EJNMMI Radiopharmacy and Chemistry最新文献

Background

The development of safer and more effective cell-based therapies requires robust methods for tracking cells in vivo. Positron emission tomography (PET) is a highly sensitive nuclear imaging technique capable of quantitatively tracking the in vivo fate of cells after administration. Here, we investigated a cell-labeling strategy based on metabolic glycoengineering (MGE) to introduce azides on the cell surface, followed by radiolabeling via the bioorthogonal perfluoroaryl azide (PFAA)-Staudinger reaction. We studied the metabolic incorporation of a tetraacetylated PFAA-derivatized mannosamine in Jurkat cells and evaluated whether three triarylphosphines bearing the REstrained Complexing Agent (RESCA), could be radiolabeled with aluminum-[F]fluoride (Al[¹⁸F]F) for use as bioorthogonal reagents in the PFAA-Staudinger ligation.

Results

Three novel triarylphosphines containing different linkers (hydrophilic ester, ethylenediamine, and cyclohexyl) between the phosphine moiety and the (+)-RESCA-chelator were synthesized and characterized. Kinetic assays showed that all compounds reacted with the PFAA-derivatized monosaccharide, exhibiting different reaction kinetics under the tested conditions. They were successfully radiolabeled with fluorine-18 under optimized mild conditions and provided key insights into the radiolabeling of small molecules bearing the RESCA-chelator. The ester derivative underwent rapid chemical decomposition while the ethylenediamine- and cyclohexyl-linked derivatives were more resistant, with the cyclohexyl analogue showing the highest stability against demetallation and/or defluorination. However, radiolabeling with Al[¹⁸F]F led to the oxidation of the phosphine moiety, with the major radiolabeled product corresponding to the oxidized form. At the same time, flow cytometry showed that the metabolic incorporation of the tetra-acetylated PFAA-derivatized mannosamine into Jurkat cells was substantially less efficient than that of the widely used tetra-acetylated N-azidoacetylmannosamine (Ac₄ManNAz) derivative at the equivalent concentration. Increased concentrations of the PFAA derivative compromised cell viability, which halted subsequent studies.

Conclusions

While the Al[¹⁸F]F radiolabeling of these (+)-RESCA-bearing small molecules offers high stability against demetallation and/or defluorination, the method cannot currently be applied to triarylphosphines due to oxidation during radiolabeling and requires further development. Among the synthesized compounds, the cyclohexyl-linked derivative exhibited the most favorable stability profile, making it a potential lead structure for future tracer development. Nevertheless, this study advanced our understanding of MGE with PFAA-derivatized monosaccharides and highlighted the need for further investigation before applying PFAA-Staudinger ligation to cell radiolabeling.

Background

Successful treatment of solid cancers relies on precise diagnosis, e.g. using noninvasive molecular imaging, followed by surgical removal and/or chemo/immunotherapy. Despite advances in pre-operative imaging, real-time intraoperative tools remain limited, which often results in high rates of tumor-positive margins and recurrence after tumor resection. To address this limitation, we aimed to develop multifunctional fibroblast activation protein alpha (FAP) targeting tracers for bimodal medical imaging, enabling both pre-operative noninvasive molecular imaging via positron emission tomography (PET) and optical visualization during intraoperative fluorescence-guided surgery.

Results

NODAGA-FAP647 and NODAGA-FAP800 targeting human FAP (hFAP) were synthesized bearing a (R)-NODAGA chelator and a fluorophore (AlexaFluor647 or IRDye800CW, respectively). Binding affinities and binding kinetics of both unlabeled and ^67/68Ga-labeled compounds were evaluated in vitro using HT1080 cells (hFAP-expressing and wild type, WT) along with respective frozen xenograft tissue sections. Using real-time binding, both compounds exhibited picomolar binding affinities to hFAP via radioactive/fluorescent detection. This was primarily driven by low dissociation rate constants in vitro. Pharmacokinetics and tumor uptake were evaluated via PET and fluorescence imaging in mice bearing xenografts from the same cells. In vivo, both compounds were rapidly distributed and accumulated in hFAP-expressing but not WT-HT1080 tumors within 10–20 min post-injection. Fluorescence imaging showed a similarly good and selective tumor uptake in the first two hours and a qualitatively visible difference compared to WT-HT1080 beyond 24 h. Both compounds were quickly cleared from normal tissue and excreted renally.

Conclusion

Two FAP-targeting bimodal ligands were synthesized and evaluated in vitro and in vivo, showing high specificity and selectivity, along with rapid and selective tumor accumulation. Their long tumor retention and high imaging contrast make them promising candidates for clinical translation.

Background

The Neuropeptide Y (NPY) system regulates mood, stress, and feeding behavior and plays a central role in neuropsychiatric and metabolic disorders. It exerts its effects primarily through a family of G-protein-coupled NPY receptors (NPYR), comprising the Y1, Y2, Y4, and Y5 subtypes. Among these, the Y2 receptor (NPY2R) has emerged as a promising imaging target through its involvement in mood regulation, anxiety, and feeding behavior. This study evaluated the in vivo performance of the selective NPY2R antagonist PET tracer N-[¹¹C]methyl-(R)-JNJ-31020028 in mice, focusing on tracer metabolism, brain uptake, and blood–brain barrier transport via P-glycoprotein (P-gp).

Results

In vitro, N-methyl-(R)-JNJ-31020028 showed nanomolar affinity and selectivity for the murine NPY2R with no observable interaction with mY1, mY4, and mY5. In vivo, brain percent injected dose per cc (%ID/cc), peaked within the first 5 min after injection and declined rapidly. Tariquidar pretreatment increased brain uptake more than threefold at 15 min post administration, particularly in hippocampus, thalamus, and striatum, but differences disappeared at 60 min. Radiometabolite analysis revealed rapid peripheral metabolism, and absence of intact tracer in the brain at 60 min in vehicle-treated mice. In plasma, the parent fraction was unaffected by tariquidar, while it was significantly higher in the brain with P-gp inhibition. Metabolite-corrected brain-to-plasma concentration ratios (K_p,brain) confirmed negligible tracer uptake without P-gp blockade.

Conclusions

N-[¹¹C]methyl-(R)-JNJ-31020028 binds selectively to NPY2R but undergoes rapid metabolism and strong P-gp–mediated efflux at the murine blood–brain barrier. Reliable data interpretation requires early imaging and metabolite correction. For preclinical NPY2R-PET, pharmacological P-gp inhibition may be essential, and future tracers should be optimized for improved metabolic stability.

Background

Radiation-induced biologic bystander effects (RIBBEs) where non-irradiated cells display radiation like responses are well established in the context of external beam radiotherapy. However, their presence and characteristics in radionuclide therapy have been explored in only a limited number of studies. With the growing application of radionuclide therapy, this study was designed to investigate RIBBEs using a targeting agent labeled with three different types of emitters, aiming to quantify the varying biological effects attributable to each radionuclide and to devise strategies for achieving maximum therapeutic effect.Trastuzumab antibody targeting human epidermal growth factor receptor 2 (HER2) was labeled with radionuclides of varying energy profile; Y-90 (β⁻emitter), Lu-177 (β⁻/γ-emitter), and I-125 (Auger electron emitter). The radiolabeled conjugates were characterized, and their specificity was evaluated. Studies to evaluate both direct and bystander effects in (HER2) receptor overexpressing cell lines were performed via a media transfer protocol.

Results

The study highlights distinct cellular responses depending on the radionuclide employed, with significant bystander induced reductions in clonogenic survival observed for all radiolabeled formulations. Notably, HER2-overexpressing SK-OV-3 and SK-BR-3 cells displayed dose-dependent variations in survival, with ⁹⁰Y and ¹⁷⁷Lu demonstrating greater bystander toxicity than ¹²⁵I. The survival fraction of recipient cells, which were exposed solely to media containing bystander factors, decreased significantly, indicating the potency of bystander factors in influencing therapeutic outcomes. Additionally, cytotoxicity assays revealed that higher doses of ⁹⁰Y and ¹⁷⁷Lu induced substantial apoptosis and necrosis, whereas ¹²⁵I exhibited lower cytotoxicity, consistent with its lower energy emission profile. Reactive oxygen species (ROS) generation assays corroborated these findings, showing elevated ROS levels in direct and donor cell groups treated with ⁹⁰Y and ¹⁷⁷Lu, while recipient cells exhibited relatively lower ROS induction.

Conclusions

This study elucidates the complex interplay of direct and bystander effects in targeted radionuclide therapy, demonstrating that the therapeutic efficacy and cellular response depend on the specific radionuclide and its energy profile. The findings emphasize the need for further exploration of RIBBEs to optimize radionuclide-based cancer therapies to enhance their clinical efficacy.

Graphical abstract

Background

⁶⁸Ga-labeled tracers are increasingly important for PET imaging and as companion diagnostics with new therapeutic radiopharmaceuticals. The ⁶⁸Ge/⁶⁸Ga generator is a common source for clinical and preclinical ⁶⁸Ga-tracer production; however, its shelf life and efficiency are critical because of the high cost of generator replacement. This study assessed whether preconcentration of [⁶⁸Ga]GaCl₃ using strong cation exchange (SCX) resin can increase yield, apparent molar activity (AMA) and also extend generator shelf-life for preclinical applications.

Results

A ⁶⁸Ge/⁶⁸Ga generator (1.8 GBq at purchase, 14–18 months old) was used. Direct elution involved elution of [⁶⁸Ga]GaCl₃ in 0.1 M HCl, followed by addition to a DOTA-conjugated affibody in acetate buffer (pH 4.6). Preconcentration involved trapping [⁶⁸Ga]GaCl₃ on a SCX (Chromafix PS-H⁺) cartridge, rinsing, and eluting with 0.12 M HCl in 5 M NaCl (300–500 µL), followed by radiolabeling. Radiolabeling was performed at 75–80 °C for 15 min, the products were purified using a NAP-5 column, and the purity was assessed by high-performance liquid chromatography (HPLC). The SCX cartridge trapping efficiency was > 99%, with a elution efficiency of 95.2 ± 1.2% (n = 8). For direct elution, the decay-corrected radiochemical yield (RCYdc) was 78.7 ± 1.5% (n = 3), the AMA was 5.6 ± 0.4 MBq/nmol, and the radiochemical purity (RCP) was 95.3 ± 0.6%. For preconcentration, RCY_dc was 69.0 ± 10.0% (n = 3), AMA was 12.6 ± 2.1 MBq/nmol, and RCP was 95.7 ± 3.0%.

Conclusions

The preconcentration technique doubled the product yield and AMA, and extended the shelf life of the generator by 9–12 months for preclinical applications. Preconcentration of [⁶⁸Ga]GaCl₃ using SCX resin is a robust, cost-effective method for maximizing ⁶⁸Ga recovery and increasing the radiotracer yield and AMA, especially with older generators. This approach extends generator shelf-life, supports sustained preclinical research, and reduces radioactive waste and operational costs.

Background

There is a renewed interest in radioligand therapy for cancer treatment since the approval of Lu-177 vipivotide tetraxetan (PSMA-617) by regulators in Europe and the United States for metastatic castrate resistant prostate cancer (mCRPC) patients. Recent expansion of indications for vipivotide tetraxetan in mCRPC by the FDA promises to increase demand for this class of radioligand therapy. The development of new targeted radioligands for mCRPC that improve binding specificity and increase radiation dose delivered to tumors is an active research space. [¹⁷⁷Lu]Lu-P17-087 and [¹⁷⁷Lu]Lu-P17-088 are new prostate specific membrane antigen (PSMA) targeted drugs under clinical investigation in China. Heretofore the preparation of these investigational drugs has been carried out manually. This report introduces a new cassette-based, radiosynthesis platform named MiniLu™ and describes the optimization and automation of the preparation of [¹⁷⁷Lu]Lu-P17-087 and [¹⁷⁷Lu]Lu-P17-088 for clinical use.

Results

Radiolabeling of [¹⁷⁷Lu]Lu-P17-087 was optimized for pH, time, temperature and ligand concentration using 0.2 – 13 MBq of Lu-177 (> 740 GBq/mg) and MiniLu™. > 99% labeling yield measured by thin layer chromatography (TLC) was achieved with a pH = 5, 50–110 °C, 10 min, 3:1 mol ratio ligand: Lu-177; ligand mass > 3 nmole in 0.5 mL reaction solution. Quantitative labeling was confirmed using these conditions (80 °C) in simulations of clinical production doses of Lu-177 (7.4 GBq; > 3000 GBq/mg) by adding cold [^natLu]LuCl₃ (14 nmoles). [¹⁷⁷Lu]Lu-P17-088 labeling yield was > 99% under the identical simulation conditions. A final product yield (activity in dose vial / starting Lu-177 activity) of ~ 95% was achieved after formulation with L-ascorbate and gentisic acid (15 mL) and sterile filtration using MiniLu™ and the simulated high activity conditions for both compounds. Final optimized concentrations for automated dose preparation were 62 µM (0.6 mL) and 70 µM (0.6 mL) for [¹⁷⁷Lu]Lu-P17-087 and [¹⁷⁷Lu]Lu-P17-088, respectively. Over 100 independent automated MiniLu™ runs were completed successfully.

Conclusions

[¹⁷⁷Lu]Lu-P17-087 and [¹⁷⁷Lu]Lu-P17-088 doses can be produced in high yield (95%) and purity (99%) using MiniLu™. Optimized, automated methods using MiniLu™ will facilitate further clinical investigations using [¹⁷⁷Lu]Lu-P17-087 and [¹⁷⁷Lu]Lu-P17-088 by standardizing preparation and reducing radiation exposure to radiochemists.

Background

Targeting the gastrin-releasing peptide receptor (GRPR) is a promising approach for radionuclide therapy in prostate and breast cancers. GRPR-targeting peptides often have limited metabolic stability, which can compromise their clinical efficacy due to rapid degradation in the bloodstream, leading to reduced tumor uptake. We previously reported the GRPR-targeting peptide AU-RM26-M2 (DOTAGA-PEG₂-Pip-[Sar¹¹]RM26), which demonstrated promising pharmacokinetics in GRPR-expressing xenografts. In this study, we aimed to enhance the metabolic stability and targeting properties of AU-RM26-M2 by incorporating α-methyl-L-tryptophan (MetTrp) at position 8 in the pharmacophore, and to investigate the influence of chelator choice (DOTAGA vs. DOTA) for labeling with Lu-177, a β-emitting therapeutic nuclide.

Results

Therefore, we designed two peptides: PKB2 (DOTAGA-PEG₂-Pip-[MetTrp⁸, Sar¹¹]RM26) and PKB3 (DOTA-PEG₂-Pip-[MetTrp⁸, Sar¹¹]RM26). For comparison, we also evaluated the DOTA-bearing analogue of AU-RM26-M2, PKB1 (DOTA-PEG₂-Pip-[Sar¹¹]RM26). PKB1, PKB2, and PKB3 were labeled with Lu-177, achieving high radiochemical yields (> 97%) and purities (> 93%). In PC-3 cells, [¹⁷⁷Lu]Lu-PKB1, [¹⁷⁷Lu]Lu-PKB2, and [¹⁷⁷Lu]Lu-PKB3 showed affinity in the sub-nanomolar range and high specificity for GRPR, with a slow internalization rate. The radiopeptides with MetTrp⁸ modification had high metabolic stability against peptidases in vivo. In PC-3 xenografts, [¹⁷⁷Lu]Lu-PKB2 and [¹⁷⁷Lu]Lu-PKB3 demonstrated rapid background clearance and high GRPR-mediated tumor activity uptake at 2 h pi, exceeding activity uptake in the kidneys. Activity uptake in the tumor was highly retained at 24 h pi.

Conclusions

This study led to the development of two metabolically stable GRPR-targeting radiopeptides, [¹⁷⁷Lu]Lu-PKB2 and [¹⁷⁷Lu]Lu-PKB3, with a high potential for targeted radionuclide therapy.

Background

The stability testing of radiopharmaceuticals is a critical aspect of drug development and regulatory approval. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) provides comprehensive guidelines for stability testing, as outlined in the ICH Harmonised Tripartite Guideline Q1A(R2). In this study, the stability of o-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET) and methyl-[¹¹C]-L-methionine ([¹¹C]MET) was evaluated under GMP conditions to ensure their quality and safety. These radiopharmaceuticals are widely used in brain tumor imaging, yet their stability remains insufficiently studied. Given the short half-life of [¹¹C]MET (20 min), rapid preparation and administration are required, whereas the longer half-life of [¹⁸F]FET (110 min) allows for transportation, making stability considerations crucial due to potential environmental effects on the injection solution.

Results

Samples from each batch were tested at different temperatures and pH values across the entire expiration period. The radiochemical purity of [¹⁸F]FET remained between 98.04 and 100% by TLC, while UPLC values ranged from 95.93 to 99.59%. Differences between these methods stem from their sensitivity and operational principles. While UPLC provides precise separation, it may trap free fluoride, leading to overestimated purity values. In contrast, TLC allows for complete sample evaluation but has lower separation efficiency. Enantiomeric purity assessments confirmed that only the L-form was present, with no detectable D-enantiomer, in the case of [¹¹C]MET a maximum of 1.7% D-enantiomer was also detected. Stability remained above the 95% threshold between − 20 and 50 °C, with slight reductions at basic pH values. Even under stressed conditions no decomposition products were detected, and enantiomeric purity exceeded 90%, confirming the robustness of [¹⁸F]FET and [¹¹C]MET stability.

Conclusion

In this report, the stability of the [¹⁸F]FET and [¹¹C]MET radiopharmaceuticals was studied within the expiration time. [¹⁸F]FET remained stable until the end of the 12-h expiration time. [¹¹C]MET samples stored even under stressed conditions did not decrease under the acceptable limit during the shelf life of the radiopharmaceutical. These findings confirm that both radiopharmaceuticals maintain their stability within the defined shelf life, ensuring their reliability for clinical use. Further studies could explore additional environmental stress factors to enhance stability assessments and optimize storage conditions.

Background

Heart-type fatty acid–binding protein (FABP3) mediates the intracellular transport of fatty acids and is highly expressed in the myocardium. During myocardial infarction, FABP3 leaks from the myocardial cytoplasm into the blood; thus, detection of FABP3 using positron emission tomography (PET) is useful in identifying the site of myocardial damage. 4-(4-Fluoro-2-(1-phenyl-5-(2-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)phenoxy)butanoic acid (LUF) is a small-molecule organic compound that specifically binds FABP3. We confirmed that fluorine-18–labeled LUF enables clear, high-quality visualization of the myocardium as well as lesions associated with myocardial injury. In this study, we performed a process validation of [¹⁸F]LUF production for clinical use and evaluated its nonclinical toxicity and radiation dosimetry estimates using mouse biodistribution data.

Results

The activity yield of [¹⁸F]LUF at the end of synthesis was > 8 GBq, with > 99% radiochemical purity, > 500 GBq/μmol molar activity, and < 1.5 μg/185 MBq total chemical content. All process validation batches complied with the product specifications. An extended single-dose intravenous toxicity study in male and female Sprague–Dawley rats indicated a no-observed adverse effect level for LUF and decay-outed [¹⁸F]LUF injection solution of ≥ 25 μg/kg and ≥ 2.5 mL/kg, respectively. These doses are > 100 times the postulated maximum dose of LUF (0.25 μg/kg) and [¹⁸F]LUF injection solution (370 MBq). Reverse mutation tests were negative for LUF and the OH form produced after β-decay of [¹⁸F]LUF. Neither LUF nor the OH form exhibited cardiotoxicity toward cardiomyocytes derived from human induced pluripotent stem cells at > 100 times the postulated maximum dose. Biodistribution study results demonstrated predominantly hepatobiliary excretion of radioactivity. The most critical organ affected was the heart wall (68.6 μGy/MBq). The estimated effective dose was calculated as 10.2 μSv/MBq. These radiation exposure doses are equivalent to 25.4 mGy (heart wall) and 3.78 mSv for a planned maximum dose of 370 MBq.

Conclusions

[¹⁸F]LUF shows acceptable pharmacological safety at the dose required for adequate PET imaging. The potential risks associated with [¹⁸F]LUF injection are well within the acceptable dose limits.

Background

Radiopharmaceuticals offer targeted treatment by combining diagnostic or therapeutic radionuclides with biologically active molecules. Auranofin is the only Food and Drug Administration (FDA) approved gold(I) complex, originally developed for the treatment of rheumatoid arthritis. Recent evidence has highlighted its potential as an anticancer agent due to its ability to disrupt redox signaling, inhibit thioredoxin reductase, and impair glycolytic metabolism. This study aims to incorporate the true theranostic radionuclide ¹⁹⁸Au into the Auranofin scaffold and evaluate its impact in-vitro on cancer cells.

Results

Carrier-added (c.a.)¹⁹⁸Au was produced via neutron activation of ¹⁹⁷Au and subsequently converted into c.a. H [¹⁹⁸Au] [AuCl₄]. Downscaled synthetic protocols were developed to sequentially generate c.a. [¹⁹⁸Au] [Au(tht)Cl], [¹⁹⁸Au] [Au(PEt₃)Cl], and [¹⁹⁸Au]Auranofin. Radiochemical purity was evaluated using radio-high performance liquid chromatography, and in vitro stability was assessed in human serum albumin (HSA) over 72 h. Cytotoxic and metabolic activity were investigated in MCF7 and PC3 cancer cell lines using the cell viability assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) and hexokinase assay, respectively. [¹⁹⁸Au]Auranofin (c.a.) was obtained with a yield of 57.0 ± 3.2% and a radiochemical purity of 96.2 ± 3.9%. The compound demonstrated stability in human serum albumin, maintaining 96.9 ± 2.5% integrity over 72 h. In vitro studies revealed that c.a. [¹⁹⁸Au]Auranofin exhibited enhanced cytotoxicity and significant hexokinase inhibition compared to its non-radioactive counterpart, while the precursor complexes remained non-toxic up to 20 µM. Viability loss was both concentration and radioactivity dependent across both cell lines.

Conclusions

[¹⁹⁸Au]Auranofin (c.a.) represents a stable and effective radiogold-based radiopharmaceutical agent, offering redox-targeted cytotoxicity alongside β⁻ emission mediated cell death and γ emission based imaging potential. These findings highlight c.a. [¹⁹⁸Au]Auranofin as a promising radiogold-based theranostic candidate, offering dual capabilities in targeted cytotoxicity and nuclear imaging. While the in vitro results are encouraging, further in vivo and translational studies are warranted to fully evaluate its clinical potential in nuclear medicine guided cancer therapy.