Enhancement of Binding Affinity for Amyloid Aggregates by Multivalent

Mar 27, 2014 - Mathis, C. A.; Dekosky, S. T.; Greenberg, S. M.; Hyman, B. T.;. Growdon ... (22) Zha, Z.; Choi, S. R.; Ploessl, K.; Lieberman, B. P.; Q...
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Enhancement of Binding Affinity for Amyloid Aggregates by Multivalent Interactions of 99mTc-Hydroxamamide Complexes Shimpei Iikuni,† Masahiro Ono,*,† Hiroyuki Watanabe,† Kenji Matsumura,† Masashi Yoshimura,† Naoya Harada,† Hiroyuki Kimura,† Morio Nakayama,‡ and Hideo Saji† †

Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan ‡ Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan S Supporting Information *

ABSTRACT: Deposition of amyloid aggregates has been regarded as an early stage of amyloidosis progression. An imaging probe that can image amyloid aggregates enables the early diagnosis of amyloidosis and contributes to the development of new medical therapies. High binding affinity for amyloid aggregates is essential to develop a useful molecular imaging probe. This article describes a new strategy to enhance the binding affinity of imaging agents targeting amyloid aggregates. We designed and synthesized novel 99mTc-hydroxamamide (99mTc-Ham) complexes with a bivalent amyloid ligand and evaluated their binding affinity for amyloid aggregates by using β-amyloid peptide (Aβ(1−42)) aggregates as a model. In vitro inhibition assay indicated that bivalent 99mTc-Ham complexes had much higher binding affinity for amyloid aggregates than monovalent complexes. In vitro autoradiography using Tg2576 mice showed the specific binding of bivalent 99mTc-Ham complexes to Aβ plaques in the mouse brain, as reflected in the results of the inhibition assay. The preliminary results suggest that a new molecular design based on bivalent 99mTc-Ham complexes may be reasonable to develop an imaging probe targeting amyloid aggregates. KEYWORDS: amyloid aggregates, multivalent interactions, technetium-99m, imaging, amyloidosis

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INTRODUCTION Amyloidosis is the general name given to diseases characterized by extracellular deposition of a specific amyloid protein that aggregates in the form of insoluble fibrils.1−3 Amyloidoses are generally classified into systemic or localized based on the chemical characterization of the precursor protein. Amyloid protein includes β-amyloid (Aβ), constituting senile plaques (SP) in Alzheimer’s disease (AD), and deposited in the walls of cerebral capillaries and arteries in cerebral amyloid angiopathy (CAA), prion protein in prion disease, and amylin in diabetes mellitus type II. Since these amyloid aggregates probably appear prior to the onset of disease,4−7 their detection in vivo may lead to an early diagnosis of the corresponding amyloidosis. Additionally, monitoring these targets in vivo may also support the development of new medical techniques such as antiamyloid therapies. Therefore, the development of a noninvasive diagnosis of amyloidosis that allows disease-specific detection is strongly needed. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) constitute major in vivo imaging techniques to carry out the noninvasive diagnosis of amyloidoses. To date, great efforts have been made to develop PET and SPECT probes that can image SP in AD © 2014 American Chemical Society

brains. Many clinical studies using PET tracers, such as [11C]PIB,8−10 [18F]AV-1,11 and [18F]AV-45,12−14 have proved their utility for the diagnosis of AD. Although most of such PET and SPECT tracers reported previously bind to core dense plaques that consist of a high degree of Aβ peptides, they are known to bind weakly to diffuse plaques that consist of a low level of Aβ peptides.15−17 Therefore, further enhancement of the binding affinity for amyloid aggregates should lead to early detection of amyloid aggregates in amyloidoses. High binding affinity for target molecules is one of the prerequisites to develop a useful molecular imaging probe. Multivalent interactions are characterized by the simultaneous binding of a ligand to multiple ligand recognition sites.18 These multivalent interactions enhance the binding of a multivalent ligand compared to the corresponding monovalent ligand. Therefore, multivalent interactions have been used as one of the strategies to enhance the binding affinity of many molecular imaging probes for their target molecules.19 For example, to Received: Revised: Accepted: Published: 1132

August 21, 2013 February 3, 2014 March 14, 2014 March 27, 2014 dx.doi.org/10.1021/mp400499y | Mol. Pharmaceutics 2014, 11, 1132−1139

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improve the binding affinity for integrin αvβ3, multimeric RGD peptides have been utilized to develop radiotracers targeting integrin αvβ3.20,21 Such multivalent interactions have also been noted by others for RGD peptides. Although Zha et al. reported imaging probes with a multivalent ligand targeting amyloid aggregates, no enhancement of the binding affinity for amyloid aggregates using a multivalent ligand was observed.22 We have previously reported that hydroxamamide (Ham) is a thiol-free chelating agent that generates 99mTc-labeled compounds with high stability and high radiochemical yield.23,24 Since Ham is a bidentate ligand, it provides a 99m Tc-Ham complex with a metal-to-ligand ratio of 1:2, indicating that a 99mTc-labeled compound with two kinds of Ham ligand can form.25 Such properties suggest that Ham would be useful in the synthesis of a 99mTc-labeled compound with a bivalent amyloid ligand. In the present study, we designed new 99mTc-Ham complexes with a bivalent amyloid ligand in order to increase the binding affinity for amyloid aggregates. We selected stilbene (SB) and benzothiazole (BT) derivatives as amyloid ligands based on the structure of AV-1 and PIB, respectively, and synthesized 99mTcSB1, 99mTc-SB2, 99mTc-BT1, and 99mTc-BT2 (Figure 1). To verify whether the binding affinity for amyloid aggregates can be enhanced by using bivalent 99mTc complexes, we systematically evaluated their binding affinity using Aβ(1−42)

Figure 1. Proposed structure of the

99m

aggregates as a model of amyloid aggregates. We also designed and synthesized 99mTc-DAB with bivalent Ham-based dimethylaminobenzene and evaluated its binding affinity for amyloid aggregates to compare the affinity for amyloid aggregates with 99m Tc-SB1, 99mTc-SB2, 99mTc-BT1, and 99mTc-BT2.

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MATERIALS AND METHODS General. All reagents were obtained commercially and used without further purification unless otherwise indicated. 1H NMR and 13C NMR spectra were recorded on a JNM-ECS400 (JEOL, Tokyo, Japan) with tetramethylsilane (TMS) as an internal standard. Coupling constants are reported in Hertz. Multiplicity was defined as singlet (s), doublet (d), triplet (t), or multiplet (m). High-resolution mass spectrometry (HRMS) was conducted with a JMS-GC mate mass spectrometer (JEOL). High-performance liquid chromatography (HPLC) was performed with a Shimadzu system (SHIMADZU, Kyoto, Japan, a LC-20AT pump with a SPD-20A UV detector, λ = 254 nm) with a Cosmosil C18 column (Nacalai Tesque, Kyoto, Japan, 5C18-AR-II, 4.6 mm × 150 mm) using a mobile phase (10 mM phosphate buffer (pH 7.4)/acetonitrile: 0 min 3/2 to 30 min 3/7) delivered at a flow rate of 1.0 mL/min. Materials. 2-Bromobenzo[d]thiazole-6-carbonitrile was purchased from Ark Pharm (Illinois, USA). Acetonitrile, triethylamine, 4-(dimethylamino)benzaldehyde, tetrakis(triphenylphosphine)palladium, sodium carbonate, dioxane, and acetate were purchased from Nacalai Tesque. Tin(II) tartrate hydrate was purchased from Strem Chemicals (Massachusetts, USA). Diethyl (4-cyanobenzyl)phosphonate, hydroxylamine hydrochloride, sodium methoxide, and N,Ndimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline were purchased from Tokyo Chemical Industry (Tokyo, J a p a n ) . N, N - D i m e t h y l f o r m a m i d e ( D M F ) an d 4 (dimethylamino)benzonitrile were purchased from Wako (Osaka, Japan). Chemistry. Synthesis of Diethyl (Z)-(4-(N′Hydroxycarbamimidoyl)benzyl)phosphonate (1). To a solution of diethyl (4-cyanobenzyl)phosphonate (759 mg, 3.00 mmol) in ethanol (20 mL) were added hydroxylamine hydrochloride (625 mg, 9.00 mmol) and triethylamine (1.25 mL, 9.00 mmol). The reaction mixture was heated to reflux for 2 h. After evaporation of the solvent, water was added. The reaction mixture was extracted with chloroform. The organic layers were combined and dried over Mg2SO4 and filtered. Evaporation of the filtrate gave a residue, which was purified by silica gel chromatography (chloroform/methanol = 10:1) to give 790 mg of 1 (92% yield). Rf = 0.37 (chloroform/methanol = 10:1). 1H NMR (400 MHz, DMSO-d6) δ 1.17 (t, J = 7.0 Hz, 6H), 3.24 (d, J = 21.8 Hz, 2H), 3.91−3.98 (m, 4H), 5.76 (s, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 9.58 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 16.2 (2C), 32.0, 61.4 (2C), 125.3 (2C), 129.5 (2C), 131.6, 133.1, 150.7. HRMS (EI) m/z calcd for C12H19N2O4P (M+) 286.1082, found 286.1086. Synthesis of (Z)-4-((E)-4-(Dimethylamino)styryl)-N′-hydroxybenzimidamide (2). To a solution of 1 (100 mg, 0.350 mmol) and 4-(dimethylamino)benzaldehyde (52 mg, 0.350 mmol) in DMF (10 mL) was slowly added sodium methoxide (5 M in MeOH, 0.14 mL, 0.700 mmol). The reaction mixture was stirred at room temperature for 3 h. The precipitate was filtered and washed with water, and the solid was purified by silica gel chromatography (chloroform/methanol = 10:1) to give 30 mg of 2 (31% yield). Rf = 0.44 (chloroform/methanol = 10:1). 1H NMR (400 MHz, DMSO-d6) δ 2.94 (s, 6H), 5.77 (s,

Tc-Ham complexes. 1133

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Scheme 1. Synthetic Route for Ham Compounds

(2C), 120.8, 126.1 (2C), 150.7, 151.0. HRMS (EI) m/z calcd for C9H13N3O (M+) 179.1058, found 179.1061. 99m Tc Labeling Reaction. To solutions of 2, 4, and 5 (0.2 mg) in acetate/ethanol (1:4, 200 μL) were added 100 μL of Na99mTcO4 solution and 15 μL of tin(II) tartrate hydrate solution [2 mg of tin(II) tartrate hydrate (7.50 μmol) dissolved in water (2.5 mL)]. The reaction mixtures were incubated at room temperature for 30 min and purified by reversed-phase HPLC (RP-HPLC). The 99mTc-Ham complexes were analyzed by analytical RP-HPLC on a Cosmosil C18 column (5C18-AR-II, 4.6 mm × 150 mm) with a solvent of phosphate buffer (10 mM, pH 7.4)/acetonitrile (0 min 3/2 to 30 min 3/7) as the mobile phase at a flow rate of 1.0 mL/min. The absorption of the complexes was measured at 254 nm, and the radioactivity of the 99mTc-Ham complexes was recorded for 30 min (Figure 2). Binding Assay Using Aβ Aggregates in Solution. A solid form of Aβ(1−42) was purchased from the Peptide Institute (Osaka, Japan). Aggregation was carried out by gently dissolving the peptide (0.25 mg/mL) in phosphate-buffered saline (PBS) (pH 7.4). The solution was incubated at 37 °C for 42 h with gentle and constant shaking. The binding assay was performed by mixing 50 μL of Aβ(1−42) aggregates (final conc., 1.25 μg/mL), 50 μL of 99mTc-Ham complex (final conc., 8.3 kBq/mL), and 900 μL of 30% EtOH. After incubation at room temperature for 3 h, the mixture was filtered through Whatman GF/B filters (Whatman, Kent, U.K.) using a Brandel M-24 cell harvester (NEUROSCIENCE, Tokyo, Japan), and the radioactivity of the filters containing the bound 99mTc-Ham complex was measured using a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, USA) (Figure 3). Inhibition Assay Using Aβ Aggregates in Solution. A mixture containing 50 μL of Aβ(1−42) aggregates (final conc., 1.25 μg/mL), 50 μL of 99mTc-Ham complex (final conc., 8.3 kBq/mL), 50 μL of PIB (final conc., 64 pM−125 μM in 30% EtOH), and 850 μL of 30% EtOH was incubated at room temperature for 3 h. The mixture was filtered through Whatman GF/B filters using a Brandel M-24 cell harvester, and the radioactivity of the filters containing the bound 99mTcHam complex was measured using a γ counter. Values for the half-maximal inhibitory concentration (IC50) were determined

2H), 6.73 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 16.2 Hz, 1H), 7.17 (d, J = 16.2 Hz, 1H), 7.44 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 9.60 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 39.9 (2C), 112.2 (2C), 122.9, 124.8, 125.4 (2C), 125.5 (2C), 127.6 (2C), 129.2, 131.2, 138.4, 150.0, 150.6. HRMS (EI) m/z calcd for C17H19N3O (M+) 281.1528, found 281.1532. Synthesis of 2-(4-(Dimethylamino)phenyl)benzo[d]thiazole-6-carbonitrile (3). A solution of 2-bromobenzo[d]thiazole-6-carbonitrile (1165 mg, 5.00 mmol), N,N-dimethyl-4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1483 mg, 6.00 mmol), and Pd(PPh3)4 (577 mg, 0.500 mmol) in 2 M Na2CO3 (aq)/dioxane (1:1, 40 mL) was stirred for 2 h under reflux. The reaction mixture was allowed to cool to room temperature, and ethyl acetate (150 mL) and water (150 mL) were added. The precipitate was filtered to give 1061 mg of 3 (76% yield). Rf = 0.36 (ethyl acetate/hexane = 1:4). 1H NMR (400 MHz, DMSO-d6) δ 3.05 (s, 6H), 6.84 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 1H), 8.64 (s, 1H). 13C NMR (100 MHz, CD2Cl2-d2) δ 40.3 (2C), 107.5, 112.0 (2C), 119.4, 120.6, 122.8, 126.4, 129.6 (2C), 129.8, 135.4, 153.3, 157.4, 173.1. HRMS (EI) m/z calcd for C16H13N3S (M+) 279.0830, found 279.0827. Synthesis of (Z)-2-(4-(Dimethylamino)phenyl)-N′hydroxybenzo[d]thiazole-6-carboximidamide (4). The same reaction as described above to prepare 1 was employed, and 4 was obtained in 58% yield from 3. Rf = 0.23 (chloroform/ methanol = 10:1). 1H NMR (400 MHz, DMSO-d6) δ 3.03 (s, 6H), 5.89 (s, 2H), 6.83 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 8.29 (s, 1H), 9.71 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 39.7 (2C), 111.8 (2C), 118.7, 120.0, 121.2, 123.9, 128.5 (2C), 129.6, 133.7, 150.5, 152.3, 154.2, 168.5. HRMS (EI) m/z calcd for C16H16N4OS (M+) 312.1045, found 312.1043. Synthesis of (Z)-4-(Dimethylamino)-N′-hydroxybenzimidamide (5). The same reaction as described above to prepare 1 was employed, and 5 was obtained in 34% yield from 4(dimethylamino)benzonitrile. Rf = 0.14 (ethyl acetate/hexane = 1:1). 1H NMR (400 MHz, DMSO-d6) δ 2.91 (s, 6H), 5.58 (s, 2H), 6.67 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 9.25 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 39.9 (2C), 111.4 1134

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from displacement curves using GraphPad Prism 5.0 (Figure 4 and Table 1). In Vitro Autoradiography of Tg2576 Mouse Brain Sections. The experiments with animals were conducted in accordance with our institutional guidelines and approved by Kyoto University Animal Care Committee. A Tg2576 transgenic mouse (female, 28 months old) and a wild-type mouse (female, 28 months old) were used as an Alzheimer’s model and an age-matched control, respectively. After the animal was sacrificed by decapitation, the brain was removed and sliced into serial sections 10 μm thick. Each slide was incubated with a 50% EtOH solution of 99mTc-Ham complex (370 kBq/mL) at room temperature for 1 h. For blocking experiments, adjacent sections were incubated with a 50% EtOH solution of 99mTcHam complex (370 kBq/mL) in the presence of nonradioactive PIB (500 μM). The sections were washed in 50% EtOH for 1.5 min two times and exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan) for 6 h. Autoradiographic images were obtained using a BAS5000 scanner system (Fuji Film) (Figure 5A, B, D− F, H−J, L−N, P, and Q). After autoradiographic examination, the same sections were stained by thioflavin-S to confirm the presence of Aβ plaques. For thioflavin-S staining, the sections were immersed in a 100 μM thioflavin-S solution containing 50% EtOH for 3 min, washed in 50% EtOH for 1 min two times, and examined using a microscope (BIOREVO BZ-9000; Keyence Corp., Osaka, Japan) equipped with a GFP-BP filter set (Figure 5C, G, K, O, and R).

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RESULTS AND DISCUSSION Chemistry. The synthesis of the Ham compounds is outlined in Scheme 1. A Ham group was introduced into diethyl (4-cyanobenzyl)phosphonate, 3, and 4(dimethylamino)benzonitrile by reacting them with hydroxylamine according to a previously reported method,24 which afforded 1, 4, and 5 in yields of 92, 58, and 34%, respectively. The SB derivative 2 was synthesized by the Horner-Wadsworth-Emmons reaction of the phosphonate 1 with 4(dimethylamino)benzaldehyde in 31% yield.26 The phenyl BT backbone was formed by the Suzuki coupling reaction between 2-bromobenzo[d]thiazole-6-carbonitrile and N,N-dimethyl-4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, which afforded 3 in 76% yield.27,28 99m Tc Labeling Reaction. The 99mTc labeling reaction was performed by the complexation reaction using the Ham precursor, 99mTc-pertechnetate, and tin(II) tartrate hydrate as a reducing agent. Figure 2 shows the RP-HPLC radiochromatograms of 99mTc-SB1, 99mTc-SB2, 99mTc-BT1, 99mTc-BT2, and 99m Tc-DAB analyzed under similar conditions. 99mTc-SB2, 99m Tc-BT2, and 99mTc-DAB were prepared from the precursors 2, 4, and 5, respectively. When the resulting mixture was analyzed by RP-HPLC, two radioactive complexes formed as reported in previous papers on 99mTc-Ham complexes, which suggested that two isomers were generated in the 99mTc complexation reaction with Ham.25,29 Hereafter, the specific isomers with shorter retention times on RP-HPLC are defined as A-form (99mTc-SB1A, 99mTc-SB2A, 99mTc-BT1A, 99mTcBT2A, and 99mTc-DABA), and the others as B-form (99mTcSB1B, 99mTc-SB2B, 99mTc-BT1B, 99mTc-BT2B, and 99mTcDABB). Characterization of these 99mTc-Ham complexes has been limited because synthesis using 99Tc is very challenging and no corresponding rhenium complexes, surrogate complexes of

Figure 2. Radiochromatograms of 99mTc-Ham complexes with SB (A) and BT (B) derivatives.

technetium, have been obtained so far. However, a recent paper reported the 99Tc complexation reaction of N-substituted Ham, and the reaction product was characterized by X-ray diffraction, nuclear magnetic resonance, and infrared spectroscopy.29 This report showed that a single 99Tc-Ham complex consisted of two N-substituted Ham ligands at the trans-position forming a square-pyramid. According to this previous report,29 we presume that the two isomers generated in the 99mTc complexation reactions with Ham are 99mTc complexes with two Ham ligands at the cis- or trans-position. The complexation reaction of 99mTc with 2 and 5 provided six radioactivity peaks, which were suggested to derive from 99m Tc-SB1, 99mTc-SB2, and 99mTc-DAB. The radioactivity peaks at retention times of 3.0 and 3.5, and 15.3 and 18.9 min corresponded with those of 99mTc-DAB and 99mTc-SB2, respectively. Therefore, two peaks at 7.7 and 10.4 min were inferred to represent the generation of 99mTc-SB1. Similarly, the complexation reaction of 99mTc with 4 and 5 gave six radioactivity peaks, which proposed that 99mTc-BT1, 99mTcBT2, and 99mTc-DAB were synthesized simultaneously. It was suggested that the pairs of peaks at retention times of 3.1 and 3.6, 5.8 and 8.1, and 11.5 and 14.3 min derived from 99mTcDAB, 99mTc-BT1, and 99mTc-BT2, respectively. 1135

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Binding Assay Using Aβ Aggregates in Solution. We utilized binding experiments in solution to determine whether a series of 99mTc-Ham complexes can bind Aβ(1−42) aggregates. In this assay, we evaluated the binding affinity for amyloid aggregates of only the A-form of 99mTc-Ham complexes. Figure 3 shows the binding affinity of 99mTc-Ham complexes as

Figure 4. Displacement curves of A-form (A) and B-form (B) of 99m Tc-Ham complexes from the inhibition assay for binding PIB to Aβ(1−42) aggregates. Values are the mean ± standard error of six independent experiments.

Figure 3. Binding assay of 99mTc-Ham complexes with SB (A) and BT (B) derivatives using Aβ(1−42) aggregates. Values are the mean ± standard error of three independent experiments.

Table 1. Half-Maximal Inhibitory Concentration (IC50, μM) for the Binding of PIB to Aβ(1−42) Aggregates Determined Using 99mTc-Ham Complexes as Ligands

specific Aβ(1−42) aggregate-bound radioactivity (%) at a fixed concentration of Aβ(1−42) aggregates. The percent radioactivity of 99mTc-SB1A and 99mTc-SB2A bound to aggregates displayed moderate affinity at 22.2 and 42.6% for Aβ(1−42) aggregates, respectively, while 99mTc-DABA, which does not include any amyloid ligands, showed no marked affinity (0.4%) (Figure 3A). Similarly, 99mTc-BT1A and 99mTc-BT2A exhibited affinity for Aβ(1−42) aggregates at 4.6 and 38.7%, respectively, while no marked binding to amyloid aggregates was observed for 99mTc-DABA (0.4%) (Figure 3B). In terms of Aβ(1−42) aggregate-bound radioactivity, the 99mTc-Ham complexes ranked in the following order: 99mTc-SB2A > 99mTc-SB1A > 99m Tc-DABA and 99mTc-BT2A > 99mTc-BT1A > 99mTc-DABA. The result indicated that the multivalent interactions induced by using bivalent 99mTc complexes could enhance the binding affinity for amyloid aggregates. Inhibition Assay Using Aβ Aggregates in Solution. To further evaluate the quantitative binding affinity for Aβ(1−42) aggregates, we carried out an inhibition binding assay with PIB as a competitive ligand (Figure 4). We evaluated the binding affinity for Aβ(1−42) aggregates of both isomers of 99mTc-Ham complexes (A-form and B-form). A fixed concentration of Aβ(1−42) aggregates and the 99mTc-Ham complex were incubated with increasing concentrations of nonradioactive PIB. As shown in Figure 4, PIB competed well with the binding of eight 99mTc-Ham complexes to amyloid aggregates. PIB showed IC50 values of 0.72, 0.38, 16.40, 2.55, 0.26, 0.47, 2.80, and 5.78 μM in the presence of 99mTc-SB1A, 99mTc-SB1B, 99m Tc-SB2A, 99mTc-SB2B, 99mTc-BT1A, 99mTc-BT1B, 99mTcBT2A, and 99mTc-BT2B, respectively (Table 1). 99mTc-DAB, which did not bind to amyloid aggregates (Figure 3), showed constant radioactivity regardless of the concentration of PIB. Therefore, its IC50 value was not able to be calculated. The result of this assay was consistent with that of the binding assay described in Figure 3, indicating that the amyloid ligand dimers (99mTc-SB2 and 99mTc-BT2) had much higher binding affinity

compd 99m

Tc-SB1A Tc-SB1B 99m Tc-SB2A 99m Tc-SB2B 99m Tc-BT1A 99m Tc-BT1B 99m Tc-BT2A 99m Tc-BT2B 99m

IC50 (μM)a 0.72 0.38 16.40 2.55 0.26 0.47 2.80 5.78

± ± ± ± ± ± ± ±

0.10 0.08 2.47 0.45 0.02 0.05 0.32 0.53

Values are the mean ± standard error of the mean of six independent experiments.

a

for Aβ(1−42) aggregates than their monomers (99mTc-SB1 and Tc-BT1). Additionally, the A-form of 99mTc-Ham complexes with SB derivatives showed higher binding affinity than B-form, while the A-form of 99mTc-Ham complexes with BT derivatives showed lower binding affinity than B-form. Thus, significant differences between IC50 values with two isomers were displayed, suggesting that geometric isomerism affected the binding affinity for aggregated amyloid peptides. Moreover, the results indicated that the 99mTc-Ham complexes bound to a similar binding site to PIB. We also synthesized 99mTc-Ham complexes composed of both SB and BT (99mTc-SBT) (Figures S1 and S2, Supporting Information). The inhibition assay was performed, and PIB showed IC50 values of 9.91 and 2.86 μM in the presence of 99m Tc-SBTA and 99mTc-SBTB, respectively (Table S1, Supporting Information). For both isomers (A-form and B-form), 99m Tc-SBT had the binding affinity for Aβ(1−42) aggregates between those of 99mTc-SB2 and 99mTc-BT2. In Vitro Autoradiography of Tg2576 Mouse Brain Sections. To confirm the affinity of the 99mTc-Ham complexes for Aβ plaques in the mouse brain, in vitro autoradiography was 99m

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Figure 5. In vitro autoradiograms of wild-type (A, E, I, and M) and Tg2576 (B, F, J, N, and Q) mouse brain sections labeled with 99mTc-Ham complexes. The same brain sections were stained with thioflavin-S (C, G, K, O, and R). Blocking studies with nonradioactive PIB were also performed using adjacent brain sections (D, H, L, and P).

performed by incubating 99mTc-Ham complexes with brain sections from Tg2576 and wild-type mice (Figure 5). Tg2576 mice are specifically engineered to overproduce Aβ plaques in the brain. Therefore, they have been frequently used to evaluate the specific binding of Aβ plaques in experiments in vitro and in vivo.11,28,30 Autoradiography of only the specific isomer of 99m Tc-Ham complex with higher binding affinity in the inhibition assay was carried out. As shown in Figure 5A, E, I, and M, no spots of radioactivity were observed in the wild-type mouse brain sections. In the Tg2576 mouse brain sections, 99m Tc-SB1A, 99mTc-SB2A, and 99mTc-BT2B intensively labeled Aβ plaques showing a strong signal in the cortex region and a low background level in the white matter (Figure 5B, F, and N, respectively). 99mTc-BT1B displayed moderate binding and labeled only a few plaques (Figure 5J). Conversely, 99mTcDABA showed no marked binding to Aβ plaques (Figure 5Q). Furthermore, the labeling pattern was partially consistent with the staining pattern observed in the same sections with thioflavin-S, a dye commonly used to stain Aβ plaques (Figure 5C, G, K, and O). The results of in vitro autoradiography well reflected those of in vitro binding assays. Moreover, the labeling of Aβ plaques with 99mTc-SB1A, 99mTc-SB2A, 99mTc-BT1B, and 99m Tc-BT2B was blocked to a large extent with an excess of nonradioactive PIB, confirming the specific binding of 99mTcHam complexes to Aβ plaques in the mouse brain (Figure 5D, H, L, and P, respectively). Additionally, when in vitro autoradiography of Tg2576 and wild-type mice brain sections with 99mTc-SBTA was carried out, it intensely labeled Aβ plaques similarly to the other 99mTc-Ham complexes (Figure S3, Supporting Information).

As for multivalent interactions, several mechanisms can explain the observed binding enhancements. Generally, the highest binding enhancement is observed when the multivalent ligand extends over adjacent binding sites.31 It is considered that simultaneous binding of multivalent ligands is important for large enhancement of binding affinity. However, significant binding enhancements are also observed in cases where the distance between ligands is too short to extend over adjacent binding sites. In these cases, one assumed mechanism is rebinding of the ligand before complete dissociation of the complex due to high local concentration of the ligand.31 Significant binding enhancement using the bivalent 99mTc-Ham complexes may be achieved by one or some of these events. To evaluate the brain uptake of the 99mTc-Ham complex, a biodistribution experiment of 99mTc-SB2A with the highest binding affinity in the inhibition assay was performed in normal mice (Table S2, Supporting Information). It displayed very low brain uptake (0.28% ID/g) at 2 min postinjection. Additionally, we reported a 99mTc-Ham complex that was uncharged and did not have very high molecular weight (presumed MW: 384); however, it showed very low brain uptake (0.10% ID) at 5 min postinjection.24 Thus, 99mTc-Ham complexes are considered hardly to cross the blood-brain barrier. Although these compounds are not suitable to diagnose AD, they may be used as imaging agents for amyloid aggregates deposited in CAA and peripheral amyloidoses. Moreover, a 99mTc-Ham complex with very low brain uptake may be useful for the development of CAA-specific imaging agents to differentiate CAA from AD because they cannot bind to Aβ plaques in cortex regions of AD brains. 1137

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CONCLUSIONS We successfully designed and synthesized novel 99mTc-Ham complexes with a bivalent amyloid ligand and evaluated their binding affinity for amyloid aggregates. In vitro inhibition assay indicated that enhancement of the binding affinity by application of bivalent 99mTc complexes is feasible. It was also verified by in vitro autoradiography, which showed specific binding of 99mTc-Ham complexes to Aβ plaques in the mouse brain, as reflected by the strength of the affinity for amyloid aggregates in vitro. Taken together, the findings in the present study strongly suggest that a new molecular design based on bivalent 99mTc-Ham complexes may be reasonable to develop an imaging probe targeting amyloid aggregates. The new strategy in this study to enhance the binding affinity for amyloid aggregates will provide useful insight into the molecular design for imaging various amyloidoses in the future.

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ASSOCIATED CONTENT

S Supporting Information *

Procedures and results of the preparation, the inhibition assays using Aβ(1−42) aggregates, and in vitro autoradiography of Tg2576 and wild-type mouse brain sections with 99mTc-SBTA and 99mTc-SBTB, and the biodistribution experiment with 99m Tc-SB2A in normal mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.O.) Phone: +81-75-753-4608. Fax: +81-75-753-4568. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (NEXT Program),” initiated by the Council for Science and Technology Policy (CSTP).

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