High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2'-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells.

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn2+, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2'-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2'-bipyridine scaffold, which acts as both the chelating agent for Zn2+ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (Kd = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn2+. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn2+ in living systems.

Development of novel radioiodinated exendin-4 derivatives targeting GLP-1 receptor for detection of ß-cell mass.

In subjects with type 2 diabetes mellitus (T2DM), pancreatic ß-cell mass decreases; however, it is unknown to what extent this decrease contributes to the pathophysiology of T2DM. Therefore, the development of a method for noninvasive detection of ß-cell mass is underway. We previously reported that glucagon-like peptide-1 receptor (GLP-1R) is a promising target molecule for ß-cell imaging. In this study, we attempted to develop a probe targeting GLP-1R for ß-cell imaging using single-photon emission computed tomography (SPECT). For this purpose, we selected exendin-4 as the lead compound and radiolabeled lysine at residue 12 in exendin-4 or additional lysine at the C-terminus using [123I]iodobenzoylation. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Biodistribution study was performed in normal ddY mice. Ex vivo autoradiography was performed in transgenic mice expressing green fluorescent protein under control of the mouse insulin I gene promoter. Additionally, SPECT imaging was performed in normal ddY mice. The affinity of novel synthesized derivatives toward pancreatic ß-cells was not affected by iodobenzoylation. The derivatives accumulated in the pancreas after intravenous administration specifically via GLP-1R expressed on the pancreatic ß-cells. Extremely high signal-to-noise ratio was observed during evaluation of biodistribution of [123I]IB12-Ex4. SPECT images using normal mice showed that [123I]IB12-Ex4 accumulated in the pancreas with high contrast between the pancreas and background. These results indicate that [123I]IB12-Ex4 for SPECT is useful for clinical applications because of its preferable kinetics in vivo.

Selective Cadmium Fluorescence Probe Based on Bis-heterocyclic Molecule and its Imaging in Cells.

Fluorescence probes that selectively image cadmium are useful for detecting and tracking the amount of Cd2+ in cells and tissues. In this study, we designed and synthesized a novel Cd2+ fluorescence probe based on the pyridine-pyrimidine structure, 4-(methylsulfanyl)-6-(pyridin-2-yl)pyrimidin-2-amine (3), as a low-molecular-weight fluorescence probe for Cd2+. Compound 3 could successfully discriminate between Cd2+ and Zn2+ and exhibited a highly selective turn-on response toward Cd2+ over biologically related metal ions. The dissociation constant (Kd) and the limit of detection (LOD) of 5.4 × 10- 6 mol L- 1 and 4.4 × 10- 7 mol L- 1, respectively, were calculated using fluorescence titration experiments. Studies with closely related analogs showed that the bis-heterocyclic moiety of 3 acted as both a coordination site for Cd2+ and a fluorophore. Further, the methylsulfanyl group of compound 3 is essential for achieving selective and sensitive Cd2+ detection. Fluorescence microscopy studies using living cells revealed that the cell membrane permeability of compound 3 is sufficient to detect intracellular Cd2+. These results indicate that novel bis-heterocyclic molecule 3 has considerable potential as a fluorescence probe for Cd2+ in biological applications.

Noninvasive longitudinal quantification of ß-cell mass with [111In]-labeled exendin-4.

Currently, quantifying ß-cell mass (BCM) requires harvesting the pancreas. In this study, we investigated a potential noninvasive method to quantify BCM changes longitudinally using [Lys12(111In-BnDTPA-Ahx)]exendin-4 ([111In]-Ex4) and single-photon emission computed tomography (SPECT). We used autoradiography and transgenic mice expressing green fluorescent protein under the control of mouse insulin 1 gene promotor to evaluate the specificity of [111In]-Ex4 toward ß cells. Using nonobese diabetic (NOD) mice, we injected [111In]-Ex4 (3.0 MBq) intravenously and performed SPECT 30 min later, repeating this at a 2-wk interval. After the second scan, we harvested the pancreas and calculated BCM from immunohistochemically stained pancreatic sections. Specific accumulation of [111In]-Ex4 in ß cells was confirmed by autoradiography, with a significant correlation (r = 0.94) between the fluorescent and radioactive signal intensities. The radioactive signal from the pancreas in the second SPECT scan significantly correlated (r = 0.89) with BCM calculated from the immunostained pancreatic sections. We developed a regression formula to estimate BCM from the radioactive signals from the pancreas in SPECT scans. BCM can be quantified longitudinally and noninvasively by SPECT imaging with [111In]-Ex4. This technique successfully demonstrated longitudinal changes in BCM in NOD mice before and after onset of hyperglycemia.-Fujita, N., Fujimoto, H., Hamamatsu, K., Murakami, T., Kimura, H., Toyoda, K., Saji, H., Inagaki, N. Noninvasive longitudinal quantification of ß-cell mass with [111In]-labeled exendin-4.

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvß6 integrin-positive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin αVß6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets αVß6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvß6 integrin, as a probe scaffold, and 68Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with 68Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVß6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([natGa]CG6 and [natGa]Ac-CG6) showed high affinity to αVß6 integrin. Both probes could be labeled by 67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [67Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [67Ga]CG6. The αVß6 integrin-positive xenografts were clearly visualized by PET imaging with [68Ga]Ac-CG6. The intratumoral distribution of [68Ga]Ac-CG6 coincided with the αVß6 integrin-positive regions detected by immunohistochemistry. Thus, [68Ga]Ac-CG6 is a useful peptide probe for the imaging of αVß6 integrin in PDAC.

Development of Gold Nanorods Conjugated with Radiolabeled Anti-human Epidermal Growth Factor Receptor 2 (HER2) Monoclonal Antibody as Single-Photon Emission Computed Tomography/Photoacoustic Dual-Imaging Probes Targeting HER2-Positive Tumors.

Surgery remains one of the main treatments of cancer and both precise pre- and intraoperative diagnoses are crucial in order to guide the operation. We consider that using an identical probe for both pre- and intra-operative diagnoses would bridge the gap between surgical planning and image-guided resection. Therefore, in this study, we developed gold nanorods (AuNRs) conjugated with radiolabeled anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody, and investigated their feasibility as novel HER2-targeted dual-imaging probes for both single photon emission computed tomography (SPECT) (preoperative diagnosis) and photoacoustic (PA) imaging (intraoperative diagnosis). To achieve the purpose, AuNRs conjugated with different amount of trastuzumab (Tra) were prepared, and Tra-AuNRs were labeled with indium-111. After the evaluation of binding affinity to HER2, cell binding assay and biodistribution studies were carried out for optimization. AuNRs with moderate trastuzumab conjugation (Tra2-AuNRs) were proposed as the novel probe and demonstrated significantly higher accumulation in NCI-N87 (HER2 high-expression) tumors than in SUIT2 (low-expression) tumors 96 h post-injection along with good affinity towards HER2. Thereafter, in vitro PA imaging and in vivo SPECT imaging studies were performed. In in vitro PA imaging, Tra2-AuNRs-treated N87 cells exhibited significant PA signal increase than SUIT2 cells. In in vivo SPECT, signal increase in N87 tumors was more notable than that in SUIT2 tumors. Herein, we report that the Tra2-AuNRs enabled HER2-specific imaging, suggesting the potential as a robust HER2-targeted SPECT and PA dual-imaging probe.

Initial evaluation of PET/CT with 18 F-FSU-880 targeting prostate-specific membrane antigen in prostate cancer patients.

This first-in-man study was carried out to evaluate the safety, whole-body distribution, dose estimation, and lesion accumulation of 18 F-FSU-880, a newly developed probe targeting prostate-specific membrane antigen. Six prostate cancer patients with known metastatic lesions underwent serial whole-body PET/computed tomography (CT) with 18 F-FSU-880. Blood and urine were analyzed before and after PET/CT. Accumulation of 18 F-FSU-880 in organs and metastatic lesions in serial PET images were evaluated by measuring the standardized uptake values. From the biodistribution data, the organ doses and whole-body effective dose were calculated using OLINDA/EXM software was developed by Dr. Michael Stabin of Vanderbilt University, Nashville, Tennessee, USA. 18 F-FSU-880 PET/CT could be carried out without significant adverse effects. High physiological uptake was observed in the salivary/lachrymal glands and kidneys. The effective dose was calculated to be 0.921 × 10-2 mSv/MBq. Known metastatic lesions were clearly visualized with high image contrast that increased with time, except in 1 patient, whose bone metastases were well-controlled and inactive. The PET/CT with 18 F-FSU-880 could be carried out safely and could clearly visualize active metastatic lesions. The present results warrant further clinical studies with a larger number of cases to verify the clinical utility of 18 F-FSU-880 PET/CT in the management of prostate cancer patients.

Development of the 99mTc-Hydroxamamide Complex as a Probe Targeting Carbonic Anhydrase IX.

Carbonic anhydrase IX (CA-IX) is regarded as a favorable target for in vivo imaging because of its specific expression in hypoxic regions of tumors. Hypoxia assists tumor propagation and growth and is resistant to chemotherapy and radiotherapy. Here, we designed and synthesized [99mTc]hydroxamamide ([99mTc]Ham) and [99mTc]methyl-substituted-hydroxamamide ([99mTc]MHam) complexes including a bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR). In a cell binding assay, [99mTc]Ham complexes with bivalent SA ([99mTc]SAB2A and [99mTc]SAB2B) and UR ([99mTc]URB2A and [99mTc]URB2B) showed significantly greater uptake into CA-IX high-expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [99mTc]URB2A and [99mTc]URB2B for CA-IX was significantly higher than that of [99mTc]SAB2A and [99mTc]SAB2B, we additionally synthesized [99mTc]MURB2 (a [99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of [99mTc]URB2A, [99mTc]URB2B, and [99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice was carried out using [99mTc]URB2A and [99mTc]MURB2 with the highest specificity for HT-29 cells. [99mTc]URB2A showed moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.

Synthesis and biological evaluation of F-18 labeled tetrahydroisoquinoline derivatives targeting orexin 1 receptor.

Orexin 1 receptor (OX1R) is thought to be involved in various body functions, including arousal maintenance and emotional control, but the full details of its function remain unknown. OX1R imaging with positron emission tomography (PET) would be useful in elucidating the orexin system including OX1R, but no PET probes targeting OX1R have been reported. We, therefore, designed and synthesized tetrahydroisoquinoline (THIQ) derivatives as novel PET probes targeting OX1R, and evaluated their utility. In an in vitro competitive binding assay, THIQ-1 and THIQ-2 showed significantly higher binding to OX1R (IC50 = 30 and 31 nM, respectively) than OX2R (IC50 = 160 and 332 nM, respectively). These features were also observed in a cell binding assay using [18F]THIQ-1 and [18F]THIQ-2, demonstrating their OX1R-specific binding property in vitro. In a biodistribution study using normal mice, the brain uptake of [18F]THIQ-1 was higher than that of [18F]THIQ-2, but further improvement is required for in vivo imaging with PET. Taken together, [18F]THIQ-1 and [18F]THIQ-2 have the potential to become useful imaging probes for PET targeting the OX1R, but require additional structural changes to improve their brain uptake.

Development of 99mTc radiolabeled A85380 derivatives targeting cerebral nicotinic acetylcholine receptor: Novel radiopharmaceutical ligand 99mTc-A-YN-IDA-C4.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been implicated in higher brain functions. To elucidate the functional mechanisms underlying nAChRs and contribute significantly to development of drugs targeting neurological and neuropsychiatric diseases, non-invasive nuclear medical imaging can be used for evaluation. In addition, technetium-99m (99mTc) is a versatile radionuclide used clinically as a tracer in single-photon emission computed tomography. Because A85380 is known as a potent α4ß2-nAChR agonist, we prepared A85380 derivatives labeled with 99mTc using a bifunctional chelate system. A computational scientific approach was used to design the probe efficiently. We used non-radioactive rhenium (Re) for a 99mTc analog and found that one of the derivatives, Re-A-YN-IDA-C4, exhibited high binding affinity at α4ß2-nAChR in both the docking simulation (-19.3 kcal/mol) and binding assay (Ki = 0.4 ±â€¯0.04 nM). Further, 99mTc-A-YN-IDA-C4 was synthesized using microwaves, and its properties were examined. Consequently, we found that 99mTc-A-YN-IDA-C4, with a structure optimized by using computational chemistry techniques, maintained affinity and selectivity for nAChR in vitro and possessed efficient characteristics as a nuclear medicine molecular imaging probe, demonstrated usefulness of computational scientific approach for molecular improvement strategy.

Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain.

We have designed (S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate ([99mTc]CPTT-A-E) with high affinity for nicotinic acetylcholine receptors (nAChRs) using (2(S)-azetidinylmethoxy)-pyridine (A-85380) as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-technetium (99mTc)-labeled nAChR imaging probe. Because technetium does not contain a stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized by coordinating rhenium, which is a homologous element having the same coordination structure as technetium. Further, the binding affinity to nAChR was evaluated. CPTR-A-E exhibited a high binding affinity to nAChR (Ki = 0.55 nM). Through the radiosynthesis of [99mTc]CPTT-A-E, an objective compound could be obtained with a radiochemical yield of 33% and a radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain exhibited that the local nAChR density strongly correlated with the amount of [99mTc]CPTT-A-E that was accumulated in each region of interest. Further, the in vivo evaluation of biodistribution revealed a higher accumulation of [99mTc]CPTT-A-E in the thalamus (characterized by the high nAChR density) when compared with that in the cerebellum (characterized by the low nAChR density). Although additional studies will be necessary to improve the uptake of [99mTc]CPTT-A-E to the brain, [99mTc]CPTT-A-E met the basic requirements for nAChR imaging.

18F-labeled benzimidazopyridine derivatives for PET imaging of tau pathology in Alzheimer's disease.

Hyperphosphorylated tau proteins are one of the neuropathological hallmarks in the Alzheimer's disease (AD) brain. The in vivo imaging of tau aggregates with nuclear medical imaging probes is helpful for the further comprehension of and medical intervention in the AD pathology. For tau-selective PET imaging, we newly designed and synthesized 18F-labeled benzimidazopyridine (BIP) derivatives with fluoroalkylamino groups, [18F]IBIPF1 and [18F]IBIPF2, and evaluated their utilities as tau imaging probes. They both bound selectively to tau against amyloid ß (Aß) aggregates in AD brain sections in vitro, and showed good pharmacokinetics in mouse brains in vivo. Notably, [18F]IBIPF1 exhibited high tau-selectivity (Tau/Aß ratio = 34.8), high brain uptake (6.22% ID/g at 2 min postinjection), and subsequent washout (2.77% ID/g at 30 min postinjection). In vivo analysis of radiometabolites indicated that [18F]IBIPF1 was stable against metabolism in the mouse brain. These encouraging preclinical results suggest that further structural optimization based on the BIP scaffold may lead to the development of more useful tau imaging probes.

Synthesis of a Novel Pyrazine⁻Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells.

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine-pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure-fluorescence relationship analysis of the substituent effects with/without Zn2+, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn2+ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn2+ toward understanding its functions in living systems.

Development of Technetium-99m-Labeled BODIPY-Based Probes Targeting Lipid Droplets Toward the Diagnosis of Hyperlipidemia-Related Diseases.

Hyperlipidemia causes systemic lipid disorder, which leads to hepatic steatosis and atherosclerosis. Thus, it is necessary to detect these syndromes early and precisely to improve prognosis. In the affected regions, abnormal formation and growth of lipid droplets is observed; therefore, lipid droplets may be a suitable target for the diagnosis of hyperlipidemia-related syndromes. In this study, we designed and synthesized [99mTc]Tc-BOD and [99mTc]Tc-MBOD composed of one technetium-99m and two BODIPY scaffolds with hydroxamamide (Ham) or N-methylated hydroxamamide (MHam) in radiochemical yields of 54 and 35%, respectively, with a radiochemical purity of over 95%. [99mTc]Tc-BOD showed significantly higher accumulation levels in foam cells than in non-foam cells (foam cells: 213.8 ± 64.8, non-foam cell: 126.2 ± 26.9 %dose/mg protein, p < 0.05) 2 h after incubation. In contrast, [99mTc]Tc-MBOD showed similar accumulation levels in foam cells and non-foam cells (foam cells: 92.2 ± 23.3, non-foam cell: 83.8 ± 19.8 %dose/mg protein). In normal mice, [99mTc]Tc-BOD exhibited gradual blood clearance (0.5 h: 4.98 ± 0.35, 6 h: 1.94 ± 0.12 %ID/g) and relatively high accumulation in the liver 6 h after administration (15.22 ± 1.72 %ID/g). Therefore, [99mTc]Tc-BOD may have potential as an imaging probe for detecting lipid droplets in disease lesions of hyperlipidemia.

Flumioxazin metabolism in pregnant animals and cell-based protoporphyrinogen IX oxidase (PPO) inhibition assay of fetal metabolites in various animal species to elucidate the mechanism of the rat-specific developmental toxicity.

Flumioxazin, an N-phenylimide herbicide, inhibits protoporphyrinogen oxidase (PPO), a key enzyme in heme biosynthesis in mammals, and causes rat-specific developmental toxicity. The mechanism has mainly been clarified, but no research has yet focused on the contribution of its metabolites. We therefore conducted in vivo metabolism studies in pregnant rats and rabbits, and found 6 major known metabolites in excreta. There was no major rat-specific metabolite. The most abundant component in rat fetuses was APF, followed by flumioxazin and 5 identified metabolites. The concentrations of flumioxazin and these metabolites in fetuses were lower in rabbits than in rats. In vitro PPO inhibition assays with rat and human liver mitochondria showed that flumioxazin is a more potent PPO inhibitor than the metabolites. There were no species differences in relative intensity of PPO inhibition among flumioxazin and these metabolites. Based on the results of these in vivo and in vitro experiments, we concluded that flumioxazin is the causal substance of the rat-specific developmental toxicity. As a more reliable test system for research on in vitro PPO inhibition, cell-based assays with rat, rabbit, monkey, and human hepatocytes were performed. The results were consistent with those of the mitochondrial assays, and rats were more sensitive to PPO inhibition by flumioxazin than humans, while rabbits and monkeys were almost insensitive. From these results, the species difference in the developmental toxicity was concluded to be due to the difference in sensitivity of PPO to flumioxazin, and rats were confirmed to be the most sensitive of these species.

Enhanced Delivery of Radiolabeled Polyoxazoline into Tumors via Self-Aggregation under Hyperthermic Conditions.

In order to develop a radiopharmaceutical for internal radiotherapy that had a high anticancer effect while exposing normal tissues to low radiation levels, we synthesized a radiolabeled polyoxazoline (POZ), a thermoresponsive polymer, and established a novel drug delivery system for targeting tumors by accelerating the accumulation of the radiolabeled POZ via self-aggregation under hyperthermic (42-43 °C) conditions. By living-cationic polymerization using 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, POZ derivatives (Et-IspPOZ) (10, 20, and 30 kDa) with lower critical solution temperatures (LCSTs) of 37-38 °C were synthesized; the POZ derivatives were soluble at the body temperature but self-aggregated upon heat treatment (42-43 °C). Next, the indium-111 (111In)-labeled Et-IspPOZ was prepared, and the effect of molecular weight and injected POZ dose on the accumulation of radioactivity in the tumors was investigated upon intravenous injection of probes under hyperthermic conditions in colon 26-bearing mice. The uptake of radioactivity in tumors was increased when the molecular weight of POZ was greater than 20 kDa, while it was independent of the injected POZ dose (4-40 nmol). The amount of radioactivity retained in the tumor did not change for up to 3 h after exposure to heat treatment was stopped. Furthermore, the tumor uptake of the Et-IspPOZ derivative with an LCST greater than 42 °C was significantly lower than that of Et-IspPOZ, which had an LCST of 37-38 °C, suggesting the involvement of the self-aggregation of POZ on tumor uptake. Finally, the intratumoral localization of fluorescence-labeled Et-IspPOZ was evaluated using in vivo confocal laser microscopy. Many bright fluorescence spots were observed in the heat-treated tumors nearby and within blood vessels. In conclusion, the high tumor uptake of radiolabeled Et-IspPOZ was elucidated under hyperthermic conditions; thereby, the possibility of developing a novel internal radiotherapy using radiolabeled POZ derivatives was demonstrated.

Characterization of Novel 18F-Labeled Phenoxymethylpyridine Derivatives as Amylin Imaging Probes.

Deposition of islet amyloid consisting of amylin constitutes one of pathological hallmarks of type 2 diabetes mellitus (T2DM), and it may be involved in the development and progression of T2DM. However, the details about the relationship between the deposition of islet amyloid and the pathology of T2DM remain unclear, since no useful imaging tracer enabling the visualization of pancreatic amylin is available. In the present study, we synthesized and evaluated six novel 18F-labeled phenoxymethylpyridine (PMP) derivatives as amylin imaging probes. All 18F-labeled PMP derivatives showed not only affinity for islet amyloid in the post-mortem T2DM pancreatic sections but also excellent pharmacokinetics in normal mice. Furthermore, ex vivo autoradiographic studies demonstrated that [18F]FPMP-5 showed intense labeling of islet amyloids in the diabetes model mouse pancreas in vivo. The preclinical studies suggested that [18F]FPMP-5 may have potential as an imaging probe that targets amylin aggregates in the T2DM pancreas.

Novel fluorescence probes based on the chalcone scaffold for in vitro staining of ß-amyloid plaques.

The ß-amyloid (Aß) plaque is one of the neuropathological hallmarks in the Alzheimer's disease brain. The detection of Aß plaques with fluorescence probes is useful for preclinical studies of Alzheimer's disease. In this study, we developed four novel fluorescence probes based on chalcone scaffold. In an in vitro binding study, all FCH derivatives showed moderate binding affinity for Aß(1-42) aggregates (Ki = 72-114 nM). The fluorescence intensities of FCH-3 and FCH-4 dramatically changed in the presence of Aß(1-42) aggregates (6.7 and 14.2 fold), but the changes of FCH-1 and FCH-2 were minor (2.0 and 2.4 fold). In a fluorescence staining study using Tg2576 mouse brain sections, FCH-3 and FCH-4 clearly visualized Aß plaques, but FCH-1 and FCH-2 did not stain. Taken together, all FCH derivatives could bind to Aß aggregates, but only FCH-3 and FCH-4 may be useful fluorescence probes for in vitro staining of Aß plaques.

Synthesis and evaluation of a [18F]formyl-Met-Leu-Phe derivative: A positron emission tomography imaging probe for bacterial infections.

The tripeptide formyl-Met-Leu-Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an 18F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X = Nle) has a high affinity for FPR (Ki = 0.62 ±â€¯0.13 nM). The radiochemical yield and purity of [18F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [18F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60 min after [18F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [18F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60 min after injection.

Conversion of iodine to fluorine-18 based on iodinated chalcone and evaluation for ß-amyloid PET imaging.

In the amyloid cascade hypothesis, ß-amyloid (Aß) plaques is one of the major pathological biomarkers in the Alzheimer's disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [18F]4-dimethylamino-4'-fluoro-chalcone ([18F]DMFC) and [18F]4'-fluoro-4-methylamino-chalcone ([18F]FMC), as Aß imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by 18F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [18F]DMFC and [18F]FMC. In a biodistribution study using normal mice, [18F]DMFC and [18F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2 min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30 min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aß imaging agent ([18F]Florbetapir), meaning the improvement of the probability of detecting Aß plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aß peptides, [18F]DMFC and [18F]FMC showed high binding affinity to recombinant Aß aggregates at the Kd values of 4.47 and 6.50 nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [18F]DMFC and [18F]FMC markedly accumulated only in a region with abundant Aß plaques, indicating that they clearly recognized human Aß plaques in vitro. These encouraging results suggest that [18F]DMFC and [18F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.