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The transition-metal-free At-211-astatination of spirocyclic aryliodonium ylides via a nucleophilic aromatic substitution reaction is described. This method enables the preparation of At-211-radiolabeled compounds derived from multi-functionalized molecules and heteroarenes in good to excellent radiochemical yields.

Ovarian cancer peritoneal metastases (OCPMs) are a pathophysiologically heterogeneous group of tumors that are rarely curable. αvβ3 integrin (αvβ3) is overexpressed on tumoral neovessels and frequently on ovarian cancer cells. Here, using two clinically relevant αvβ3-positive OCPM mouse models, we studied the theranostic potential of an αvβ3-specific radiopeptide, 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RaftRGD), and its intra- and intertumoral distribution in relation to the tumor microenvironment.

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 reported that the interface peptides from programmed death-ligand 1 (PD-L1) can be used in positron emission tomography (PET) imaging of the 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 of the significance of PET imaging as a companion diagnostics 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.

Synovial sarcoma (SS) is a rare yet refractory soft-tissue sarcoma that predominantly affects young adults. We show in a mouse model that radioimmunotherapy (RIT) with an α-particle emitting anti-Frizzled homolog 10 (FZD10) antibody, synthesized using the α-emitter radionuclide astatine-211 (211At-OTSA101), suppresses the growth of SS xenografts more efficiently than the corresponding β-particle emitting anti-FZD10 antibody conjugated with the β-emitter yettrium-90 (90Y-OTSA101). In biodistribution analysis, 211At was increased in the SS xenografts but decreased in other tissues up to 1 day after injection as time proceeded, albeit with a relatively higher uptake in the stomach. Single 211At-OTSA101 doses of 25 and 50 μCi significantly suppressed SS tumor growth in vivo, whereas a 50 μCi dose of 90Y-OTSA101 was needed to achieve this. Importantly, 50 μCi of 211At-OTSA101 suppressed tumor growth immediately after injection, whereas this effect required several days in the case of 90Y-OTSA101. Both radiolabeled antibodies at the 50 μCi dosage level significantly prolonged survival. Histopathologically, severe cellular damage accompanied by massive cell death was evident in the SS xenografts at even 1 day after the 211At-OTSA101 injection but these effects were relatively milder with 90Y-OTSA101 at the same timepoint, even though the absorbed doses were comparable (3.3 and 3.0 Gy, respectively). We conclude that α-particle RIT with 211At-OTSA101 is a potential new therapeutic option for SS.

Pancreatic cancer (PC) has a very poor prognosis. Surgery is the primary treatment for patients with resectable PC; however, local recurrence, hepatic metastasis, and peritoneal dissemination often occur even after extensive surgery. Adjuvant chemotherapy, typically with gemcitabine, has been used clinically but with only a modest survival benefit. To achieve a better outcome, we investigated the efficacy of 64Cu-intraperitoneal radioimmunotherapy (ipRIT) with 64Cu-labeled antiepidermal growth factor receptor antibody cetuximab as an adjuvant treatment after PC surgery using an orthotopic xenografted mouse model. Methods: The efficacy of adjuvant 64Cu-ipRIT was investigated in a human PC mouse model harboring orthotopic xenografts of xPA-1-DC cells. To reproduce the clinical situation, PC xenografts were surgically resected when pancreatic tumors were readily visible but not metastatic tumors. Increasing doses of 64Cu-cetuximab were intraperitoneally injected, and the mice were monitored for toxicity to determine the safe therapeutic dose. For adjuvant 64Cu-ipRIT, the day after tumor resection, the mice were intraperitoneally administered 22.2 MBq of 64Cu-PCTA-cetuximab and the survival was compared with that in surgery-only controls. For comparison, adjuvant chemotherapy with gemcitabine was also examined using the same model. Results: The mouse model not only developed primary tumors in the pancreas but also subsequently reproduced local recurrence, hepatic metastasis, and peritoneal dissemination after surgery, which is similar to the manifestations that occur with human PC. Adjuvant 64Cu-ipRIT with 64Cu-labeled cetuximab after surgery effectively suppressed local recurrence, hepatic metastasis, and peritoneal dissemination in this model. Significant improvement of the survival with minimal toxicity was achieved by adjuvant 64Cu-ipRIT compared with that in control mice that underwent surgery only. Adjuvant chemotherapy with gemcitabine nominally prolonged the survival, but the effect was not statistically significant. Conclusion: 64Cu-ipRIT with cetuximab can be an effective adjuvant therapy after PC surgery.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed two 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 two radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results: 131I-IITM and 211At-AITM were obtained with radiochemical purity of >99% and radiochemical yields of 42.7% ± 10.4% and 28.9% ± 9.9%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited maximum uptakes of 4.66% ± 0.70% ID/g and 7.68% ± 0.71% ID/g in the targeted melanomas, respectively, and were rapidly cleared from non-target 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 non-toxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

The α-emitter 211At-labeled meta-astatobenzylguanidine (211At-MABG) has a strong antitumor effect on pheochromocytoma xenograft tumors and holds great promise as a new therapeutic option for malignant pheochromocytoma. To evaluate the acute radiation-related toxicity of 211At-MABG, we conducted biodistribution and dosimetry studies of 211At-MABG in ICR mice to estimate the doses absorbed by organs. We determined the maximum tolerated doses (MTD) of 211At-MABG on the basis of body weight loss and assessed the acute radiation-related toxicity induced by MTD administration on the basis of organ weights, histologic features, hematologic indices, and biochemical indices. The biodistribution and dosimetry studies of α-emitting 211At-MABG revealed high doses absorbed by most organs except the brain in ICR mice. The administration of 1.1, 2.2, and 3.3 MBq of 211At-MABG induced transient body weight loss, and 4.4 MBq of 211At-MABG induced unrecoverable body weight loss; thus, the MTD was 3.3 MBq for ICR mice. Although by day 5 the administration of 3.3 MBq had induced some radiation-related toxicity symptoms—such as body weight loss and leucopenia, which are generally observed in radiation therapy including β−-emitting radiopharmaceuticals—the mice had recovered by day 28. We observed no unexpected severe toxicity in ICR mice despite the high absorbed doses in most organs, especially the thyroid, heart, stomach, and adrenal glands. Our findings suggest that therapeutic treatments with appropriate doses of 211At-MABG estimated by dosimetry in each patient could be tolerated, although lower doses may initially be necessary to ensure patient safety in the first-in-human study.

Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found, however, that locoregionally administered trastuzumab armed with astatine-211 (211At-trastuzumab) is effective against human epidermal growth factor receptor 2 (HER2)-positive PMGC in a xenograft mouse model. We first observed that 211At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in s.c. tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test a-particle radioimmunotherapy with 211At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the i.p. administration of 211At-trastuzumab (1 MBq) was a more efficient means of delivery of 211At into metastatic tumors than i.v.injection; the maximum tumor uptake with i.p. administration was over 60% injected dose per gram of tissue (%ID/g) compared to approximately 18%ID/g with i.v. injection. Surprisingly, a single i.p. injection of 211At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA doublestrand breaks, and to drastically reduce the tumor burden in another three mice. No bodyweight loss, leukocytopenia, or significant biochemical changes in liver or kidney function were observed in the treatment group. Accordingly, locoregionally administered 211At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211At-trastuzumab may offer a new treatment option for HER2-positive PMGC.

Arsenic contamination is a major environmental issue, as it may lead to serious health hazard. The reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Root transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots compared with the wild-type. At the cellular level, arsenite specifically targets intracellular sorting of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 function results in arsenite hypersensitivity in roots. XFI coupled with XAS further revealed that loss of PIN2 function results in specific accumulation of arsenical species, but not the other metals such as iron, zinc, or calcium in the root tip. Collectively, these results suggest that PIN2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta.

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.

Supplementary materials and methods. (PDF 95 kb)

(a) Tumor growth ratios of the same set of treated groups as described in Fig. 3. *, †, ‡P

Intratumoral distribution of 64Cu-ATSM and Cy5.5-RaftRGD. Mice bearing U87MG tumors with sizes of 50–1065 mm3 were co-injected with 64Cu-ATSM and Cy5.5-RaftRGD and euthanized 3 h later. Autoradiography, fluorescence imaging and HE staining were sequentially performed in the same tumor sections. Merged images showing 64Cu autoradiogram in green, Cy5.5 fluorescence in red, and HE stains. Yellow, red/green overlay. The necrotic regions are surrounded by dotted lines. Scale bars, 1, 2 mm. (PDF 235 kb)

Tumor growth curves (a) and body weight changes (b) of the same set of experimental groups as described in Fig. 3. Values are the means ± standard deviations (n = 6/group). The final data points shown for each group of mice (b) represent the results obtained at survival endpoint days (represented by a mean value). *, †, ‡P

Hematology (a) and hepatorenal functions (b) of U87MG tumor-bearing mice after co-administration of 64Cu-RaftRGD and 64Cu-ATSM at 111 or 148 MBq. (PDF 100 kb)

Tumor growth curves of U87MG tumor-bearing mice after single injection of the vehicle solution (control) or 18.5 MBq of 64Cu-RaftRGD (a) or 64Cu-ATSM (b). n = 5–6/group. (PDF 116 kb)

Intratumoral distribution of 64Cu-ATSM and microvasculature. An adjacent slice to that presented in Fig. 2b was examined by autoradiography, CD31 immunofluorescence staining, and HE staining. Merged image showing 64Cu-ATSM distribution in green, CD31-stained microvessels in red, and HE stains. High-resolution pictures shown by dotted rectangles clearly depicting the stained microvessels in smaller size in 64Cu-ATSM high- vs. low-accumulated areas. Nuclei were stained with DAPI (blue). Scale bars, 2 mm, 200 μm. (PDF 285 kb)

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. \n