[18F]DAA1106: Automated radiosynthesis using spirocyclic iodonium ylide and preclinical evaluation for positron emission tomography imaging of translocator protein (18 kDa)

DAA1106 (N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide), is a potent and selective ligand for the translocator protein (18?kDa, TSPO) in brain mitochondrial fractions of rats and monkey (K_i?=?0.043 and 0.188?nM, respectively). In this study, to translate [¹⁸F]DAA1106 for clinical studies, we performed automated syntheses of [¹⁸F]DAA1106 using the spirocyclic iodonium ylide (1) as a radiolabelling precursor and conducted preclinical studies including positron emission tomography (PET) imaging of TSPO in ischemic rat brains. Radiofluorination of the ylide precursor 1 with [¹⁸F]F^?, followed by HPLC separation and formulation, produced the [¹⁸F]DAA1106 solution for injection in 6% average (n?=?10) radiochemical yield (based on [¹⁸F]F^?) with >98% radiochemical purity and molar activity of 60–100?GBq/μmol at the end of synthesis. The synthesis time was 87?min from the end of bombardment. The automated synthesis achieved [¹⁸F]DAA1106 with sufficient radioactivity available for preclinical and clinical use. Biodistribution study of [¹⁸F]DAA1106 showed a low uptake of radioactivity in the mouse bones. Metabolite analysis showed that >96% of total radioactivity in the mouse brain at 60?min after the radiotracer injection was unmetabolized [¹⁸F]DAA1106. PET study of ischemic rat brains visualized ischemic areas with a high uptake ratio (1.9?±?0.3) compared with the contralateral side. We have provided evidence that [¹⁸F]DAA1106 could be routinely produced for clinical studies.     

Design,synthesis and evaluation of 18F-labeled cationic carbonic anhydrase IX inhibitors for PET imaging

Carbonic anhydrase IX (CA-IX) is a marker for tumor hypoxia, and its expression is negatively correlated with patient survival. CA-IX represents a potential target for eliminating hypoxic cancers. We synthesized fluorinated cationic sulfonamide inhibitors 1–3 designed to target CA-IX. The binding affinity for CA-IX ranged from 0.22 to 0.96?μM. We evaluated compound 2 as a diagnostic PET imaging agent. Compound 2 was radiolabeled with ¹⁸F in 10?±?4% decay-corrected radiochemical yield with 85.1?±?70.3 GBq/μmol specific activity and >98% radiochemical purity. ¹⁸F-labeled 2 was stable in mouse plasma at 37?°C after 1?h incubation. PET/CT imaging was conducted at 1?h post-injection in a human colorectal cancer xenograft model. ¹⁸F-labeled 2 cleared through hepatobiliary and renal pathways. Tumor uptake was approximately 0.41?±?0.06% ID/g, with a tumor-to-muscle ratio of 1.99?±?0.25. Subsequently, tumor xenografts were visualized with moderate contrast. This study demonstrates the use of a cationic motif for conferring isoform selectively for CA-IX imaging agents.     

Synthesis and preliminary evaluation of 18F-icotinib for EGFR-targeted PET imaging of lung cancer

Epidermal growth factor receptor (EGFR) has emerged as an attracting target in the field of imaging and treatment for non-small cell lung cancer (NSCLC). Radiolabeled EGFR-tyrosine kinase inhibitors (EGFR-TKIs) specifically targeting EGFR are deemed as promising probes for the imaging of NSCLC. This study aimed to label icotinib (one kind of EGFR-TKI) with ¹⁸F through click reaction to develop a new EGFR-targeting PET probe-¹⁸F-icotinib. ¹⁸F-icotinib was obtained in 44.81% decay-corrected yield in 100?min synthesis time with 34?GBq/μmol specific activity and >99% radiochemical purity at the end of synthesis. The identity of the product was confirmed by co-injection with ¹⁸F-icotinib and ¹⁹F-icotinib. The Log P was 1.28?±?0.04 (n?=?6). The tracer displayed excellent stability after incubation for 4?h in vitro. ¹⁸F-icotinib showed satisfying binding ability to A549 NSCLC cells, which could be inhibited by icotinib. PET imaging studies demonstrated a specific uptake of the radiotracer (0.90?±?0.24% ID/g) in A549 tumor-bearing mice, while lower uptake was observed in heart, lung and spleen at 1.5?h post injection. Inmunohistochemical staining confirmed that the A549 tumor was EGFR-positive. Therefore, we considered that ¹⁸F-icotinib was a highly promising compound for EGFR-based tumor PET imaging.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Development of a Novel PET Tracer [18F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

Background and Objective

The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.

Methods

C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested in vitro and in vivo.

Results

The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.

Conclusions

[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.     

Radiosynthesis and evaluation of [11C]EMPA as a potential PET tracer for orexin 2 receptors

EMPA is a selective antagonist of orexin 2 (OX₂) receptors. Previous literature with [³H]-EMPA suggest that it may be used as an imaging agent for OX₂ receptors; however, brain penetration is known to be modest. To evaluate the potential of EMPA as a PET radiotracer in non-human primate (as a step to imaging in man), we radiolabeled EMPA with carbon-11. Radiosynthesis of [¹¹C]N-ethyl-2-(N-(6-methoxypyridin-3-yl)-2-methylphenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide ([¹¹C]EMPA), and evaluation as a potential PET tracer for OX₂ receptors is described. Synthesis of an appropriate non-radioactive O-desmethyl precursor was achieved from EMPA with sodium iodide and chlorotrimethylsilane. Selective O-methylation using [¹¹C]CH₃I in the presence of cesium carbonate in DMSO at room temp afforded [¹¹C]EMPA in 1.5–2.5% yield (non-decay corrected relative to trapped [¹¹C]CH₃I at EOS) with ?95% chemical and radiochemical purities. The total synthesis time was 34–36 min from EOB. Studies in rodent suggested that uptake in tissue was dominated by nonspecific binding. However, [¹¹C]EMPA also showed poor uptake in both rats and baboon as measured with PET imaging.     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

Optimization of Multimodal Imaging of Mesenchymal Stem Cells Using the Human Sodium Iodide Symporter for PET and Cerenkov Luminescence Imaging

Purpose

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

Methods

First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, ¹²⁴I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using ^99mTcO₄ ⁻ radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell''s differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI.

Results

The expression of both imaging reporter genes was functional and specific. An elution of ^99mTcO₄ ⁻ from the cells was observed, with 31% retention after 3 h. After labeling cells with ¹²⁴I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using ¹²⁴I small-animal PET, CLI and BLI.

Conclusions

This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.     

Ascorbic acid analogue 6-Deoxy-6-[18F] fluoro-L-ascorbic acid as a tracer for identifying human colorectal cancer with SVCT2 overexpression

L-ascorbic acid (AA) was reported to have an anti-cancer effect over 40 years. In recent years, several ongoing clinical trials are exploring the safety and efficacy of intravenous high-dose AA for cancer treatment. The lack of appropriate imaging modality limits the identification of potentially suitable patients for AA treatment. This study focuses on identifying AA-sensitive tumor cells using molecular imaging. 6-Deoxy-6-[¹⁸F] fluoro-L-ascorbic Acid (¹⁸F-DFA), a structural analog of AA, was synthesized and labeled to visualize the metabolism of AA in vivo. Colorectal cancer (CRC) cell lines with high and low expression of sodium-dependent vitamin C transporters 2 (SVCT2) were used for a series of cellular uptake tests. PET imaging was performed on xenograft tumor-bearing mice. More AA uptake was observed in CRC cells with high SVCT2 expression than in cells with low SVCT2 expression. The substrate (unlabeled AA) can competitively inhibit the ¹⁸F-DFA tracer uptake by CRC cells. The biodistribution of ¹⁸F-DFA in mice showed high radioactivity was seen in organs such as adrenal glands, kidneys, and liver that were known to have high concentrations of AA. Both PET imaging and tissue distribution showed that cancer cells with high SVCT2 expression enhanced the accumulation of ¹⁸F-DFA in mice after tumor formation. Immunohistochemistry was used to verify the corresponding results. As a radiotracer, ¹⁸F-DFA can provide powerful imaging information to identify tumor with high affinity of AA, and SVCT2 can be a potential biomarker in this process.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.     

Radiosynthesis and evaluation of [11C]CMP,a high affinity GSK3 ligand

Dysfunction of GSK3 is implicated in the etiology of many brain, inflammatory, cardiac diseases, and cancer. PET imaging would enable in vivo detection and quantification of GSK3 and can impact the choice of therapy, allow non-invasive monitoring of disease progression and treatment effects. In this report, the synthesis and evaluation of a high affinity GSK3 ligand, [¹¹C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide, ([¹¹C]CMP, (3), (IC₅₀?=?3.4?nM, LogP?=?1.1) is described. [¹¹C]CMP was synthesized in 25?±?5% yield by radiomethylating the corresponding phenolate using [¹¹C]CH₃I. The radioligand exhibited modest uptake in U251 human glioblastoma cell lines with ～50% specific binding. MicroPET studies in rats indicated negligible blood–brain barrier (BBB) penetration of [¹¹C]CMP, despite its high affinity and suitable logP value for BBB penetration. However, administration of cyclosporine prior to [¹¹C]CMP injection showed significant improvement in brain radioactivity uptake and the tracer binding. This finding indicates that [¹¹C]CMP might be a P-gp efflux substrate and therefore has some limitations for routine in vivo PET evaluations in brain.     

Synthesis and preliminary biological evaluation of <Emphasis Type="Italic">S</Emphasis>-<Superscript>11</Superscript>C-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-cysteine as a new amino acid PET tracer for cancer imaging

S-¹¹C-methyl-l-cysteine (LMCYS) is an attractive amino acid tracer for clinical tumor positron emission tomography (PET) imaging. d-isomers of some radiolabeled amino acids are potential PET tracers for tumor imaging. In this work, S-¹¹C-methyl-d-cysteine (DMCYS), a d-amino acid isomer of S-¹¹C-methyl-cysteine for tumor imaging was developed and evaluated. DMCYS was prepared by ¹¹C-methylation of the precursor d-cysteine, with an uncorrected radiochemical yield over 50 % from ¹¹CH₃I within a total synthesis time from ¹¹CO₂ about 12 min. In vitro competitive inhibition studies showed that DMCYS uptake was primarily transported through the Na⁺-independent system L, and also the Na⁺-dependent system B^0,+ and system ASC, with almost no system A. In vitro incorporation experiments indicated that almost no protein incorporation was found in Hepa 1–6 hepatoma cell lines. Biodistribution studies demonstrated higher uptake of DMCYS in pancreas and liver at 5 min post-injection, relatively lower uptake in brain and muscle, and faster radioactivity clearance from most tissues than those of l-isomer during the entire observation time. In the PET imaging of S180 fibrosarcoma–bearing mice and turpentine-induced inflammatory model mice, 2-¹⁸F-fluoro-2-deoxy-d-glucose (FDG) exhibited significantly high accumulation in both tumor and inflammatory lesion with low tumor-to-inflammation ratio of 1.40, and LMCYS showed low tumor-to-inflammation ratio of 1.64 at 60 min post-injection. By contrast, DMCYS showed moderate accumulation in tumor and very low uptake in inflammatory lesion, leading to relatively higher tumor-to-inflammation ratio of 2.25 than ¹¹C-methyl-l-methionine (MET) (1.85) at 60 min post-injection. Also, PET images of orthotopic transplanted glioma models demonstrated that low uptake of DMCYS in normal brain tissue and high uptake in brain glioma tissue were observed. The results suggest that DMCYS is a little better than the corresponding l-isomers as a potential PET tumor-detecting agent and is superior to MET and FDG in the differentiation of tumor from inflammation.     

Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.     

Radiosynthesis and evaluation of a novel monoacylglycerol lipase radiotracer: 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[11C]carboxylate

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, the inhibition of MAGL is an attractive therapeutic target for neurodegenerative diseases. In this study, to visualize MAGL via positron emission tomography (PET), we report a new carbon-11-labeled radiotracer, namely 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[¹¹C]carboxylate ([¹¹C]6). Compound 6 exhibited high in vitro binding affinity (IC₅₀ = 0.41 nM) to MAGL in the brain with a suitable lipophilicity (cLogD = 3.29). [¹¹C]6 was synthesized by reacting 1,1,1,3,3,3-hexafluoropropanol (7) with [¹¹C]phosgene ([¹¹C]COCl₂), followed by a reaction with 3-(1-benzyl-1H-pyrazol-3-yl)azetidine hydrochloride (8), which resulted in a 15.0 ± 6.8% radiochemical yield (decay-corrected, n = 7) based on [¹¹C]CO₂ and a 45 min synthesis time from the end of bombardment. A biodistribution study in mice showed high uptake of radioactivity in MAGL-rich organs, including the lungs, heart, and kidneys. More than 90% of the total radioactivity was irreversibly bound in the brain homogenate of rats 5 min and 30 min after the radiotracer injection. PET summation images of rat brains showed high radioactivity in all brain regions. Pretreatment with 6 or MAGL-selective inhibitor JW642 significantly reduced the uptake of radioactivity in the brain. [¹¹C]6 is a promising PET tracer which offers in vivo specific binding and selectivity for MAGL in rodent brains.     

Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

Purpose

Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.

Methods

HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.

Results

cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.

Conclusions

These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.     

PET imaging of cardiomyocyte apoptosis in a rat myocardial infarction model

Cardiomyocyte apoptosis has been observed in several cardiovascular diseases and contributes to the subsequent cardiac remodeling processes and progression to heart failure. Consequently, apoptosis imaging is helpful for noninvasively detecting the disease progression and providing treatment guidance. Here, we tested ¹⁸F-labeled 2-(5-fluoropentyl)-2-methyl-malonic acid (¹⁸F-ML-10) and ¹⁸F-labeled 2-(3-fluoropropyl)-2-methyl-malonic acid (¹⁸F-ML-8) for apoptosis imaging in rat models of myocardial infarction (MI) and compared them with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG). MI was induced in Sprague-Dawley rats by permanent left coronary artery ligation. Procedural success was confirmed by echocardiography and positron emission tomography (PET) imaging with ¹⁸F-FDG. In vivo PET imaging with ¹⁸F-ML-10 and ¹⁸F-ML-8 was performed in the MI models at different time points after operation. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemical analyses were used to evaluate myocardial apoptosis. In vitro cell binding assays were performed to validate ¹⁸F-ML-8 binding to apoptotic cardiomyocytes. PET imaging demonstrated high ¹⁸F-ML-10 and ¹⁸F-ML-8 uptake where ¹⁸F-FDG uptake was absent. The focal accumulation of the two tracers was high on days 1 and 3 but was not notable on days 5 and 7 after surgery. The infarct-to-lung uptake ratio was 4.29?±?0.30 for ¹⁸F-ML-10 and 3.51?±?0.18 for ¹⁸F-ML-8 (n?=?6, analyzed by averaging the uptake ratios on postoperative days 1 and 3, P?<?0.05). The TUNEL results showed that myocardial cell apoptosis was closely related to the focal uptake of the apoptotic tracers in the infarct area. In addition, the apoptosis rates calculated from the TUNEL results were better correlated with ¹⁸F-ML-8 uptake than with ¹⁸F-ML-10 uptake. Ex vivo cell binding assays demonstrated that ¹⁸F-ML-8 accumulated in apoptotic cells but not in necrotic or normal cells. PET imaging using ¹⁸F-ML-10 or ¹⁸F-ML-8 allows the noninvasive detection of myocardial apoptosis in the early phase. In addition, ¹⁸F-ML-8 may be better than ¹⁸F-ML-10 for apoptosis imaging. We propose that PET imaging with ¹⁸F-ML-10 or ¹⁸F-ML-8 combined with ¹⁸F-FDG is an alternative for detecting and assessing MI.     

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [¹⁸F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([¹⁸F]FSL25.1188; [¹⁸F]6), as a candidate ¹⁸F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [¹⁸F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [¹⁸F]6 binding in MAO-B rich regions. PET imaging study of [¹⁸F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [¹⁸F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [¹⁸F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [¹⁸F]6 is a promising MAO-B PET radioligand. Further evaluation of [¹⁸F]6 and structurally related ¹⁸F-analogs are underway to identify an optimized candidate for clinical research studies.     

Preclinical comparison study between [18F]fluoromethyl-PBR28 and its deuterated analog in a rat model of neuroinflammation

We designed and synthesized deuterium-substituted [¹⁸F]fluoromethyl-PBR28 ([¹⁸F]1-d₂) as a novel translocator protein 18?kDa (TSPO)-targeted radioligand with enhanced in vivo stability. The comparison studies between [¹⁸F]fluoromethyl-PBR28 ([¹⁸F]1) and its deuterate analog ([¹⁸F]1-d₂) were investigated in terms of in vitro binding affinity, lipophilicity and in vivo stability. In addition, the accuracies of both radioligands were determined by comparing the PET imaging data in the same LPS-induced neuroinflammation rat model. Both aryloxyanilide analogs showed similar lipophilicity and in vitro affinity for TSPO. However, [¹⁸F]1-d₂ provided significantly lower femur uptake than [¹⁸F]1 (1.5?±?1.2 vs. 4.1?±?1.7%ID/g at 2?h post-injection) in an ex vivo biodistribution study. [¹⁸F]1-d₂ was also selectively accumulated in the inflammatory lesion with the binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in tissue (BP_ND?=?3.17?±?0.48), in a LPS-induced acute neuroinflammation rat model, comparable to that of [¹⁸F]1 (BP_ND?=?2.13?±?0.51). These results indicate that [¹⁸F]1-d₂ had higher in vivo stability, which resulted in an enhanced target-to-background ratio compared to that induced by [¹⁸F]1.