A thiourea-bridged 99mTc(CO)3-dipicolylamine-2-nitroimidazole complex for targeting tumor hypoxia: Utilizing metabolizable thiourea-bridge to improve pharmacokinetics.

The 2-nitroimidazole based 99mTc-radiopharmaceuticals are widely explored for imaging tumor hypoxia. Radiopharmaceuticals for targeting hypoxia are often lipophilic and therefore, show significant uptake in liver and other vital organs. In this context, lipophilic radiopharmaceuticals with design features enabling faster clearance from liver may be more desirable. A dipicolylamine-NCS bifunctional chelator that could generate a thiourea-bridge up on conjugation to primary amine bearing molecule was used to synthesize a 2-nitroimidazole-dipicolyl amine ligand for radiolabeling with 99mTc(CO)3 core. Corresponding Re(CO)3-analogue was prepared to establish the structure of 2-nitroimidazole-99mTc(CO)3 complex prepared in trace level. The 2-nitroimidazole-99mTc(CO)3 complex showed a hypoxic to normoxic ratio of ~2.5 in CHO cells at 3 h. In vivo, the complex showed accumulation and retention in tumor with high tumor to blood and tumor to muscle ratio. The study demonstrated the utility of metabolizable thiourea-bridge in 2-nitroimidazole-99mTc(CO)3 complex in inducing faster clearance of the radiotracer from liver. The dipicolylamine-NCS bifunctional chelator reported herein can also be used for radiolabeling other class of target specific molecules with 99mTc(CO)3 core.

Radiosynthesis and preclinical evaluation of a 68Ga-labeled tetrahydroisoquinoline-based ligand for PET imaging of C-X-C chemokine receptor type 4 in an animal model of glioblastoma.

BACKGROUND: This study aimed to develop a novel positron emission tomography (PET) tracer, [68Ga]Ga-TD-01, for CXCR4 imaging. To achieve this goal, the molecular scaffold of TIQ15 was tuned by conjugation with the DOTA chelator to make it suitable for 68Ga radiolabeling. METHODS: A bifunctional chelator was prepared by conjugating the amine group of TIQ15 with p-NCS-Bz-DOTA, yielding TD-01, with a high yield (68.92%). TD-01 was then radiolabeled with 68Ga using 0.1 M ammonium acetate at 60 °C for 10 min. A 1-h dynamic small animal PET/MRI study of the labeled compound in GL261-luc2 tumor-bearing mice was performed, and brain tumor uptake was assessed. Blocking studies involved pre-administration of TIQ15 (10 mg/kg) 10 min before the PET procedure started. RESULTS: [68Ga]Ga-TD-01 exhibited a radiochemical yield (RCY) of 36.33 ± 1.50% (EOS), with a radiochemical purity > 99% and a molar activity of 55.79 ± 1.96 GBq/µmol (EOS). The radiotracer showed in vitro stability in PBS and human plasma for over 4 h. Biodistribution studies in healthy animals revealed favorable kinetics for subsequent PET pharmacokinetic modeling with low uptake in the brain and moderate uptake in lungs, intestines and spleen. Elimination could be assigned to a renal-hepatic pathway as showed by high uptake in kidneys, liver, and urinary bladder. Importantly, [68Ga]Ga-TD-01 uptake in glioblastoma (GBM)-bearing mice significantly decreased upon competition with TIQ15, with a baseline tumor-to-background ratios > 2.5 (20 min p.i.), indicating high specificity. CONCLUSION: The newly developed CXCR4 PET tracer, [68Ga]Ga-TD-01, exhibited a high binding inhibition for CXCR4, excellent in vitro stability, and favorable pharmacokinetics, suggesting that the compound is a promising candidate for full in vivo characterization of CXCR4 expression in GBM, with potential for further development as a tool in cancer diagnosis.

Synthesis, Quality Control, and Bench-to-Bed Translation of a New [68Ga]Ga-Labeled NOTA-Conjugated Bisphosphonate for Imaging Skeletal Metastases by Positron Emission Tomography.

Background: Early detection of skeletal metastasis is of great interest to determine the prognosis of cancer. Positron emission tomography-computed tomography (PET-CT) imaging provides a better temporal and spectral resolution than single photon emission computed tomography-computed tomography (SPECT-CT) imaging, and hence is more suitable to detect small metastatic lesions. Although [18F]NaF has been approved by U.S. FDA for a similar purpose, requirement of a medical cyclotron for its regular formulation restricts its extensive utilization. Efforts have been made to find suitable alternative molecules that can be labeled with 68Ga and used in PET-CT imaging. Objective: The main objective of this study is to synthesize and evaluate a new [68Ga]Ga-labeled NOTA-conjugated geminal bisphosphonate for its potential use in early detection of skeletal metastases using PET-CT. Methods: The authors performed a multistep synthesis of a new NOTA-conjugated bisphosphonic acid using thiourea linker and radiolabeled the molecule with 68Ga. The radiolabeled formulation was evaluated for its in vitro stability, affinity for hydroxyapatite (HA) particles, preclinical biodistribution in animal models, and PET-CT imaging in patients. Results: The bifunctional chelator (NOTA)-conjugated bisphosphonate was synthesized with 97.8% purity and radiolabeled with 68Ga in high yield (>98%). The radiolabeled formulation was found to retain its stability in vitro to the extent of >95% up to 4 h in physiological saline and human serum. The formulation also showed high affinity for HA particles in vitro with Kd = 907 ± 14 mL/g. Preclinical biodistribution studies in normal Wistar rats demonstrated rapid and almost exclusive skeletal accumulation of the complex. PET-CT imaging in a patient confirmed its ability to detect small metastatic skeletal lesions. Conclusions: The newly synthesized [68Ga]Ga-labeled NOTA-conjugated bisphosphonate is a promising radiotracer for PET-CT imaging for skeletal metastases.

Synthesis of novel DOTA-/AAZTA-based bifunctional chelators: Solution thermodynamics, peptidomimetic conjugation, and radiopharmaceutical evaluation.

Bifunctional chelators (BFCs), which link metallic radionuclide and a targeting vector, are some of the most crucial components of metallic radionuclide-based radiopharmaceuticals for positron-emission computed tomography (PET) imaging. In this study, we designed and synthesized two versatile BFCs, p-NCS-Ph-DE4TA and p-NCS-Ph-AAZ4TA, and we conjugated them with a prostate-specific membrane antigen (PSMA) inhibitor. These two chelators showed high affinity for Ga (III) according to a study of the thermodynamics and kinetics and DFT calculations. The labeled PSMA targeted probes, [68Ga]Ga-p-NCS-Ph-DE4TA-PSMA and [68Ga]Ga-p-NCS-Ph-AAZ4TA-PSMA, maintained excellent stability in vitro, and they exhibited high specific activity when binding to PSMA. A PET/CT imaging study in mice bearing SMMC-7721 hepatocellular carcinoma xenografts demonstrated clear visualization of tumors with a high tumor uptake and low background level, indicating the excellent performance in vivo and specific activity when targeting hepatocellular carcinomas. In summary, p-NCS-Ph-DE4TA and p-NCS-Ph-AAZ4TA are leading developmental candidates for PET imaging for tumor diagnosis.

Radiochemical and Biological Evaluation of 3p-C-NETA-ePSMA-16, a Promising PSMA-Targeting Agent for Radiotheranostics.

Bifunctional chelators (BFCs) are a key element in the design of radiopharmaceuticals. By selecting a BFC that efficiently complexes diagnostic and therapeutic radionuclides, a theranostic pair possessing almost similar biodistribution and pharmacokinetic properties can be developed. We have previously reported 3p-C-NETA as a promising theranostic BFC, and the encouraging preclinical outcomes obtained with [18F]AlF-3p-C-NETA-TATE led us to conjugate this chelator to a PSMA-targeting vector for imaging and treatment of prostate cancer. In this study, we synthesized 3p-C-NETA-ePSMA-16 and radiolabeled it with different diagnostic (111In, 18F) and therapeutic (177Lu, 213Bi) radionuclides. 3p-C-NETA-ePSMA-16 showed high affinity to PSMA (IC50 = 4.61 ± 1.33 nM), and [111In]In-3p-C-NETA-ePSMA-16 showed specific cell uptake (1.41 ± 0.20% ID/106 cells) in PSMA expressing LS174T cells. Specific tumor uptake of [111In]In-3p-C-NETA-ePSMA-16 was observed up to 4 h p.i. (1.62 ± 0.55% ID/g at 1 h p.i.; 0.89 ± 0.58% ID/g at 4 h p.i.) in LS174T tumor-bearing mice. Only a faint signal could be seen at 1 h p.i. in the SPECT/CT scans, whereas dynamic PET/CT scans performed after administration of [18F]AlF-3p-C-NETA-ePSMA-16 in PC3-Pip tumor xenografted mice resulted in a better tumor visualization and imaging contrast. Therapy studies with short-lived radionuclides such as 213Bi could further elucidate the therapeutic potential of 3p-C-NETA-ePSMA-16 as a radiotheranostic.

Bismuth chelation for targeted alpha therapy: Current state of the art.

Current interest in the α-emitting bismuth radionuclides, bismuth-212 (212Bi) and bismuth-213 (213Bi), stems from their great potential for targeted alpha therapy (TAT), an expanding and promising approach for the treatment of micrometastatic disease and the eradication of single malignant cells. To selectively deliver their emission to the cancer cells, these radiometals must be firmly coordinated by a bifunctional chelator (BFC) attached to a tumour-seeking vector. This review provides a comprehensive overview of the current state-of-the-art chelating agents for bismuth radioisotopes. Several aspects are reported, from their 'cold' chelation chemistry (thermodynamic, kinetic, and structural properties) and radiolabelling investigations to the preclinical and clinical studies performed with a variety of bioconjugates. The aim of this review is to provide both a guide for the rational design of novel optimal platforms for the chelation of these attractive α-emitters and emphasize the prospects of the most encouraging chelating agents proposed so far.

AAZTA-Derived Chelators for the Design of Innovative Radiopharmaceuticals with Theranostic Applications.

With the development of 68Ga and 177Lu radiochemistry, theranostic approaches in modern nuclear medicine enabling patient-centered personalized medicine applications have been growing in the last decade. In conjunction with the search for new relevant molecular targets, the design of innovative chelating agents to easily form stable complexes with various radiometals for theranostic applications has gained evident momentum. Initially conceived for magnetic resonance imaging applications, the chelating agent AAZTA features a mesocyclic seven-membered diazepane ring, conferring some of the properties of both acyclic and macrocyclic chelating agents. Described in the early 2000s, AAZTA and its derivatives exhibited interesting properties once complexed with metals and radiometals, combining a fast kinetic of formation with a slow kinetic of dissociation. Importantly, the extremely short coordination reaction times allowed by AAZTA derivatives were particularly suitable for short half-life radioelements (i.e., 68Ga). In view of these particular characteristics, the scope of this review is to provide a survey on the design, synthesis, and applications in the nuclear medicine/radiopharmacy field of AAZTA-derived chelators.

Modern Developments in Bifunctional Chelator Design for Gallium Radiopharmaceuticals.

The positron-emitting radionuclide gallium-68 has become increasingly utilised in both preclinical and clinical settings with positron emission tomography (PET). The synthesis of radiochemically pure gallium-68 radiopharmaceuticals relies on careful consideration of the coordination chemistry. The short half-life of 68 min necessitates rapid quantitative radiolabelling (≤10 min). Desirable radiolabelling conditions include near-neutral pH, ambient temperatures, and low chelator concentrations to achieve the desired apparent molar activity. This review presents a broad overview of the requirements of an efficient bifunctional chelator in relation to the aqueous coordination chemistry of gallium. Developments in bifunctional chelator design and application are then presented and grouped according to eight categories of bifunctional chelator: the macrocyclic chelators DOTA and TACN; the acyclic HBED, pyridinecarboxylates, siderophores, tris(hydroxypyridinones), and DTPA; and the mesocyclic diazepines.

Novel theranostic agent for PET imaging and targeted radiopharmaceutical therapy of tumour-infiltrating immune cells in glioma.

BACKGROUND: Malignant gliomas are deadly tumours with few therapeutic options. Although immunotherapy may be a promising therapeutic strategy for treating gliomas, a significant barrier is the CD11b+ tumour-associated myeloid cells (TAMCs), a heterogeneous glioma infiltrate comprising up to 40% of a glioma's cellular mass that inhibits anti-tumour T-cell function and promotes tumour progression. A theranostic approach uses a single molecule for targeted radiopharmaceutical therapy (TRT) and diagnostic imaging; however, there are few reports of theranostics targeting the tumour microenvironment. METHODS: Utilizing a newly developed bifunctional chelator, Lumi804, an anti-CD11b antibody (αCD11b) was readily labelled with either Zr-89 or Lu-177, yielding functional radiolabelled conjugates for PET, SPECT, and TRT. FINDINGS: 89Zr/177Lu-labeled Lumi804-αCD11b enabled non-invasive imaging of TAMCs in murine gliomas. Additionally, 177Lu-Lumi804-αCD11b treatment reduced TAMC populations in the spleen and tumour and improved the efficacy of checkpoint immunotherapy. INTERPRETATION: 89Zr- and 177Lu-labeled Lumi804-αCD11b may be a promising theranostic pair for monitoring and reducing TAMCs in gliomas to improve immunotherapy responses. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Exploring the Application of Bifunctional Metal Chelators in Treating Triple-Negative Breast Cancer.

Purpose: In this study, we independently synthesised and labelled a novel bidentate bifunctional chelating agent, 177Lu-3,4-HOPO-Cetuximab, that achieved tight binding between targeting and radioactivity, and evaluated its targeted killing ability of cells in vitro and in vivo. Method: 3,4-HOPO was successfully synthesised through a series of chemical steps using malt phenol as the raw material, which was then coupled with Cetuximab labelled with 177Lu. 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab was tested for its cell viability and cell-binding rate after different times and at different doses by CCK-8 and cell-binding experiments. 177Lu-3,4-HOPO-Cetuximab (~500 µCi) and 177Lu-DOTA-Cetuximab (~500 µCi) were injected into the tail vein of a subcutaneous metastasis mouse model of triple-negative breast cancer with a single injection, and tumour volume growth and body weight changes were regularly monitored for 20 days. The radioactivity distribution in nude mice was analysed after sacrifice, and the treated and untreated tumour tissues were analysed by HE staining. Result: The cell viability of 177Lu-3,4-HOPO-Cetuximab declined exponentially after treatment for 48 h at 50 Bq/mL to 500 kBq/mL, respectively; the cell activity was slowed down from 8 to 96 h at a dose of 500 kBq; while the binding rate of 4T1 cells in 177Lu-3,4-HOPO-Cetuximab from 1 to 24 h, respectively, increased logarithmically, which was similar with 177Lu-DOTA-Cetuximab. After 20 days of treatment, the body weight of nude mice with 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were hardly changed, while the body weight with physiological saline decreased significantly. The tumour inhibition rate of the 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were (37.03 ± 11.16)% and (38.7 ± 5.1)%; HE staining showed that tumour cells were affected by the action of 177Lu causing necrosis. Conclusion: The experiments showed that 177Lu-3,4-HOPO-Cetuximab has a certain targeted therapeutic ability for triple-negative breast cancer, and it is expected to become a potential targeted nuclear medicine treatment for triple-negative breast cancer.

Bismuth-213 for Targeted Radionuclide Therapy: From Atom to Bedside.

In contrast to external high energy photon or proton therapy, targeted radionuclide therapy (TRNT) is a systemic cancer treatment allowing targeted irradiation of a primary tumor and all its metastases, resulting in less collateral damage to normal tissues. The α-emitting radionuclide bismuth-213 (213Bi) has interesting properties and can be considered as a magic bullet for TRNT. The benefits and drawbacks of targeted alpha therapy with 213Bi are discussed in this review, covering the entire chain from radionuclide production to bedside. First, the radionuclide properties and production of 225Ac and its daughter 213Bi are discussed, followed by the fundamental chemical properties of bismuth. Next, an overview of available acyclic and macrocyclic bifunctional chelators for bismuth and general considerations for designing a 213Bi-radiopharmaceutical are provided. Finally, we provide an overview of preclinical and clinical studies involving 213Bi-radiopharmaceuticals, as well as the future perspectives of this promising cancer treatment option.

Proof-of-Concept Study of the NOTI Chelating Platform: Preclinical Evaluation of 64Cu-Labeled Mono- and Trimeric c(RGDfK) Conjugates.

PURPOSE: We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for the preparation of 68Ga- and 64Cu-based radiopharmaceuticals. Based on this platform, the chelator scaffold NOTI-TVA with three additional carboxylic acid groups for bioconjugation was synthesized and characterized. The primary aims of this proof-of-concept study were (1) to evaluate if trimeric radiotracers on the basis of the NOTI-TVA 6 scaffold can be developed, (2) to determine if the additional substituents for bioconjugation at the non-coordinating NH atoms of the imidazole residues of the building block NOTI influence the metal binding properties, and (3) what influence multiple targeting vectors have on the biological performance of the radiotracer. The cyclic RGDfK peptide that specifically binds to the αvß3 integrin receptor was selected as the biological model system. PROCEDURES: Two different synthetic routes for the preparation of NOTI-TVA 6 were explored. Three c(RGDfK) peptide residues were conjugated to the NOTI-TVA 6 building block by standard peptide chemistry providing the trimeric bioconjugate NOTI-TVA-c(RGDfK)3 9. Labeling of 9 with [64Cu]CuCl2 was performed manually at pH 8.2 at ambient temperature. Binding affinities of Cu-8, the Cu2+ complex of the previously described monomer NODIA-Me-c(RGDfK) 8, and the trimer Cu-9 to integrin αvß3 were determined in competitive cell binding experiments in the U-87MG cell line. The pharmacokinetics of both 64Cu-labeled conjugates [64Cu]Cu-8 and [64Cu]Cu-9 were determined by small-animal PET imaging and ex vivo biodistribution studies in mice bearing U-87MG xenografts. RESULTS: Depending on the synthetic route, NOTI-TVA 6 was obtained with an overall yield up to 58 %. The bioconjugate 9 was prepared in 41 % yield. Both conjugates [64Cu]Cu-8 and [64Cu]Cu-9 were radiolabeled quantitatively at ambient temperature in high molar activities of Am ~ 20 MBq nmol-1 in less than 5 min. Competitive inhibitory constants IC50 of c(RDGfK) 7, Cu-8, and Cu-9 were determined to be 159.5 ± 1.3 nM, 256.1 ± 2.1 nM, and 99.5 ± 1.1 nM, respectively. In small-animal experiments, both radiotracers specifically delineated αvß3 integrin-positive U-87MG tumors with low uptake in non-target organs and rapid blood clearance. The trimer [64Cu]Cu-9 showed a ~ 2.5-fold higher tumor uptake compared with the monomer [64Cu]Cu-8. CONCLUSIONS: Functionalization of NOTI at the non-coordinating NH atoms of the imidazole residues for bioconjugation was straightforward and allowed the preparation of a homotrimeric RGD conjugate. After optimization of the synthesis, required building blocks to make NOTI-TVA 6 are now available on multi-gram scale. Modifications at the imidazole groups had no measurable impact on metal binding properties in vitro and in vivo suggesting that the NOTI scaffold is a promising candidate for the development of 64Cu-labeled multimeric/multifunctional radiotracers.

Design of immunogens: The effect of bifunctional chelator on immunological response to chelated copper.

To produce specific antibodies for the detection and quantification of copper ions, bifunctional chelators (BFCs) are commonly applied in the preparation of copper conjugates. However, some copper-chelator complexes exhibit limited stability under in vivo conditions. In this study, Cu2+ was coupled with carrier proteins via three different macrocyclic BFCs: p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, and p-SCN-Bn-TETA. The stability in plasma and the immunogenicity of three copper immunoconjugates were compared. The chelators other than p-SCN-Bn-DOTA were very stable in plasma, with <9% dissociation of Cu2+ over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu2+-NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu2+. p-SCN-Bn-NOTA, which showed significant advantages over the other chelators, was used for antibody production. The efficiency of immune-positive hybridoma production was satisfactory, and the resultant monoclonal antibodies (McAbs) 4B7 showed sensitivity (half-maximal inhibitory concentration (IC50) of 8.9 ng/mL) to chelated Cu2+, with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu2+ from water samples was 85.7-108%, and the intra- and inter-assay coefficients of variation were 4.0-10.1% and 7.1-11.4%, respectively. Compared with previously reported McAb specific to Cu2+, DF4, the sensitivity of the newly developed assay was improved 100-fold. The results of this study indicate the utility of NOTA for the efficient generation of highly sensitive McAbs against Cu2+.

Preparation of a Dithiol-Reactive Probe for PET Imaging of Cell Death.

Conjugates of 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO) with optical or radionuclide probes are able to image cell death in vivo. GSAO conjugates are retained in the cytosol of dying and dead cells via the formation of covalent bonds between the As(III) ion and the thiol groups of proximal cysteine residues. Here we describe the method for preparing a NODAGA-GSAO conjugate and its radiolabeling with gallium-68 (68Ga-NODAGA-GSAO) for positron-emission tomography (PET) imaging of cell death.

Rational Design, Synthesis and Preliminary Evaluation of Novel Fusarinine C-Based Chelators for Radiolabeling with Zirconium-89.

Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for 89Zr. Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for 89Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of 89Zr-complexes by saturating the 8-coordination sphere of [89Zr] Zr4+, and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)2 and FSC(succ)3. FSC(succ)2 was further derivatized to FSC(succ)2 AA by reacting with acetic anhydride (AA). The Zr4+ complexation properties of these chelators were studied by reacting with ZrCl4. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of 89Zr-complexes were carried out in vitro and the results were compared with those for 89Zr-desferrioxamine B ([89Zr]Zr-DFO) and 89Zr-triacetylfusarinine C ([89Zr]Zr-TAFC). The in vivo properties of [89Zr]Zr-FSC(succ)3 were further compared with [89Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)2AA and FSC(succ)3 were synthesized with satisfactory yields. Complexation with ZrCl4 was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)2AA and Zr-FSC(succ)3 with 1:1 stoichiometry. Distribution coefficients of 89Zr-complexes revealed increased hydrophilic character compared to [89Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [89Zr]Zr-FSC(succ)3 > [89Zr]Zr-TAFC > [89Zr]Zr-FSC(succ)2AA >> [89Zr]Zr-DFO. Biodistribution studies of [89Zr]Zr-FSC(succ)3 revealed a slower excretion pattern compared to [89Zr]Zr-TAFC. In conclusion, [89Zr]Zr-FSC(succ)3 showed the best stability and inertness. The promising results obtained with [89Zr]Zr-FSC(succ)2AA highlight the potential of FSC(succ)2 as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.

Radionuclide generators: the prospect of availing PET radiotracers to meet current clinical needs and future research demands.

Targeted molecular imaging with positron emission tomography (PET) constitutes a successful technique for detecting and diagnosing disease conditions promptly and accurately, and for effectively prognosticating outcomes and treating patients with a tailored and more individualized intervention. In order to expand the success of PET in nuclear medicine, it is important to assure access to radiotracers of desired quantities and qualities. In this context, the benefit of accessing PET radiotracers through a radionuclide generator (RNG) cannot be overstated, as generators offer the potential of enriching the PET radiotracer arsenal at the medical centers both with and without onsite cyclotrons. While RNG technology to avail PET tracers is in its infancy, their use is expected to revitalize current PET practices and seems poised to broaden the palette of PET in nuclear medicine in the foreseeable future. In this review, we discuss the principles of RNGs, assess major parent/daughter pairs of interest for PET, RNGs currently in use in clinical PET, and identify the potentially useful RNGs which have made substantial progress or are likely to be used in daily clinical practices in the near future. Availability of the parent radionuclides required for PET RNGs is an important criterion and hence their production will also be reviewed. This overview outlines a critical assessment of RNGs to avail PET tracers, the contemporary status of RNGs, and key challenges and apertures to the near future.

Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent.

The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst2-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)3]+ and [99mTc][Tc(CO)3]+. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst2-ANT peptide (3), to yield NSO-sst2-ANT (4). Two synthetic methods were used to prepare Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)3]+ complexation by 2. The resulting products demonstrated the expected molecular mass and nanomolar in vitro SSTR2 affinity (IC50 values under 30 nM, AR42J cells, [125I]iodo-Tyr11-somatostatin-14 radioligand standard). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [99mTc][Tc(OH2)3(CO)3]+ (99mTc; t½â€¯= 6 h, 141 keV γ) with 4 formed a third product, distinct from the Re analogues, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24 h), along with 75% stability in mouse serum through 4 h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [99mTc][Tc(CO)3]-labeled sst2-ANT derivative previously studied. Yet despite having adequate tumor uptake at 1 h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-saturating dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)3]+ core (M = Re, 99mTc) by disfavoring involvement of the N-triazole.

Preparation and preclinical evaluation of a 68Ga-labelled c(RGDfK) conjugate comprising the bifunctional chelator NODIA-Me.

BACKGROUND: We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three, five-membered azaheterocyclic arms for the development of 68Ga- and 64Cu-based radiopharmaceuticals. Here, a 68Ga-labelled conjugate comprising the bifunctional chelator NODIA-Me in combination with the αvß3-targeting peptide c(RGDfK) has been synthesized and characterized. The primary aim was to evaluate further the potential of our NODIA-Me chelating system for the development of 68Ga-labelled radiotracers. RESULTS: The BFC NODIA-Me was conjugated to c(RGDfK) by standard peptide chemistry to obtain the final bioconjugate NODIA-Me-c(RGDfK) 3 in 72% yield. Labelling with [68Ga]GaCl3 was accomplished in a fully automated, cGMP compliant process to give [68Ga]3 in high radiochemical yield (98%) and moderate specific activity (~ 8 MBq nmol- 1). Incorporation of the Ga-NODIA-Me chelate to c(RGDfK) 2 had only minimal influence on the affinity to integrin αvß3 (IC50 values [natGa]3 = 205.1 ± 1.4 nM, c(RGDfK) 2 = 159.5 ± 1.3 nM) as determined in competitive cell binding experiments in U-87 MG cell line. In small-animal PET imaging and ex vivo biodistribution studies, the radiotracer [68Ga]3 showed low uptake in non-target organs and specific tumor uptake in U-87 MG tumors. CONCLUSION: The results suggest that the bifunctional chelator NODIA-Me is an interesting alternative to existing ligands for the development of 68Ga-labelled radiopharmaceuticals.

Syntheses and evaluation of 68 Ga- and 153 Sm-labeled DOTA-conjugated bisphosphonate ligand for potential use in detection of skeletal metastases and management of pain arising from skeletal metastases.

This article reports the syntheses and evaluation of 68 Ga- and 153 Sm-complexes of a new DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-conjugated geminal bisphosphonate, DOTA-Bn-SCN-BP, for their potential uses in the early detection of skeletal metastases by imaging and palliation of pain arising from skeletal metastases, respectively. The conjugate was synthesized in high purity following an easily adaptable three-step reaction scheme. Gallium-68- and 153 Sm-complexes were prepared in high yield (>98%) and showed excellent in vitro stability in phosphate-buffered saline (PBS) and human serum. Both the complexes showed high affinity for hydroxyapatite particles in in vitro binding study. In biodistribution studies carried out in normal Wistar rats, both the complexes exhibited rapid skeletal accumulation with almost no retention in any other major organ. The newly synthesized molecule DOTA-Bn-SCN-BP would therefore be a promising targeting ligand for the development of radiopharmaceuticals for both imaging skeletal metastases and palliation of pain arising out of it in patients with cancer when radiolabeled with 68 Ga and 153 Sm, respectively. A systematic comparative evaluation, however, showed that there was no significant improvement of skeletal accumulation of the 153 Sm-DOTA-Bn-SCN-BP complex over 153 Sm-DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid) as the later itself demonstrated optimal properties required for an agent for bone pain palliation.

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector.

INTRODUCTION: We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for complexation of the PET nuclides gallium-68 and copper-64. The main objective of this study was to evaluate the stability and pharmacokinetics of 68Ga- and 64Cu-complexes of the bifunctional chelator NODIA-Me 1 covalently bound to a PSMA targeting vector in vivo. METHODS: NODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the bioconjugate NODIA-Me-NaI-Ahx-PSMA 4. The stability of [68Ga]4 and [64Cu]4 was assessed in vitro by serum stability studies. The PSMA binding affinity was determined in competitive cell experiments in LNCaP cells using 68Ga-PSMA-HBED-CC as radioligand. The stability and pharmacokinetics of [68Ga]4 and [64Cu]4 was evaluated by PET imaging and ex vivo biodistribution studies in mice bearing subcutaneous LNCaP tumors. RESULTS: In human serum, [68Ga]4 and [64Cu]4 remained intact to 85% (3 h) and 92% (24 h), respectively. Nature of the metal chelate influenced PSMA binding affinity with IC50 of 233 ±â€¯10 nM for uncomplexed 4, 681 ±â€¯7 nM for Cu-4 and 176 ±â€¯10 nM for Ga-4. In animal studies, [68Ga]4 and [64Cu]4 revealed low uptake (≤1% IA g-1) in the majority of organs. Kidney uptake at 1 h p.i. was 6.28 ±â€¯0.92% IA g-1 and 4.96 ±â€¯0.79% IA g-1 and specific tumor uptake was 1.33 ±â€¯0.46% IA g-1 and 2.15 ±â€¯0.38% IA g-1 for [68Ga]4 and [64Cu]4, respectively. CONCLUSION: The bifunctional chelator NODIA-Me 1 was successfully conjugated to a PSMA targeting moiety. In small-animal PET imaging and ex vivo biodistribution studies, 68Ga- and 64Cu-labelled conjugates specifically delineated PSMA-positive LNCaP tumors and exhibited rapid renal clearance from non-target tissues with no significant demetallation/transchelation in vivo. The results support further development of this novel chelating platform for production of 68Ga- and 64Cu-labelled radiopharmaceuticals.