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Background: Automated [ 89 Zr]Zr-radiolabeling processes have the potential to streamline the production of [ 89 Zr]Zr-labelled PET imaging agents. Most radiolabeling protocols use [ 89 Zr][Zr(ox) 4 ] 4- as the starting material and oxalate is removed after radiolabeling. In some instances, radiolabeling with [ 89 Zr]ZrCl 4 as starting material gives better radiochemical yields at lower reaction temperatures. In this work, a fully-automated process for production of [ 89 Zr]ZrCl 4 is reported and its use for the synthesis of [ 89 Zr]ZrDFOSq-bisPhPSMA and [ 89 Zr]ZrDFOSq-TATE., Results: A simple automated process for the isolation of [ 89 Zr]ZrCl 4 by trapping [ 89 Zr][Zr(ox) 4 ] 4- on a bicarbonate-activated strong anion exchange cartridge followed by elution with 0.1 M HCl in 1 M NaCl was developed. [ 89 Zr]ZrCl 4 was routinely recovered from [ 89 Zr][Zr(ox) 4 ] 4- in > 95% yield in mildly acidic solution of 0.1 M HCl in 1 M NaCl using a fully-automated process. The [ 89 Zr]ZrCl 4 was neutralized with sodium acetate buffer (0.25 M) removing the requirement for cumbersome manual neutralization with strong base. The mixture of [ 89 Zr]ZrCl 4 was used for direct automated radiolabeling reactions to produce [ 89 Zr]Zr-DFOSquaramide-bisPhPSMA and [ 89 Zr]ZrDFOSquaramide-TATE in 80-90% over all RCY in > 95% RCP., Conclusions: This method for the production of [ 89 Zr]ZrCl 4 does not require removal of HCl by evaporation making this process relatively fast and efficient. The fully automated procedures for the production of [ 89 Zr]ZrCl 4 and its use in radiolabeling are well suited to support the centralized and standardized manufacture of multiple dose preparations of zirconium-89 based radiopharmaceuticals., (© 2024. The Author(s).)

Peptides are ideal for theranostic development as they afford rapid target accumulation, fast clearance from background tissue, and exhibit good tissue penetration. Previously, we developed a novel series of peptides that presented discreet folding propensity leading to an optimal candidate [ 68 Ga]Ga-DOTA- GA1 ([D-Glu] 6 -Ala-Tyr- N MeGly-Trp- N MeNle-Asp-Nal-NH 2 ) with 50 pM binding affinity against cholecystokinin-2 receptors (CCK 2 R). However, we were confronted with challenges of unfavorably high renal uptake. Methods: A structure activity relationship study was undertaken of the lead theranostic candidate. Prudent structural modifications were made to the peptide scaffold to evaluate the contributions of specific N -terminal residues to the overall biological activity. Optimal candidates were then evaluated in nude mice bearing transfected A431-CCK 2 tumors, and their biodistribution was quantitated ex vivo . Results: We identified and confirmed that D-Glu 3 to D-Ala 3 substitution produced 2 optimal candidates, [ 68 Ga]Ga-DOTA- GA12 and [ 68 Ga]Ga-DOTA- GA13 . These radiopeptides presented with high target/background ratios, enhanced tumor retention, excellent metabolic stability in plasma and mice organ homogenates, and a 4-fold reduction in renal uptake, significantly outperforming their non-alanine counterparts. Conclusions: Our study identified novel radiopharmaceutical candidates that target the CCK 2 R. Their high tumor uptake and reduced renal accumulation warrant clinical translation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Peptide receptor radionuclide therapy (PRRT) is a promising form of systemic radiation therapy designed to eradicate cancer. Cholecystokinin-2 receptor (CCK 2 R) is an important molecular target that is highly expressed in a range of cancers. This study describes the synthesis and in vivo characterization of a novel series of 177 Lu-labeled peptides ([ 177 Lu]Lu- 2b - 4b ) in comparison with the reference CCK 2 R-targeting peptide CP04 ([ 177 Lu]Lu- 1b ). [ 177 Lu]Lu- 1b-4b showed high chemical purity (HPLC ≥ 94%), low Log  D 7.4 (-4.09 to -4.55) with strong binding affinity to CCK 2 R ( K D 0.097-1.61 nM), and relatively high protein binding (55.6-80.2%) and internalization (40-67%). Biodistribution studies of the novel 177 Lu-labeled peptides in tumors (AR42J and A431-CCK 2 R) showed uptake one- to eight-fold greater than the reference compound CP04 at 1, 24, and 48 h. Rapid clearance and high tumor uptake and retention were established for [ 177 Lu]Lu- 2b - 4b , making these compounds excellent candidates for theranostic applications against CCK 2 R-expressing tumors.

(1) Background: [18F]Flumazenil 1 ([18F]FMZ) is an established positron emission tomography (PET) radiotracer for the imaging of the gamma-aminobutyric acid (GABA) receptor subtype, GABAA in the brain. The production of [18F]FMZ 1 for its clinical use has proven to be challenging, requiring harsh radiochemical conditions, while affording low radiochemical yields. Fully characterized, new methods for the improved production of [18F]FMZ 1 are needed. (2) Methods: We investigate the use of late-stage copper-mediated radiofluorination of aryl stannanes to improve the production of [18F]FMZ 1 that is suitable for clinical use. Mass spectrometry was used to identify the chemical by-products that were produced under the reaction conditions. (3) Results: The radiosynthesis of [18F]FMZ 1 was fully automated using the iPhase FlexLab radiochemistry module, affording a 22.2 ± 2.7% (n = 5) decay-corrected yield after 80 min. [18F]FMZ 1 was obtained with a high radiochemical purity (>98%) and molar activity (247.9 ± 25.9 GBq/µmol). (4) Conclusions: The copper-mediated radiofluorination of the stannyl precursor is an effective strategy for the production of clinically suitable [18F]FMZ 1.

Radiolabeled derivatives of Tyr 3 -octreotide and Tyr 3 -octreotate, synthetic analogues of the peptide hormone somatostatin, can be used for positron emission tomography (PET) imaging of somatostatin receptor expression in neuroendocrine tumors. In this work, a squaramide ester derivative of desferrioxamine B (H 3 DFOSq) was used attach either Tyr 3 -octreotide or Tyr 3 -octreotate to the metal binding ligand to give H 3 DFOSq-TIDE and H 3 DFOSq-TATE. These new peptide-H 3 DFOSq conjugates form stable complexes with either of the positron-emitting radionuclides gallium-68 ( t 1/2 = 68 min) or zirconium-89 ( t 1/2 = 3.3 days). The new complexes were evaluated in an AR42J xenograft model that has endogenous expression of SSTR2. All four agents displayed good tumor uptake and produced high-quality PET images. For both radionuclides, the complexes formed with H 3 DFOSq-TATE performed better, with higher tumor uptake and retention than the complexes formed with H 3 DFOSq-TIDE. The versatile ligands presented here can be radiolabeled with either gallium-68 or zirconium-89 at room temperature. The long radioactive half-life of zirconium-89 makes distribution of pre-synthesized tracers produced to certified standards feasible and could increase the number of clinical centers that can perform diagnostic PET imaging of neuroendocrine tumors.

Radionuclide therapy targeting prostate-specific membrane antigen (PSMA) is promising for prostate cancer. We previously reported a ligand, 64 Cu-CuSarbisPSMA, featuring 2 lysine-ureido-glutamate groups. Here, we report the therapeutic potential of 67 Cu-CuSarbisPSMA. Methods: Growth of PSMA-positive xenografts was evaluated after treatment with 67 Cu-CuSarbisPSMA or 177 Lu-LuPSMA imaging and therapy (I&T). Results: At 13 d after injection, tumor growth was similarly inhibited by the 2 tracers in a dose-dependent manner. Survival was comparable after single (30 MBq) or fractionated (2 × 15 MBq, 2 wk apart) administrations. Conclusion: 67 Cu-CuSarbisPSMA is efficacious in a PSMA-expressing model of prostate cancer., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Proteins adopt unique folded secondary and tertiary structures that are responsible for their remarkable biological properties. This structural complexity is key in designing efficacious peptides that can mimic the three-dimensional structure needed for biological function. In this study, we employ different chemical strategies to induce and stabilize a β-hairpin fold of peptides targeting cholecystokinin-2 receptors for theranostic application (combination of a targeted therapeutic and a diagnostic companion). The newly developed peptides exhibited enhanced folding capacity as demonstrated by circular dichroism (CD) spectroscopy, ion-mobility spectrometry-mass spectrometry, and two-dimensional (2D) NMR experiments. Enhanced folding characteristics of the peptides led to increased biological potency, affording four optimal Ga-68 labeled radiotracers ([ 68 Ga]Ga- 4b , [ 68 Ga]Ga- 11b-13b ) targeting CCK-2R. In particular, [ 68 Ga]Ga- 12b and [ 68 Ga]Ga- 13b presented improved metabolic stability, enhanced cell internalization, and up to 6 fold increase in tumor uptake. These peptides hold great promise as next-generation theranostic radiopharmaceuticals.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled octreotate is an effective treatment for somatostatin receptor 2-expressing neuroendocrine tumors. The diagnostic and therapeutic potential of 64 Cu and 67 Cu, respectively, offers the possibility of using a single somatostatin receptor-targeted peptide conjugate as a theranostic agent. A sarcophagine cage amine ligand, MeCOSar (5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid), conjugated to (Tyr 3 )-octreotate, called 64 Cu-CuSarTATE, was demonstrated to be an imaging agent and potential prospective dosimetry tool in 10 patients with neuroendocrine tumors. This study aimed to explore the antitumor efficacy of 67 Cu-CuSarTATE in a preclinical model of neuroendocrine tumors and compare it with the standard PRRT agent, 177 Lu-LuDOTA-Tyr 3 -octreotate ( 177 Lu-LuTATE). Methods: The antitumor efficacy of various doses of 67 Cu-CuSarTATE in AR42J (rat pancreatic exocrine) tumor-bearing mice was compared with 177 Lu-LuTATE. Results: Seven days after a single administration of 67 Cu-CuSarTATE (5 MBq), tumor growth was inhibited by 75% compared with vehicle control. Administration of 177 Lu-LuTATE (5 MBq) inhibited tumor growth by 89%. Survival was extended from 12 d in the control group to 21 d after treatment with both 67 Cu-CuSarTATE and 177 Lu-LuTATE. In a second study, the efficacy of fractionated delivery of PRRT was assessed, comparing the efficacy of 30 MBq of 67 Cu-CuSarTATE or 177 Lu-LuTATE, either as a single intravenous injection or as two 15-MBq fractions 2 wk apart. Treatment of tumors with 2 fractions significantly improved survival over delivery as a single fraction ( 67 Cu-CuSarTATE: 47 vs. 36 d [ P = 0.036]; 177 Lu-LuTATE: 46 vs. 29 d [ P = 0.040]). Conclusion: This study demonstrates that 67 Cu-CuSarTATE is well tolerated in BALB/c nude mice and highly efficacious against AR42J tumors in vivo. Administration of 67 Cu-CuSarTATE and 177 Lu-LuTATE divided into 2 fractions over 2 wk was more efficacious than administration of a single fraction. The antitumor activity of 67 Cu-CuSarTATE in the AR42J tumor model demonstrated the suitability of this novel agent for clinical assessment in the treatment of somatostatin receptor 2-expressing neuroendocrine tumors., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Prostate-specific membrane antigen (PSMA) is a carboxypeptidase that is overexpressed in prostate cancer and is an excellent candidate for targeted diagnostic imaging and therapy. Lysine-ureido-glutamate inhibitors of PSMA radiolabeled with positron-emitting radionuclides can be used for diagnostic imaging with positron emission tomography (PET). A squaramide ester derivative of desferrioxamine B (H 3 DFOSq) was used to prepare four new agents with either one or two lysine-ureido-glutamate pharmacophores. The H 3 DFOSq ligand can be used to form stable complexes with either of the positron-emitting radionuclides gallium-68 ( t 1/2 = 68 min) or zirconium-89 ( t 1/2 = 3.3 days). The complexes were evaluated in PSMA-positive xenograft mouse models. Bivalent inhibitors, where two pharmacophores are tethered to a single DFOSq ligand, have better tumor uptake than their monovalent analogues. The ligands presented here, which can be labeled with either gallium-68 or zirconium-89, have the potential to increase the number of clinical sites that can perform diagnostic PET imaging.

Alzheimer's disease is characterized by the presence of extracellular amyloid-β plaques. Positron emission tomography (PET) imaging with tracers radiolabeled with positron-emitting radionuclides that bind to amyloid-β plaques can assist in the diagnosis of Alzheimer's disease. With the goal of designing new imaging agents radiolabeled with positron-emitting copper-64 radionuclides that bind to amyloid-β plaques, a family of bis(thiosemicarbazone) ligands with appended substituted stilbenyl functional groups has been prepared. The ligands form charge-neutral and stable complexes with copper(II). The new ligands can be radiolabeled with copper-64 at room temperature. Two lead complexes were demonstrated to bind to amyloid-β plaques present in post-mortem brain tissue from subjects with clinically diagnosed Alzheimer's disease and crossed the blood-brain barrier in mice. The work presented here provides strategies to prepare compounds with radionuclides of copper that can be used for targeted brain PET imaging.

Indirect radiolabelling has for a long time been the mainstay strategy for radiofluorination of biomolecules. Acylation of biomolecules through the use of an 18 F-labelled activated ester is a standard method for indirect radiolabelling. However, the preparation of 18 F-labelled activated esters is typically a complex and multistep procedure. Herein, we describe the use of 4-nitrophenyl (PNP) activated esters to rapidly prepare 18 F-labelled acylation synthons in one step. Furthermore, we present a comparative study of PNP activated esters and the commonly utilised 2,3,5,6-tetrafluorphenyl (TFP) activated esters under direct radiofluorination conditions and demonstrate their relative acylation behaviour. We demonstrate the superiority of PNP esters under direct radiofluorination conditions with favourable acylation kinetics., (This journal is © The Royal Society of Chemistry 2020.)

Molecules containing lysine-ureido-glutamate functional groups bind to the active site of prostate specific membrane antigen, which is overexpressed in prostate cancer. To prepare copper radiopharmaceuticals for the diagnosis and therapy of prostate cancer, macrobicyclic sarcophagine ligands tethered to either one or two lysine-ureido-glutamate functional groups through an appropriate linker have been prepared. Sarcophagine ligands can be readily radiolabeled with positron-emitting copper-64 at room temperature. The bivalent agent, in which two targeting groups are tethered to a single copper complex, dramatically outperforms the monomeric agent with respect to tumor uptake and retention. The high tumor uptake, low background, and prolonged tumor retention, even at 24 hours post injection, suggest the bivalent agent is a promising diagnostic for prostate cancer and could be used for prospective dosimetry for therapy with a copper-67 variant., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Gallium-68 ([ 68 Ga]Ga) labelled radiopharmaceuticals have become a valuable tool in clinical practice using Positron Emission Tomography (PET). These agents are typically produced on-site owing to the short half-life of [ 68 Ga]Ga (68 min), which hinders distant transportation and often cannot comply with Good Manufacturing Practice (GMP) in hospital environments due to limited resources or infrastructure constraints. Moreover, full blown GMP production of radiopharmaceuticals under development can be prohibitively expensive. [ 68 Ga]Ga-DOTA-CP04 is a promising peptide for imaging neuroendocrine tumors overexpressing the cholecyctokinin-2 receptor. Automation is an integral process in ensuring the radiopharmaceuticals produced under non-GMP conditions are of a uniform quality for routine clinical use. Herein, we describe the development of an automation platform, the iPHASE MultiSyn radiosynthesizer, to produce 68 Ga-labelled DOTA-CP04 for routine clinical provision., Results: The use of the MultiSyn module for 68 Ga-labelling of DOTA-CP04 was investigated. [ 68 Ga]Ga-DOTA-CP04, was reproducibly prepared in high (> 70%) decay-corrected yields. [ 68 Ga]Ga-DOTA-CP04 passed all predetermined acceptance criteria for human injection., Conclusions: [ 68 Ga]Ga-DOTA-CP04 was produced effectively using the MultiSyn module in a consistent and reproducible manner suitable for human injection.

The synthesis of new bis(thiosemicarbazonato)copper(II) complexes featuring polyamine substituents via selective transamination reactions is presented. Polyamines of different lengths, with different ionizable substituent groups, were used to modify and adjust the hydrophilic/lipophilic balance of the copper complexes. The new analogues were radiolabeled with copper-64 and their lipophilicities estimated using distribution coefficients. The cell uptake of the new polyamine complexes was investigated with preliminary in vitro biological studies using a neuroblastoma cancer cell line. The in vivo biodistribution of three of the new analogues was investigated in vivo in mice using positron-emission tomography imaging, and one of the new complexes was compared to [ 64 Cu]Cu(atsm) in an A431 squamous cell carcinoma xenograft model. Modification of the copper complexes with various amine-containing functional groups alters the biodistribution of the complexes in mice. One complex, with a pendent ( N, N-dimethylamino)ethane functional group, displayed tumor uptake similar to that of [ 64 Cu]Cu(atsm) but higher brain uptake, suggesting that this compound has the potential to be of use in the diagnostic brain imaging of tumors and neurodegenerative diseases.

Radiolabelled peptides are vital tools used in positron emission tomography imaging for the diagnosis of disease, drug discovery, and biomedical research. Peptides are typically labeled through conjugation to a radiolabelled prosthetic group, which usually necessitates complex, multi-step procedures, especially for fluorine-18 labeled peptides. Herein, we describe the automated synthesis and formulation of 2-[ 18 F]fluoropropionate labeled RGD-peptides through use of the iPHASE Flexlab as an effective dual-stage radiochemical synthesis module. The fully automated preparation of the monomeric RGD-peptides, [ 18 F]FP-GalactoRGD and [ 18 F]FP-c(RGDy(SO 3 )K), was accomplished in under 90 minutes with n.d.c. radiochemical yields ca. 7% from fluoride. Similarly, the automated preparation of the dimeric RGD-peptides, [ 18 F]F-PRGD 2 and [ 18 F]FP-E(RGDy(SO 3 )K) 2 , was accomplished in under 105 minutes with n.d.c. yields ca. 4% from fluoride., (Copyright © 2017 John Wiley & Sons, Ltd.)

Control of the biodistribution of radiolabeled peptides has proven to be a major challenge in their application as imaging agents for positron emission tomography (PET). Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[ 18 F]fluoropropionate, and the biodistribution of the radiolabeled peptides was compared with that of their nonsulfonated, clinically relevant counterparts, [ 18 F]GalactoRGD and [ 18 F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da toward the MW, compared with 189 Da for both the "Galacto" and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabeled peptides.

The versatile (18) F-labeled prosthetic group, 4-nitrophenyl 2-[(18) F]fluoropropionate ([(18) F]NFP), was synthesized in a single step in 45 min from 4-nitrophenyl 2-bromopropionate, with a decay corrected radiochemical yield of 26.2% ± 2.2%. Employing this improved synthesis of [(18) F]NFP, [(18) F]GalactoRGD - the current 'gold standard' tracer for imaging the expression of αV β3 integrin - was prepared with high specific activity in 90 min and 20% decay corrected radiochemical yield from [(18) F]fluoride., (Copyright © 2013 John Wiley & Sons, Ltd.)

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.

Triple helices containing C+xGxC triplets are destabilised at physiological pH due to the requirement for base protonation of 2'-deoxycytidine (dC), which has a pKa of 4.3. The C nucleoside 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (beta-AP) is structurally analogous to dC but is considerably more basic, with a pKa of 5.93. We have synthesised 5'-psoralen linked oligodeoxyribonucleotides (ODNs) containing thymidine (dT) and either beta-AP or its alpha-anomer (alpha-AP) and have assessed their ability to form triplexes with a double-stranded target derived from standard deoxynucleotides (i.e. beta-anomers). Third strand ODNs derived from dT and beta-AP were found to have considerably higher binding affinities for the target than the corresponding ODNs derived from dT and either dC or 5-methyl-2'-deoxycytidine (5-Me-dC). ODNs containing dT and alpha-AP also showed enhanced triplex formation with the duplex target and, in addition are more stable in serum-containing medium than standard oligopyrimidine-derived ODNs or ODNs derived from dT and beta-AP. Molecular modelling studies showed that an alpha-anomeric AP nucleotide can be accommodated within an otherwise beta-anomeric triplex with only minor perturbation of the triplex structure. Molecular dynamics (MD) simulations on triplexes containing either the alpha- or beta-anomer of (N1-protonated) AP showed that in both cases the base retained two standard hydrogen bonds to its associated guanine when the 'A-type' model of the triplex was used as the start-point for the simulation, but that bifurcated hydrogen bonds resulted when the alternative 'B-type' triplex model was used. The lack of a differential stability between alpha-AP- and beta-AP-containing triplexes at pH >7, predicted from the behaviour of the B-type models, suggests that the A-type models are more appropriate.