One-Step Synthesis of [18F]Aromatic Electrophile Prosthetic Groups via Organic Photoredox Catalysis.

To avoid the harsh conditions that are oftentimes adopted in direct radiofluorination reactions, conjugation of bioactive ligands with 18F-labeled prosthetic groups has become an important strategy to construct novel PET agents under mild conditions when the ligands are structurally sensitive. Prosthetic groups with [18F]fluoroarene motifs are especially appealing because of their stability in physiological environments. However, their preparation can be intricate, often requiring multistep radiosynthesis with functional group conversions to prevent the decomposition of unprotected reactive prosthetic groups during the harsh radiofluorination. Here, we report a general and simple method to generate a variety of highly reactive 18F-labeled electrophiles via one-step organophotoredox-mediated radiofluorination. The method benefits from high step-economy, reaction efficiency, functional group tolerance, and easily accessible precursors. The obtained prosthetic groups have been successfully applied in PET agent construction and subsequent imaging studies, thereby demonstrating the feasibility of this synthetic method in promoting imaging and biomedical research.

First-in-Human Evaluation of [18F]FDOPA Produced by Organo-Photoredox Reactions.

Photoredox is a powerful synthetic tool in organic chemistry and has been widely used in various fields, including nuclear medicine and molecular imaging. In particular, acridinium-based organophotoredox radiolabeling has significantly impacted the production and discovery of positron emission tomography (PET) agents. Despite their extensive use in preclinical research, no PET agents synthesized by acridinium photoredox labeling have been tested in humans. [18F]FDOPA is clinically used for tumor diagnosis and the evaluation of neuropsychiatric disorders, but its application is limited by complex synthesis methods, the need for expensive modules, and/or the high cost of consumable materials/cassettes. In this report, we integrated a photoredox labeling unit with an automated module and produced [18F]FDOPA for human study. This research not only represents the first human study of a PET agent generated by acridinium-based organophotoredox reactions but also demonstrates the safety of this novel labeling method, serving as a milestone/reference for the clinical translation of other PET agents generated by this technique in the future.

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting In Vivo Distribution Using Multiamine Macrocycles.

The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

Manufacturing 6-[18F]Fluoro-L-DOPA via Flow Chemistry-Enhanced Photoredox Radiofluorination.

In this study, we introduce a practical methodology for the synthesis of PET probes by seamlessly combining flow chemistry with photoredox radiofluorination. The clinical PET tracer 6-[18F]FDOPA was smoothly prepared in a 24.3% non-decay-corrected yield with over 99.0% radiochemical purity (RCP) and enantiomeric excess (ee), notably by a simple cartridge-based purification. The flow chemistry-enhanced photolabeling method supplies an efficient and versatile solution for the synthesis of 6-[18F]FDOPA and for more PET tracer development.

Chelator boosted tumor-retention and pharmacokinetic properties: development of 64Cu labeled radiopharmaceuticals targeting neurotensin receptor.

PURPOSE: Accumulating evidence suggests that neurotensin (NTS) and neurotensin receptors (NTSRs) play key roles in lung cancer progression by triggering multiple oncogenic signaling pathways. This study aims to develop Cu-labeled neurotensin receptor 1 (NTSR1)-targeting agents with the potential for both imaging and therapeutic applications. METHOD: A series of neurotensin receptor antagonists (NRAs) with variable propylamine (PA) linker length and different chelators were synthesized, including [64Cu]Cu-CB-TE2A-iPA-NRA ([64Cu]Cu-4a-c, i = 1, 2, 3), [64Cu]Cu-NOTA-2PA-NRA ([64Cu]Cu-4d), [64Cu]Cu-DOTA-2PA-NRA ([64Cu]Cu-4e, also known as [64Cu]Cu-3BP-227), and [64Cu]Cu-DOTA-VS-2PA-NRA ([64Cu]Cu-4f). The series of small animal PET/CT were conducted in H1299 lung cancer model. The expression profile of NTSR1 was also confirmed by IHC using patient tissue samples. RESULTS: For most of the compounds studied, PET/CT showed prominent tumor uptake and high tumor-to-background contrast, but the tumor retention was strongly influenced by the chelators used. For previously reported 4e, [64Cu]Cu-labeled derivative showed initial high tumor uptake accompanied by rapid tumor washout at 24 h. The newly developed [64Cu]Cu-4d and [64Cu]Cu-4f demonstrated good tumor uptake and tumor-to-background contrast at early time points, but were less promising in tumor retention. In contrast, our lead compound [64Cu]Cu-4b demonstrated 9.57 ± 1.35, 9.44 ± 2.38 and 9.72 ± 4.89%ID/g tumor uptake at 4, 24, and 48 h p.i., respectively. Moderate liver uptake (11.97 ± 3.85, 9.80 ± 3.63, and 7.72 ± 4.68%ID/g at 4, 24, and 48 h p.i.) was observed with low uptake in most other organs. The PA linker was found to have a significant effect on drug distribution. Compared to [64Cu]Cu-4b, [64Cu]Cu-4a had a lower background, including a greatly reduced liver uptake, while the tumor uptake was only moderately reduced. Meanwhile, [64Cu]Cu-4c showed increased uptake in both the tumor and the liver. The clinical relevance of NTSR1 was also demonstrated by the elevated tumor expression in patient tissue samples. CONCLUSIONS: Through the side-by-side comparison, [64Cu]Cu-4b was identified as the lead agent for further evaluation based on its high and sustained tumor uptake and moderate liver uptake. It can not only be used to efficiently detect NTSR1 expression in lung cancer (for diagnosis, patient screening, and treatment monitoring), but also has the great potential to treat NTSR-positive lesions once chelating to the beta emitter 67Cu.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Expression of the Calcium-Sensing Receptor on Normal and Abnormal Parathyroid and Thyroid Tissue.

INTRODUCTION: Current imaging techniques have several limitations in detecting parathyroid glands. We have investigated the calcium-sensing receptor (CaSR) as a potential target for specifically labeling parathyroid glands for radiologic detection. For accurate imaging it is vital that a large differential expression exists between the target tissue and adjacent structures. We sought to investigate the relative abundance of the CaSR in normal and abnormal parathyroid tissue, as well as normal and abnormal thyroid. METHODS: Existing clinical specimens were selected that represented a wide variety of pathologically and clinically confirmed malignant and benign thyroid and parathyroid specimens. Sections were stained for the CaSR using immunohistochemistry and scored for intensity and abundance of expression. (H score = intensity scored from 0 to 3 multiplied by the % of cells at each intensity. Range 0-300). RESULTS: All parathyroid specimens expressed the CaSR to a high degree. Normal parathyroid had the highest H score (271, s.d. 25.4). Abnormal parathyroid specimens were slightly lower but still much higher than normal thyroid (H score 38.3, s.d. 23.3). Medullary thyroid cancer also expressed the CaSR significantly higher than normal thyroid (H score 182, s.d. 69.1, P < 0.001) but below parathyroid levels. Hürthle cell carcinoma expressed the CaSR to a lesser degree but higher than normal thyroid (H score 101, s.d. 46.4, P = 0.0037). CONCLUSIONS: The CaSR is differentially expressed on parathyroid tissue making it a feasible target for parathyroid imaging. False positives might be anticipated with medullary and Hürthle cell cancers.

Development of 18F-Labeled hydrophilic trans-cyclooctene as a bioorthogonal tool for PET probe construction.

We report the development of a hydrophilic 18F-labeled a-TCO derivative [18F]3 (log P = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [18F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.

Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy-lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition.

Development of [18F]F-5-OMe-Tryptophans through Photoredox Radiofluorination: A New Method to Access Tryptophan-Based PET Agents.

Although various radiolabeled tryptophan analogs have been developed to monitor tryptophan metabolism using positron emission tomography (PET) for various human diseases including melanoma and other cancers, their application can be limited due to the complicated synthesis process. In this study, we demonstrated that photoredox radiofluorination represents a simple method to access novel tryptophan-based PET agents. In brief, 4-F-5-OMe-tryptophans (l/d-T13) and 6-F-5-OMe-tryptophans (l/d-T18) were easily synthesized. The 18F-labeled analogs were produced by photoredox radiofluorination with radiochemical yields ranging from 2.6 ± 0.5% to 32.4 ± 4.1% (3 ≤ n ≤ 5, enantiomeric excess ≥ 99.0%) and over 98.0% radiochemical purity. Small animal imaging showed that l-[18F]T13 achieved 9.58 ± 0.26%ID/g tumor uptake and good contrast in B16F10 tumor-bearing mice (n = 3). Clearly, l-[18F]T13 exhibited prominent tumor uptake, warranting future evaluations of its potential usage in precise immunotherapy monitoring.

11C, 12C and 13C-Cyanation of Electron-Rich Arenes via Organic Photoredox Catalysis.

As a non-invasive imaging technology, positron emission tomography (PET) plays a crucial role in personalized medicine, including early diagnosis, patient screening, and treatment monitoring. The advancement of PET research depends on the discovery of new PET agents, which requires the development of simple and efficient radiolabeling methods in many cases. As bioisosteres for halogen and carbonyl moieties, nitriles are important functional groups in pharmaceutical and agrochemical compounds. Here, we disclose a mild organophotoredox-catalyzed method for efficient cyanation of a broad spectrum of electron-rich arenes, including abundant and readily available veratroles and pyrogallol trimethyl ethers. Notably, the transformations not only are compatible with various affordable 12C and 13C-cyanide sources, but also could be applied to carbon-11 synthons to incorporate [11C]nitriles into arenes. The aryl [11C]nitriles can be further derivatized to [11C]carboxylic acids, [11C]amides, and [11C]alkyl amines. The newly developed reaction can serve as a powerful tool for generating new PET agents.

Direct C-H Radiocyanation of Arenes via Organic Photoredox Catalysis.

Innovative labeling methods to incorporate the short-lived positron emitter carbon-11(11C) into bioactive molecules are attractive for positron emission tomography (PET) tracer discovery. Herein, we report a direct C-H radiocyanation method that incorporates [11C]cyanide (11CN-) to a series of functional electron-rich arenes via photoredox catalysis. This photoredox-mediated radiocyanation can proceed in an aerobic environment and is not moisture sensitive, which allows for ease of reaction setup and for scalable synthesis of 11C-aryl nitriles from readily available precursors.

Modular PET Agent Construction Strategy through Strain-Promoted Double-Click Reagent with Efficient Photoclick Step.

An efficient modular strategy for rapid assembly of positron emission tomography (PET) agents has been developed. The use of a sequential, rapid, and selective double-click reaction allows for a combinatorial approach to the cross-linking of positron emitter-bearing prosthetic groups with various ligands. The strain-promoted azide alkyne cyclization (SPAAC) coupling of 18F-labeled azide synthon with MC-DIBOD, a cyclooctadiyne with one of the triple bonds caged as a cyclopropenone moiety, produces a stable intermediate. A brief exposure of the latter to 350-420 nm light removes protection of the second triple bond allowing for the addition of an azide-tagged biomolecule. The utility of this strategy has been demonstrated by the construction of several PET agents. The value of modularity was demonstrated in the preparation of PSMA PET agents, where the hydrophilicity was easily modified to improve tumor to background contrast.

The Synthesis and Initial Evaluation of MerTK Targeted PET Agents.

MerTK (Mer tyrosine kinase), a receptor tyrosine kinase, is ectopically or aberrantly expressed in numerous human hematologic and solid malignancies. Although a variety of MerTK targeting therapies are being developed to enhance outcomes for patients with various cancers, the sensitivity of tumors to MerTK suppression may not be uniform due to the heterogeneity of solid tumors and different tumor stages. In this report, we develop a series of radiolabeled agents as potential MerTK PET (positron emission tomography) agents. In our initial in vivo evaluation, [18F]-MerTK-6 showed prominent uptake rate (4.79 ± 0.24%ID/g) in B16F10 tumor-bearing mice. The tumor to muscle ratio reached 1.86 and 3.09 at 0.5 and 2 h post-injection, respectively. In summary, [18F]-MerTK-6 is a promising PET agent for MerTK imaging and is worth further evaluation in future studies.

Arene radiofluorination enabled by photoredox-mediated halide interconversion.

Positron emission tomography (PET) is a powerful imaging technology that can visualize and measure metabolic processes in vivo and/or obtain unique information about drug candidates. The identification of new and improved molecular probes plays a critical role in PET, but its progress is somewhat limited due to the lack of efficient and simple labelling methods to modify biologically active small molecules and/or drugs. Current methods to radiofluorinate unactivated arenes are still relatively limited, especially in a simple and site-selective way. Here we disclose a method for constructing C-18F bonds through direct halide/18F conversion in electron-rich halo(hetero)arenes. [18F]F- is introduced into a broad spectrum of readily available aryl halide precursors in a site-selective manner under mild photoredox conditions. Notably, our direct 19F/18F exchange method enables rapid PET probe diversification through the preparation and evaluation of an [18F]-labelled O-methyl tyrosine library. This strategy also results in the high-yielding synthesis of the widely used PET agent L-[18F]FDOPA from a readily available L-FDOPA analogue.

Biodistribution of Biomimetic Drug Carriers, Mononuclear Cells, and Extracellular Vesicles, in Nonhuman Primates.

Discovery of novel drug delivery systems to the brain remains a key task for successful treatment of neurodegenerative disorders. Herein, the biodistribution of immunocyte-based carriers, peripheral blood mononuclear cells (PBMCs), and monocyte-derived EVs are investigated in adult rhesus macaques using longitudinal PET/MRI imaging. 64 Cu-labeled drug carriers are introduced via different routes of administration: intraperitoneal (IP), intravenous (IV), or intrathecal (IT) injection. Whole body PET/MRI (or PET/CT) images are acquired at 1, 24, and 48 h post injection of 64 Cu-labeled drug carriers, and standardized uptake values (SUVmean and SUVmax ) in the main organs are estimated. The brain retention for both types of carriers increases based on route of administration: IP < IV < IT. Importantly, a single IT injection of PBMCs produces higher brain retention compared to IT injection of EVs. In contrast, EVs show superior brain accumulation compared to the cells when administered via IP and IV routes, respectively. Finally, a comprehensive chemistry panel of blood samples demonstrates no cytotoxic effects of either carrier. Overall, living cells and EVs have a great potential to be used for drug delivery to the brain. When identifying the ideal drug carrier, the route of administration could make big differences in CNS drug delivery.

Development of 18F-Labeled Vinyl Sulfone-PSMAi Conjugates as New PET Agents for Prostate Cancer Imaging.

Radiolabeled prostate-specific membrane antigen (PSMA) ligands have been rapidly adopted as part of patient care for prostate cancer. In this study, a new series of 18F-labeled PSMA-targeting agents was developed based on the high-affinity Glu-ureido-Lys scaffold and 18F-vinyl sulfones (VSs), the tumor uptake and tumor/major organ contrast of which could be tuned by pharmacokinetic linkers within the molecules. In particular, 18F-PEG3-VS-PSMAi showed the highest tumor uptake (12.1 ± 2.2%ID/g at 0.5 h p.i.) and 18F-PEG2-VS-PSMAi showed the highest tumor-to-liver ratio (T/L = 3.7 ± 1.0, 4.8 ± 1.2, and 6.3 ± 1.1 at 0.5, 1.5, and 3 h p.i. respectively). Significantly, compared with the FDA-approved 68Ga-PSMA-11, the newly developed 18F-PEG3-VS-PSMAi has an almost double tumor uptake (P < 0.0001) when tested in the same animal model. In conclusion, 18F-VS-labeled PSMA ligands are promising PET agents with prominent tumor uptake and high contrast. The lead agents 18F-PEG2-VS-PSMAi and 18F-PEG3-VS-PSMAi warrant further evaluation in prostate cancer patients.

Image-guided selection of Gd@C-dots as sensitizers to improve radiotherapy of non-small cell lung cancer.

BACKGROUND: Recently, gadolinium-intercalated carbon dots (Gd@C-dots) have demonstrated potential advantages over traditional high-Z nanoparticles (HZNPs) as radiosensitizers due to their high stability, minimal metal leakage, and remarkable efficacy. RESULTS: In this work, two Gd@C-dots formulations were fabricated which bore carboxylic acid (CA-Gd@C-dots) or amino group (pPD-Gd@C-dots), respectively, on the carbon shell. While it is critical to develop innovative nanomateirals for cancer therapy, determining their tumor accumulation and retention is equally important. Therefore, in vivo positron emission tomography (PET) was performed, which found that 64Cu-labeled pPD-Gd@C-dots demonstrated significantly improved tumor retention (up to 48 h post injection) compared with CA-Gd@C-dots. Indeed, cell uptake of 64Cu-pPD-Gd@C-dots reached close to 60% of total dose compared with ~ 5% of 64Cu-CA-Gd@C-dots. pPD-Gd@C-dots was therefore further evaluated as a new radiosensitizer for non-small cell lung cancer treatment. While single dose radiation plus intratumorally injected pPD-Gd@C-dots did lead to improved tumor suppression, the inhibition effect was further improved with two doses of radiation. The persistent retention of pPD-Gd@C-dots in tumor region eliminates the need of reinjecting radiosensitizer for the second radiation. CONCLUSIONS: PET offers a simple and straightforward way to study nanoparticle retention in vivo, and the selected pPD-Gd@C-dots hold great potential as an effective radiosensitizer.