Oligoethyleneoxy-Modified 99mTc-Labeled ß-Amyloid Imaging Probes with Improved Brain Pharmacokinetics for Single-Photon Emission Computed Tomography.

An oligoethyleneoxy linker was introduced for conjugation between 99mTc/Re-bis(aminoethanethiol) (BAT) and ß-amyloid (Aß) binding scaffolds. Rhenium complexes exhibited high to moderate binding affinity to Aß1-42 aggregates and efficient fluorescent staining to Aß plaques in brain tissue. After radiolabeling, the 99mTc-labeled probes revealed improved brain pharmacokinetics in normal ICR mice. Probe [99mTc]15 with potent binding affinity (Ki = 13.4 nM) and the highest initial brain uptake (2.10% ID/g at 2 min) in normal ICR mice was evaluated further. In vitro autoradiography showed specific labeling of Aß plaques by [99mTc]15 in transgenic (Tg) mouse brain tissue. Ex vivo autoradiography further demonstrated its efficient labeling of Aß plaques in a living Tg mouse. In vivo single photon emission computed tomography (SPECT)/CT imaging in six rhesus monkeys revealed remarkably improved brain uptakes (1.94-2.63% ID within 20 min) of [99mTc]15, making it highly potential to be used in humans for Aß plaques imaging in the brain.

In vivo near-infrared and Cerenkov luminescence imaging of amyloid-ß deposits in the brain: a fluorinated small molecule used for dual-modality imaging.

Three fluorinated (19F or 18F) small molecules were evaluated as fluorescent or radiolabeled probes for Aß deposits in the brain. In vivo near-infrared imaging suggested that probe 4c could efficiently distinguish between transgenic mice and wild-type controls. Furthermore, the in vivo Cerenkov luminescence imaging with [18F]4c showed modest differences between the two groups.

99mTc-Labeled 2-Arylbenzothiazoles: Aß Imaging Probes with Favorable Brain Pharmacokinetics for Single-Photon Emission Computed Tomography.

A series of 2-arylbenzothiazole derivatives conjugated with bis(aminoethanethiol) (BAT) chelating groups were designed and synthesized. A competitive binding assay-based screening was used to select seven rhenium complexes with potent binding affinity toward Aß1-42 aggregates (Ki < 50 nM) for 99mTc labeling and further evaluation. The 99mTc-labeled probes showed good affinity and specificity to Aß plaques in Tg mouse brain tissue in in vitro autoradiography studies. Moreover, [99mTc]14b exhibited favorable brain pharmacokinetics in normal mice (2.11% ID/g at 2 min and 0.62% ID/g at 60 min). Ex vivo autoradiography revealed extensive labeling of Aß plaques by [99mTc]14b in the brain of Tg mice. Furthermore, we performed the first single-photon emission computed tomography (SPECT) imaging study in nonhuman primates with 99mTc-labeled Aß probes. The semiquantitative data showed that [99mTc]14b penetrated the brains of rhesus monkeys. These results indicate that [99mTc]14b could be utilized as a SPECT imaging probe for Aß plaques.

2-Arylbenzothiazoles labeled with [CpRe/99mTc(CO)3] and evaluated as ß-amyloid imaging probes.

An array of complexes, 99mTc/Re-labeled cyclopentadienyl tricarbonyl and 2-phenyl/pyridylbenzothiazoles, conjugated through an ester linkage were tested as potential ß-amyloid (Aß) probes for SPECT imaging. The [CpRe/99mTc(CO)3] complexes were prepared by double ligand transfer reactions from ferrocene precursors, and the X-ray structure of one rhenium surrogate revealed a classical "piano stool" like geometry. Several of the rhenium complexes displayed high affinity for Aß1-42 aggregates in in vitro inhibition assays, and they could intensely stain Aß deposits on brain sections from transgenic mice and Alzheimer's disease (AD) patients. Complex [99mTc]4h strongly binds to Aß deposits in blood vessels of the brain section of AD patients in in vitro autoradiography. Biodistribution experiments in normal mice revealed that 99mTc-labeled tracers exhibited moderate degree of initial brain uptake (0.54%-1.06% ID/g at 2 min). These tracers targeting Aß plaques of AD patients warrant further structure optimization to improve their ability to penetrate blood-brain barrier, moreover, they may be suitable for developing imaging probes for targeting amyloid aggregates outside of the brain.

Optically Pure Diphenoxy Derivatives as More Flexible Probes for ß-Amyloid Plaques.

The highly rigid and planar scaffold with π-conjugated systems has been widely considered to be indispensable for Aß binding probes. However, the flexible benzyloxybenzene derivative [(125)I]BOB-4 represents an excellent lead candidate for targeting Aß in AD brains. Based on that, we designed two pairs of more flexible and optically pure diphenoxy derivatives with a chiral center as novel Aß probes. These compounds possessed high affinity (Ki = 15.8-45.0 nM) for Aß1-42 aggregates, and (R)-enantiomers showed slightly better binding ability than (S)-enantiomers. In addition, the competition binding assay implied that the optically pure diphenoxy derivatives with more flexible geometry shared the same binding site as IMPY, a classical rigid and planar Aß probe. For (125)I-radiolabeled enantiomers, (S)-[(125)I]5 and (R)-[(125)I]5, specific plaque labeling on brain sections of Tg mice and AD patients were observed in in vitro autoradiography, persuasively proving the excellent affinity of the probes. In biodistribution, (S)-[(125)I]5 and (R)-[(125)I]5 with relatively low lipophilicity exhibited moderate initial brain uptake (4.37% and 3.72% ID/g at 2 min, respectively) and extremely fast washout from normal mice brain (brain2min/brain60min = 19.0 and 17.7, respectively). In summary, the separate enantiomers displayed similar properties in vitro and in vivo, and (S/R)-[(123)I]5 may be potential SPECT probes for recognizing Aß plaques in AD brains.

(99m)Tc-Cyclopentadienyl Tricarbonyl Chelate-Labeled Compounds as Selective Sigma-2 Receptor Ligands for Tumor Imaging.

We have designed and synthesized a series of cyclopentadienyl tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ2 receptor ligands. Rhenium compound 20a possessed low nanomolar σ2 receptor affinity (K(i) = 2.97 nM) and moderate subtype selectivity (10-fold). Moreover, it showed high selectivity toward vesicular acetylcholine transporter (2374-fold), dopamine D2L receptor, NMDA receptor, opiate receptor, dopamine transporter, norepinephrine transporter, and serotonin transporter. Its corresponding radiotracer [(99m)Tc]20b showed high uptake in a time- and dose-dependent manner in DU145 prostate cells and C6 glioma cells. In addition, this tracer exhibited high tumor uptake (5.92% ID/g at 240 min) and high tumor/blood and tumor/muscle ratios (21 and 16 at 240 min, respectively) as well as specific binding to σ receptors in nude mice bearing C6 glioma xenografts. Small animal SPECT/CT imaging of [(99m)Tc]20b in the C6 glioma xenograft model demonstrated a clear visualization of the tumor at 180 min after injection.

Amyloid-ß Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

The formation of extracellular amyloid-ß (Aß) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aß plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aß plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aß aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aß plaques in the brain.

Preliminary evaluation of fluoro-pegylated benzyloxybenzenes for quantification of ß-amyloid plaques by positron emission tomography.

A new series of fluoro-pegylated benzyloxybenzenes were designed, synthesized and evaluated as PET probes for early detection of Aß plaques. Molecular docking revealed that all of the flexible benzyloxybenzenes inserted themselves into the hydrophobic Val18_Phe20 cleft on the flat spine of the Aß fiber, in a manner similar to that of IMPY molecule. The most potent probe, [(18)F]9a, exhibited a combination of high binding affinity to Aß aggregates (Ki = 21.0 ± 4.9 nM), high initial brain uptake (9.14% ID/g at 2 min), fast clearance from normal brain tissue (1.79% ID/g at 60 min), and satisfactory in vivo biostability in the brain (95% of intact form at 2 min). [(18)F]9a clearly labeled Aß plaques in in vitro autoradiography of postmortem AD patients and Tg mice brain sections. Ex vivo autoradiography further demonstrated that [(18)F]9a did penetrate the intact BBB and specifically bind to Aß plaques in vivo. Overall, [(18)F]9a may be a potential PET probe for imaging Aß plaques in AD brains.

Highly Sensitive Near-Infrared Fluorophores for in Vivo Detection of Amyloid-ß Plaques in Alzheimer's Disease.

Alzheimer's disease (AD) is pathologically characterized by the accumulation of ß-amyloid (Aß) deposits in the parenchymal and cortical brain. In this work, we designed, synthesized, and evaluated a series of near-infrared (NIR) probes with electron donor-acceptor end groups interacting through a π-conjugated system for the detection of Aß deposits in the brain. Among these probes, 3b and 3c had excellent fluorescent properties (emission maxima > 650 nm and high quantum yields) and displayed high sensitivity and high affinities to Aß aggregates (3b, Kd = 8.8 nM; 3c, Kd = 1.9 nM). Both 3b and 3c could readily penetrate the blood-brain barrier with high initial brain uptake and fast to moderate washout from the brain. In vivo NIR imaging revealed that 3b and 3c could efficiently differentiate transgenic and wild-type mice. In summary, our research provides new hints for developing smarter and more activatable NIR probes targeting Aß.

Preliminary Characterization and In Vivo Studies of Structurally Identical (18)F- and (125)I-Labeled Benzyloxybenzenes for PET/SPECT Imaging of ß-Amyloid Plaques.

With the assistance of molecular docking and 3D-QSAR models established previously, structurally identical (18)F- and (125)I-labeled benzyloxybenzene derivatives were designed to achieve the early detection of Aß plaques by PET/SPECT imaging. In competition binding assay, ligands 7a and 12a displayed high binding affinities to Aß42 aggregates with Ki values of 19.5 nM and 23.9 nM, respectively. Specific plaque labeling was observed on the in vitro autoradiography of brain sections from AD patients and Tg mice. In biodistribution, [(125)I]7a, [(18)F]7a, [(125)I]12a and [(18)F]12a all exhibited high initial brain uptakes (>5% ID/g at 2 min). [(125)I]7a and [(125)I]12a cleared fast from the normal brain regions, while corresponding [(18)F]7a and [(18)F]12a showed slow washout rates. Dynamic microPET/CT and microSPECT/CT imaging data in normal ICR mice were in accordance with in vivo biodistribution results. In vivo metabolism results indicated that the different clearance profiles between the structurally identical (18)F- and (125)I-labeled tracers could be attributed to different biochemical characteristics of the radiometabolites. Radioiodinated benzyloxybenzene derivatives exhibited good in vivo biostability in brain. Ex vivo autoradiography further confirmed the strong in vivo Aß labeling ability of [(125)I]7a. These new fluorinated and iodinated benzyloxybenzenes can develop into PET/SPECT dual imaging agents targeting Aß plaques.

99mTc(CO)3-Labeled Benzothiazole Derivatives Preferentially Bind Cerebrovascular Amyloid: Potential Use as Imaging Agents for Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a disorder affecting the elderly that is characterized by amyloid-ß (Aß) deposition in blood vessel walls of the brain. A series of 99mTc(CO)3-labeled benzothiazole derivatives as potential SPECT imaging probes for cerebrovascular Aß deposition is reported. Rhenium surrogate displayed high affinities to Aß aggregates with Ki values ranging from 106 to 42 nM, and they strongly stained Aß deposits in transgenic mice (Tg) and Alzheimer's disease (AD) patients. In vitro autoradiography on brain sections of Tg and AD patients confirmed that [99mTc]24 possessed sufficient affinity for Aß plaques, and [99mTc]24 could only label Aß deposition in blood vessels but not Aß plaques in the parenchyma of the brain of AD patients. Moreover, [99mTc]24 possessed favorable initial uptake (1.21% ID/g) and fast blood washout (blood2 min/blood60 min=23) in normal mice. These preliminary results suggest that [99mTc]24 may be used as an Aß imaging probe for the detection of CAA.

The synthesis and evaluation of near-infrared probes with barbituric acid acceptors for in vivo detection of amyloid plaques.

A new array of near-infrared probes containing barbituric acid acceptors has been developed as Aß imaging agents. These probes displayed long-emission wavelengths and large Stokes shifts, as well as high affinities for Aß aggregates. In vivo and ex vivo studies demonstrated that BBTOM-3 could intensely label Aß plaques in the brains of transgenic mice.

(18)F-Labeled 1,4-Dioxa-8-azaspiro[4.5]decane Derivative: Synthesis and Biological Evaluation of a σ1 Receptor Radioligand with Low Lipophilicity as Potent Tumor Imaging Agent.

We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ1 receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K(i) = 5.4 ± 0.4 nM) for σ1 receptors and selectivity for σ2 receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [(18)F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/µmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [(18)F]5a to σ1 receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ1 receptors could be used for tumor imaging.

2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, (99m)Tc) complexes as potential imaging probes for ß-amyloid plaques.

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates , , and were demonstrated to have moderate to high affinities for Aß1-42 aggregates with Ki values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aß plaques. Moreover, in in vitro autoradiography, (99m)Tc-labeled complexes clearly detected ß-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aß plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g(-1)), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these (99m)Tc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aß plaques.

(99m)Tc-labeled-2-arylbenzoxazole derivatives as potential Aß imaging probes for single-photon emission computed tomography.

Four neutral (99m)Tc/Re-labeled 2-arylbenzoxazole derivatives conjugated to bis (aminoethanethiol) (BAT) chelating ligand via a short propoxy spacer were synthesized and evaluated. In vitro binding assay showed that they displayed binding affinities to Aß1-42 aggregates (Ki = 15.86-393.18 nM). In vitro autoradiography studies further confirmed the high and specific binding of [(99m)Tc]20 to ß-amyloid plaques on brain sections of transgenic mice. Biodistribution study of [(99m)Tc]17-20 in normal mice displayed moderate initial brain uptake (0.96-1.55%ID/g at 2 min), and fast washed out from the brain (0.14-0.40%ID/g at 60 min), especially for [(99m)Tc]20 with a brain2min/brain60min ratio of 8.86. Taken together, these preliminary data suggested that [(99m)Tc]20 may be a potential imaging probe for detecting amyloid plaques in the brain.

Synthesis and evaluation of a 18F-labeled spirocyclic piperidine derivative as promising σ1 receptor imaging agent.

Several spirocyclic piperidine derivatives were designed and synthesized as σ1 receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ1 receptors and subtype selectivity. Particularly for ligand 1'-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofuran-1,4'-piperidine] (2), high σ1 receptor affinity (Ki=2.30 nM) and high σ1/σ2 subtype selectivity (142-fold) as well as high σ1/VAChT selectivity (234-fold) were observed. [18F]2 was synthesized using an efficient one-pot, two-step reaction method in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8-10%, a radiochemical purity of higher than 99%, and specific activity of 56-78GBq/µmol. Biodistribution studies of [18F]2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 µmol/kg) 5 min prior to injection of [18F]2 significantly decreased the accumulation of radiotracer in organs known to contain σ1 receptors. Ex vivo autoradiography in Sprague-Dawley rats demonstrated high accumulation of radiotracer in brain areas with high expression of σ1 receptors. These encouraging results prove that [18F]2 is a suitable candidate for σ1 receptor imaging with PET in humans.

Evaluation of molecules based on the electron donor-acceptor architecture as near-infrared ß-amyloidal-targeting probes.

A novel class of near-infrared molecules based on the donor-acceptor architecture were synthesized and evaluated as Aß imaging probes. In vivo imaging studies suggested that MCAAD-3 could penetrate the blood-brain barrier and label Aß plaques in the brains of transgenic mice. Computational studies could reproduce the experimental trends well.

Novel cyclopentadienyl tricarbonyl (99m)tc complexes containing 1-piperonylpiperazine moiety: potential imaging probes for sigma-1 receptors.

We report the design, synthesis, and evaluation of a series of novel cyclopentadienyl tricarbonyl (99m)Tc complexes as potent σ1 receptor radioligands. Rhenium compounds 3-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)propylcarbonylcyclopentadienyl tricarbonyl rhenium (10a) and 4-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)butylcarbonylcyclopentadienyl tricarbonyl rhenium (10b) possessed high in vitro affinity for σ1 receptors and moderate to high selectivity for σ2 receptors and the vesicular acetylcholine transporter. Biodistribution studies in mice demonstrated high initial brain uptake for corresponding (99m)Tc derivatives [(99m)Tc]23 and [(99m)Tc]24 of 2.94 and 2.13% injected dose (ID)/g, respectively, at 2 min postinjection. Pretreatment of haloperidol significantly reduced the radiotracer accumulation of [(99m)Tc]23 or [(99m)Tc]24 in the brain. Studies of the cellular uptake of [(99m)Tc]23 in C6 and DU145 tumor cells demonstrated a reduction of accumulation by incubation with haloperidol, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine (SA4503), or 1,3-di-o-tolyl-guanidine (DTG). Furthermore, blocking studies in C6 glioma-bearing mice confirmed the specific binding of [(99m)Tc]23 to σ1 receptors in the tumor.

Novel ¹8F-labeled dibenzylideneacetone derivatives as potential positron emission tomography probes for in vivo imaging of ß-amyloid plaques.

A series of dibenzylideneacetones were synthesized and evaluated as imaging probes for ß-amyloid plaques. They displayed high binding affinity to Aß(1-42) aggregates (K(i) = 6.4 for 8, K(i) = 3.0 for 9), and the high binding were confirmed by in vitro autoradiography with AD human and transgenic mouse brain sections. Two of them were selected for (18)F-labeling directly on the benzene ring. In biodistribution experiments, [(18)F]8 and [(18)F]9 displayed high initial uptakes (9.29 ± 0.41 and 5.38 ± 0.68% ID/g) and rapid washouts from the normal brain (brain(2 min)/brain(60 min) ratios of 21.6 and 13.4). These preliminary results suggest that [(18)F]8 and [(18)F]9 may be used as potential PET imaging agents for the detection of Aß plaques in the brain.

Building a Ni3S2 nanotube array and investigating its application as an electrode for lithium ion batteries.

A Ni3S2 nanotube array has been synthesized on Ni foam using a template-free hydrothermal method. The Ni foam acts as both the reactant and support. The resulting architecture as an electrode for lithium ion batteries benefits from the unique morphology and exhibits excellent electrochemical performance with high capacity, long cycle life and superior rate capability.