Novel Bivalent 99mTc-Complex with N-Methyl-Substituted Hydroxamamide as Probe for Imaging of Cerebral Amyloid Angiopathy

Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid aggregates in the walls of the cerebral vasculature. Recently, the development of molecular imaging probes targeting CAA has been attracting much attention. We previously reported the 99mTc-hydroxamamide (99mTc-Ham) complex with a bivalent benzothiazole scaffold as a binding moiety for amyloid aggregates ([99mTc]BT2) and its utility for CAA-specific imaging. However, the simultaneous generation of two radiolabeled complexes derived from the geometric isomers was observed in the 99mTc-labeling reaction. It was recently reported that the complexation reaction of 99Tc with N-methyl-substituted Ham provided a single 99Tc-Ham complex consisting of two N-methylated Ham ligands with marked stability. In this article, we designed and synthesized a novel N-methylated bivalent 99mTc-Ham complex ([99mTc]MBT2) and evaluated its utility for CAA-specific imaging. N-Methyl substitution of [99mTc]BT2 prevented the generation of its isomer in the 99mTc-labeling reaction. Enhanced in vitro stability of [99mTc]MBT2 as compared with [99mTc]BT2 was observed. [99mTc]MBT2 showed very low brain uptake, which is favorable for CAA-specific imaging. An in vitro inhibition assay using β-amyloid aggregates and in vitro autoradiographic examination of brain sections from a Tg2576 mouse and a CAA patient showed a decline in the binding affinity for amyloid aggregates due to N-methylation of the 99mTc-Ham complex. These results suggest that the scaffold of the 99mTc-Ham complex may play important roles in the in vitro stability and the binding affinity for amyloid aggregates.


Introduction
Cerebral amyloid angiopathy (CAA) is defined as the deposition of amyloid aggregates, most commonly β-amyloid peptide (Aβ), in the media and adventitia of arteries and, less often, capillaries of the brain [1][2][3]. CAA is a sporadic or familial disorder and is common in the elderly brain, with an age-related prevalence and significant increase with age [4]. CAA is present in nearly all Alzheimer's disease (AD) brains [4], and severe CAA is present in approximately 25% of AD brains [5], while fewer than 50% of CAA cases meet the pathologic criteria for AD [6]. CAA can cause fatal intracerebral hemorrhage (ICH) and vascular cognitive impairment [3,7,8], and is also associated with small vessel diseases such as white matter hyperintensity and cerebral microbleeds [3,9,10].
A definitive CAA diagnosis can be performed based on only histological findings of brain tissue, obtained by autopsy or brain biopsy [11]. Although computed tomography (CT) and magnetic resonance imaging (MRI) without any probe have commonly been used as noninvasive and useful modalities to diagnose CAA-associated ICH [12,13], they detect intracerebral bleeding as a surrogate marker of CAA. These indirect diagnostic techniques cannot shed light on the etiology of the sign, making it unlikely to perform disease-specific diagnoses. In contrast, positron emission tomography (PET) and single photon emission computed tomography (SPECT) can provide information on the localization of amyloid aggregates, while CT and MRI provide the anatomical information. Therefore, PET and SPECT have been utilized as major in vivo imaging techniques to carry out the noninvasive diagnosis of amyloidoses. Over the past few decades, research on imaging of Aβ aggregates constituting senile plaque in AD brains using PET and SPECT tracers has made marked progress [14][15][16][17][18][19].
Several groups have reported on the detection of cerebrovascular amyloid depositions using [ 11 C]PIB, which is the most commonly used Aβ-imaging probe [20][21][22]. However, due to highlevel efficiency with the penetration of the blood-brain barrier (BBB), [ 11 C]PIB is believed to visualize amyloid depositions in the whole brain, indicating that it detects senile plaque as a background signal in cases of CAA diagnosis.
With the aim of CAA-specific imaging, some groups, in addition to our group, have reported PET and SPECT imaging probes targeting CAA [23][24][25][26][27]. We previously reported a series of 99m Tc-hydroxamamide ( 99m Tc-Ham) complexes with a bivalent amyloid ligand as imaging probes targeting CAA [28]. The bivalent 99m Tc-Ham complex, [ 99m Tc]BT2 including two benzothiazole scaffolds as the binding moiety, showed the specific detection of CAA on ex vivo autoradiographic examination. However, the radiolabeling reaction of [ 99m Tc]BT2 caused the simultaneous generation of two geometric isomers that have different biological features, including binding affinity for Aβ aggregates. Thipyapong et al. recently reported that the 99 Tc complexation reaction of N-methyl-substituted Ham rendered a single 99 Tc-Ham complex consisting of two N-methylated Ham ligands with marked stability [29]. On the basis of this previous report, we applied the concept to the novel design of 99m Tc-Ham complexes.
We herein designed and synthesized N-methyl-substituted [ 99m Tc]BT2 ([ 99m Tc]MBT2), and characterized its potential for the development of a novel imaging probe targeting CAA (Fig 1).

General
All reagents were obtained commercially and used without further purification unless otherwise indicated. PIB was purchased from ABX (Saxony, Germany). Na 99m TcO 4 was purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan) or obtained from a commercial 99 Mo/ 99m Tc generator (Ultra-Techne Kow; FUJIFILM RI Pharma Co., Ltd., Tokyo, Japan).

Animals
Animal experiments were conducted in accordance with our institutional guidelines and were approved by the Kyoto University Animal Care Committee. Male ddY mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Female Tg2576 mice and wild-type mice were purchased from Taconic Farms, Inc. (New York, USA). Animals were fed standard chow and had free access to water. All efforts were made to minimize suffering.

Human brain tissues
Experiments involving human subjects were performed in accordance with the relevant guidelines and regulations and were approved by the ethics committee of Kyoto University. Informed consent was obtained from all subjects in this study. Postmortem brain tissues from autopsyconfirmed cases of CAA (female, 67 years old) were obtained from the Graduate School of Medicine, Kyoto University.

Assessment of the BBB permeability
A saline solution (100 μL) of the 99m Tc-Ham complex (20 kBq) containing EtOH (10 μL) was injected directly into the tail vein of ddY mice (male, 5 weeks old). The mice were sacrificed at 2, 10, 30, and 60 min postinjection. The brain was removed and weighed, and radioactivity was measured using a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, USA). The % injected dose (ID)/g of samples was calculated by comparing the sample counts with the count of the diluted initial dose. In vitro autoradiography of Tg2576 mouse brain sections A Tg2576 transgenic mouse (female, 28 months old) and a wild-type mouse (female, 28 months old) were used as an AD model and an age-matched control, respectively. After each animal had been sacrificed by decapitation, the brain was immediately removed, embedded in Super Cryoembedding Medium (SCEM) compound (SECTION-LAB Co., Ltd., Hiroshima, Japan), and then frozen in a dry ice/hexane bath. Frozen sections were prepared at a 10-μm thickness. Each slide was incubated with a 50% EtOH solution of the 99m Tc-Ham complex (370 kBq/mL) at room temperature for 1 h. For blocking experiments, adjacent sections were incubated with a 50% EtOH solution of the 99m Tc-Ham 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). 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.

In vitro autoradiography of human CAA brain sections
Six-micrometer-thick serial human brain sections of paraffin-embedded blocks were used for autoradiography. To completely deparaffinize the sections, they were incubated in xylene for 30 min two times and in 100% EtOH for 1 min two times. Subsequently, they were subjected to 1-min incubation in 90% EtOH and 1-min incubation in 70% EtOH, followed by a 5-min wash in water. Each slide was incubated with a 50% EtOH solution of the 99m Tc-Ham complex (370 kBq/mL) at room temperature for 1 h. The sections were washed in 50% EtOH for 3 min two times and exposed to a BAS imaging plate (Fuji Film) for 2 h. Autoradiographic images were obtained using a BAS5000 scanner system (Fuji Film). After autoradiographic examination, the adjacent section was immunostained by an antibody against Aβ(1-40) to confirm the presence of Aβ depositions. For immunohistochemical staining of Aβ(1-40), the section was autoclaved for 15 min in 0.01 M citric acid buffer (pH 6.0) to activate the antigen. After three 5-min incubations in PBS-Tween 20 (PBST), it was incubated with anti-Aβ(1-40) primary antibody (BA27; Wako, Osaka, Japan) at room temperature overnight. Subsequently, it was incubated in PBST for 5 min three times, and incubated with biotinylated goat anti-mouse IgG (Wako) at room temperature for 3 h. After three 5-min incubations in PBST, the section was incubated with Streptavidin-Peroxidase complex at room temperature for 30 min. After three 5-min incubations in PBST, it was incubated with diaminobenzidine (Merck, Hesse, Germany) as a chromogen for 5 min. After washing with water, the section was observed under a microscope (BIOREVO BZ-9000).

Results and Discussion Chemistry
The precursor for [ 99m Tc]MBT2 (MBTHam, 5) was prepared in four steps from the precursor for [ 99m Tc]BT2 (BTHam, 1) according to a previous report (Fig 2) [31]. BTHam was prepared according to our previous report [30]. Reaction of BTHam with ethyl chlorocarbonate provided compound 2, which was cyclized to compound 3 in alkaline solution. After methylation with iodomethane to give compound 4, it was decyclized by heating in alkaline solution to MBTHam.

Radiolabeling
The 99m Tc labeling reaction was performed by the complexation reaction using the Ham precursor, 99m Tc-pertechnetate, and tin(II) tartrate hydrate as a reducing agent (Fig 3)[30]. The Tc complexation reaction with MBTHam rendered a single peak at the retention time of 13.6 min on RP-HPLC, while the reaction with BTHam provided two peaks at the retention times of 11.5 and 14.3 min (Fig 4). This suggests that N-methylation of the Ham ligand would be a useful strategy to obtain a single 99m Tc-Ham complex by preventing the isomerism, as described in a previous report [29]. We defined the specific isomer of [ 99m Tc]BT2 with a shorter retention time on RP-HPLC as [ 99m Tc]BT2A, and the other as [ 99m Tc]BT2B.

Stability in murine plasma
The in vitro stability of [ 99m Tc]MBT2 and [ 99m Tc]BT2 was evaluated by incubating them in murine plasma at 37°C for 0.5, 1, and 2 h (Fig 5 and Table 1). For [ 99m Tc]BT2, the radiochemical  Tc-Ham complex enhanced its stability, as demonstrated in a previous report [29].

Assessment of the BBB permeability
Biodistribution experiments were performed in order to assess the brain uptake of [ 99m Tc] MBT2 in normal mice (Table 2). To reduce the binding to Aβ aggregates in the brain cortex, CAA-specific imaging probes need not to show high-level penetration of the BBB [28,29].
[ 99m Tc]MBT2 displayed very low brain uptake (0.35%ID/g at 2 min postinjection), as well as [ 99m Tc]BT2B (0.37%ID/g at that time) [28], indicating that [ 99m Tc]MBT2 has a favorable property for CAA imaging. It was suggested that the methyl group introduced into the hydroxamamide moiety of [ 99m Tc]MBT2 may not contribute to its brain entry.

Inhibition assay using Aβ aggregates in solution
To evaluate the binding affinity for Aβ aggregates of [ 99m Tc]MBT2 and [ 99m Tc]BT2, we performed an inhibition binding assay with PIB as a competitive ligand. A fixed concentration of Aβ aggregates and the 99m Tc-Ham complex were incubated with increasing concentrations of nonradioactive PIB in solution. The binding affinity of 99m Tc-complexes was expressed as values for the half-maximal inhibitory concentration (IC 50 ) determined from displacement curves (Fig 6). When calculating the IC 50 values from the displacement curves, PIB showed IC 50 values of 0.56, 2.80, and 5.78 μM in the presence of [ 99m Tc]MBT2, [ 99m Tc]BT2A, and [ 99m Tc] BT2B, respectively (Table 3). A decrease in the binding affinity for Aβ aggregates due to Nmethylation of the 99m Tc-Ham complex was observed.

In vitro autoradiography of Tg2576 mouse brain sections
The binding of [ 99m Tc]MBT2 and [ 99m Tc]BT2 to Aβ plaques in brain sections from Tg2576 mice was evaluated by in vitro autoradiography. Since Tg2576 mice overproduce Aβ aggregates in the brain, they have been commonly used to evaluate the specific binding affinity of probes for Aβ aggregates in a variety of experiments in vitro and in vivo [19,28,30]. As shown in  7B, 7F and 7J). These radioactive spots were consistent with Aβ depositions confirmed by the fluorescent staining in the same sections with thioflavin-S, a dye commonly used to stain Aβ plaques (Fig 7C, 7G  The results of in vitro autoradiography well reflected those of the inhibition assay. Moreover, the labeling of Aβ plaques with 99m Tc-labeled compounds was blocked to a large extent with an excess of nonradioactive PIB, confirming the specific binding to Aβ plaques in the mouse brain (Fig 7D, 7H and 7L). A decline in the binding affinity for Aβ plaques by chemical modification of the 99m Tc-Ham complex was suggested in autoradiograms, as observed in the inhibition assay.
In vitro autoradiography of human CAA brain sections An in vitro autoradiographic examination of human CAA brain sections was carried out in order to confirm binding affinity for Aβ plaques deposited in the human brain (Fig 8). The distribution of Aβ deposits was confirmed by immunohistochemical staining of Aβ (Fig 8B), showing that [ 99m Tc]MBT2 moderately labeled Aβ deposits in a brain section from a CAA patient. However, as observed in the inhibition assay and autoradiographic examination using  the mouse brain sections, it was suggested that [ 99m Tc]MBT2 showed lower binding affinity as compared with that of the parent 99m Tc-Ham complex. These results suggest that the core of chelate in the 99m Tc-Ham complex may contribute to the binding affinity for aggregated amyloid peptides.

Conclusions
Herein, we designed and synthesized a novel N-methyl-substituted 99m Tc-Ham complex with a bivalent amyloid ligand and evaluated its fundamental utility as an imaging probe targeting CAA. In the 99m Tc complexation reaction, N-methyl substitution of the Ham ligand abolished the generation of the geometric isomer, forming a single 99m Tc-labeled compound. The in vitro stability in murine plasma of [ 99m Tc]MBT2 was higher than that of [ 99m Tc]BT2. An ex vivo biodistribution study showed that both [ 99m Tc]MBT2 and [ 99m Tc]BT2 could hardly penetrate the BBB in normal mice. An in vitro inhibition assay with Aβ aggregates and in vitro autoradiography using brain sections from a Tg2576 mouse and a CAA patient showed a decrease in the binding affinity for Aβ aggregates due to chemical modification of the 99m Tc-Ham complex. These results suggest that the scaffold of the 99m Tc-Ham complex may play crucial roles in the stability in murine plasma and binding affinity for amyloid aggregates. These findings also provide important information for the molecular design of novel imaging probes based on the 99m Tc-Ham complex targeting amyloid aggregates in the future.