A 211At-labelled mGluR1 inhibitor induces cancer senescence to elicit long-lasting anti-tumor efficacy.

Metabotropic glutamate receptor 1 (mGluR1), a key mediator of glutamatergic signaling, is frequently overexpressed in tumor cells and is an attractive drug target for most cancers. Here, we present a targeted radiopharmaceutical therapy strategy that antagonistically recognizes mGluR1 and eradicates mGluR1+ human tumors by harnessing a small-molecule alpha (α)-emitting radiopharmaceutical, 211At-AITM. A single dose of 211At-AITM (2.96 MBq) in mGluR1+ cancers exhibits long-lasting in vivo antitumor efficacy across seven subtypes of four of the most common tumors, namely, breast cancer, pancreatic cancer, melanoma, and colon cancers, with little toxicity. Moreover, complete regression of mGluR1+ breast cancer and pancreatic cancer is observed in approximate 50% of tumor-bearing mice. Mechanistically, the functions of 211At-AITM are uncovered in downregulating mGluR1 oncoprotein and inducing senescence of tumor cells with a reprogrammed senescence-associated secretory phenotype. Our findings suggest α-radiopharmaceutical therapy with 211At-AITM can be a useful strategy for mGluR1+ pan-cancers, regardless of their tissue of origin.

Trace Metal Impurities Effects on the Formation of [64Cu]Cu-diacetyl-bis(N4-methylthiosemicarbazone) ([64Cu]Cu-ATSM).

[64Cu]Cu-diacetyl-bis(N4-methylthiosemicarbazone) ([64Cu]Cu-ATSM) is a radioactive hypoxia-targeting therapeutic agent being investigated in clinical trials for malignant brain tumors. For the quality management of [64Cu]Cu-ATSM, understanding trace metal impurities' effects on the chelate formation of 64Cu and ATSM is important. In this study, we conducted coordination chemistry studies on metal-ATSM complexes. First, the effects of nonradioactive metal ions (Cu2+, Ni2+, Zn2+, and Fe2+) on the formation of [64Cu]Cu-ATSM were evaluated. When the amount of Cu2+ or Ni2+ added was 1.2 mol or 288 mol, equivalent to ATSM, the labeling yield of [64Cu]Cu-ATSM fell below 90%. Little effect was observed even when excess amounts of Zn2+ or Fe2+ were added to the ATSM. Second, these metals were reacted with ATSM, and chelate formation was measured using ultraviolet-visible (UV-Vis) absorption spectra. UV-Vis spectra showed a rapid formation of Cu2+ and the ATSM complex upon mixing. The rate of chelate formation by Ni2+ and ATSM was lower than that by Cu-ATSM. Zn2+ and Fe2+ showed much slower reactions with the ATSM than Ni2+. Trace amounts of Ni2+, Zn2+, and Fe2+ showed little effect on [64Cu]Cu-ATSM' quality, while the concentration of impurity Cu2+ must be controlled. These results can provide process management tools for radiopharmaceuticals.

Whole-body PET tracking of a d-dodecapeptide and its radiotheranostic potential for PD-L1 overexpressing tumors.

Peptides that are composed of dextrorotary (d)-amino acids have gained increasing attention as a potential therapeutic class. However, our understanding of the in vivo fate of d-peptides is limited. This highlights the need for whole-body, quantitative tracking of d-peptides to better understand how they interact with the living body. Here, we used mouse models to track the movement of a programmed death-ligand 1 (PD-L1)-targeting d-dodecapeptide antagonist (DPA) using positron emission tomography (PET). More specifically, we profiled the metabolic routes of [64Cu]DPA and investigated the tumor engagement of [64Cu/68Ga]DPA in mouse models. Our results revealed that intact [64Cu/68Ga]DPA was primarily eliminated by the kidneys and had a notable accumulation in tumors. Moreover, a single dose of [64Cu]DPA effectively delayed tumor growth and improved the survival of mice. Collectively, these results not only deepen our knowledge of the in vivo fate of d-peptides, but also underscore the utility of d-peptides as radiopharmaceuticals.

Automated radiosynthesis of [11 C]MTP38-a phosphodiesterase 7 imaging tracer-using [11 C]hydrogen cyanide for clinical applications.

We have developed 8-amino-3-(2S,5R-dimethyl-1-piperidyl)-[1,2,4]triazolo[4,3-a]pyrazine-5-[11 C]carbonitrile ([11 C]MTP38) as a positron emission tomography (PET) tracer for the imaging of phosphodiesterase 7. For the fully automated production of [11 C]MTP38 routinely and efficiently for clinical applications, we determined the radiosynthesis procedure of [11 C]MTP38 using [11 C]hydrogen cyanide ([11 C]HCN) as a PET radiopharmaceutical. Radiosynthesis of [11 C]MTP38 was performed using an automated 11 C-labeling synthesizer developed in-house within 40 min after the end of irradiation. [11 C]MTP38 was obtained with a relatively high radiochemical yield (33 ± 5.5% based on [11 C]CO2 at the end of irradiation, decay-corrected, n = 15), radiochemical purity (>97%, n = 15), and molar activity (47 ± 12 GBq/µmol at the end of synthesis, n = 15). All the results of the quality control (QC) testing for the [11 C]MTP38 injection complied with our in-house QC and quality assurance specifications. We successfully automated the radiosynthesis of [11 C]MTP38 for clinical applications using an 11 C-labeling synthesizer and sterile isolator. Taken together, this protocol provides a new radiopharmaceutical [11 C]MTP38 suitable for clinical applications.

Development of a Stable Peptide-Based PET Tracer for Detecting CD133-Expressing Cancer Cells.

CD133 has been recognized as a prominent biomarker for cancer stem cells (CSCs), which promote tumor relapse and metastasis. Here, we developed a clinically relevant, stable, and peptide-based positron emission tomography (PET) tracer, [64Cu]CM-2, for mapping CD133 protein in several kinds of cancers. Through the incorporation of a 6-aminohexanoic acid (Ahx) into the N terminus of a CM peptide, we constructed a stable peptide tracer [64Cu]CM-2, which exhibited specific binding to CD133-positive CSCs in multiple preclinical tumor models. Both PET imaging and ex vivo biodistribution verified the superb performance of [64Cu]CM-2. Furthermore, the matched physical and biological half-life of [64Cu]CM-2 makes it a state-of-the-art PET tracer for CD133. Therefore, [64Cu]CM-2 PET may not only enable the longitudinal tracking of CD133 dynamics in the cancer stem cell niche but also provide a powerful and noninvasive imaging tool to track down CSCs in refractory cancers.

Automated radiosynthesis of two 18F-labeled tracers containing 3-fluoro-2-hydroxypropyl moiety, [18F]FMISO and [18F]PM-PBB3, via [18F]epifluorohydrin.

BACKGROUND: [18F]Fluoromisonidazole ([18F]FMISO) and 1-[18F]fluoro-3-((2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dien-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol ([18F]PM-PBB3 or [18F]APN-1607) are clinically used radiotracers for imaging hypoxia and tau pathology, respectively. Both radiotracers were produced by direct 18F-fluorination using the corresponding tosylate precursors 1 or 2 and [18F]F-, followed by the removal of protecting groups. In this study, we synthesized [18F]FMISO and [18F]PM-PBB3 by 18F-fluoroalkylation using [18F]epifluorohydrin ([18F]5) for clinical applications. RESULTS: First, [18F]5 was synthesized by the reaction of 1,2-epoxypropyl tosylate (8) with [18F]F- and was purified by distillation. Subsequently, [18F]5 was reacted with 2-nitroimidazole (6) or PBB3 (7) as a precursor for 18F-labeling, and each reaction mixture was purified by preparative high-performance liquid chromatography and formulated to obtain the [18F]FMISO or [18F]PM-PBB3 injection. All synthetic sequences were performed using an automated 18F-labeling synthesizer. The obtained [18F]FMISO showed sufficient radioactivity (0.83 ± 0.20 GBq at the end of synthesis (EOS); n = 8) with appropriate radiochemical yield based on [18F]F- (26 ± 7.5 % at EOS, decay-corrected; n = 8). The obtained [18F]PM-PBB3 also showed sufficient radioactivity (0.79 ± 0.10 GBq at EOS; n = 11) with appropriate radiochemical yield based on [18F]F- (16 ± 3.2 % at EOS, decay-corrected; n = 11). CONCLUSIONS: Both [18F]FMISO and [18F]PM-PBB3 injections were successfully synthesized with sufficient radioactivity by 18F-fluoroalkylation using [18F]5.

211At-Labeled Polymer Nanoparticles for Targeted Radionuclide Therapy of Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR)-Overexpressed Cancer.

Targeted radionuclide therapy (TRT) provides new and safe opportunities for cancer treatment and management with high precision and efficiency. Here we have designed a novel semiconducting polymer nanoparticle (SPN)-based radiopharmaceutical (211At-MeATE-SPN-GIP) for TRT against glucose-dependent insulinotropic polypeptide receptor (GIPR)-positive cancers to further explore the applications of nanoengineered TRT. 211At-MeATE-SPN-GIP was engineered via nanoprecipitation, followed by its functionalization with a glucose-dependent insulinotropic polypeptide (GIP) to target GIPR and deliver 211At for α therapy. The therapeutic effect and biological safety of 211At-MeATE-SPN-GIP were investigated using GIPR-overexpressing human pancreatic cancer CFPAC-1 cells and CFPAC-1-bearing mice. In this work, 211At-MeATE-SPN-GIP was produced with a radiochemical yield of 43% and radiochemical purity of 98%, which exhibited a specifically high uptake in CFPAC-1 cells, inducing cell cycle arrest at the G2/M phase and extensive DNA damage. In the CFPAC-1-bearing tumor model, 211At-MeATE-SPN-GIP exhibited high therapeutic efficiency, with no obvious side effects. The GIPR-specific binding of 211At-MeATE-SPN-GIP combined with effective inhibition of tumor growth and fewer side effects compared to control suggests that 211At-MeATE-SPN-GIP TRT holds great potential as a novel nanoengineered TRT strategy for patients with GIPR-positive cancer.

Marriage of black phosphorus and Cu2+ as effective photothermal agents for PET-guided combination cancer therapy.

The use of photothermal agents (PTAs) in cancer photothermal therapy (PTT) has shown promising results in clinical studies. The rapid degradation of PTAs may address safety concerns but usually limits the photothermal stability required for efficacious treatment. Conversely, PTAs with high photothermal stability usually degrade slowly. The solutions that address the balance between the high photothermal stability and rapid degradation of PTAs are rare. Here, we report that the inherent Cu2+-capturing ability of black phosphorus (BP) can accelerate the degradation of BP, while also enhancing photothermal stability. The incorporation of Cu2+ into BP@Cu nanostructures further enables chemodynamic therapy (CDT)-enhanced PTT. Moreover, by employing 64Cu2+, positron emission tomography (PET) imaging can be achieved for in vivo real-time and quantitative tracking. Therefore, our study not only introduces an "ideal" PTA that bypasses the limitations of PTAs, but also provides the proof-of-concept application of BP-based materials in PET-guided, CDT-enhanced combination cancer therapy.

PET Imaging of VEGFR with a Novel 64Cu-Labeled Peptide.

Vascular endothelial growth factor receptors (VEGFRs) are well recognized as significant biomarkers of tumor angiogenesis. Herein, we have developed a first-of-its-kind peptide-based VEGFR positron emission tomography (PET) tracer. The novel [64Cu]VEGF125-136 peptide possessed satisfactory radio-characteristics and showed good specificity for the visualization of VEGFR in various mouse models, in which the tumor-specific radioactivity uptake was highly correlated to the VEGFR expression level. Moreover, the tracer showed high tumor uptake (ca. 5.89 %ID/g at 20 min postinjection in B16F10 mice) and excellent pharmacokinetics, achieving the maximum imaging quality within 1 h after injection. These features convey [64Cu]VEGF125-136 as a promising, clinically translatable PET tracer for the imaging of tumor angiogenesis.

Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice.

[Thiocarbonyl-11C]disulfiram ([11C]DSF) was synthesized via iodine oxidation of [11C]diethylcarbamodithioic acid ([11C]DETC), which was prepared from [11C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [11C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (Am, 0.27 GBq/µmol). On the other hand, [11C]DSF was obtained in 0.4% RCY with a high Am value (95 GBq/µmol) in the absence of carbon disulfide. The radiochemical purity of [11C]DSF was always >98%. The first PET study on [11C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the Am value of the [11C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [11C]DSF in mouse plasma.

Radiosynthesis and evaluation of 4-(6-[18F]Fluoro-4-(5-isopropoxy-1H-indazol-3-yl)pyridin-2-yl)morpholine as a novel radiotracer candidate targeting leucine-rich repeat kinase 2.

Mutations that increase leucine-rich repeat kinase 2 (LRRK2) activity in the brain are associated with Parkinson's disease. Here, we synthesized a novel compound 4-(6-fluoro-4-(5-isopropoxy-1H-indazol-3-yl)pyridin-2-yl)morpholine (FIPM) and labeled it with fluorine-18 (18F), to develop a positron emission tomography (PET) tracer for in vivo visualization of LRRK2 in the brain. FIPM showed high in vitro binding affinity for LRRK2 (IC50 = 8.0 nM). [18F]FIPM was prepared in 5% radiochemical yield (n = 5), by inserting 18F into a pyridine ring, followed by removal of the protecting group. After HPLC separation and formulation, [18F]FIPM was acquired with >97% radiochemical purity and 103-300 GBq µmol-1 of molar activity at the end of radiosynthesis. Biodistribution and small-animal PET studies in mice indicated a low in vivo specific binding of [18F]FIPM. While [18F]FIPM presented limited potential as an in vivo PET tracer for LRRK2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.

Harnessing the PD-L1 interface peptide for positron emission tomography imaging of the PD-1 immune checkpoint.

Interface peptides that mediate protein-protein interactions (PPI) are a class of important lead compounds for designing PPI inhibitors. However, their potential as precursors for radiotracers has never been exploited. Here we report that the interface peptides from programmed death-ligand 1 (PD-L1) can be used in positron emission tomography (PET) imaging of programmed cell death 1 (PD-1) with high accuracy and sensitivity. Moreover, the performance differentiation between murine PD-L1 derived interface peptide (mPep-1) and human PD-L1 derived interface peptide (hPep-1) as PET tracers for PD-1 unveiled an unprecedented role of a non-critical residue in target binding, highlighting the significance of PET imaging as a companion diagnostic in drug development. Collectively, this study not only provided a first-of-its-kind peptide-based PET tracer for PD-1 but also conveyed a unique paradigm for developing imaging agents for highly challenging protein targets, which could be used to identify other protein biomarkers involved in the PPI networks.

131I-IITM and 211At-AITM: Two Novel Small-Molecule Radiopharmaceuticals Targeting Oncoprotein Metabotropic Glutamate Receptor 1.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed 2 new radiopharmaceuticals, 4-131I-iodo- and 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (131I-IITM and 211At-AITM), targeting the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanomas for TRT studies. Methods:131I-IITM and 211At-AITM were synthesized by reacting a stannyl precursor with 131I-NaI and 211At in the presence of an oxidizing agent. The therapeutic efficacy and safety of the 2 radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results:131I-IITM and 211At-AITM were obtained with a radiochemical purity of greater than 99% and radiochemical yields of 42.7% ± 10.4% and 45.7% ± 6.5%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited a maximum uptake of 4.66% ± 0.70 and 7.68% ± 0.71 percentage injected dose per gram (%ID/g) in the targeted melanomas, respectively, and were rapidly cleared from nontarget organs after intravenous injection. Both agents markedly inhibited melanoma growth compared with the controls (61.00% and 95.68%, respectively). In the melanoma model, considerably greater therapeutic efficacy with negligible toxicity was observed using 211At-AITM. Conclusion: The nontoxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

Correction: Developing native peptide-based radiotracers for PD-L1 PET imaging and improving imaging contrast by pegylation.

Correction for 'Developing native peptide-based radiotracers for PD-L1 PET imaging and improving imaging contrast by pegylation' by Kuan Hu et al., Chem. Commun., 2019, DOI: 10.1039/c9cc00445a.

Development of a remote purification apparatus with disposable evaporator for the routine production of high-quality 64Cu for clinical use.

We developed a new apparatus for the routine production of 64Cu in clinical use. The apparatus has many disposable parts that stabilize the product quality (such that there is a low deviation of the concentrations of impurity metals in the product) and reduce the work load of preparation for routine production. We also developed a new evaporator using near-infrared heaters for disposable use. We conducted a production test using the new apparatus and evaluated product quality. The product yield was 6.3 ±â€¯0.32 GBq (end of bombardment) (N = 4), the product quality in terms of the concentrations of impurity metals (Cu2+, Ni2+, Fe3+, Zn2+, Mn2+) was as good as that usually achieved, likely on the order of parts per billion, and the preparation time was reduced from 2 days to 1 day.

Developing native peptide-based radiotracers for PD-L1 PET imaging and improving imaging contrast by pegylation.

Herein, a PD-L1 targeted native peptide was developed for PET imaging. 18F and 64Cu were utilized to label the peptide. To improve the pharmacokinetics and biodistribution of the tracers, the peptide was further pegylated to form star-like tetramers. Consequently, four tracers were synthesized with acceptable radiochemical characteristics and their in vivo pharmacokinetics and PD-L1 imaging capability were systematically evaluated. This proof-of-principle study may provide new possibilities for PD-L1 PET imaging in cancers.

[18F]DAA1106: Automated radiosynthesis using spirocyclic iodonium ylide and preclinical evaluation for positron emission tomography imaging of translocator protein (18 kDa).

DAA1106 (N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide), is a potent and selective ligand for the translocator protein (18 kDa, TSPO) in brain mitochondrial fractions of rats and monkey (Ki = 0.043 and 0.188 nM, respectively). In this study, to translate [18F]DAA1106 for clinical studies, we performed automated syntheses of [18F]DAA1106 using the spirocyclic iodonium ylide (1) as a radiolabelling precursor and conducted preclinical studies including positron emission tomography (PET) imaging of TSPO in ischemic rat brains. Radiofluorination of the ylide precursor 1 with [18F]F-, followed by HPLC separation and formulation, produced the [18F]DAA1106 solution for injection in 6% average (n = 10) radiochemical yield (based on [18F]F-) with >98% radiochemical purity and molar activity of 60-100 GBq/µmol at the end of synthesis. The synthesis time was 87 min from the end of bombardment. The automated synthesis achieved [18F]DAA1106 with sufficient radioactivity available for preclinical and clinical use. Biodistribution study of [18F]DAA1106 showed a low uptake of radioactivity in the mouse bones. Metabolite analysis showed that >96% of total radioactivity in the mouse brain at 60 min after the radiotracer injection was unmetabolized [18F]DAA1106. PET study of ischemic rat brains visualized ischemic areas with a high uptake ratio (1.9 ±â€¯0.3) compared with the contralateral side. We have provided evidence that [18F]DAA1106 could be routinely produced for clinical studies.

Automated Synthesis of (rac)-, (R)-, and (S)-[18 F]Epifluorohydrin and Their Application for Developing PET Radiotracers Containing a 3-[18 F]Fluoro-2-hydroxypropyl Moiety.

To introduce the 3-[18 F]fluoro-2-hydroxypropyl moiety into positron emission tomography (PET) radiotracers, we performed automated synthesis of (rac)-, (R)-, and (S)-[18 F]epifluorohydrin ([18 F]1) by nucleophilic displacement of (rac)-, (R)-, or (S)-glycidyl tosylate with 18 F- and purification by distillation. The ring-opening reaction of (R)- or (S)-[18 F]1 with phenol precursors gave enantioenriched [18 F]fluoroalkylated products without racemisation. We then synthesised (rac)-, (R)-, and (S)- 2-{5-[4-(3-[18 F]fluoro-2-hydroxypropoxy)phenyl]-2-oxobenzo[d]oxazol-3(2H)-yl}-N-methyl-N-phenylacetamide ([18 F]6) as novel radiotracers for the PET imaging of translocator protein (18 kDa) and showed that (R)- and (S)-[18 F]6 had different radioactivity uptake in mouse bone and liver. Thus, (rac)-, (R)-, and (S)-[18 F]1 are effective radiolabelling reagents and can be used to develop PET radiotracers by examining the effects of chirality on their in vitro binding affinities and in vivo behaviour.

Small-scale production of 67Cu for a preclinical study via the 64Ni(α, p)67Cu channel.

INTRODUCTION: Copper-67 is an attractive beta emitter for targeted radionuclide therapy. However, the availability of 67Cu limits its potential use in a wide range of applications. In this study, we propose an easy small-scale production of 67Cu using 64Ni target for a preclinical study. METHODS: 67Cu was produced from an electrodeposited 64Ni target via the 64Ni(α, p)67Cu-reaction with a 36 MeV alpha beam at 15 eµA (electrical microampere) conducted for 7 h. The chemical separation process of 67Cu from the 64Ni target was performed following by our routine procedure of 64Cu production using cation exchange resin, AG50W-X8, with minor modification. The target and its holder were redesigned in the preparation. RESULTS: The 67Cu product was obtained with a yield of 55 ±â€¯10 MBq at the end of bombardment (EOB), and the yield was 527 ±â€¯96 kBq/µAh at the EOB. The copper impurity in the product was low (0.71 ±â€¯0.21 µg) and the product was suitable for a preclinical study. CONCLUSIONS: We produced 67Cu with sufficient activity and quality for a preclinical study using a 64Ni-target. This production method also showed advantages as a routine method, i.e., shorten the processing time, reducing the radiation exposure and ready target recycling, when compared with that of a conventional Zn-target used for 67Cu production.

Synthesis and evaluation of [64Cu]PSMA-617 targeted for prostate-specific membrane antigen in prostate cancer.

We radiolabeled a ligand, PSMA-617, of prostate-specific membrane antigen (PSMA) with copper-64 (64Cu), to evaluate the metabolism, biodistribution, and potential of [64Cu]PSMA-617 for PET imaging of prostate cancer. [64Cu]PSMA-617 was synthesized by heating PSMA-617 with [64Cu]CuCl2 in buffer solution at 90°C for 5 min. In vitro uptake was determined in two cell lines of prostate cancer. In vivo regional distributions were determined in normal and tumor-bearing mice. High radiolabeling efficiency of 64Cu for PSMA-617 yielded [64Cu]PSMA-617 with >99% radiochemical purity. In vitro cellular uptake experiments demonstrated the specificity of [64Cu]PSMA-617 for PSMA-positive LNCaP cells. Biodistribution observations of normal mice revealed high uptake of radioactivity in the kidney and liver. PET with [64Cu]PSMA-617 visualized tumor areas implanted by PSMA-positive LNCaP cells in the mice. Two hours after the injection of [64Cu]PSMA-617 into mice, a radiolabeled metabolite was observed in the blood, liver, urine, and LNCaP tumor tissues. [64Cu]PSMA-617 was easily synthesized, and exhibited a favorable biodistribution in PSMA-positive tumors. Although this radioligand shows slow clearance for kidney and high liver uptake, change of its chelator moiety and easy radiolabeling may enable development of new 64Cu or 67Cu-labeled PSMA ligands for imaging and radiotherapy.