The Influence of Size on the Intracranial Distribution of Biomedical Nanoparticles Administered by Convection-enhanced Delivery in Minipigs.

Because of the blood-brain barrier (BBB), successful drug delivery to the brain has long been a key objective for the medical community, calling for pioneering technologies to overcome this challenge. Convection-enhanced delivery (CED), a form of direct intraparenchymal microinfusion, shows promise but requires optimal infusate design and real-time distribution monitoring. The size of the infused substances appears to be especially critical, with current knowledge being limited. Herein, we examined the intracranial administration of polyethylene glycol (PEG)-coated nanoparticles (NPs) of various sizes using CED in groups of healthy minipigs (n = 3). We employed stealth liposomes (LIPs, 130 nm) and two gold nanoparticle designs (AuNPs) of different diameters (8 and 40 nm). All were labeled with copper-64 for quantitative and real-time monitoring of the infusion via positron emission tomography (PET). NPs were infused via two catheters inserted bilaterally in the putaminal regions of the animals. Our results suggest CED with NPs holds promise for precise brain drug delivery, with larger LIPs exhibiting superior distribution volumes and intracranial retention over smaller AuNPs. PET imaging alongside CED enabled dynamic visualization of the process, target coverage, timely detection of suboptimal infusion, and quantification of distribution volumes and concentration gradients. These findings may augment the therapeutic efficacy of the delivery procedure while mitigating unwarranted side effects associated with nonvisually monitored delivery approaches. This is of vital importance, especially for chronic intermittent infusions through implanted catheters, as this information enables informed decisions for modulating targeted infusion volumes on a catheter-by-catheter, patient-by-patient basis.

In vivo biodistribution and tumor uptake of [64Cu]-FAU nanozeolite via positron emission tomography Imaging.

Nanoparticles have emerged as promising theranostic tools for biomedical applications, notably in the treatment of cancers. However, to fully exploit their potential, a thorough understanding of their biodistribution is imperative. In this context, we prepared radioactive [64Cu]-exchanged faujasite nanosized zeolite ([64Cu]-FAU) to conduct positron emission tomography (PET) imaging tracking in preclinical glioblastoma models. In vivo results revealed a rapid and gradual accumulation over time of intravenously injected [64Cu]-FAU zeolite nanocrystals within the brain tumor, while no uptake in the healthy brain was observed. Although a specific tumor targeting was observed in the brain, the kinetics of uptake into tumor tissue was found to be dependent on the glioblastoma model. Indeed, our results showed a rapid uptake in U87-MG model while in U251-MG glioblastoma model tumor uptake was gradual over the time. Interestingly, a [64Cu] activity, decreasing over time, was also observed in organs of elimination such as kidney and liver without showing a difference in activity between both glioblastoma models. Ex vivo analyses confirmed the presence of zeolite nanocrystals in brain tumor with detection of both Si and Al elements originated from them. This radiolabelling strategy, performed for the first time using nanozeolites, enables precise tracking through PET imaging and confirms their accumulation within the glioblastoma. These findings further bolster the potential use of zeolite nanocrystals as valuable theranostic tools.

Diagnosis of Melanoma with 61Cu-Labeled PET Tracer.

Until the recent years, substances containing radioactive 61Cu were strongly considered as potential positron-emitting radiopharmaceuticals for use in positron emission tomography (PET) applications; however, due to their suitably long half-life, and generator-independent and cost-effective production, they seem to be economically viable for human imaging. Since malignant melanoma (MM) is a major public health problem, its early diagnosis is a crucial contributor to long-term survival, which can be achieved using radiolabeled α-melanocyte-stimulating hormone analog NAPamide derivatives. Here, we report on the physicochemical features of a new CB-15aneN5-based Cu(II) complex ([Cu(KFTGdiac)]-) and the ex vivo and in vivo characterization of its NAPamide conjugate. The rigid chelate possesses prompt complex formation and suitable inertness (t1/2 = 18.4 min in 5.0 M HCl at 50 °C), as well as excellent features in the diagnosis of B16-F10 melanoma tumors (T/M(SUVs) (in vivo): 12.7, %ID/g: 6.6 ± 0.3, T/M (ex vivo): 22).

Customizable Porphyrin Platform Enables Folate Receptor PET Imaging Using Copper-64.

Folate receptors including folate receptor α (FRα) are overexpressed in up to 90% of ovarian cancers. Ovarian cancers overexpressing FRα often exhibit high degrees of drug resistance and poor outcomes. A porphyrin chassis has been developed that is readily customizable according to the desired targeting properties. Thus, compound O5 includes a free base porphyrin, two water-solubilizing groups that project above and below the macrocycle plane, and a folate targeting moiety. Compound O5 was synthesized (>95% purity) and exhibited aqueous solubility of at least 0.48 mM (1 mg/mL). Radiolabeling of O5 with 64Cu in HEPES buffer at 37 °C gave a molar activity of 1000 µCi/µg (88 MBq/nmol). [64Cu]Cu-O5 was stable in human serum for 24 h. Cell uptake studies showed 535 ± 12% bound/mg [64Cu]Cu-O5 in FRα-positive IGROV1 cells when incubated at 0.04 nM. Subcellular fractionation showed that most radioactivity was associated with the cytoplasmic (39.4 ± 2.7%) and chromatin-bound nuclear (53.0 ± 4.2%) fractions. In mice bearing IGROV1 xenografts, PET imaging studies showed clear tumor uptake of [64Cu]Cu-O5 from 1 to 24 h post injection with a low degree of liver uptake. The tumor standardized uptake value at 24 h post injection was 0.34 ± 0.16 versus 0.06 ± 0.07 in the blocking group. In summary, [64Cu]Cu-O5 was synthesized at high molar activity, was stable in serum, exhibited high binding to FRα-overexpressing cells with high nuclear translocation, and gave uptake that was clearly visible in mouse tumor xenografts.

64Cu tumor labeling with hexadentate picolinic acid-based bispidine immunoconjugates.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities >200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed specific accumulation of the 64Cu-labeled TMs in PSCA- and FAP-overexpressing tumors (standardized uptake value (SUV) for PC3: 2.7±0.6 and HT1080: 7.2±1.25) with almost no uptake in wild type tumors.

Development of New CD38 Targeted Peptides for Cancer Imaging.

PURPOSE: Multiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges. PROCEDURES: Using phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems. RESULTS: The CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 - 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h). CONCLUSIONS: The novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Comparison of PET tracing and biodistribution between 64Cu-labeled micro-and nano-polystyrene in a murine inhalation model.

INTRODUCTION: Recent studies showed the presence of microplastic in human lungs. There remains an unmet need to identify the biodistribution of microplastic after inhalation. In this study, we traced the biodistribution of inhaled micro-sized polystyrene (mPS) and/or nano-sized PS (nPS) using 64Cu with PET in mice. METHODS: We used 0.2-0.3-µm sized mPS and 20-nm sized nPS throughout. 64Cu-DOTA-mPS, 64Cu-DOTA-nPS and/or 64CuCl2 were used to trace the distribution in the murine inhalation model. PET images were acquired using an INVEON PET scanner at 1, 12, 24, 48, and 72 h after intratracheal instillation, and the SUVmax for interesting organs were determined, biodistribution was then determined in terms of percentage injected dose/gram of tissue (%ID/g). Ex vivo tissue-radio thin-layer chromatography (Ex vivo-radioTLC) was used to demonstrate the existence of 64Cu-DOTA-PS in tissue. RESULTS: PET image demonstrated that the amount of 64Cu-DOTA-mPS retained within the lung was significantly higher than 64Cu-DOTA-nPS until 72 h; SUVmax values of 64Cu-DOTA-mPS in lungs was 11.7 ± 5.0, 48.3 ± 6.2, 65.5 ± 2.3, 42.2 ± 13.1, and 13.2 ± 2.3 at 1, 12, 24, 48, and 72 h respectively whereas it was 31.2 ± 3.1, 17.3 ± 5.9, 10.0 ± 3.4, 8.1 ± 2.4 and 8.9 ± 3.6 for 64Cu-DOTA-nPS at the corresponding timepoints. The biodistribution data supported the PET data with a similar pattern of clearance of the radioactivity from the lung. nPS cleared rapidly post instillation in comparison to mPS within the lungs. Higher accumulation of %ID/g for nPS (roughly 2 times) were observed compared to mPS in spleen, liver, intestine, thymus, kidney, brain, salivary gland, ovary, and urinary bladder. Ex vivo-radioTLC was used to demonstrate that the detected gamma rays originated from 64Cu-DOTA-mPS or nPS. CONCLUSION: PET image demonstrated the differences in accumulations of mPS and/or nPS between lungs and other interesting organs. The information provided may be used as the basis for future studies on the toxicity of mPS and/or nPS.

Coordination Chemistry of Sulfur-Containing Bifunctional Chelators: Toward in Vivo Stabilization of 64Cu PET Imaging Agents for Alzheimer's Disease.

The broader utilization of 64Cu positron emission tomography (PET) imaging agents has been hindered by the unproductive demetalation induced by bioreductants. To advance the development of 64Cu-based PET imaging tracers for Alzheimer's Disease (AD), there is a need for novel ligand design strategies. In this study, we developed sulfur-containing dithiapyridinophane (N2S2) bifunctional chelators (BFCs) as well as all nitrogen-based diazapyridinophane (N4) BFCs to compare their abilities to chelate Cu and target Aß aggregates. Through spectrophotometric titrations and electrochemical measurements, we have demonstrated that the N2S2-based BFCs exhibit >10 orders of magnitude higher binding affinity toward Cu(I) compared to their N4-based counterparts, while both types of BFCs exhibit high stability constants toward Cu(II). Notably, solid state structures for both Cu(II) and Cu(I) complexes supported by the two ligand frameworks were obtained, providing molecular insights into their copper chelating abilities. Aß binding experiments were conducted to study the structure-affinity relationship, and fluorescence microscopy imaging studies confirmed the selective labeling of the BFCs and their copper complexes. Furthermore, we investigated the potential of these ligands for the 64Cu-based PET imaging of AD through radiolabeling and autoradiography studies. We believe our findings provide molecular insights into the design of bifunctional Cu chelators that can effectively stabilize both Cu(II) and Cu(I) and, thus, can have significant implications for the development of 64Cu PET imaging as a diagnostic tool for AD.

Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging.

Biomolecules labeled with positron-emitting radionuclides like fluorine-18 or radiometals like copper-64 and zirconium-89 are increasingly employed in nuclear medicine for diagnosis purposes. Given the fragility and complexity of these compounds, their labeling requires mild conditions. Besides, it is essential to develop methods inducing minimal modification of the tertiary structure, as it is fundamental for the biological activity of such complex entities. Given these requirements, disulfide rebridging represents a promising possibility since it allows protein modification as well as conservation of the tertiary structure. In this context, we have developed an original radiofluorinated dibromopyridazine dione prosthetic group for labeling of disulfide-containing biomolecules via rebridging. We employed it to radiolabel octreotide, a somatostatin analogue, and to radiolabel fragment antigen binding (Fab) targeting programmed death-ligand 1 (PD-L1), whose properties were then evaluated in vitro and in vivo by positron emission tomography (PET) imaging. We next extended our strategy to the radiolabeling of cetuximab, a monoclonal antibody, with various radiometals commonly used in PET imaging (zirconium-89, copper-64) by developing various rebridging molecules bearing the appropriate chelators. The stabilities of the radiolabeled antibody conjugates were assessed in biological conditions.

HNODThia: A Promising Chelator for the Development of 64Cu Radiopharmaceuticals.

Herein, we present the synthesis and coordination chemistry of copper(II) and zinc(II) complexes of two novel heterocyclic triazacyclononane (tacn)-based chelators (HNODThia and NODThia-AcNHEt). The chelator HNODThia was further derivatized to obtain a novel PSMA-based bioconjugate (NODThia-PSMA) and a bifunctional photoactivatable azamacrocyclic analogue, NODThia-PEG3-ArN3, for the development of copper-64 radiopharmaceuticals. 64Cu radiolabeling experiments were performed on the different metal-binding chelates, whereby quantitative radiochemical conversion (RCC) was obtained in less than 10 min at room temperature. The in vitro stability of NODThia-PSMA in human plasma was assessed by ligand-challenge and copper-exchange experiments. Next, we investigated the viability of the photoactivatable analog (NODThia-PEG3-ArN3) for the light-induced photoradiosynthesis of radiolabeled proteins. One-pot photoconjugation reactions to human serum albumin (HSA) as a model protein and the clinically relevant monoclonal antibody formulation MetMAb were performed. [64Cu]Cu-7-azepin-HSA and [64Cu]Cu-7-azepin-onartuzumab were prepared in less than 15 min by irradiation at 395 nm, with radiochemical purities (RCP) of >95% and radiochemical yields (RCYs) of 42.7 ± 5.3 and 49.6%, respectively. Together, the results obtained here open the way for the development of highly stable 64Cu-radiopharmaceuticals by using aza-heterocyclic tacn-based chelators, and the method can easily be extended to the development of 67Cu radiopharmaceuticals for future applications in molecularly targeted radio(immuno)therapy.

Antibody and Nanobody Radiolabeling with Copper-64: Solid vs. Liquid Target Approach.

Antibody and nanobody-based copper-64 radiopharmaceuticals are increasingly being proposed as theranostic tools in multiple human diseases. While the production of copper-64 using solid targets has been established for many years, its use is limited due to the complexity of solid target systems, which are available in only a few cyclotrons worldwide. In contrast, liquid targets, available in virtually in all cyclotrons, constitute a practical and reliable alternative. In this study, we discuss the production, purification, and radiolabeling of antibodies and nanobodies using copper-64 obtained from both solid and liquid targets. Copper-64 production from solid targets was performed on a TR-19 cyclotron with an energy of 11.7 MeV, while liquid target production was obtained by bombarding a nickel-64 solution using an IBA Cyclone Kiube cyclotron with 16.9 MeV on target. Copper-64 was purified from both solid and liquid targets and used to radiolabel NODAGA-Nb, NOTA-Nb, and DOTA-Trastuzumab conjugates. Stability studies were conducted on all radioimmunoconjugates in mouse serum, PBS, and DTPA. Irradiation of the solid target yielded 13.5 ± 0.5 GBq with a beam current of 25 ± 1.2 µA and an irradiation time of 6 h. On the other hand, irradiation of the liquid target resulted in 2.8 ± 1.3 GBq at the end of bombardment (EOB) with a beam current of 54.5 ± 7.8 µA and an irradiation time of 4.1 ± 1.3 h. Successful radiolabeling of NODAGA-Nb, NOTA-Nb, and DOTA-Trastuzumab with copper-64 from both solid and liquid targets was achieved. Specific activities (SA) obtained with the solid target were 0.11, 0.19, and 0.33 MBq/µg for NODAGA-Nb, NOTA-Nb, and DOTA-trastuzumab, respectively. For the liquid target, the corresponding SA values were 0.15, 0.12, and 0.30 MBq/µg. Furthermore, all three radiopharmaceuticals demonstrated stability under the testing conditions. While solid targets have the potential to produce significantly higher activity in a single run, the liquid process offers advantages such as speed, ease of automation, and the feasibility of back-to-back production using a medical cyclotron. In this study, successful radiolabeling of antibodies and nanobodies was achieved using both solid and liquid targets approaches. The radiolabeled compounds exhibited high radiochemical purity and specific activity, rendering them suitable for subsequent in vivo pre-clinical imaging studies.

Two-Chain Mature Hepatocyte Growth Factor-Specific Positron Emission Tomography Imaging in Tumors Using 64Cu-Labeled HiP-8, a Nonstandard Macrocyclic Peptide Probe.

Two-chain hepatocyte growth factor (tcHGF), the mature form of HGF, is associated with malignancy and anticancer drug resistance; therefore, its quantification is an important indicator for cancer diagnosis. In tumors, activated tcHGF hardly discharges into the systemic circulation, indicating that tcHGF is an excellent target for molecular imaging using positron emission tomography (PET). We recently discovered HGF-inhibitory peptide-8 (HiP-8) that binds specifically to human tcHGF with nanomolar affinity. The purpose of this study was to investigate the usefulness of HiP-8-based PET probes in human HGF knock-in humanized mice. 64Cu-labeled HiP-8 molecules were synthesized using a cross-bridged cyclam chelator, CB-TE1K1P. Radio-high-performance liquid chromatography-based metabolic stability analyses showed that more than 90% of the probes existed in intact form in blood at least for 15 min. In PET studies, significantly selective visualization of hHGF-overexpressing tumors versus hHGF-negative tumors was observed in double-tumor-bearing mice. The accumulation of labeled HiP-8 into the hHGF-overexpressing tumors was significantly reduced by competitive inhibition. In addition, the radioactivity and distribution of phosphorylated MET/HGF receptor were colocalized in tissues. These results demonstrate that the 64Cu-labeled HiP-8 probes are suitable for tcHGF imaging in vivo, and secretory proteins like tcHGF can be a target for PET imaging.

Pre-targeting amyloid-ß with antibodies for potential molecular imaging of Alzheimer's disease.

With the aim of developing the concept of pretargeted click chemistry for the diagnosis of Alzheimer's disease two antibodies specific for amyloid-ß were modified to incorporate trans-cyclooctene functional groups. Two bis(thiosemicarbazone) compounds with pendant 1,2,4,5-tetrazine functional groups were prepared and radiolabelled with positron emitting copper-64. The new copper-64 complexes rapidly react with the trans-cyclooctene functionalized antibodies in a bioorthogonal click reaction and cross the blood-brain barrier in mice.

N,N-Alkylation Clarifies the Role of N- and O-Protonated Intermediates in Cyclen-Based 64Cu Radiopharmaceuticals.

Radioisotopes of Cu, such as 64Cu and 67Cu, are alluring targets for imaging (e.g., positron emission tomography, PET) and radiotherapeutic applications. Cyclen-based macrocyclic polyaminocarboxylates are one of the most frequently examined bifunctional chelators in vitro and in vivo, including the FDA-approved 64Cu radiopharmaceutical, Cu(DOTATATE) (Detectnet); however, connections between the structure of plausible reactive intermediates and their stability under physiologically relevant conditions remain to be established. In this study, we share the synthesis of a cyclen-based, N,N-alkylated spirocyclic chelate, H2DO3AC4H8, which serves as a model for N-protonation. Our combined experimental (in vitro and in vivo) and computational studies unravel complex pH-dependent speciation and enable side-by-side comparison of N- and O-protonated species of relevant 64Cu radiopharmaceuticals. Our studies suggest that N-protonated species are not inherently unstable species under physiological conditions and demonstrate the potential of N,N-alkylation as a tool for the rational design of future radiopharmaceuticals.

Cross-section measurement for an optimized 61Cu production at an 18 MeV medical cyclotron from natural Zn and enriched 64Zn solid targets.

The availability of novel medical radionuclides is a key point in the development of personalised nuclear medicine. In particular, copper radioisotopes are attracting considerable interest as they can be used to label various molecules of medical interest, such as proteins and peptides, and offer two of the most promising true theranostic pairs, namely 61Cu/67Cu and 64Cu/67Cu. Although 64Cu (t1/2 = 12.7006 h, ß+: 17.6%, ß-: 38.5%) is nowadays the most commonly used as a diagnostic radionuclide, 61Cu (t1/2 = 3.339 h, ß+: 61%) features more favourable nuclear properties, such as a higher positron decay fraction and the absence of ß- emissions. To date, the production of 61Cu has been carried out irradiating highly enriched 61Ni targets with a low energy proton beam. However, the use of the very expensive 61Ni targets requires an efficient recovery of the target material and makes this method quite inconvenient. Another promising production route is the proton irradiation of natural Zn or enriched 64Zn targets, exploiting the (p,α) nuclear reaction. Along this line, a research program is ongoing at the Bern medical cyclotron, equipped with an external beam transfer line and a solid target station. In this paper, we report on cross-section measurements of the 64Zn(p,α)61Cu nuclear reaction using natural Zn and enriched 64Zn material, which served as the basis to perform optimized 61Cu production tests with solid targets.

A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections.

Tracking the pathogen of coronavirus disease 2019 (COVID-19) in live subjects may help estimate the spatiotemporal distribution of SARS-CoV-2 infection in vivo. This study developed a positron emission tomography (PET) tracer of the S2 subunit of spike (S) protein for imaging SARS-CoV-2. A pan-coronavirus inhibitor, EK1 peptide, was synthesized and radiolabeled with copper-64 after being conjugated with 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA). The in vitro stability tests indicated that [64Cu]Cu-NOTA-EK1 was stable up to 24 h both in saline and in human serum. The binding assay showed that [64Cu]Cu-NOTA-EK1 has a nanomolar affinity (Ki = 3.94 ± 0.51 nM) with the S-protein of SARS-CoV-2. The cell uptake evaluation used HEK293T/S+ and HEK293T/S- cell lines that showed that the tracer has a high affinity with the S-protein on the cellular level. For the in vivo study, we tested [64Cu]Cu-NOTA-EK1 in HEK293T/S+ cell xenograft-bearing mice (n = 3) and pseudovirus of SARS-CoV-2-infected HEK293T/ACE2 cell bearing mice (n = 3). The best radioactive xenograft-to-muscle ratio (X/Nxenograft 8.04 ± 0.99, X/Npseudovirus 6.47 ± 0.71) was most evident 4 h postinjection. Finally, PET imaging in the surrogate mouse model of beta-coronavirus, mouse hepatic virus-A59 infection in C57BL/6 J mice showed significantly enhanced accumulation in the liver than in the uninfected mice (1.626 ± 0.136 vs 0.871 ± 0.086 %ID/g, n = 3, P < 0.05) at 4 h postinjection. In conclusion, our experimental results demonstrate that [64Cu]Cu-NOTA-EK1 is a potential molecular imaging probe for tracking SARS-CoV-2 in extrapulmonary infections in living subjects.

Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.

64Cu-labeling of small extracellular vesicle surfaces via a cross-bridged macrocyclic chelator for pharmacokinetic study by positron emission tomography imaging.

We developed a method of labeling the surfaces of small extracellular vesicles (sEVs) with 64Cu using a cross-bridged, macrocyclic chelator (CB-TE1A1P) and applied to pharmacokinetics study with positron emission tomography (PET). After incubation in 20% plasma for 10 min, approximately a half of the 64Cu was desorbed from 64Cu-labeled sEVs purified by phosphate-buffered saline wash, suggesting partly weak interaction without coordinating to CB-TE1A1P. After subsequent purification with albumin, 64Cu desorption was greatly reduced, resulting in a radiochemical stability of 95.7%. Notably, labeling did not alter the physicochemical and biological properties of sEVs. After intravenous injection, 64Cu-labeled sEVs rapidly disappeared from the systemic blood circulation and accumulated mainly in the liver and spleen of macrophage-competent mice. In macrophage-depleted mice, 64Cu-labeled sEVs remained in the blood circulation for a longer period and gradually accumulated in the liver and spleen, suggesting mechanisms of hepatic and splenic accumulation other than macrophage-dependent phagocytosis. The comparison of tissue uptake clearance between macrophage-competent and macrophage-depleted mice suggests that macrophages contributed to 67% and 76% of sEV uptake in the liver and spleen, respectively. The application of this method in pharmacokinetics PET studies can be useful in preclinical and clinical research and the development of sEV treatment modalities.

Photoactivatable bis(thiosemicarbazone) derivatives for copper-64 radiotracer synthesis.

In recent years, copper-64 and copper-67 have been considered as a useful theranostic pair in nuclear medicine, due to their favourable and complementary decay properties. As 67Cu and 64Cu are chemically identical, development of both existing and new bifunctional chelators for 64Cu imaging agents can be readily adapted for the 67Cu-radionuclide. In this study, we explored the use of photoactivatable copper chelators based on the asymmetric bis(thiosemicarbazone) scaffold, H2ATSM/en, for the photoradiolabelling of protein. Photoactivatable 64CuATSM-derivatives were prepared by both direct synthesis and transmetallation from the corresponding natZn complex. Then, irradiation with UV light in the presence of a protein of interest in a pH buffered aqueous solution afforded the 64Cu-labelled protein conjugates in decay-corrected radiochemical yield of 86.9 ± 1.0% via the transmetallation method and 35.3 ± 1.7% from the direct radiolabelling method. This study successfully demonstrates the viability of photochemically induced conjugation methods for the development of copper-based radiotracers for potential applications in diagnostic positron emission tomography (PET) imaging and targeted radionuclide therapy.

A comparison of 64Cu-labeled bi-terminally PEGylated A20FMDV2 peptides targeting integrin ανß6.

Expression of epithelial-specific integrin ανß6 is up-regulated in various aggressive cancers and serves as a prognostic marker. Integrin-targeted PET imaging probes have been successfully developed and tested in the clinic. Radiotracers based on the peptide A20FMDV2 derived from foot-and-mouth disease virus represent specific and selective PET ligands for imaging ανß6-positive cancers. The present study aims to describe the radiolabeling, in vitro and in vivo evaluation of a bi-terminally PEGylated A20FMDV2 conjugated with DOTA or PCTA for 64Cu radiolabeling. Stability studies showed radiolabeled complexes remained stable up to 24 h in PBS and human serum. In vitro cell assays in CaSki cervical cancer cells and BxPC-3 pancreatic cancer cells confirmed that the peptides displayed high affinity for αvß6 with Kd values of ~50 nM. Biodistribution studies revealed that [64Cu] Cu-PCTA-(PEG28)2-A20FMDV2 exhibited higher tumor uptake (1.63 ± 0.53 %ID/g in CaSki and 3.86 ± 0.58 %ID/g in BxPC-3 at 1 h) when compared to [64Cu]Cu-DOTA-(PEG28)2-A20FMDV2 (0.95 ± 0.29 %ID/g in CaSki and 2.12 ± 0.83 %ID/g in BxPC-3 at 1 h) . However, higher tumor uptake was accompanied by increased radioactive uptake in normal organs. Therefore, both peptides are appropriate for imaging ανß6-positive lesions although further optimization is needed to improve tumor-to-normal-tissue ratios.