[18F]Difluorocarbene for positron emission tomography.

The advent of total-body positron emission tomography (PET) has vastly broadened the range of research and clinical applications of this powerful molecular imaging technology1. Such possibilities have accelerated progress in fluorine-18 (18F) radiochemistry with numerous methods available to 18F-label (hetero)arenes and alkanes2. However, access to 18F-difluoromethylated molecules in high molar activity is mostly an unsolved problem, despite the indispensability of the difluoromethyl group for pharmaceutical drug discovery3. Here we report a general solution by introducing carbene chemistry to the field of nuclear imaging with a [18F]difluorocarbene reagent capable of a myriad of 18F-difluoromethylation processes. In contrast to the tens of known difluorocarbene reagents, this 18F-reagent is carefully designed for facile accessibility, high molar activity and versatility. The issue of molar activity is solved using an assay examining the likelihood of isotopic dilution on variation of the electronics of the difluorocarbene precursor. Versatility is demonstrated with multiple [18F]difluorocarbene-based reactions including O-H, S-H and N-H insertions, and cross-couplings that harness the reactivity of ubiquitous functional groups such as (thio)phenols, N-heteroarenes and aryl boronic acids that are easy to install. The impact is illustrated with the labelling of highly complex and functionalized biologically relevant molecules and radiotracers.

A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level.

"AquaF" Building Blocks for Water-Compatible SN2 18F-Fluorination of Small-Molecule Radiotracers.

Despite the widespread use of hydrophilic building blocks to incorporate 18F and improve tracer pharmacokinetics, achieving effective leaving group-mediated nucleophilic 18F-fluorination in water (excluding 18F/19F-exchange) remains a formidable challenge. Here, we present a water-compatible SN2 leaving group-mediated 18F-fluorination method employing preconjugated "AquaF" (phosphonamidic fluorides) building blocks. Among 19 compact tetracoordinated pentavalent P(V)-F candidates, the "AquaF" building blocks exhibit superior water solubility, sufficient capacity for 18F-fluorination in water, and excellent in vivo metabolic properties. Two nitropyridinol leaving groups, identified from a pool of leaving group candidates that further enhance the precursor water solubility, enable 18F-fluorination in water with a 10-2 M-1 s-1 level reaction rate constant (surpassing the 18F/19F-exchange) at room temperature. With the exergonic concerted SN2 18F-fluorination mechanism confirmed, this 18F-fluorination method achieves ∼90% radiochemical conversions and reaches a molar activity of 175 ± 40 GBq/µmol (using 12.2 GBq initial activity) in saline for 12 "AquaF"-modified proof-of-concept functional substrates and small-molecule 18F-tracers. [18F]AquaF-Flurpiridaz demonstrates significantly improved radiochemical yield and molar activity compared to 18F-Flurpiridaz, alongside enhanced cardiac uptake and heart/liver ratio in targeted myocardial perfusion imaging, providing a comprehensive illustration of "AquaF" building blocks-assisted water-compatible SN2 18F-fluorination of small-molecule radiotracers.

Construction and Preliminary Evaluation of Two 18F-Labeled Radiopharmaceuticals for Myocardial Perfusion Imaging.

AIMS: PET myocardial perfusion imaging (MPI) is the gold standard for the noninvasive diagnosis of ischemic myocardial. Construction of 18F-labeled PET MPI probe showed benefits to reduce the imaging cost, and enhance the image quality and patient-friendliness. METHODS: Two 18F-labeled MPI probes (18F-BoMPI) were developed. Detailed in vitro/vivo evaluation including photophysical properties, in vitro stability, myocardial cell uptake kinetics and mechanisms, cytotoxicity and IC50, biodistribution and plasma clearance curve were investigated. Resting and stressing myocardial perfusion PET imaging were performed in healthy and myocardial ischemic mice. RESULTS: 18F-BoMPI could be quickly labeled and easily postprocessed, and demonstrated excellent in vitro stability. Cell assays indicated that 18F-BoMPI exhibited mitochondria-targeting but potential-independent myocardial uptake. In vivo evaluation revealed the effective myocardial uptake and rapid background clearance. PET MPI confirmed effective probe accumulation in the healthy heart, but rapidly clearance in the background, making heart clearly delineated in the images. Ischemic myocardial could be clearly distinguished as the region of radioactivity sparsity in PET MPI. CONCLUSION: The 18F-labeled probes showed great potentials to reduce the practicability threshold of PET MPI.

Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.

Legumain-Triggered Macrocyclization of Radiofluorinated Tracer for Enhanced PET Imaging.

Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an 18F-labeled radiotracer [18F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([18F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient 18F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.

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.

Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies.

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (18F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[18F]FH3RESCA)-NH2) or the unlabeled chelator (H-GGGK((±)-H3RESCA)-NH2) followed by labeling with Al[18F]F, respectively. The overall radiochemical yields were 15-43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18F-labeling method to prepare nanobodies (Nb-Al[18F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

Cyclohexanediamine Triazole (CHDT) Functionalization Enables Labeling of Target Molecules with Al18F/68Ga/111In.

The Al18F-labeling approach offers a one-step access to radiofluorinated biomolecules by mimicking the labeling process for radiometals. Although these labeling conditions are considered to be mild compared to classic radiofluorinations, improvements of the chelating units have led to the discovery of (±)-H3RESCA, which allows Al18F-labeling already at ambient temperature. While the suitability of (±)-H3RESCA for functionalization and radiofluorination of proteins is well established, its use for small molecules or peptides is less explored. Herein, we advanced this acyclic pentadentate ligand by introducing an alkyne moiety for the late-stage functionalization of biomolecules via click chemistry. We show that in addition to Al18F-labeling, the cyclohexanediamine triazole (CHDT) moiety allows stable complexation of 68Ga and 111In. Three novel CHDT-functionalized PSMA inhibitors were synthesized and their Al18F-, 68Ga-, and 111In-labeled analogs were subjected to a detailed in vitro radiopharmacological characterization. Stability studies in vitro in human serum revealed among others a high kinetic inertness of all radiometal complexes. Furthermore, the Al18F-labeled PSMA ligands were characterized for their biodistribution in a LNCaP derived tumor xenograft mouse model by PET imaging. One radioligand, Al[18F]F-CHDT-PSMA-1, bearing a small azidoacetyl linker at the glutamate-urea-lysine motif, provided an in vivo performance comparable to that of [18F]PSMA-1007 but with even higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. Overall, our results highlight the suitability of the novel CHDT moiety for functionalization and radiolabeling of small molecules or peptides with Al18F, 68Ga, and 111In and the triazole ring seems to entail favorable pharmacokinetic properties for molecular imaging purposes.

18F-Radiolabeling and Evaluation of an AMD3465 Derivative for PET Imaging of CXCR4 in a Mouse Breast Tumor Model.

The exploration of pharmaceutically active agents and positron emission tomography (PET) tracers targeting CXCR4 has been a focal point in cancer research given its pivotal role in the development and progression of various cancers. While significant strides have been made in PET imaging with radiometal-labeled tracers, the landscape of 18F-labeled small molecule tracers remains relatively limited. Herein, we introduce a novel and promising derivative, [18F]SFB-AMD3465, as a targeted PET tracer for CXCR4. The compound was synthesized by modifying the pyridine ring of AMD3465, which was subsequently labeled with 18F using [18F]SFB. The study provides comprehensive insights into the design, synthesis, and biological evaluation of [18F]SFB-AMD3465. In vitro and in vivo assessments demonstrated the CXCR4-dependent, specific, and sensitive uptake of [18F]SFB-AMD3465 in the CXCR4-overexpressing 4T1 cell line and the corresponding xenograft-bearing mouse model. These findings contribute to bridging the gap in 18F-labeled PET tracers for CXCR4 and underscore the potential of [18F]SFB-AMD3465 as a PET radiotracer for in vivo CXCR4 imaging.

Synthesis and preclinical evaluation of novel 18F-vancomycin-based tracers for the detection of bacterial infections using positron emission tomography.

INTRODUCTION: Bacterial infections are a major problem in medicine, and the rapid and accurate detection of such infections is essential for optimal patient outcome. Bacterial infections can be diagnosed by nuclear imaging, but most currently available modalities are unable to discriminate infection from sterile inflammation. Bacteria-targeted positron emission tomography (PET) tracers have the potential to overcome this hurdle. In the present study, we compared three 18F-labelled PET tracers based on the clinically applied antibiotic vancomycin for targeted imaging of Gram-positive bacteria. METHODS: [18F]FB-NHS and [18F]BODIPY-FL-NHS were conjugated to vancomycin. The resulting conjugates, together with our previously developed [18F]PQ-VE1-vancomycin, were tested for stability, lipophilicity, selective binding to Gram-positive bacteria, antimicrobial activity and biodistribution. For the first time, the pharmacokinetic properties of all three tracers were compared in healthy animals to identify potential binding sites. RESULTS: [18F]FB-vancomycin, [18F]BODIPY-FL-vancomycin, and [18F]PQ-VE1-vancomycin were successfully synthesized with radiochemical yields of 11.7%, 2.6%, and 0.8%, respectively. [18F]FB-vancomycin exhibited poor in vitro and in vivo stability and, accordingly, no bacterial binding. In contrast, [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin showed strong and specific binding to Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which was outcompeted by unlabeled vancomycin only at concentrations exceeding clinically relevant vancomycin blood levels. Biodistribution showed renal clearance of [18F]PQ-VE1-vancomycin and [18F]BODIPY-FL-vancomycin with low non-specific accumulation in muscles, fat and bones. CONCLUSION: Here we present the synthesis and first evaluation of the vancomycin-based PET tracers [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin for image-guided detection of Gram-positive bacteria. Our study paves the way towards real-time bacteria-targeted diagnosis of soft tissue and implant-associated infections that are oftentimes caused by Gram-positive bacteria, even after prophylactic treatment with vancomycin.

A comparison of [18F]AlF- and 68Ga-labeled dual targeting heterodimer FAPI-RGD in malignant tumor: preclinical evaluation and pilot clinical PET/CT imaging.

Due to the heterogeneity of tumors, strategies to improve the effectiveness of dual-targeting tracers in tumor diagnostics have been intensively practiced. In this study, the radiolabeled [18F]AlF-NOTA-FAPI-RGD (denoted as [18F]AlF-LNC1007), a dual-targeting heterodimer tracer targeting both fibroblast activation protein (FAP) and integrin αvß3 to enhance specific tumor uptake and retention, was synthesized and evaluated. The tracer was compared with [68Ga]Ga-LNC1007 in preclinical and clinical settings. METHODS: The preparation of [18F]AlF- and 68Ga-labeled FAPI-RGD was carried out with an optimized protocol. The stability was tested in PBS and fetal bovine serum (FBS). Cellular uptake and in vivo distribution of the two products were compared and carried out on the U87MG cell line and its xenograft model. The safety and dosimetry of [18F]AlF-LNC1007 PET/CT scan were evaluated in six patients with malignant tumors. RESULTS: Two radiolabeling protocols of [18F]AlF-/[68Ga]Ga-LNC1007 were developed and optimized to give a high yield of tracers with good stability. In vivo microPET images showed that the two tracers exhibited comparable pharmacokinetic characteristics, with high tumor uptake and prolonged tumor retention. In vivo distribution data showed that the target-to-non-target ratios of [18F]AlF-LNC1007 were similar to[68Ga]Ga-LNC1007. A total of six patients underwent [18F]AlF-LNC1007 PET/CT evaluation while two had head-to-head [18F]FDG PET/CT scans. The total body effective dose was 9.94E-03 mSv/MBq. The biodistribution curve showed optimal normal organ uptake with high tumor uptake and long retention of up to 3h p.i., and notably, the tumor-to-background ratio increased over time. CONCLUSION: We successfully prepared an [18F]AlF-LNC1007 dual-targeting PET probe with comparable performances as [68Ga]Ga-LNC1007. With prolonged tumor retention and tumor specificity, it produced good imaging quality in preclinical and clinical translational studies, indicating that [18F]AlF-LNC1007 is a promising non-invasive tracer for detecting tumors expressing FAP and/or integrin avß3, with the prospect of clinical implementation.

First-in-human study of PSMA-targeting agent, [18F]AlF-P16-093: dosimetry and initial evaluation in prostate cancer patients.

PURPOSE: This is a first-in-human study to evaluate the radiation dosimetry of a new prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical, [18F]AlF-P16-093, and also initial investigation of its ability to detect PSMA-positive tumors using PET scans in a cohort of prostate cancer (PCa) patients. METHODS: The [18F]AlF-P16-093 was automatically synthesized with a GE TRACERlab. A total of 23 patients with histopathologically proven PCa were prospectively enrolled. Dosimetry and biodistribution study investigations were carried out on a subset of six (6) PCa patients, involving multiple time-point scanning. The mean absorbed doses were estimated with PMOD and OLINDA software. RESULTS: [18F]AlF-P16-093 was successfully synthesized, and radiochemical purity was > 95%, and average labeling yield was 36.5 ± 8.3% (decay correction, n = 12). The highest tracer uptake was observed in the kidneys, spleen, and liver, contributing to an effective dose of 16.8 ± 1.3 µSv/MBq, which was ~ 30% lower than that of [68Ga]Ga-P16-093. All subjects tolerated the PET examination well, and no reportable side-effects were observed. The PSMA-positive tumors displayed rapid uptake, and they were all detectable within 10 min, and no additional lesions were observed in the following multi-time points scanning. Each patient had at least one detectable tumor lesion, and a total of 356 tumor lesions were observed, including intraprostatic, lymph node metastases, bone metastases, and other soft tissue metastases. CONCLUSIONS: We report herein a streamlined method for high yield synthesis of [18F]AlF-P16-093. Preliminary study in PCa patients has demonstrated its safety and acceptable radiation dosimetry. The initial diagnostic study indicated that [18F]AlF-P16-093 PET/CT is efficacious and potentially useful for a widespread application in the diagnosis of PCa patients.

ImmunoPET/CT imaging of clear cell renal cell carcinoma with [18F]RCCB6: a first-in-human study.

PURPOSE: The cluster of differentiation (CD70) is a potential biomarker of clear cell renal cell carcinoma (ccRCC). This study aims to develop CD70-targeted immuno-positron emission tomography/computed tomography (immunoPET/CT) imaging tracers and explore the diagnostic value in preclinical studies and the potential value in detecting metastases in ccRCC patients. METHODS: Four novel CD70-specific single-domain antibodies (sdAbs) were produced and labelled with 18F by the aluminium fluoride restrained complexing agent (AlF-RESCA) method to develop radiotracers. The visualisation properties of the tracers were evaluated in a subcutaneous ccRCC patient-derived xenograft (PDX) model. In a registered prospective clinical trial (NCT06148220), six patients with pathologically confirmed RCC were included and underwent immunoPET/CT examination exploiting one of the developed tracers (i.e., [18F]RCCB6). RESULTS: We engineered four sdAbs (His-tagged RCCB3 and RCCB6, His-tag-free RB3 and RB6) specifically targeting recombinant human CD70 without cross-reactivity to murine CD70. ImmunoPET/CT imaging with [18F]RCCB3 and [18F]RCCB6 demonstrated a high tumour-to-background ratio in a subcutaneous ccRCC PDX model, with the latter showing better diagnostic potential supported by higher tumour uptake and lower bone accumulation. In comparison, [18F]RB6, developed by sequence optimisation, has significantly lower kidney accumulation than that of [18F]RCCB6. In a pilot translational study, [18F]RCCB6 immunoPET/CT displayed ccRCC metastases in multiple patients and demonstrated improved imaging contrast and diagnostic value than 18F-FDG PET/CT in a patient with ccRCC. CONCLUSION: The work successfully developed a series of CD70-targeted immunoPET/CT imaging tracers. Of them, [18F]RCCB6 clearly and specifically identified inoculated ccRCCs in preclinical studies. Clinical translation of [18F]RCCB6 suggests potential for identifying recurrence and/or metastasis in ccRCC patients.

KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis.

Pancreatic ductal adenocarcinoma (PDAC), which has a poor prognosis and nonspecific symptoms and progresses rapidly, is the most common pancreatic cancer type. Inhibitors targeting KRAS G12D and G12C mutations have been pivotal in PDAC treatment. Cancer cells with different KRAS mutations exhibit various degrees of glutamine dependency; in particular, cells with KRAS G12D mutations exhibit increased glutamine uptake. (2S,4R)-4-[18F]FGln has recently been developed for clinical cancer diagnosis and tumor cell metabolism analysis. Thus, we verified the heterogeneity of glutamine dependency in PDAC models with different KRAS mutations by a visual and noninvasive method with (2S,4R)-4-[18F]FGln. Two tumor-bearing mouse models (bearing the KRAS G12D or G12C mutation) were injected with (2S,4R)-4-[18F]FGln, and positron emission tomography (PET) imaging features and biodistribution were observed and analyzed. The SUVmax in the regions of interest (ROI) was significantly higher in PANC-1 (G12D) tumors than in MIA PaCa-2 (G12C) tumors. Biodistribution analysis revealed higher tumor accumulation of (2S,4R)-4-[18F]FGln and other metrics, such as T/M and T/B, in the PANC-1 mouse models compared to those in the MIAPaCa-2 mouse models. In conclusion, PDAC cells with the KRAS G12D and G12C mutations exhibit various degrees of (2S,4R)-4-[18F]FGln uptake, indicating that (2S,4R)-4-[18F]FGln might be applied to detect KRAS G12C and G12D mutations and provide treatment guidance.

Preclinical Evaluation of a Radiolabeled Pan-RAF Inhibitor for RAF-Specific PET/CT Imaging.

Abnormalities in the RAS-RAF signaling pathway occur in many solid tumors, leading to aberrant tumor proliferation, invasion, and metastasis. Due to the elusive pharmacology of RAS, RAF inhibitors have become the main targeted therapeutic drugs. Naporafenib (LXH-254) is a high-affinity pan-RAF inhibitor with FDA Fast Track Qualification. We sought to develop an 18F-labeled molecular probe from LXH-254 for PET imaging of tumors overexpressing RAF to noninvasively screen patients for susceptibility to targeted RAF therapy. To reduce the lipid solubility, LXH-254 was designed with triethylene glycol di(p-toluenesulfonate) (TsO-PEG3-OTs) to obtain the precursor (LXH-254-OTs) and a nucleophilic substitution reaction with 18F to obtain the tracer ([18F]F-LXH-254). [18F]F-LXH-254 exhibited good molar activity (7.16 ± 0.81 GBq/µmol), radiochemical purity (>95%), and stability. Micro-PET imaging revealed distinct radioactivity accumulation of [18F]F-LXH-254 in tumors in the imaging groups, whereas in the blocked group, the tumor radioactivity level was consistent with the background tissue, illustrating the affinity and specificity of [18F]F-LXH-254 in targeting RAF. Overall, [18F]F-LXH-254 is a promising radiotracer for screening and diagnosing patients with RAF-related disease and monitoring their treatment. This is the first attempt at using an 18F-labeled RAF-specific radiotracer.

Development and Preclinical Evaluation of 18F-Labeled PEGylated Sansalvamide A Decapeptide for Noninvasive Evaluation of Hsp90 Status in Pancreas Cancer.

Heat shock protein 90 (Hsp90) is a promising target for cancer therapy and imaging. Accurate detection of Hsp90 levels in tumors via noninvasive PET imaging might be beneficial for management. To achieve this, the precursor compound Dimer-Sansalvamide A (Dimer-San A) was PEGylated and modified by conjugating it with the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The 18F-labeled PEGylated Dimer-SanA decapeptide (18F-PEGylated San A) was completed within 30 min using a two-step process. In vitro stability and specificity were assessed, including competition studies with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). MicroPET imaging was performed on PL45 tumor-bearing mice to evaluate probe accumulation and tumor-to-muscle ratios. Biodistribution studies determined the route of excretion. The probe resulted in a radiochemical yield of 23.11% with a purity exceeding 95%. In vitro, 18F-PEGylated San A exhibited high stability and selectively accumulated in Hsp90-positive PL45 cells, with binding effectively blocked by the Hsp90 inhibitor 17AAG, confirming its specificity. MicroPET imaging of PL45 tumor-bearing mice showed significant probe accumulation in tumor tissues at 1 and 2 h postinjection (4.06 ± 0.30 and 3.72 ± 0.61%ID/g, respectively), with optimal tumor-to-muscle ratios observed at 2 h postinjection (6.09 ± 1.92). While 18F-PEGylated San A demonstrates enhanced water solubility, as indicated by increased kidney uptake relative to liver accumulation. The study successfully incorporated PEG units to create the novel probe 18F-PEGylated San A targeting to Hsp90 without affecting its targeting capability, aimed at improving the pharmacokinetics and PET imaging of Hsp90 expression noninvasively.

Radiosynthesis and Preclinical Evaluation of m-[18F]FET and [18F]FET-OMe as Novel [18F]FET Analogs for Brain Tumor Imaging.

O-([18F]Fluoroethyl)-l-tyrosine ([18F]FET) is actively transported into the brain and cancer cells by LAT1 and possibly other amino acid transporters, which enables brain tumor imaging by positron emission tomography (PET). However, tumor delivery of this probe in the presence of competing amino acids may be limited by a relatively low affinity for LAT1. The aim of the present work was to evaluate the meta-substituted [18F]FET analog m-[18F]FET and the methyl ester [18F]FET-OMe, which were designed to improve tumor delivery by altering the physicochemical, pharmacokinetic, and/or transport properties. Both tracers could be prepared with good radiochemical yields of 41-56% within 66-90 min. Preclinical evaluation with [18F]FET as a reference tracer demonstrated reduced in vitro uptake of [18F]FET-OMe by U87 glioblastoma cells and no advantage for in vivo tumor imaging. In contrast, m-[18F]FET showed significantly improved in vitro uptake and accelerated in vivo tumor accumulation in an orthotopic glioblastoma model. As such, our work identifies m-[18F]FET as a promising alternative to [18F]FET for brain tumor imaging that deserves further evaluation with regard to its transport properties and in vivo biodistribution.

Switching the Chemoselectivity in the Preparation of [18F]FNA-N-CooP, a Free Thiol-Containing Peptide for Targeted Positron Emission Tomography Imaging of Fatty Acid Binding Protein 3.

Fatty acid binding protein 3 (FABP3) is expressed both in tumor cells and in the tumor vasculature, making it a potential target for medical imaging and therapy. In this study, we aimed to radiolabel a CooP peptide with a free amino and thiol group, and evaluate the radiolabeled product [18F]FNA-N-CooP for imaging FABP3 expression in breast cancer brain metastases by positron emission tomography. [18F]FNA-N-CooP was prepared by highly chemoselective N-acylation and characterized using different chemical approaches. We validated its binding to the target using in vitro tissue section autoradiography and performed stability tests in vitro and in vivo. [18F]FNA-N-CooP was successfully synthesized in 16.8% decay-corrected radiochemical yield with high radiochemical purity (98.5%). It exhibited heterogeneous binding on brain metastasis tissue sections from a patient with breast cancer, with foci of radioactivity binding corresponding to FABP3 positivity. Furthermore, the tracer binding was reduced by 55% in the presence of nonradioactive FNA-N-CooP a blocker, indicating specific tracer binding and that FABP3 is a viable target for [18F]FNA-N-CooP. Favorably, the tracer did not bind to necrotic tumor tissue. However, [18F]FNA-N-CooP displayed limited stability both in vitro in mouse plasma or human serum and in vivo in mouse, therefore further studies are needed to improve the stability [18F]FNA-N-CooP to be used for in vivo applications.

Difluoromethoxide Is a Strong Leaving Group in the Photoredox Deoxyradiofluorination of 2-Phenylpyridines.

A 2-phenyl-3-difluoromethoxy-pyridinyl moiety features in potent phosphodiesterase 4D inhibitors that are considered to be candidate radiotracers for positron emission tomography if they are labeled with fluorine-18. Fluorine-18 could be installed as desired at the 3'-phenyl position with acridinium-mediated photoredox radiodeoxyfluorination in homologues bearing variously substituted 3'-aryloxy groups. However, a distal 3-difluoromethoxide (-OCHF2) group strongly competes as a leaving group, especially when an electron-deficient aryloxy group is present at position 3'. A yield of up to 50% may occur without observable 19F for 18F exchange.