A pyrogallol-formaldehyde resin (PGR) column was developed for one-step pre-purification of 68Ge/68Ga generator eluates. The resin selectively adsorbs tetravalent cations (Ge4+, Sn4+, Ti4+) with efficiencies of 81.8%, 42.4%, and 29.8% in standards, reducing Sn4+ and Ti4+ to undetectable levels in generator eluates. Formaldehyde leaching was negligible, and cell viability remained 98% ± 1.7%, confirming biocompatibility. 68Ge breakthrough decreased >20-fold (0.0008 ± 0.0001% to 0.00003 ± 0.00002%). Manual [68Ga]Ga-DOTA-TATE labeling yield increased from 58.9 ± 6% to 75.9 ± 1.8% (n = 10). The column is reusable for ≥10 runs. PGR enables high-purity, high-yield 68Ga radiopharmaceutical synthesis without additional processing time, resolving pH-dependent tetravalent cation interference via colloid mitigation.
Introduction Platelet-derived growth factor receptor beta (PDGFRβ) is a widely used and important marker for mesenchymal cells, including fibroblasts, vascular smooth muscle cells, and pericytes. Dysfunction of the mesenchymal compartment is fundamentally linked to fibrotic disease and cancer. ATH001 is an Affibody molecule being developed for Positron Emission Tomography (PET) imaging for improved diagnosis and therapy follow-up in disease involving aberrant mesenchymal regulation. Here we present a novel NOTA-conjugated variant of ATH001 for PET imaging of PDGFRβ. Methods Transcription of PDGFRβ in human cells and tissues was investigated in the reference scRNAseq database Tabula Sapiens. ATH001 was generated by custom chemical solid phase peptide synthesis, followed by conjugation with NOTA via maleimide chemistry at a C-terminal cysteine. The resulting precursor NOTA-ATH001 was radiolabeled with Gallium-68. Affinity towards recombinant PDGFRβ was determined using a Surface Plasmon Resonance assay. [68Ga]Ga-NOTA-ATH001 binding and biodistribution was evaluated by in vitro autoradiography and in vivo PET scanning of U87 xenografted immunodeficient mice. Results PDGFRβ transcription was restricted to mesenchymal components and was particularly high in pericytes and stellate cells. NOTA-ATH001 demonstrated picomolar affinity to human PDGFRβ. [68Ga]Ga-NOTA-ATH001 bound to PDGFRβ-positive U87 cells and fibrotic tissues, with low background binding in PDGFRβ-negative cells and tissues. [68Ga]Ga-NOTA-ATH001 exhibited high in vivo plasma stability, rapid tissue clearance and strong, blockable binding to U87 xenografts. However, [68Ga]Ga-NOTA-ATH001 was not superior to the previously reported analogue [68Ga]Ga-DOTA-ATH001. Conclusions [68Ga]Ga-NOTA-ATH001 is a novel, high affinity PDGFRβ-targeting PET tracer that enables in vivo PET imaging of mesenchymal cells in health and disease. Although it does not outperform its DOTA-conjugated analogue, the NOTA chelator offers practical advantages, such as convenient Al18F or 64Cu radiolabeling.
Introduction 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is a promising chelator for radiotheranostic applications. Recently, we developed a DOTA-based radiolabeled antibody fragment, [111In]In-DO3AiBu-Bn-FGK-Fab, which contains a cleavable linkage recognized by renal brush border membrane (BBM) enzymes. Molecular design of radiometabolites that rapidly escape from the coated vesicles in renal cells during the early stage of endocytosis is crucial for reducing renal radioactivity levels. In the present study, we prepared [111In]In-DO3AEt-Bn-FGK-Fab by replacing the isobutyl group of [111In]In-DO3AiBu-Bn-FGK-Fab with an ethyl group. We assessed the reduction effect of [111In]In-DO3AEt-Bn-FGK-Fab on renal radioactivity levels to investigate the importance of the choice of alkyl group inserted into the DOTA backbone. Methods Density functional theory (DFT) calculations were performed for three In-complexes, In-DO3AEt-Bn, In-DO3AiBu-Bn, and In-DOTA-Bn. Enzymatic recognition of the cleavable FGK linkage was assessed by incubating a model low-molecular-weight (LMW) model substrate, [111In]In-DO3AEt-Bn-FGK(Boc) with brush border membrane vesicles. Then, DO3AEt-Bn-FGK-Fab was prepared by conjugating DO3AEt-Bn-FGK(Mal) to thiolated Fab and was radiolabeled with 111In. [111In]In-DO3AEt-Bn-FGK-Fab was administered to normal mice, and biodistribution studies and metabolic analyses of the kidney homogenate and urine samples were performed with comparison to an 111In-labeled Fab prepared by a conventional method ([111In]In-DOTA-Bn-SCN-Fab). Biodistribution studies and SPECT imaging were also performed for both [111In]In-DO3AEt-Bn-FGK-Fab and [111In]In-DOTA-Bn-SCN-Fab in tumor-bearing mice. Results High similarity in the coordination environments was observed for the structures of In-DO3AEt-Bn and In-DO3AiBu-Bn predicted by DFT calculations. The release of [111In]In-DO3AEt-Bn-F was observed in an in vitro system using renal BBM vesicles and was inhibited by an angiotensin-converting enzyme inhibitor. [111In]In-DO3AEt-Bn-FGK-Fab showed a moderate reduction in renal radioactivity levels compared to the reference and higher renal radioactivity levels than [111In]In-DO3AiBu-Bn-FGK-Fab. Metabolic studies revealed that the release of [111In]In-DO3AEt-Bn-F also occurred in vivo. Contrary to the reduction in renal radioactivity levels, tumor accumulation was not impaired when [111In]In-DO3AEt-Bn-FGK-Fab was compared to the reference. Conclusion Since the difference in the radiometabolites liberated from [111In]In-DO3AEt-Bn-FGK-Fab and [111In]In-DO3AiBu-Bn-FGK-Fab was the alkyl (ethyl and isobutyl) group inserted into the DOTA structure, slight differences in the structure of the radiometabolites severely affected their renal residence time. These results provide a warning for further applications of the renal brush border strategy to other radiometal-chelator complexes. The radiometabolites would need to be carefully designed to ensure rapid elimination from renal cells.
Radionuclides emitting beta- particles and Auger electrons present real or perspective radioactive components of radiopharmaceuticals used or developed for radionuclide therapy. Two important representatives of such radionuclides, lutetium-177 and terbium-161 have been applied as radiotoxic components of biomolecules and low-molecular agents developed for targeted radionuclide therapy. However, limited knowledge exists regarding the pharmacokinetics of potential contaminants or their final metabolic forms, such as chelate-radionuclide complexes or free radionuclides. These chemical forms can result from the radiolabeling process or the metabolism of radioimmunoconjugates in the organism. Since the pharmacokinetics of the undesired ionic forms of terbium-161 and lutetium-177 have not been fully explored using one experimental model and appropriate chemical forms of the radionuclides, this head-to-head study in mice was conducted. The experiment compared the biodistribution of a model 161Tb- or 177Lu-labeled antibody ramucirumab, 161Tb- or 177Lu-labeled DOTA-complexes, and free metal forms (161Tb3+ and 177Lu3+). Organ distribution was examined at selected time points (4 or 6, 24, 72, 144, and 240 h) following administration. Plasma binding was determined as an important distribution parameter. [161Tb]Tb-DOTA-ramucirumab and [177Lu]Lu-DOTA-ramucirumab distribution parameters exhibited typical pharmacokinetic characteristics of radioimmunoconjugates, such as a long elimination half-life and long-term retention in the liver or spleen. The general pharmacokinetic behavior of the congeners was very similar. A significant accumulation of free cationic form in bone and liver was observed. Whereas liver accumulation was transient, bone radioactivity did not decrease up to the last experimental interval. In contrast, [161Tb]Tb-DOTA and [177Lu]Lu-DOTA were eliminated rapidly from the organism without any significant organ retention. The high elimination rate of DOTA complexes was also supported by negligible binding to plasma. The obtained data demonstrate similar pharmacokinetic behavior for 161Tb- and 177Lu-labeled congeners, which may be useful for the development of future radiopharmaceuticals.
PURPOSE:The PET tracer 4‑borono-2-[18F]-fluoro-phenylalanine fructose ([18F]FBPA-Fr) is commonly used to estimate the biodistribution of L-4-dihydroxy-borylphenylalanine fructose (BPA-Fr). However, its limited regulatory approval has prompted the exploration of alternative imaging agents. In this study, we evaluated the feasibility of [18F]fluciclovine and [18F]fluoroethyl-tyrosine ([18F]FET) PET for pre-BNCT assessment, with direct comparison to [18F]FBPA-Fr. METHODS:In vitro cellular uptake studies were performed using two different cancer cell lines. In vivo microPET/MR imaging and biodistribution analyses were conducted in mice bearing wild-type and L-type amino acid transporter 1 (LAT1)-knockdown GBM8401 tumors. The therapeutic efficacy of BPA-Fr-based BNCT was evaluated in tumor-bearing mice. In addition, a pilot clinical study compared [18F]fluciclovine and [18F]FBPA-Fr PET imaging in participants with brain and head-and-neck tumors. RESULTS:[18F]FBPA-Fr uptake was strongly dependent on LAT1 expression, decreasing by approximately 60 % in LAT1-knockdown cells, whereas [18F]fluciclovine uptake was reduced by only ~10 %. In vivo, [18F]FET showed the highest absolute tumor uptake but the lowest tumor-to-normal brain (T/N) ratio due to high background activity. Mice with high [18F]fluciclovine tumor uptake exhibited significantly prolonged survival following BNCT (15-17 days) compared with controls (~10 days). Clinically, [18F]fluciclovine and [18F]FBPA-Fr showed comparable T/N ratios in patients with pontine glioma and maxillary sinus carcinoma. Moreover, [18F]fluciclovine PET visualized metabolic changes after BNCT, which were consistent with subsequent disease progression. CONCLUSION:Although [18F]FBPA-Fr remains the most LAT1-specific tracer, [18F]fluciclovine provides comparable tumor-to-normal tissue contrast and reliable imaging performance, supporting its role as a clinically feasible alternative for pre-BNCT patient selection and treatment response assessment.
Fibroblast activation protein (FAP) is an established target for molecular imaging of the tumor stroma. In this study, we report the development and preclinical evaluation of a novel FAP-targeted positron emission tomography (PET) tracer, [68Ga]Ga-CB6NT-FAPI. [68Ga]Ga-CB6NT-FAPI was efficiently radiolabeled under mild conditions (room temperature, 10 min), yielding high radiochemical purity (>99.9%) and demonstrating excellent in vitro stability. In vitro studies showed significantly higher uptake in FAP-positive cells than in FAP-negative cells, which was reduced by excess unlabeled ligand, confirming FAP-specific binding. In vivo PET/MR imaging in a U87MG xenograft model demonstrated clear tumor uptake with favorable contrast. Blocking studies resulted in a marked reduction in tumor signal, and ex vivo biodistribution confirmed specific tumor accumulation with an approximately 99.9% reduction under blocking conditions. The tracer exhibited partial hepatobiliary involvement in addition to renal clearance. These findings indicate that CB6NT-FAPI represents a pharmacokinetically differentiated FAPI tracer that expands the diversity of FAP-targeted PET imaging by providing an alternative balance between renal and hepatobiliary clearance.
BACKGROUND:Angiostatin (ANG) is an endogenously cleaved protein of the plasminogen-plasmin conversion pathway which is activated during tumor angiogenesis and oxidative stress. One of the hallmarks of sub-clinical oxidative stress are persistent aggregation of platelets and neutrophils (and other immune cells) leading to endothelial activation. The goal of the study was to develop and test the specificity of a highly glycosylated form of ANG, derived from neutrophil elastase, as an in vivo radiopharmaceutical marker of oxidative stress and to monitor the progression of vascular and platelet activation. METHODS:Sequential PET imaging of radiolabeled ANG ([89Zr]Zr-ANG) in a combined ozone and LPS induced murine model of oxidative stress (n = 8 per group) was followed by whole-body, fractionated blood, aortic ring and platelet biodistribution studies. In vitro, platelets were exposed to H2O2 and/or LPS to induce oxidative stress. RESULTS:Compared with sham mice, the uptake of [89Zr]Zr-ANG in the exposed lungs was 4.8 to 8.5-fold (male mice) and 4.2 to 11.6-fold (female mice) higher over 72 h after ozone and LPS exposure (p < 0.0001). Ex vivo biodistribution and microPET/CT imaging revealed accumulation of [89Zr]Zr-ANG in platelets and multiple organs including lungs highlighting vascular inflammation and platelet activation in response to ozone and LPS. Uptake of ANG in aorta and platelets was confirmed by injecting ozone and LPS exposed mice with fluorescent angiostatin. Lastly, platelets subjected to oxidative stress preferentially bind ANG compared to untreated platelets and split them into ATP-rich ultra-small (0.7-10 nm) vesicles and large (1-10 μm) micro-particles. This binding is dependent upon ATP inhibitory factor, IF1's displacement to bind with ATP synthase subunit β (ATPβ). CONCLUSIONS:[89Zr]Zr-ANG is a stable, ATP synthase directed endothelial cell and platelet-specific imaging tool for detection of acute murine oxidative stress-induced lung as well as systemic inflammation. Thus, the current study provides evidence towards potential applications of [89Zr]Zr-ANG in understanding the fate of platelets in chronic disease models.
Background The cystine/glutamate antiporter xCT has attracted attention due to its role in maintaining redox homeostasis. Noninvasive techniques for visualizing xCT expression in tumors provide valuable diagnostic information for evaluating tumor biology. Therefore, we developed and evaluated a novel SPECT radiopharmaceutical to visualize xCT expression in cancer cells based on inhibitors. Methods We designed and synthesized a radioiodinated SSZ derivative ([125I]I-SSZd), based on the structure of sulfasalazine (SSZ)—an xCT inhibitor. We radioiodinated 4-hydroxy-L-phenylglycine (L-HPG) and 4-hydroxy-D-phenylglycine (D-HPG) to synthesize [125I]I-L-HPG and [125I]I-D-HPG, respectively. The affinities of [125I]I-SSZd, [125I]I-L-HPG, and [125I]I-D-HPG to xCT were evaluated using SSZ and cystine inhibition assays in two human-derived colon cancer cell lines (LS180 and DLD-1). xCT expression was quantified using real-time polymerase chain reaction. In vivo, the biodistribution of [125I]I-L-HPG was examined in tumor-bearing mice. Stability analysis was performed in mice liver and kidney homogenates. Results [125I]I-L-HPG showed high affinity for xCT. [125I]I-D-HPG exhibited some affinity for xCT. xCT was not involved in [125I]I-SSZd accumulation. The gene expression levels of xCT were higher in LS180 than in DLD-1 cells. [125I]I-L-HPG and [125I]I-D-HPG showed high accumulation in LS180 cells. [125I]I-L-HPG showed higher accumulation than [125I]I-D-HPG in both cell lines. In the kidneys, [125I]I-L-HPG accumulation decreased over time. In the thyroid, [125I]I-L-HPG accumulation increased over time; however, deiodination did not occur in mice. Tumor-to-muscle ratio was >2.0 at almost all timepoints for both types of tumor-bearing mice, and the maximum tumor-to-large intestine ratio reached 4.0 in LS180 tumors with high xCT expression. Conclusion In tumors, [125I]I-L-HPG shows promise as a radiopharmaceutical by exhibiting high affinity for xCT.
Introduction [18F]fluoro-L-α-methyltyrosine ([18F]FAMT) has been reported as a positron-emission tomography (PET) probe that has high specificity for L-type amino acid transporter 1 (LAT1), which is overexpressed in various malignant tumors. However, [18F]FAMT showed rapid washout from the tumor and high retention in the kidney. This study aimed to develop and evaluate a novel LAT1-targeting PET probe, [18F]FAMT-OMe, and compare its performance with [18F]FAMT in glioma xenograft mice. Methods [18F]-FAMT-OMe was synthesized via nucleophilic substitution. The uptake of [18F]FAMT-OMe and [18F]FAMT was compared in in vitro studies using C6 glioma and U-87MG cells. PET scans were performed on C6 glioma- and U-87MG tumor-bearing mice (n = 20 each) following intravenous administration of either [18F]FAMT-OMe or [18F]FAMT. After PET/computed tomography (CT) imaging, the organs were weighed and the radioactivity present was measured using a gamma counter. Results In vitro analyses demonstrated higher uptake of [18F]FAMT-OMe compared with [18F]FAMT in both C6 and U-87MG cells. PET imaging demonstrated significantly greater tumor retention of [18F]FAMT-OMe than [18F]FAMT (SUVmax at 60 min in C6 glioma: 2.13 ± 0.39 vs. 1.09 ± 0.79, P < 0.05). The kidneys and urine showed significantly lower uptake and excretion of [18F]FAMT-OMe than [18F]FAMT (kidney uptake: SUVmean 3.75 ± 0.89 vs. 5.55 ± 2.44, P < 0.05 urine excretion: SUVmean 16.50 ± 7.65 vs. 34.38 ± 8.74, P < 0.05), while blood retention of [18F]FAMT-OMe was significantly increased (SUVmean 1.78 ± 0.85 vs. 1.20 ± 0.82, P < 0.05). Conclusion [18F]FAMT-OMe showed improved tumor retention on PET compared with [18F]FAMT in the C6 glioma tumor model, suggesting its potential utility for future applications in LAT1-targeted PET.
INTRODUCTION:Endometriosis, affecting up to 10% of women, is a chronic estrogen dependent disorder where ectopic endometrial-like tissue causes pelvic pain and infertility. Endometriosis is challenging to diagnose due to symptom overlap and limited imaging accuracy, often requiring surgical visualization. Positron emission tomography (PET) potentially offers a non-invasive, molecular-based approach for highly specific diagnosis and monitoring of endometriosis. Aromatase, typically low in normal endometrial tissue, is elevated in endometriotic lesions, while neutrophils, which are scarce in normal tissue, are increased in these lesions. Fibrosis, resulting from activated platelet-derived growth factor receptor β (PDGFR-β)-expressing myofibroblasts, has been observed in endometriosis lesions. In this study, three PET tracers; [11C]cetrozole ([11C]CET), [11C]GW457427 ([11C]NES), and [68Ga]Ga-ATH001, targeting aromatase, neutrophil elastase (NE), and PDGFR-β, respectively, were tested in endometriosis patient biopsies through autoradiography (ARG) combined with histological examinations. A pilot PET/Magnetic resonance imaging (PET/MR) study was performed in endometriosis patients with [11C]NES. METHODS:Ten biopsies from patients with endometriosis were collected. Frozen tissue was sectioned at 20 μm for ARG and 4 μm for immunohistochemistry (IHC). Total binding and non-specific binding, with quantification using ImageJ (fmol/mm3) was determined. Specific binding was calculated as total minus non-specific binding and expressed as a percentage. Sections were stained with Hematoxylin and Eosin (H/E) and Cytokeratin7 (CK7) for morphology, Sirius Red (SIR) for fibrosis, antibodies against PDGFR-β and NE. Neutrophil extracellular traps (NETs) were identified by combined staining for NE and Histone H3. Three endometriosis patients were scanned with [11C]NES by PET/MR. RESULTS:All three tracers showed high in vitro binding to endometriotic tissue in ARG. [11C]NES and [68Ga]Ga-ATH001 had high degree of specific binding to elastase and PDGFR-β, respectively, whereas no specific binding could be shown for [11C]CET. ARG results were validated by IHC: CK7 confirmed epithelial lesions, NE and Histone H3 verified neutrophils and NETs, and PDGFR-β/SIR indicated fibrosis. However, in vivo, no [11C]NES uptake was detected on the PET/MR scans of the three endometriosis patients. CONCLUSION:In ARG on endometriotic tissue samples, [68Ga]Ga-ATH001 and [11C]NES showed specific binding to their respective targets. However, tracer delivery potentially forms a challenge in visualizing fibrotic endometriosis lesions in vivo, as seen by [11C]NES PET/MR in patients.
Histone deacetylases (HDACs) play an important role in the regulation of cellular functions and have attracted highly attention as promising therapeutic targets for the treatment of neurodegenerative diseases. Among the various HDAC isoforms, HDAC2 have shown significantly to be involved in the emergence and progression of neurodegenerative disorders. However, research on the biological mechanism of HDAC2 requires more advanced tools. In this study, we present the radiosynthesis of [18F]HYF005 with high radiochemical purity and satisfactory molar activity. In vitro autoradiographic studies shown [18F]HYF005 has good binding specificity in mice brain tissues. In vivo evaluation of PET imaging in rodent models confirmed that [18F]HYF005 exhibits good BBB penetration, with a highest brain peak uptake SUV = 1.2 in mice. Our study indicates that [18F]HYF005 can be used as a potential PET probe for imaging the HDAC2 system in the brain and provides a foundation for the future development of more potential HDAC2 targeting PET probes.
OBJECTIVE:This study aims to characterize and compare the saturation limits, spatial resolution, and image quality of various conventional and emerging positron-emitting radionuclides using a preclinical PET/CT scanner. By characterizing the performance of these radionuclides, the study sought to provide insights into their utility in high-resolution PET imaging. METHODS:Radionuclides (18F, 43Sc, 45Ti, 48V, 52Mn, 55Co, 64Cu, 68Ga, 89Zr) were evaluated on a GNEXT PET/CT scanner (Xodus Imaging, Torrance, CA) using saturation and Derenzo phantoms. Saturation was assessed by measuring the deviation between the actual and the region of interest (ROI) activity at varying concentrations of each radionuclide. Spatial resolution was quantified using full-width half maximum (FWHM) measurements from intensity profiles across six Derenzo phantom diameter sizes (1.2 mm-4.8 mm). Signal-to-noise ratios (SNRs) were calculated as a measure of image quality and Bland-Altman plots were used to assess the repeatability of resolution measurements. Statistical comparisons of test-retest were done to evaluate differences in accuracy and consistency across radionuclides. RESULTS:Saturation analysis revealed a broad range of limits across radionuclides, with 64Cu having the highest saturation threshold near 2 mCi (74 MBq), while 52Mn exhibited the lowest at approximately 250 μCi (9.25 MBq). Spatial resolution was inversely related to positron energy, with radionuclides like 18F and 64Cu producing clear images down to rod sizes of 1.6 mm compared to 68Ga and 55Co, which showed blurring at the same rod size. SNR analysis confirmed the superior image quality of lower-energy radionuclides, particularly for smaller structures, visually resolvable to 1.6 mm. Bland-Altman analysis showed that across the combination of rod sizes, 18F displayed improved repeatability in resolution measurements compared to 68Ga (standard errors of 0.03 and 0.15, respectively). CONCLUSION:This study demonstrates that the physical properties of radionuclides, particularly positron energy, significantly affected PET image quality, spatial resolution, and saturation thresholds. Lower-energy radionuclides like 18F and 52Mn are optimal for high-resolution applications, while higher energy radionuclides are better suited for high-activity imaging. These findings provide valuable guidance for optimizing radionuclide selection in preclinical and clinical PET imaging studies.
INTRODUCTION:Aldosterone-producing adenomas (APAs) account for most cases of primary aldosteronism (PA), a major cause of secondary hypertension and hypokalemia. Although adrenal vein sampling (AVS) is the diagnostic gold standard, its invasiveness and limited availability underscore the need for less invasive diagnostic alternatives such as positron emission tomography (PET). We recently developed 6-chloro-5-fluoro-1-(2-[18F]fluoroethyl)-2-(pyridin-3-yl)-1H-benzo[d]imidazole ([18F]ApaScan), a highly CYP11B2-selective PET tracer for imaging APAs. This study characterizes the metabolism, biodistribution, dosimetry, and automated synthesis of [18F]ApaScan in preparation for first-in-human studies. METHODS:Metabolism was evaluated in vitro using mouse, rat, and human liver microsomes and in vivo in mouse blood following the administration of [18F]ApaScan. Biodistribution (%ID/g) was measured in mice, and human radiation doses were estimated using the MIRD schema. The automated radiosynthesis process was optimized on a cassette-based FASTlab module integrated with preparative HPLC to afford the final formulation (ca. 5% ethanol). This formulation was validated in three consecutive production runs in accordance with the in-house manufactured PET drug standards in Japan. RESULTS:A single radiometabolite, identified as the pyridine N-oxide derivative of ApaScan, was observed in both in vitro liver microsome assays and in vivo mouse studies. Biodistribution studies revealed adrenal uptake peaked at 5.9%ID/g at 2 min post-injection and cleared rapidly, with no significant retention in other organs or in bone. The extrapolated human radiation doses were 13.4 μSv/MBq for males and 15.9 μSv/MBq for females, comparable to other clinically used 18F-labeled tracers. The automated radiosynthesis achieved a non-decay-corrected yield of 29.2 ± 0.5%, a molar radioactivity of 1070 ± 246 GBq/μmol, and a radiochemical purity >99%. The final product met all quality specifications, including sterility and endotoxin limits. CONCLUSION:[18F]ApaScan undergoes oxidative metabolism while maintaining favorable adrenal kinetics and exhibiting acceptable dosimetry. We established a reproducible automated radiosynthesis of [18F]ApaScan suitable for clinical use. These findings support proceeding to first-in-human studies to evaluate the pharmacokinetics and APA imaging performance of [18F]ApaScan in patients with PA.
Azeotropic removal of water remains a significant limitation in fluorine-18 radiochemistry, often leading to longer synthesis times, variable yields, increased solvent use and operational complexity, and, indirectly, increased resource demand due to decay- and loss-driven activity requirements and incompatibility with base- or heat-sensitive precursors. This review critically evaluates drying-free strategies that mitigate or obviate this step while sustaining high radiochemical performance and compliance with good manufacturing practice. Methods are organized into controlled hydrous fluorination, ionic-liquid media, mixed organic solvent systems, alcohol-assisted elution, copper-mediated aromatic radiofluorination, rhenium-complexation routes, and other advanced approaches. Comparative analysis addresses fluoride recovery, radiochemical yield, substrate scope, including electron-rich arenes and base-sensitive chemotypes, tolerance to residual water/alcohol, cycle time, solvent and waste metrics, and suitability for automation and clinical translation. Copper-mediated protocols currently provide broad aromatic coverage with competitive yields under minimally basic, non-dried conditions; alcohol-assisted and mixed-solvent systems offer rapid, cassette-ready workflows for many aliphatic targets; and rhenium-assisted labeling enables mild conditions for sensitive scaffolds. Remaining challenges include standardized reagent kits and quality control, management of residual metals or additives, harmonized sustainability metrics, and consistent implementation across synthesis platforms. Collectively, drying-free strategies support more robust, streamlined and resource-efficient 18F tracer synthesis and are poised to facilitate scalable production and wider clinical adoption.
A post-column derivatization high-performance liquid chromatography-ultraviolet (HPLC-UV) method was developed to determine Kryptofix 2.2.2 (K2.2.2) in PET tracers. This method utilizes the specific complexation between K2.2.2 and Pb2+, forming a stable 1:1 host-guest complex. The complexation produces a bathochromic shift in UV absorption maximum (from 210 nm to 254 nm), enabling sensitive detection. This approach addresses the limitations of poor performance and narrow applicability associated with direct single-method detection of K2.2.2. The chromatographic conditions were optimized (column flow rate: 0.7 mL·min-1; derivatization reagent flow rate: 0.7 mL·min-1; Pb2+ concentration: 50 μg·mL-1) and the method was validated. The results exhibited excellent linearity (1-100 μg·mL-1, r2 > 0.999), high reproducibility (relative standard deviation (RSD) < 5%), and a low detection limit (0.5 μg·mL-1). The method successfully quantified K2.2.2 in [18F]FDG, [18F]AV45 and [18F]DPA714, achieving spiked recoveries of 89%-105%. Additionally, the method remained unaffected by interference from common sample components such as sodium chloride and sodium ascorbate. This method provides a novel and efficient quality control tool for the simultaneous analysis of radiochemical purity and K2.2.2 content in diverse PET tracers.
AIM/INTRODUCTION:Astatine-211 is one of the alpha-particle emitting nuclides investigated for use within Targeted Alpha Therapy (TAT) of disseminated cancer. In previous studies, a difference in blood clearance has been observed when comparing astatinated compounds with their iodinated counterparts. This has been explained by a potentially prolonged retention in the blood by present free astatine. This study examined the spontaneous binding of non-conjugated astatine to albumin under physiological conditions in vitro and compared it to that of iodine. Additionally, the in vivo blood circulation patterns of free astatine were assessed and contrasted with those of iodine. MATERIALS AND METHODS:Astatine solutions were formulated following dry distillation and evaporation to dryness of astatine solvated in CHCl3. In vitro evaluation of astatine and iodine association to albumin was mainly performed using size exclusion chromatography applying both disposable columns (PD10 and NAP10) as well as an FPLC system (ÄKTA) with online UV detection and activity fraction collection. Also methanol precipitation and radio-TLC methods were used for analysis. In vivo evaluation was performed using furry BALB/C mice (3/group) with both i.v. and i.p. injection of astatine, followed by sequential blood sampling from the tail vein and biodistribution. RESULTS:Oxidized and unmodified forms of free astatine display a significantly higher and more rapid association to albumin in vitro compared to reduced forms, with >97% compared to <25% associated after 10 min. Both the corresponding oxidized and reduced forms of iodine display a very low and slow association to albumin with <5% associated after 40 min. Oxidized, unmodified and reduced astatine show very similar blood profiles over time as well as a similar uptake in biodistribution after 20-22 h following i.p. injection. Upon i.v. injection a larger difference in blood profiles between the species could be observed, which in turn was different compared to the curve obtained after i.p. injection. In addition, an unexpected uptake of astatide in stomach was found. In all cases the blood profile and biodistribution of astatine was significantly different compared to iodine, which displayed a greater and more rapid blood clearance and specific accumulation in thyroid. CONCLUSION:Different forms of unbound astatine differ in their association to albumin. However, all investigated forms of free astatine associates to albumin to a much higher degree than iodine. This behavior could explain the prolonged blood circulation of free astatine compared to iodine.
Pancreatic ductal adenocarcinoma (PDAC) continues to be deadly and resistant to traditional treatments. Overexpressed in >80% of PDACs, mesothelin is an ideal target for antibody-based α-therapy. Actinium-225 (225Ac) produces high-LET α-particles leading to irreparable DNA damage, but its utility has been compromised by unstable chelation with traditional ligands. Here, we engineered a Macropa-enabled, site-specifically [225Ac]Ac-Macropa-PEG6-Amatuximab, a radioimmunoconjugate against mesothelin. Conjugation and labeling were characterized by MALDI-TOF and SEC-HPLC. In vitro stability, immunoreactivity, and kinetics of binding were tested in mesothelin-positive AsPC-1 cells and subsequently in vivo biodistribution, dosimetry, and therapy in AsPC-1 xenograft-bearing nude mice. Conjugation had an average ratio of 3.6 ± 0.1 for chelator per antibody, radiolabeling efficiency of 96.3 ± 1.1%, and radiochemical purity ≥98%. The radioconjugate was >92% stable after 168 h in serum, with immunoreactivity (82.2 ± 2.8%) and affinity (Kd = 4.3 ± 0.9 nM). It exhibited specific, time-dependent internalization in AsPC-1 cells and minimal nonspecific uptake. In vivo, [225Ac]Ac-Macropa-PEG6-Amatuximab exhibited prolonged circulation, specific tumor localization (3.9 ± 0.5 to 16.3 ± 2.1% ID/g, 1-168 h), and enhanced tumor-to-blood ratios (0.21-3.40). Blocking with unlabeled Amatuximab decreased tumor uptake by >60%. The tumor absorbed dose (1.82 ± 0.14 Gy/MBq) was 4-20-fold greater than doses to normal organs. Therapeutically, it caused dose-dependent tumor regression (TGI: 58% at 50 kBq; 92% at 150 kBq) and prolonged survival (>60 days vs. 0-1% in controls, p < 0.001). [225Ac]Ac-Macropa-PEG6-Amatuximab is stable, selective, and therapeutically effective, demonstrating Macropa-based 225Ac chelation as a stable platform for targeted α-therapy of PDAC.
BACKGROUND:CTT1403 (177Lu-CTT2001), an irreversible phosphoramidate PSMA inhibitor developed by Cancer Targeted Technology, was initially synthesized using a two-step radiolabeling method and has previously been evaluated in first-in-human studies. This two-step approach protected the phosphoramidate pharmacophore-containing a temperature- and pH-labile PN bond-from the harsh conditions required for lutetium-177 (177Lu) chelation. Although the final chemical structure of CTT1403 is identical regardless of the radiolabeling route, it was not clear that CTT2001 could tolerate one-step labeling conditions while preserving PSMA-binding integrity. Therefore, the aim of the study was to develop, optimize, and automate a one-step radiolabeling method for CTT1403 and to confirm that the resulting product is biologically equivalent and exhibits comparable in vitro and in vivo behavior to CTT1403 produced by the original two-step process. METHODS:CTT2001 was synthesized and radiolabeled with 177Lu using an optimized one-step procedure that was subsequently automated on a Trasis AllinOne synthesizer. Radiochemical purity, stability, cellular uptake/internalization, and biodistribution in PC3-PIP tumor-bearing mice were evaluated. RESULTS:CTT1403 synthesized via the one-step method demonstrated cellular uptake and internalization in PC3-PIP cells, as well as in vivo biodistribution in PC3-PIP tumor-bearing mice, that were comparable to those of the two-step-labeled product. The one-step procedure was successfully automated on the Trasis All-in-One synthesizer, producing CTT1403 with a radiochemical yield of 86.5 ± 4.27% (n = 3), a molar activity of 30.3 ± 1.11 MBq/nmol, and a radiochemical purity of 97.6 ± 0.80% (n = 3) in a total synthesis time of 38 min. The final product remained stable for at least 24 h at -4 °C and -20 °C. CONCLUSIONS:The one-step radiolabeling method yields CTT1403 that is biologically equivalent to the two-step product and can be reliably produced using fully automated synthesis. This streamlined, efficient, and reproducible approach supports routine clinical manufacturing of CTT1403.
BACKGROUND:[18F]Fluoropivalate ([18F]FPIA), also known as 18F-pivalate or 18F-RAD101, is the fluorinated analogue of pivalic acid and has shown promise in ongoing clinical trials for the detection of brain metastases. The original synthesis of [18F]FPIA involved a two-step procedure that was not fully automated, limiting its suitability for large-scale or GMP production. A subsequent report described an improved two-step, one-pot synthesis on a cassette-based module, although with certain limitations. Here, we present an optimized two-step, one-pot synthesis of [18F]FPIA using a vial-based automated synthesizer and demonstrate its successful implementation under GMP conditions. We also report [18F]FPIA-PET imaging in prostate cancer patient-derived xenograft (PDX) models. RESULTS:[18F]FPIA was successfully produced with the optimized synthetic strategy with a total synthesis time of 75 min and a 25.4 ± 3.8% (n = 9) activity yield at end of synthesis (EOS), with >99% radiochemical purity. In a GMP setting, the scale-up synthesis was successful with a 37 ± 9% (n = 37) activity yield at EOS and a > 99% radiochemical purity. In the proof-of-concept PET imaging study of [18F]FPIA in androgen receptor (AR)-negative and -positive prostate cancer PDX animal models, uptake was observed in both groups when the tumor reached a size of 50-300 mm3. The AR-negative group showed significantly higher [18F]FPIA uptake compared to the AR-positive group, with average tumor-to-muscle ratios of 1.6 and 1.2, respectively. CONCLUSIONS:In summary, an optimized one-pot, two-step synthesis of [18F]FPIA on a vial-based automated synthesizer was successful and a seamless transition into a GMP facility is reported, enabling a streamlined transition to clinical production. Furthermore, we have demonstrated the use of [18F]FPIA for noninvasive metabolic imaging in prostate cancer and its potential to distinguish between different prostate cancer subtypes.
Four regioisomers of [68Ga]Ga-TMoS-DAZA, a PET/CT radio tracer for liver function imaging, were studied as candidates of potentially improved hepatobiliary biokinetics. Liver uptake and biliary clearance behavior were compared using an in ovo model based on ostrich eggs for PET/CT imaging and ex vivo biodistribution analysis. The tracer lipophilicity was evaluated via logD determination. The experiments showed remarkable differences between the four tracers concerning their maximum liver uptake, percentage of tracer cleared via the biliary tract and their respective logD values.
