Evaluation of (rac)-, (R)-, and (S)-18F-OF-NB1 for Imaging GluN2B Subunit-Containing N-Methyl-d-Aspartate Receptors in Nonhuman Primates.

Despite 2 decades of research, no N-methyl-d-aspartate (NMDA) glutamate receptor (GluN) subtype 2B (GluN1/2B) radioligand is yet clinically validated. Previously, we reported on (rac)-18F-OF-NB1 as a promising GluN1/2B PET probe in rodents and its successful application for the visualization of GluN2B-containing NMDA receptors in postmortem brain tissues of patients with amyotrophic lateral sclerosis. In the current work, we report on the in vivo characterization of (rac)-, (R)-, and (S)-18F-OF-NB1 in nonhuman primates. Methods: PET scans were performed on rhesus monkeys. Plasma profiling was used to obtain the arterial input function. Regional brain time-activity curves were generated and fitted with the 1- and 2-tissue-compartment models and the multilinear analysis 1 method, and the corresponding regional volumes of distribution were calculated. Blocking studies with the GluN1/2B ligand Co 101244 (0.25 mg/kg) were performed for the enantiopure radiotracers. Receptor occupancy, nonspecific volume of distribution, and regional binding potential (BP ND) were obtained. Potential off-target binding toward σ1 receptors was assessed for (S)-18F-OF-NB1 using the σ1 receptor ligand FTC-146. Results: Free plasma fraction was moderate, ranging from 12% to 16%. All radiotracers showed high and heterogeneous brain uptake, with the highest levels in the cortex. (R)-18F-OF-NB1 showed the highest uptake and slowest washout kinetics of all tracers. The 1-tissue-compartment model and multilinear analysis 1 method fitted the regional time-activity curves well for all tracers and produced reliable regional volumes of distribution, which were higher for (R)- than (S)-18F-OF-NB1. Receptor occupancy by Co 101244 was 85% and 96% for (S)-18F-OF-NB1 and (R)-18F-OF-NB1, respectively. Pretreatment with FTC-146 at both a low (0.027 mg/kg) and high (0.125 mg/kg) dose led to a similar reduction (48% and 49%, respectively) in specific binding of (S)-18F-OF-NB1. Further, pretreatment with both Co 101244 and FTC-146 did not result in a further reduction in specific binding compared with Co 101244 alone in the same monkey (82% vs. 81%, respectively). Regional BP ND values ranged from 1.3 in the semiovale to 3.4 in the cingulate cortex for (S)-18F-OF-NB1. Conclusion: Both (R)- and (S)-18F-OF-NB1 exhibited high binding specificity to GluN2B subunit-containing NMDA receptors. The fast washout kinetics, good regional BP ND values, and high plasma free fraction render (S)-18F-OF-NB1 an attractive radiotracer for clinical translation.

Synthesis of a new bifunctional NODA for bioconjugation with PSMA ligand and one-step Al18F labeling.

The Al18F labeling method is a relatively new approach that allows radiofluorination of biomolecules such as peptides and proteins in a one-step procedure and in an aqueous solution. However, instability of the complex of [AlF]2+ with hexadentate chelator NOTA may attribute to the disassociation of free 18F- and [Al18F]2+ and accumulation in bone. In this study, we designed and synthesized a new bifunctional pentadentate AlF-chelator p-SCN-PhPr-NODA as well as its nitro form p-NO2-PhPr-NODA. Chelator p-NO2-PhPr-NODA exhibited increased Al (III) complexation kinetics determined by AA III complexation kinetic studies and stronger coordination ability towards [AlF]2+ according to DFT calculation studies in comparison with hexadentate chelator NOTA. As a proof of concept, bifunctional chelator p-SCN-PhPr-NODA was furthermore conjugated to a PSMA targeting moiety Glu-urea-Lys to form NODA-PrPh-GuL. The conjugated peptide showed acceptable radiochemical yield (12.5-16.4%) and efficiency with an excellent radiochemical purity (∼100% after SPE purification) in Al18F labeling. The labeled peptide exhibited good in vitro stability and significant specificity for PSMA. Biodistribution study and MicroPET scan in healthy Kun Ming mice with the labeled peptide were performed and demonstrated excellent in vivo stability of Al18F-labeled construct. In general, the successful application of the new bifunctional chelator in labeling dipeptide Glu-urea-Lys with Al18F could facilitate its possibility in conjugating with other peptides for PET imaging with enhanced in vivo stability, thus providing better in vivo performances.

Comparison of three novel radiotracers for GluN2B-containing NMDA receptors in non-human primates: (R)-[11C]NR2B-Me, (R)-[18F]of-Me-NB1, and (S)-[18F]of-NB1.

The NMDA receptor GluN2B subunit is a target of interest in neuropsychiatric disorders but to date there is no selective radiotracer available to quantify its availability in vivo. Here we report direct comparisons in non-human primates of three GluN2B-targeting radioligands: (R)-[11C]NR2B-Me, (R)-[18F]OF-Me-NB1, and (S)-[18F]OF-NB1. Plasma free fraction, metabolism, tissue distribution and kinetics, and quantitative kinetic modeling methods and parameters were evaluated in two adult rhesus macaques. Free fraction in plasma was <2% for (R)-[11C]NR2B-Me and (R)-[18F]OF-Me-NB1 and higher for (S)-[18F]OF-NB1 (15%). All radiotracers showed good brain uptake and distribution throughout grey matter, with substantial (>68%) blockade across the brain by the GluN2B-targeting drug Co-101,244 (0.25 mg/kg), including in the cerebellum. Time-activity curves were well-fitted by the one-tissue compartment model, with volume of distribution values of 20-40 mL/cm3 for (R)-[11C]NR2B-Me, 8-16 mL/cm3 for (R)-[18F]OF-Me-NB1, and 15-35 mL/cm3 for (S)-[18F]OF-NB1. Estimates of regional non-displaceable binding potential were in the range of 2-3 for (R)-[11C]NR2B-Me and (S)-[18F]-OF-NB1, and 0.5-1 for (R)-[18F]OF-Me-NB1. Altogether, each radiotracer showed an acceptable profile for quantitative imaging of GluN2B. (S)-[18F]OF-NB1 has particularly promising imaging characteristics for potential translation into humans. However, the source of unexpected displaceable binding in the cerebellum for each of these compounds requires further investigation.

Translational PET Imaging of Spinal Cord Injury with the Serotonin Transporter Tracer [11C]AFM.

PURPOSE: The descending raphespinal serotonin (5-HT) system contributes to neural activities required for locomotion. The presynaptic serotonin transporter (SERT) is a marker of 5-HT innervation. In this study, we explored the use of PET imaging with the SERT radioligand [11C]AFM as a biomarker of 5-HT axon damage after spinal cord injury (SCI) in a rodent model and its translation to imaging SCI in humans. PROCEDURES: PET imaging with [11C]AFM was performed in healthy rats under baseline and citalopram blocking conditions and a mid-thoracic transection rat model of SCI. The lumbar-to-cervical activity (L/C) ratio was calculated for the healthy and SCI animals to assess SERT binding decrease after SCI. Finally, translation of [11C]AFM PET was attempted to explore its potential to image SCI in humans. RESULTS: Intense uptake in the brain and intact spinal cord was observed at 30-60 min post-injection of [11C]AFM in healthy rats. About 65% of [11C]AFM uptake in the spinal cord was blocked by citalopram. In the SCI rat model, the cervical uptake of [11C]AFM was similar to that in healthy rats, but the lumbar uptake was dramatically reduced, resulting in about half the L/C ratio in SCI rats compared to healthy rats. In contrast, [11C]AFM uptake in the human spinal cord showed no obvious decrease after treatment with citalopram. In the human subjects with SCI, decreases in [11C]AFM uptake were also not obvious in the section of spinal cord caudal to the injury point. CONCLUSION: [11C]AFM PET imaging of SERT provides a useful preclinical method to non-invasively visualize the rodent spinal cord and detect SERT changes in SCI rodent models. However, there appears to be little detectable specific binding signal for [11C]AFM in the human spinal cord. An SERT tracer with higher affinity and lower non-specific binding signal is needed to image the spinal cord in humans and to assess the axonal status in SCI patients.

New wine in old bottles: 68Ga-PSMA-11 PET/CT reveals COVID-19 in patients with prostate cancer.

The COVID-19 pandemic continues to influence every aspect of human life across the globe. It was reported that vascular angiogenesis of COVID-19 was elevated in patients with equally severe influenza virus infection. In this issue of AJNMMI, Farolfi et al. reported that there was lung uptake not related to prostate cancer in almost all COVID-19 patients who performed 68Ga-PSMA-11 PET/CT scans and most of the lung uptake lesions were matched with typical CT patterns of COVID-19. With the advantages of having various tracers for whole-body imaging, PET provides opportunities to study the mechanism of COVID-19 from different aspects and obtain patterns of extrapulmonary lesions in COVID-19, which helps explore more effective treatments for the patients. This case series opened the door to many future studies. Furthermore, such a multi-national/multi-institutional collaboration in the pandemic truly encouraged us that science is indeed without borders.

Preclinical Advances in Theranostics for the Different Molecular Subtypes of Breast Cancer.

Breast cancer is the most common cancer in women worldwide. The heterogeneity of breast cancer and drug resistance to therapies make the diagnosis and treatment difficult. Molecular imaging methods with positron emission tomography (PET) and single-photon emission tomography (SPECT) provide useful tools to diagnose, predict, and monitor the response of therapy, contributing to precision medicine for breast cancer patients. Recently, many efforts have been made to find new targets for breast cancer therapy to overcome resistance to standard of care treatments, giving rise to new therapeutic agents to offer more options for patients with breast cancer. The combination of diagnostic and therapeutic strategies forms the foundation of theranostics. Some of these theranostic agents exhibit high potential to be translated to clinic. In this review, we highlight the most recent advances in theranostics of the different molecular subtypes of breast cancer in preclinical studies.

Biomimetic oxygen delivery nanoparticles for enhancing photodynamic therapy in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a kind of aggressive breast cancer with a high rate of metastasis, poor overall survival time, and a low response to targeted therapies. To improve the therapeutic efficacy and overcome the drug resistance of TNBC treatments, here we developed the cancer cell membrane-coated oxygen delivery nanoprobe, CCm-HSA-ICG-PFTBA, which can improve the hypoxia at tumor sites and enhance the therapeutic efficacy of the photodynamic therapy (PDT), resulting in relieving the tumor growth in TNBC xenografts. RESULTS: The size of the CCm-HSA-ICG-PFTBA was 131.3 ± 1.08 nm. The in vitro 1O2 and ROS concentrations of the CCm-HSA-ICG-PFTBA group were both significantly higher than those of the other groups (P < 0.001). In vivo fluorescence imaging revealed that the best time window was at 24 h post-injection of the CCm-HSA-ICG-PFTBA. Both in vivo 18F-FMISO PET imaging and ex vivo immunofluorescence staining results exhibited that the tumor hypoxia was significantly improved at 24 h post-injection of the CCm-HSA-ICG-PFTBA. For in vivo PDT treatment, the tumor volume and weight of the CCm-HSA-ICG-PFTBA with NIR group were both the smallest among all the groups and significantly decreased compared to the untreated group (P < 0.01). No obvious biotoxicity was observed by the injection of CCm-HSA-ICG-PFTBA till 14 days. CONCLUSIONS: By using the high oxygen solubility of perfluorocarbon (PFC) and the homologous targeting ability of cancer cell membranes, CCm-HSA-ICG-PFTBA can target tumor tissues, mitigate the hypoxia of the tumor microenvironment, and enhance the PDT efficacy in TNBC xenografts. Furthermore, the HSA, ICG, and PFC are all FDA-approved materials, which render the nanoparticles highly biocompatible and enhance the potential for clinical translation in the treatment of TNBC patients.

pH-Triggered Assembly of Natural Melanin Nanoparticles for Enhanced PET Imaging.

Natural melanin nanoplatforms have attracted attention in molecular imaging. Natural melanin can be made into small-sized nanoparticles, which penetrate tumor sites deeply, but unfortunately, the particles continue to backflow into the blood or are cleared into the surrounding tissues, leading to loss of retention within tumors. Here, we report a pH-triggered approach to aggregate natural melanin nanoparticles by introducing a hydrolysis-susceptible citraconic amide on the surface. Triggered by pH values lower than 7.0, such as the tumor acid environment, the citraconic amide moiety tended to hydrolyze abruptly, resulting in both positive and negative surface charges. The electrostatic attractions between nanoparticles drove nanoparticle aggregation, which increased accumulation in the tumor site because backflow was blocked by the increased size. Melanin nanoparticles have the natural ability to bind metal ions, which can be labeled with isotopes for nuclear medicine imaging. When the melanin nanoparticles were labeled by 68Ga, we observed that the pH-induced physical aggregation in tumor sites resulted in enhanced PET imaging. The pH-triggered assembly of natural melanin nanoparticles could be a practical strategy for efficient tumor targeted imaging.

Synthesis and Preclinical Evaluation of a 68Ga-Radiolabeled Peptide Targeting Very Late Antigen-3 for PET Imaging of Pancreatic Cancer.

Pancreatic cancer is highly malignant and has a five-year survival rate of 5% due to an early lymph node, nerve, and vascular metastasis. Integrin α3ß1 (also called very late antigen-3, VLA-3) is overexpressed in many tumors and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we developed a 68Ga-radiolabeled peptide tracer targeting the α3 unit of VLA-3 and evaluated its potential application in positron emission computed tomography (PET) imaging of pancreatic cancer. NOTA-CK11 was prepared by solid-phase synthesis and successfully radiolabeled with 68Ga with greater than 99% radiochemical purity and a specific activity of 37 ± 5 MBq/nmol (n = 5). The expression level of integrin α3 in three human pancreatic cancer cells was evaluated with the order of SW1990, BXPC-3, and PANC-1 from high to low, while the expression level of integrin ß1 was relatively close. When SW1990 cells with the highest expression level of VLA-3 were stained with FITC-CK11, strong fluorescence was observed by flow cytometry and under a laser confocal microscope. However, no significant fluorescence was observed in the blocking group when treated with excessive CK11. 68Ga-NOTA-CK11 showed significant radioactivity accumulation in SW1990 cells and was blocked by CK11 successfully. Subsequent small-animal PET imaging and biodistribution studies in mice bearing SW1990 xenografts confirmed its high tumor uptake with a good tumor-to-blood ratio and tumor-to-muscle ratio (2.45 ± 0.31 and 3.65 ± 0.33, respectively) at 1 h post injection of the probe. In summary, we successfully developed a peptide-based imaging agent, 68Ga-NOTA-CK11, that showed a strong binding affinity with VLA-3 and good target specificity for SW1990 cells and xenografted pancreatic tumor, rending it a promising radiotracer for PET imaging of VLA-3 expression in pancreatic cancer.

A thiopyrylium salt for PET/NIR-II tumor imaging and image-guided surgery.

All tumor imaging modalities have resolution limits below which deeply situated small metastatic foci may not be identified. Moreover, incomplete lesion excision will affect the outcomes of the patients. Scintigraphy is adept in locating lesions, and second near-infrared window (NIR-II) imaging may allow precise real-time tumor delineation. To achieve complete excision of all lesions, multimodality imaging is a promising method for tumor identification and management. Here, a NIR-II thiopyrylium salt, XB1034, was first synthesized and bound to cetuximab and trans-cyclooctene (TCO) to produce XB1034-cetuximab-TCO. This probe provides excellent sensitivity and high temporal resolution NIR-II imaging in mice bearing tumors developed from human breast cancer cells MDA-MB-231. To enable PET imaging, 68 Ga-NETA-tetrazine is subsequently injected into the mice to undergo a bio-orthogonal reaction with the preinjected XB1034-cetuximab-TCO. PET images achieved in the tumor models using the pretargeting strategy are of much higher quality than those obtained using the direct radiolabeling method. Moreover, real-time NIR-II imaging allows accurate tumor excision and sentinel lymph node mapping. In conclusion, XB1034 is a promising molecular imaging probe for tumor diagnosis and treatment.

Red blood cell membrane-coated upconversion nanoparticles for pretargeted multimodality imaging of triple-negative breast cancer.

Upconversion nanoparticles (UCNPs) have been widely employed for tumor imaging using magnetic resonance imaging (MRI) and upconversion luminescence (UCL) imaging. The short blood clearance time and immunogenicity of UCNPs have limited their further application in vivo. We have designed UCNPs camouflaged with an exterior red blood cell (RBC) membrane coating (RBC-UCNPs) to solve these problems. Moreover, because of some intrinsic disadvantages of MRI and UCL imaging, we investigated the use of pretargeted RBC-UCNPs for positron-emission tomography (PET) imaging to obtain more comprehensive information. Our data showed that RBC-UCNPs retained the immunity feature from the source cells and the superior optical and chemical features from the pristine UCNP cores. The tumor-targeting ability of RBC-UCNPs was enhanced by binding 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG-FA) molecules onto the cell membranes. PET imaging with short half-life radionuclides to visualize the RBC-UCNPs was successfully realized by a combination of pre-targeting and in vivo click chemistry. Blood chemistry, hematology, and histologic analysis suggested good in vivo biocompatibility of the RBC-UCNPs. Our method provides a new potential biomedical application of biomimetic nanoparticles.

Ultra-sensitive Nanoprobe Modified with Tumor Cell Membrane for UCL/MRI/PET Multimodality Precise Imaging of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the estrogen receptor and progesterone receptor are not expressed, and human epidermal growth factor receptor 2 is not amplified or overexpressed either, which make the clinical diagnosis and treatment very challenging. Molecular imaging can provide an effective way to diagnose TNBC. Upconversion nanoparticles (UCNPs), are a promising new generation of molecular imaging probes. However, UCNPs still need to be improved for tumor-targeting ability and biocompatibility. This study describes a novel probe based on cancer cell membrane-coated upconversion nanoparticles (CCm-UCNPs), owing to the low immunogenicity and homologous-targeting ability of cancer cell membranes, and modified multifunctional UCNPs. This probe exhibits excellent performance in breast cancer molecular classification and TNBC diagnosis through UCL/MRI/PET tri-modality imaging in vivo. By using this probe, MDA-MB-231 was successfully differentiated between MCF-7 tumor models in vivo. Based on the tumor imaging and molecular classification results, the probe is also expected to be modified for drug delivery in the future, contributing to the treatment of TNBC. The combination of nanoparticles with biomimetic cell membranes has the potential for multiple clinical applications.

Comparison of Al18F- and 68Ga-labeled NOTA-PEG4-LLP2A for PET imaging of very late antigen-4 in melanoma.

Malignant melanoma is an aggressive cancer with poor prognosis. Very late antigen-4 (VLA-4) is overexpressed in melanoma and many other tumors, making it an attractive target for developing molecular diagnostic and therapeutic agents. We compared Al18F- and 68Ga-labeled LLP2A peptides for PET imaging of VLA-4 expression in melanoma. The peptidomimetic ligand LLP2A was modified with chelator 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and the resulting NOTA-PEG4-LLP2A peptide was then radiolabeled with Al18F or 68Ga. The two labeled peptides were assayed for in vitro and in vivo VLA-4 targeting efficiency. Good Al18F and 68Ga radiolabeling yields were achieved, and the resulting PET tracers showed good serum stability. In the in vivo evaluation of the B16F10 xenograft mouse model, both tracers exhibited high accumulation with good contrast in static PET images. Compared with 68Ga-NOTA-PEG4-LLP2A, Al18F-NOTA-PEG4-LLP2A resulted in relatively higher background, including higher liver uptake (1 h: 20.1 ± 2.6 vs. 15.3 ± 1.7%ID/g, P < 0.05; 2 h: 11.0 ± 1.2 vs. 8.0 ± 0.8%ID/g, P < 0.05) and lower tumor-to-blood ratios (2.5 ± 0.4 vs. 3.3 ± 0.5 at 1 h, P < 0.05; 5.1 ± 0.9 vs. 7.3 ± 0.6 at 2 h, P < 0.01) at some time points. The results obtained from the mice blocked with unlabeled peptides and VLA-4-negative A375 xenografts groups confirmed the high specificity of the developed tracers. Despite the relatively high liver uptake, both Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A exhibited high VLA-4 targeting efficacy with comparable in vivo performance, rendering them promising candidates for imaging tumors that overexpress VLA-4.

Preclinical Evaluation of a Fluorine-18 (18F)-Labeled Phosphatidylinositol 3-Kinase Inhibitor for Breast Cancer Imaging.

Breast cancer is one of the commonest malignancies in women, especially in middle-aged and elderly women. Abnormal activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKt/mTOR) pathway has been found to be involved in breast cancer proliferation. Pictilisib (GDC-0941) is a potent inhibitor of PI3K with high affinity and is undergoing phase 2 clinical trials. In this study, we aimed to develop a noninvasive PI3K radiotracer to help determine the mechanism of the PI3K/AKt/mTOR pathway to aid in diagnosis. We designed a new 18F-radiolabeled radiotracer based on the structure of pictilisib, to evaluate noninvasively abnormal activation of the PI3K/AKT/mTOR pathway. To increase the water solubility, and to decrease hepatobiliary and gastrointestinal uptake of the tracer, pictilisib was modified with triethylene glycol di(p-toluenesulfonate) (TsO-PEG3-OTs) to obtain TsO-PEG3-GDC-0941 as the precursor for 18F labeling. A nonradiolabeled reference compound [19F]-PEG3-GDC-0941 was also prepared. Breast cancer cell lines, MCF-7 and MDA-MB-231, were used as high- and low-expression PI3K models, respectively. PET imaging and ex vivo biodistribution assays of [18F]-PEG3-GDC-0941 in MCF-7 and MDA-MB-231 xenografts were also performed, and the results were compared. The precursor compound and reference standard compound were successfully synthesized and identified using NMR and mass spectroscopy. The 18F radiolabeling was achieved with a high yield (61 ± 1%) at a high molar activity (2100 ± 100 MBq/mg). MicroPET images and biodistribution studies showed a higher uptake of the radiotracer in MCF-7 tumors than in MDA-MB-231 tumors (7.56 ± 1.01%ID/g vs 4.07 ± 0.68%ID/g, 1 h postinjection). Additionally, the MCF-7 tumor uptake was significantly decreased when a blocking dose of GDC-0941 was coinjected, indicating high specificity. The liver was found to be the major excretory organ with 5.82 ± 0.88%ID/g at 30 min postinjection for MCF-7 mice. This radiotracer holds great potential for patient screening, diagnosis, and therapy prediction of PI3K-related diseases.