∗∗, Bin Ji b,∗

Research Department, Fujifilm RI Pharma Co. LTD., Chiba, JapanMolecular Imaging Center, National Institute of Radiological Sciences, Chiba, JapanClinical Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu, JapanSchool of Pharmacy, Fudan University, Shanghai, China

i g h l i g h t s

DRK092 has higher affinity for fibril-lary A� and brain uptake than IMPY.125I-DRK092 labeled A� deposited inbrains of AD patient and mouse invitro.125I-DRK092 detected A� accumula-tion more sensitively than 125I-IMPYex vivo.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 7 June 2014eceived in revised form 31 July 2014ccepted 19 August 2014vailable online 27 August 2014

eywords:lzheimer’s disease (AD)myloid-� peptide (A�)myloid precursor protein (APP) transgenic

a b s t r a c t

Non-invasive determination of amyloid-� peptide (A�) deposition has important significance for earlydiagnosis and medical intervention in Alzheimer’s disease (AD). In this study, we investigated the avail-ability of a radioiodinated imidazo[1,2-a]pyridine derivative, termed 125I-DRK092, as single photonemission computed tomography (SPECT) ligand for in vivo detection of A� deposition. DRK092 showedhigh binding affinity for either synthetic human A� fibrils or brain homogenates from amyloid precur-sor protein transgenic (Tg) mouse (PS1-ki/JU-Tg2576) and AD patient with a dissociation constant (Kd)of one-digit nM, and excellent brain permeability (peak value of uptake: approximately 0.9% of injec-tion dose/g rat brain). Ex vivo autoradiographic analysis showed that measurement with 125I-DRK092has higher sensibility for detecting A� accumulation than with 125I-IMPY, a well-known amyloid SPECT

125

ouseRK092

MPYingle photon emission computedomography (SPECT)

ligand, in Tg mice. In vitro autoradiography with I-DRK092 also confirmed higher accumulation ofradioactivity in the cortical arepared with the corresponding

above lead us to draw the concimaging in AD.

∗ Corresponding author at: Molecular Imaging Center, National Institute of Radiologicael.: +81 43 206 3251; fax: +81 43 253 0396.∗∗ Corresponding author at: The United Graduate School of Veterinary Science, Gifu Uniax: +81 155 49 5398.

E-mail addresses: kyamada@obihiro.ac.jp (K. Yamada), kihin@nirs.go.jp (B. Ji).

ttp://dx.doi.org/10.1016/j.neulet.2014.08.036304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

a, enriched with A� plaques, of Tg mouse and AD patient brains, as com-

areas in non-Tg mouse and healthy control brains. All the data presentedlusion that radioiodinated DRK092 is a potential SPECT ligand for amyloid

© 2014 Elsevier Ireland Ltd. All rights reserved.

l Sciences, 4-9-1, Anagawa, Inage-ku, Chiba, Chiba 263-8555, Japan.

versity, 1-1 Yanagido, Gifu City 501-1193, Japan. Tel.: +81 155 49 5395;

1 nce Le

1

davesbfgvlatatp1[teatppncwhipiw

2

2

apnts

2

oYcodDtmkpsmta

04 C.-J. Chen et al. / Neuroscie

. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerativeisorder characterized by the two pathological hallmarks ofmyloid-� peptide (A�) plaques and neurofibrillary tangles. Inivo non-invasive detection of A� deposition is important forarly diagnosis and medical intervention of AD at prodromaltage, since fibrillary A� has already been accumulating in therain for a few decades before onset of AD [1]. Over the pastew years, the availabilities of several positron emission tomo-raphy (PET) tracers for amyloid imaging have successfully beenerified in human patients with AD and model animals with amy-oid pathology [2–5]. Although being inferior to PET in sensitivitynd quantitative performance, single photon emission computedomography (SPECT) imaging has advantages in terms of oper-ting cost and already-installed rate in medical hospitals, and ishereby more suitable for primary screening for prodromal ADatients. To date, a number of iodinated ligands, such as dipheny-,3,4-oxadiazole (1,3,4-DPOD) derivatives [6], aurone derivatives7], 1,4-diphenyltriazole derivatives [8], pyridyl benzofuran deriva-ives [9], and imidazo[1,2-a]pyridine derivatives [10] have beenmployed for amyloid imaging with SPECT. IMPY, an imidazo[1,2-]pyridine derivative, is the only ligand for SPECT that has beenested in humans [11]. Although this ligand has shown excellentroperties as an imaging probe in preclinical studies [12,13], thereliminary clinical data for 123I-IMPY showed a poor signal-to-oise ratio, possibly making it difficult to distinguish betweenognitively normal persons and AD patients [14]. Very recently,e synthesized a series of imidazo[1,2-a]pyridine derivatives withigher affinity for synthetic human A� fibrils for amyloid SPECT

maging. In the present study, we examined the potential of oneromising candidate compound, termed DRK092, by performing

n vitro and ex vivo autoradiographic analyses of a the model mouseith AD-like amyloid pathology and postmortem human tissues.

. Materials and methods

.1. Radiosynthesis of radioligands

Radiosynthesis of 125I-DRK092 and 125I-IMPY was performeds described in previous publications [10,15]. The radiochemicalurity of 125I-DRK092 and 125I-IMPY was 99.5 ± 0.27% (mean ± SE;

= 3) and 99.6 ± 0.25% (n = 4), respectively, and specific radioac-ivity was 81.4 and 81.4 GBq/�mol, respectively, at the end ofynthesis.

.2. Experimental animals

We had obtained the JU-Tg2576 mouse, generated by backcrossf a transgenic (Tg) mouse line (Tg2576; Taconic Farms Inc. Nework, NY), which overexpressed a mutant form of amyloid pre-ursor protein (APPK670/671L), with JU strain (JU/Ct-C, A.) mousever 29 generations, under license agreement with the Mayo Foun-ation for Medical Education and Research (Rochester, MN), fromaiichi Sankyo Co. Ltd. (Tokyo, Japan) for easier daily handling. Fur-

hermore, we crossbred the JU-Tg2576 mouse with a “knock-in”ouse with the I213T preseniline-1 (PS1) missense mutation (PS1-

i) [16] to generate PS1-ki/JU-Tg2576 mouse for earlier amyloidathogenesis. Tg mice (indicated as PS1-ki/JU-Tg2576 if without

pecial description) and body-weight matched non-Tg JU strainice as control animals were used in the present study except for

he in vitro autoradiographic experiment, in which commerciallyvailable Tg2576 mouse brain was used.

tters 581 (2014) 103–108

2.3. Preparation of brain homogenates and A ̌ fibrils

The Tg mice were deeply anesthetized and euthanized by decap-itation. The neocortex was rapidly removed and homogenizedwith Potter-Elvehjem homogenizer (Thermo Fisher Scientific Inc.,Philadelphia, PA) in 5 volumes of 20 mM Tris–HCl buffer (pH 7.6)containing protease inhibitor cocktail (Nacalai Tesque Inc., Kyoto,Japan) and 0.25 M sucrose. Aliquots of the homogenates wereimmediately frozen and stored at −80 degree until the experi-ments. The homogenates of gray matter from the parahippocampalgyrus from an 89-year-old patient with AD (Harvard Brain Tis-sue Resource Center and Analytical Biological Services, Inc.) wereprepared using the same method. A� fibrils were generated byspontaneous assembly. Briefly, lyophilized synthetic A�(1-40) pep-tides (Peptide Institute Inc. Osaka, Japan, final concentration:100 �M) were gently dissolved in 10 mM phosphate buffered saline(PBS, pH7.4) containing 1 mM EDTA and incubated at 37 ◦C for4 days with gentle, constant shaking. Aggregated A� fibrils werestored at 4 ◦C and diluted into working solution (final concentra-tion: 0.5 �M) for in vitro binding assay.

2.4. In vitro binding assay

The 100-�l aliquots of A� fibrils working solutions or brainhomogenates were reacted with 900 �l of PBS containing ascorbicacid (final concentration: 20 mM) and 125I-DRK092 or 125I-IMPY(20 kBq; final concentrations were adjusted to serial concen-trations of 0.2–100 nM for synthetic A� fibril or 0.1–500 nMfor brain homogenate incubation solutions by the addition ofnon-radiolabeled DRK092 or IMPY). The reaction mixtures wereincubated at 37 ◦C for 1 h, followed by vacuum filtration throughWhatman GF/B filters using a Brandel cell harvester (Model M-24R, Biomedical Research. Lab., Gaithesburg, MD). Radioactivityon the GF/B filters was measured by gamma counter after threerapid washes with 2 ml of ice-cold PBS containing 0.1 mM ascorbicacid. The dissociation constant (Kd) and maximum specific bind-ing (Bmax) were generated by Scatchard analysis using a GraphPadPrism (GraphPad Software, version 4.3, San Diego, CA, USA).

2.5. In vitro autoradiographic analysis

Fresh frozen brain sections (20-�m thickness) from a 23-month-old Tg2576 Tg mouse and deparaffinized human brain sections (10-�m thickness; Center for Neurodegenerative Disease Research atthe University of Pennsylvania Perelman School of Medicine) werepreincubated with PBS for 30 min, followed by incubation with 125I-DRK092 (2 nM) in PBS containing 20% ethanol in the absence orpresence of non-radiolabeled DRM106, an analog of DRK092 withhigh affinity for A� fibrils [15], at room temperature for 1 h. Afterthat, the sections were rinsed with ice-cold PBS containing 20%ethanol 2 times for 2 min each time, and finally dipped into distilledwater for 10 s. After warmly blow-dried and attached to an imagingplate (BAS-MS2025; Fujifilm, Tokyo, Japan) for 3 days, radiolabelingwas detected by scanning the imaging plate using the BAS-5000system (Fujifilm).

2.6. Ex vivo autoradiographic analysis

Male Tg mice (19 months old) and age-matched non-Tg litter-mate mice received bolus injection of radioligands (1.3 MBq of125I-DRK092 or 125I-IMPY in 0.2 ml of saline containing 1% tween80and 40 mM sodium l-ascorbate) via tail vein. At indicated time

points, the animals were deeply anesthetized with isoflurane andeuthanized by decapitation. The brains were immediately removed,embedded in Tissue-Tec. O.C.T. Compound (Sakura FinetechnicalCo., Ltd., Tokyo, Japan), frozen in dry ice, and cut into 15-�m thick

C.-J. Chen et al. / Neuroscience Le

125I-IMPY

125I-DRK092

FtI

cTf(iBoot

3

3

wmdifismRIheCnTDtbsmttTsIt3fiws

ig. 1. Chemical structures of 125I-DRK092 and 125I-IMPY. Squares enclosed by dot-ed lines indicate the -NMe2 group and the oxazole ring in chemical structures ofMPY and DRK092, respectively.

oronal sections by cryostat microtome (Carl Zeiss, Jena, Germany).hese brain sections were closely attached to the imaging plateor an optimized contact period and scanned by FLA3000 systemGE Healthcare). Region of interests (ROIs) were carefully placed onndicated brain regions with reference to a brain atlas (The Mouserain in Stereotaxic Coordinates, Paxinos G and Franklin KBJ, Sec-nd Edition, Academic Press, San Diego, 2001). The counterstainingf plaques with Congo Red was performed in the same brains sec-ions according to a well-established method after autoradiogram.

. Results and discussion

.1. In vitro binding parameters

With a most advanced SPECT ligand, ex vivo autographyith 125I-IMPY has successfully visualized A� deposition in ADodel mouse [13]. However, it still lacks sufficient ability to

istinguish patients with AD from cognitively normal subjectsn SPECT scans. Metabolic instability, insufficient affinity forbrillary A� and brain permeability are considered to be respon-ible for its limited application in human subjects [14]. Thus,ore improvements are needed for further clinical applications.

eplacement of the -NMe2 group in the chemical structure ofMPY with the oxazole ring resulted in DRK092 (Fig. 1). Thealf-maximal inhibitory concentration (IC50) values of sev-ral well-known amyloid ligands including IMPY, Pittsburghompound-B (PiB), 2-(1-{6-[(2-fluoroethyl)(methyl)amino]-2-aphthyl}ethylidene)malononitrile (FDDNP), Congo Red andhioflavin T are shown in Supplementary Information, whereRK092 showed higher affinity for synthetic human A� fibrils than

hese compounds. Binding parameter analyses using a two-siteinding model also demonstrated that the affinity of DRK092 forynthetic human A�(1-40) fibrils, and brain homogenates of Tgice and AD patients was approximately 2.4–18 times higher than

hat of IMPY (Table 1). Thus, replacement of the -NMe2 group withhe oxazole ring seems to improve the affinity for A� deposition.o the best of our knowledge, no published data demonstrated thatuch replacement leaded to improve the affinity for A� deposition.n addition, such modification in chemical structure also decreasedhe lipophilic parameter (LogD) from approximately 4.03 (IMPY) to.64 (DRK092) [15]. These results provide important information

or strategies for imaging agent discovery, because many amyloidmaging agents have the -NMe2 group in their chemical structures

ith different scaffolds, such as 18F-BF227 and 18F-FACT, whichhowed limited utility for the detection of A� deposition in human

tters 581 (2014) 103–108 105

subjects likely due to their relatively high lipophilicity [17,18],and such modification may be helpful for improving affinity andlipophilicity.

3.2. Biodistribution of 125I-DRK092 in normal rats

Biodistribution analysis demonstrated that, in the initial phase,125I-DRK092 was mainly accumulated in lung, kidney and liveramong peripheral organs. Notably, no relevant abnormal accu-mulation in specific organs was measured during the observationperiod after the bolus injection, confirming that the use of DRK092should not give rise to safety concerns regarding abnormal dis-tribution. There was only a slight increase in thyroid uptakewithin 40 min, suggesting that there need be little worry aboutde-iodination of 125I-DRK092 in further SPECT imaging in short-term scans. Measured brain-to-blood ratios were approximately3.2, 3.2, 1.3, 0.6, and 0.4 at 2, 5, 20, 40, and 90 min post-injection,respectively (Table 2), much higher than those of 125I-IMPY(approximately 0.15, 0.04 and 0.02 at 2, 30 and 60 min post-injection, respectively) as reported in previous studies [13].

3.3. In vitro autoradiographic analysis

Since A� plaques in patients with AD may be structurally dif-ferent from those in Tg mouse [2,19], we performed in vitroautoradiography with 125I-DRK092 using both Tg (Tg2576) mouseand patient with AD tissues to confirm its binding to A� deposited inthe brains of Tg mouse and AD patient. More evident radio-signalswere captured in the cortical/hippocampal area and gray matterof the temporal cortical area, usually enriched with A� deposition,in Tg (Tg2576) mouse and AD brains, respectively, as comparedwith the corresponding areas in non-Tg mouse and healthy con-trol (HC). The addition of non-radiolabeled DRM106 significantlyblocked the binding of 125I-DRK092 in Tg (Tg2576) and AD brainsections, but not in non-Tg and HC brains, indicating that these wereamyloidosis-associated specific binding, although there were fewdetectable plaque-like radio-signals remaining in the subiculum ofTg mouse. In addition to those plaque-associated radiolabelings,there were also distinct lipophilic non-specific bindings to myelin-rich regions in mouse and human tissues (Fig. 2).

3.4. Ex vivo autoradiographic detection of A ̌ deposition with125I-DRK092 and 125I-IMPY

Ex vivo autoradiographic images have clearly demonstrated thatthe brain uptake of either 125I-IMPY or 125I-DRK092 decreasedwith time after an initial peak, and there was appreciable A�plaque-associated accumulation of these two radioligands in thecortices of Tg, but not non-Tg mice, within 2 h post-injection.However, the labeling of 125I-IMPY on A� plaques became veryweak and finally invisible by 5–8 h post-injection. In contrast,the labeling of 125I-DRK092 on A� plaques was still appreciableeven after 8 h. Unlike the in vitro autoradiographic images, therewas little lipophilic non-specific binding of 125I-DRK092 in thecorpus callosum (Fig. 3A). Although the ratio of signals in cortex-to-striatum (CT-to-ST ratio), as plaque-rich and reference tissues,respectively, of 125I-DRK092 seemed not to overly differentiatefrom that of 125I-IMPY in Tg mouse, radioactivity of 125I-DRK092remaining in brains of both Tg and non-Tg mice was much higherthan that of 125I-IMPY at all time points examined (Fig. 3B), likelydue to its higher brain uptake and affinity for A� aggregates. Theamyloid deposit-associated 125I-DRK092 labeling was further con-

firmed by subsequent counterstaining with Congo Red (Fig. 3C andD). The above observations provided explicit evidence that 125I-DRK092 has improved detectability of A� plaques as comparedwith 125I-IMPY. Higher brain retention without negative effect on

106 C.-J. Chen et al. / Neuroscience Letters 581 (2014) 103–108

Table 1Binding parameters.

Sample High-affinity binding site Low-affinity binding site

125I-IMPY 125I-DRK092 125I-IMPYc 125I-DRK092

aKdbBmax Kd Bmax Kd Bmax Kd Bmax

A�1-40 fibrils 1.14 ± 0.36 0.14 ± 0.03 0.28 ± 0.20 0.041 ± 0.03 24.65 ± 1.27 1.47 ± 0.02 2.56 ± 0.13 1.01 ± 0.02Tg brain 13.18 ± 2.58 8.40 ± 2.25 0.73 ± 0.73 0.44 ± 0.63 1107 ± 451 7848 ± 2648 14.90 ± 3.06 59.30 ± 10.80AD brain 4.58 ± 0.65 1.99 ± 0.11 1.87 ± 0.17 1.66 ± 0.08 – – 97.40 ± 11.28 3.56 ± 0.08

The data of A� fibrils, Tg brain (20-month-old), and AD brain (89-year-old) were mean ± standard errors of duplicate experiments.a Kd was expressed as nM.b Bmax for A� fibrils and mouse/human tissues were expressed as pmol/nmol of A� and pmol/g tissue, respectively.c Only one binding site was detected in curve-fitting analysis of 125I-IMPY.

Table 2Biodistribution of [125I]DRK092 in normal rats.

Organ 2 min 5 min 20 min 40 min 90 min 180 min 360 min 1440 min

% ID/g tissue (mean ± SD; n = 3)Blood 0.29 ± 0.03 0.25 ± 0.00 0.23 ± 0.02 0.24 ± 0.01 0.22 ± 0.03 0.24 ± 0.02 0.31 ± 0.15 0.12 ± 0.01Brain 0.92 ± 0.10 0.81 ± 0.05 0.30 ± 0.04 0.14 ± 0.00 0.08 ± 0.01 0.05 ± 0.00 0.05 ± 0.02 0.02 ± 0.00Heart 0.82 ± 0.07 0.57 ± 0.06 0.36 ± 0.03 0.27 ± 0.01 0.22 ± 0.04 0.21 ± 0.02 0.21 ± 0.09 0.09 ± 0.01Lung 1.36 ± 0.18 1.27 ± 0.11 0.76 ± 0.11 0.59 ± 0.04 0.49 ± 0.07 0.42 ± 0.05 0.42 ± 0.19 0.20 ± 0.01Liver 0.94 ± 0.22 1.48 ± 0.27 1.10 ± 0.16 0.74 ± 0.05 0.54 ± 0.05 0.40 ± 0.01 0.38 ± 0.17 0.20 ± 0.01Spleen 0.61 ± 0.08 0.53 ± 0.03 0.31 ± 0.03 0.24 ± 0.00 0.18 ± 0.01 0.17 ± 0.01 0.20 ± 0.07 0.08 ± 0.01Kidney 2.89 ± 0.07 2.02 ± 0.07 1.29 ± 0.16 0.79 ± 0.04 0.55 ± 0.07 0.42 ± 0.03 0.40 ± 0.18 0.16 ± 0.02

% ID (mean ± SD; n = 3)Thyroid gland 0.08 ± 0.00 0.07 ± 0.00 0.10 ± 0.02 0.18 ± 0.01 0.40 ± 0.01 0.83 ± 0.10 1.56 ± 0.35 3.75 ± 0.34

Normal Sprague-Dawley (SD) rats (8 weeks old; Charles River Laboratories Japan Inc., Yokohama, Japan) were administered with [125I]DRK092 (1.85 MBq/kg body weight)v us pu

stifiwihbArp

Ftm

ia the tail vein, and following processes were performed as described in our previo

ignal-to-noise ratio would provide more intense radio-signals andherefore be beneficial for clearer imaging, and also allow lowernjection doses and resultant attenuation of radiation exposureor acquirement of similar-quality images. Very recently, SPECTmaging with a radioiodine-labeled pyridyl benzofuran derivative

as reported to successfully visualize amyloid deposited in liv-ng Tg2576 mice [9]. This compound showed approximately 3-foldigher affinity for fibrillary A� than IMPY as estimated with inhi-

ition constants for the binding of 125I-IMPY to human synthetic� aggregate [9]. Although we have not validated the utility ofadiolabeled DRK092 for SPECT imaging in living brains, brain-ermeability and affinity for A� deposits (approximately from

ig. 2. In vitro autoradiographic images of 125I-DRK092. The brain sections were from 2he absence (upper) and presence (lower) of non-radiolabeled DRM106 (10 �M). Non-sp

atter.

blication [15].

2.4- to 24-fold higher than IMPY as estimated with Kd or IC50 invarious samples; Table 1 and Supplementary Information) seemsufficient for in vivo SPECT imaging. Additionally, the ratio ofradioactivity remaining in normal brain at 2 min to that at 60 min(brain2/60 min ratio) post-injection of radioiodinated pyridyl ben-zofuran derivative was approximately 0.3 [9], much higher thanthat of 125I-DRK092 (brain2/60 min ratio was estimated at between0.15 and 0.09; Table 2). These numerical data were consistent with

high- and low-level lipophilic non-specific bindings to myelin-richareas, such as the corpus callosum and internal capsule, in ex vivoautoradiographic images with radioiodinated pyridyl benzofuranderivative [9], and 125I-DRK092 (Fig. 3A), respectively.

3-month-old non-Tg, Tg mice, healthy control (HC) and AD from left to right, inecific binding was defined in the presence of non-radiolabeled DRM106. W: white

C.-J. Chen et al. / Neuroscience Letters 581 (2014) 103–108 107

Fig. 3. Ex vivo autoradiographic analysis of 125I-DRK092 and 125I-IMPY in Tg mouse brains. (A and B) The representative coronal images (approximately 0.2–0.8 mm anteriorto the bregma) of 19-month-old male Tg and age-matched non-Tg mice having received bolus injection of radiolabeled 125I-DRK092 and 125I-IMPY at 1, 2, 5, and 8 h post-injection are shown in Panel A. Quantitative analyses of radioactivity in cortical areas (left; radioactivity was estimated as photostimulated luminescence (PSL) per mm2) andcortex-to-striatum (CT-to-ST) ratios of radioactivity (right) of Tg (closed symbols, n = 2 for data at 2 and 5 h, n = 1 for data at 1 and 8 h post-injection) and non-Tg mice (opensymbols, n = 1 for each time point) administered with 125I-DRK092 (squares) and 125I-IMPY (circles) are shown in Panel B. Vertical bars in graph denote SD. (C and D) Co-l ongo RR pectivr

ti

A

MvwMeAo

ocalization of 125I-DRK092 accumulation (C) and counterstaining of plaques with Ced and blue arrowheads indicate A� plaques and cerebrovascular A� deposits, reseferred to the web version of the article.)

All the data presented above lead us to draw the conclusionhat radioiodinated DRK092 is a potential SPECT ligand for amyloidmaging in AD.

cknowledgments

The authors thank Prof. John Q. Trojanowski and Prof. Virginia.-Y. Lee (Center for Neurodegenerative Disease Research, Uni-

ersity of Pennsylvania) for kindly providing human tissue. Thisork was supported in part by Grants-in-Aid for Japan Advanced

olecular Imaging Program and Core Research for Evolutional Sci-

nce and Technology (T. S.), and Scientific Research on Innovativereas (“Brain Environment”) 23111009 (M. H.) from the Ministryf Education, Culture, Sports, Science and Technology, Japan.

ed (D). The same brain section was stained with Congo Red after autoradiography.ely. (For interpretation of the references to color in this figure legend, the reader is

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.neulet.2014.08.036.

References

[1] H. Braak, E. Braak, Neuropathological stageing of Alzheimer-related changes,Acta Neuropathol. (Berl) 82 (1991) 239–259.

[2] J. Maeda, B. Ji, T. Irie, T. Tomiyama, M. Maruyama, T. Okauchi, M. Staufenbiel, N.Iwata, M. Ono, T.C. Saido, K. Suzuki, H. Mori, M. Higuchi, T. Suhara, Longitudi-nal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloidtreatment in a living mouse model of Alzheimer’s disease enabled by positronemission tomography, J. Neurosci. 27 (2007) 10957–10968.

1 nce Le

[

[

[

[

[

[

[

[

[

[19] W.E. Klunk, B.J. Lopresti, M.D. Ikonomovic, I.M. Lefterov, R.P. Koldamova, E.E.

08 C.-J. Chen et al. / Neuroscie

[3] W.E. Klunk, H. Engler, A. Nordberg, Y. Wang, G. Blomqvist, D.P. Holt, M.Bergstrom, I. Savitcheva, G.F. Huang, S. Estrada, B. Ausen, M.L. Debnath, J. Bar-letta, J.C. Price, J. Sandell, B.J. Lopresti, A. Wall, P. Koivisto, G. Antoni, C.A. Mathis,B. Langstrom, Imaging brain amyloid in Alzheimer’s disease with PittsburghCompound-B, Ann. Neurol. 55 (2004) 306–319.

[4] V. Camus, P. Payoux, L. Barre, B. Desgranges, T. Voisin, C. Tauber, R. La Joie,M. Tafani, C. Hommet, G. Chetelat, K. Mondon, V. de La Sayette, J.P. Cottier, E.Beaufils, M.J. Ribeiro, V. Gissot, E. Vierron, J. Vercouillie, B. Vellas, F. Eustache,D. Guilloteau, Using PET with 18F-AV-45 (florbetapir) to quantify brain amy-loid load in a clinical environment, Eur. J. Nucl. Med. Mol. Imaging 39 (2012)621–631.

[5] Z. Cselenyi, M.E. Jonhagen, A. Forsberg, C. Halldin, P. Julin, M. Schou, P. John-strom, K. Varnas, S. Svensson, L. Farde, Clinical validation of 18F-AZD4694, anamyloid-beta-specific PET radioligand, J. Nucl. Med. 53 (2012) 415–424.

[6] H. Watanabe, M. Ono, R. Ikeoka, M. Haratake, H. Saji, M. Nakayama, Synthesisand biological evaluation of radioiodinated 2,5-diphenyl-1,3,4-oxadiazoles fordetecting beta-amyloid plaques in the brain, Bioorg. Med. Chem. 17 (2009)6402–6406.

[7] Y. Maya, M. Ono, H. Watanabe, M. Haratake, H. Saji, M. Nakayama, Novelradioiodinated aurones as probes for SPECT imaging of beta-amyloid plaquesin the brain, Bioconjug. Chem. 20 (2009) 95–101.

[8] W. Qu, M.P. Kung, C. Hou, S. Oya, H.F. Kung, Quick assembly of 1,4-diphenyltriazoles as probes targeting beta-amyloid aggregates in Alzheimer’sdisease, J. Med. Chem. 50 (2007) 3380–3387.

[9] M. Ono, Y. Cheng, H. Kimura, H. Watanabe, K. Matsumura, M. Yoshimura,S. Iikuni, Y. Okamoto, M. Ihara, R. Takahashi, H. Saji, Development of novel123I-labeled pyridyl benzofuran derivatives for SPECT imaging of beta-amyloidplaques in Alzheimer’s disease, PLOS ONE 8 (2013) e74104.

10] Z.P. Zhuang, M.P. Kung, A. Wilson, C.W. Lee, K. Plossl, C. Hou, D.M. Holtzman, H.F.Kung, Structure–activity relationship of imidazo[1,2-a]pyridines as ligands for

detecting beta-amyloid plaques in the brain, J. Med. Chem. 46 (2003) 237–243.

11] A.B. Newberg, N.A. Wintering, K. Plossl, J. Hochold, M.G. Stabin, M. Watson,D. Skovronsky, C.M. Clark, M.P. Kung, H.F. Kung, Safety, biodistribution, anddosimetry of 123I-IMPY: a novel amyloid plaque-imaging agent for the diag-nosis of Alzheimer’s disease, J. Nucl. Med. 47 (2006) 748–754.

tters 581 (2014) 103–108

12] M.P. Kung, C. Hou, Z.P. Zhuang, B. Zhang, D. Skovronsky, J.Q. Trojanowski, V.M.Lee, H.F. Kung, IMPY: an improved thioflavin-T derivative for in vivo labelingof beta-amyloid plaques, Brain Res. 956 (2002) 202–210.

13] M.P. Kung, C. Hou, Z.P. Zhuang, A.J. Cross, D.L. Maier, H.F. Kung, Character-ization of IMPY as a potential imaging agent for beta-amyloid plaques indouble transgenic PSAPP mice, Eur. J. Nucl. Med. Mol. Imaging 31 (2004) 1136–1145.

14] M.P. Kung, C.C. Weng, K.J. Lin, I.T. Hsiao, T.C. Yen, S.P. Wey, Amyloid plaqueimaging from IMPY/SPECT to AV-45/PET, Chang Gung Med. J. 35 (2012)211–218.

15] C-J. Chen, K. Bando, H. Ashino, K. Taguchi, H. Shiraishi, O. Fujimoto, C. Kitamura,S. Mastushima, M Fujinaga, M-R. Zhang, H. Kasahara, T. Minamizawa, C. Jiang, T.Suhara, M. Higuchi, K. Yamada, B. Ji, Synthesis and biological evaluation of novelradioiodinated imidazopyridine derivatives for amyloid- imaging in Alzheimerdisease, Bioorg. Med. Chem. 22 (2014) 4189–4197.

16] Y. Nakano, G. Kondoh, T. Kudo, K. Imaizumi, M. Kato, J.I. Miyazaki, M. Tohyama,J. Takeda, M. Takeda, Accumulation of murine amyloidbeta42 in a gene-dosage-dependent manner in PS1 ‘knock-in’ mice, Eur. J. Neurosci. 11 (1999)2577–2581.

17] H. Ito, H. Shinotoh, H. Shimada, M. Miyoshi, K. Yanai, N. Okamura, H. Takano, H.Takahashi, R. Arakawa, F. Kodaka, M. Ono, Y. Eguchi, M. Higuchi, T. Fukumura, T.Suhara, Imaging of amyloid deposition in human brain using positron emissiontomography and [18F]FACT: comparison with [11C]PIB, Eur. J. Nucl. Med. Mol.Imaging 41 (2014) 745–754.

18] S. Furumoto, N. Okamura, K. Furukawa, M. Tashiro, Y. Ishikawa, K. Sugi, N.Tomita, M. Waragai, R. Harada, T. Tago, R. Iwata, K. Yanai, H. Arai, Y. Kudo,A 18F-labeled BF-227 derivative as a potential radioligand for imaging denseamyloid plaques by positron emission tomography, Mol. Imaging Biol. 15(2013) 497–506.

Abrahamson, M.L. Debnath, D.P. Holt, G.F. Huang, L. Shao, S.T. DeKosky, J.C. Price,C.A. Mathis, Binding of the positron emission tomography tracer Pittsburghcompound-B reflects the amount of amyloid-beta in Alzheimer’s disease brainbut not in transgenic mouse brain, J. Neurosci. 25 (2005) 10598–10606.