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Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rationale: Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. Methods: In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for in vivo PET quantification, whereas results for ex vivo assays are reported as percent injected dose per gram (%ID/g). Results: In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed in vivo by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[ 18 F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Conclusion: Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: The aim of this study was to develop a positron emission tomography (PET) radiotracer for measuring pyruvate kinase M2 (PKM2) with improved physicochemical and pharmacokinetic properties compared to [ 18 F]DASA-23., Experimental Design: First, we synthesized [ 18 F]DASA-10 and tested its uptake and retention compared to [ 18 F]DASA-23 in human and mouse glioma cell lines. We then confirmed the specificity of [ 18 F]DASA-10 by transiently modulating the expression of PKM2 in DU145 and HeLa cells. Next, we determined [ 18 F]DASA-10 pharmacokinetics in healthy nude mice using PET imaging and subsequently assessed the ability of [ 18 F]DASA-10 versus [ 18 F]DASA-23 to enable in vivo detection of intracranial gliomas in syngeneic C6 rat models of glioma., Results: [ 18 F]DASA-10 demonstrated excellent cellular uptake and retention with values significantly higher than [ 18 F]DASA-23 in all cell lines and timepoints investigated. [ 18 F]DASA-10 showed a 73 % and 65 % reduced uptake respectively in DU145 and HeLa cells treated with PKM2 siRNA as compared to control siRNA treated cells. [ 18 F]DASA-10 showed favorable biodistribution and pharmacokinetic properties and a significantly improved tumor-to-brain ratio in rat C6 glioma models relative to [ 18 F]DASA-23 (3.2 ± 0.8 versus 1.6 ± 0.3, p = 0.01)., Conclusion: [ 18 F]DASA-10 is a new PET radiotracer for molecular imaging of PKM2 with potential to overcome the prior limitations observed with [ 18 F]DASA-23. [ 18 F]DASA-10 shows promise for clinical translation to enable imaging of brain malignancies owing to its low background signal in the healthy brain., Competing Interests: Declaration of competing interest CB and MLJ hold a patent (US20180043040A1) describing the use of radiopharmaceuticals to measure PKM2. The authors declare no other conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Positron emission tomography (PET) is a powerful tool for studying neuroinflammatory diseases; however, current PET biomarkers of neuroinflammation possess significant limitations. We recently reported a promising dendrimer PET tracer ([ 18 F]OP-801), which is selectively taken up by reactive microglia and macrophages. Here, we describe further important characterization of [ 18 F]OP-801 in addition to optimization and validation of a two-step clinical radiosynthesis. [ 18 F]OP-801 was found to be stable in human plasma for 90 min post incubation, and human dose estimates were calculated for 24 organs of interest; kidneys and urinary bladder wall without bladder voiding were identified as receiving the highest absorbed dose. Following optimization detailed herein, automated radiosynthesis and quality control (QC) analyses of [ 18 F]OP-801 were performed in triplicate in suitable radiochemical yield (6.89 ± 2.23% decay corrected), specific activity (37.49 ± 15.49 GBq/mg), and radiochemical purity for clinical imaging. Importantly, imaging mice with tracer (prepared using optimized methods) 24 h following the intraperitoneal injection of liposaccharide resulted in the robust brain PET signal. Cumulatively, these data enable clinical translation of [ 18 F]OP-801 for imaging reactive microglia and macrophages in humans. Data from three validation runs of the clinical manufacturing and QC were submitted to the Food and Drug Administration (FDA) as part of a Drug Master File (DMF). Subsequent FDA approval to proceed was obtained, and a phase 1/2 clinical trial (NCT05395624) for first-in-human imaging in healthy controls and patients with amyotrophic lateral sclerosis is underway.

Intro: Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate., Methods: First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [ 11 C]AZD1283 with radioactive precursor [ 11 C]NH 4 CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [ 11 C]AZD1283 via dynamic PET imaging of naïve wild-type mice, P2Y12R knockout mouse, and a rhesus macaque., Results: We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [ 11 C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/μmol (average of n = 3). Of note, we found [ 11 C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV))., Conclusion: Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [ 11 C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [ 11 C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (∼15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[ 18 F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([ 18 F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM., Experimental Design: [ 18 F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/μmol and radiochemical purity >95%. We performed initial testing of [ 18 F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [ 18 F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma., Results: In mouse imaging studies, [ 18 F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [ 18 F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [ 18 F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [ 18 F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation., Conclusions: We developed and translated [ 18 F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [ 18 F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism., (©2021 American Association for Cancer Research.)

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R 2  = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form of alternating electric fields therapy on the aberrant glycolysis of hGBM. [ 18 F]FDG is the most common radiotracer in cancer metabolic imaging, but its utility in hGBM is impaired due to high glucose uptake in normal brain tissue. With TTFields, radiochemistry, Western blot, and immunofluorescence microscopy, we identified pyruvate kinase M2 (PKM2) as a biomarker of hGBM response to therapeutic TTFields. We used [ 18 F]DASA-23, a novel radiotracer that measures PKM2 expression and which has been shown to be safe in humans, to detect a shift away from hGBM aberrant glycolysis in response to TTFields. Compared to unexposed hGBM, [ 18 F]DASA-23 uptake was reduced in hGBM exposed to TTFields (53%, P< 0.05) or temozolomide chemotherapy (33%, P > 0.05) for 3 d. A 6-d TTFields exposure resulted in a 31% reduction (P = 0.043) in 60-min uptake of [ 18 F]DASA-23. [ 18 F]DASA-23 was retained after a 10 but not 30-min wash-out period. Compared to [ 18 F]FDG, [ 18 F]DASA-23 demonstrated a 4- to 9-fold greater uptake, implying an improved tumor-to-background ratio. Furthermore, compared to no-TTFields exposure, a 6-d TTFields exposure caused a 35% reduction in [ 18 F]DASA-23 30-min uptake compared to only an 8% reduction in [ 18 F]FDG 30-min uptake. Quantitative Western blot analysis and qualitative immunofluorescence for PKM2 confirmed the TTFields-induced reduction in PKM2 expression. This is the first study to demonstrate that TTFields impairs hGBM aberrant glycolytic metabolism through reduced PKM2 expression, which can be non-invasively detected by the [ 18 F]DASA-23 radiotracer., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[ 18 F]-fluoro-3-[hydroxymethyl]butyl)guanine ([ 18 F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells., (©2020 American Association for Cancer Research.)

Purpose: To assess the safety, biodistribution, and radiation dosimetry of the novel positron emission tomography (PET) radiopharmaceutical 1-((2-fluoro-6-[[ 18 F]]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([ 18 F]DASA-23) in healthy volunteers., Methods: We recruited 5 healthy volunteers who provided a written informed consent. Volunteers were injected with 295.0 ± 8.2 MBq of [ 18 F]DASA-23 intravenously. Immediately following injection, a dynamic scan of the brain was acquired for 15 min. This was followed by serial whole-body PET/MRI scans acquired up to 3 h post-injection. Blood samples were collected at regular intervals, and vital signs monitored pre- and post-radiotracer administration. Regions of interest were drawn around multiple organs, time-activity curves were calculated, and organ uptake and dosimetry were estimated with OLINDA/EXM (version 1.1) software., Results: All subjects tolerated the PET/MRI examination, without adverse reactions to [ 18 F]DASA-23. [ 18 F]DASA-23 passively crossed the blood-brain barrier, followed by rapid clearance from the brain. High accumulation of [ 18 F]DASA-23 was noted in organs such as the gallbladder, liver, small intestine, and urinary bladder, suggesting hepatobiliary and urinary clearance. The effective dose of [ 18 F]DASA-23 was 23.5 ± 5.8 μSv/MBq., Conclusion: We successfully completed a pilot first-in-human study of [ 18 F]DASA-23. Our results indicate that [ 18 F]DASA-23 can be used safely in humans to evaluate pyruvate kinase M2 levels. Ongoing studies are evaluating the ability of [ 18 F]DASA-23 to visualize intracranial malignancies, NCT03539731., Trial Registration: ClinicalTrials.gov , NCT03539731 (registered 28 May 2018).

Recent advances in novel immune strategies, particularly chimeric antigen receptor (CAR)-bearing T-cells, have shown limited efficacy against glioblastoma (GBM) in clinical trials. We currently have an incomplete understanding of how these emerging therapies integrate with the current standard of care, specifically radiation therapy (RT). Additionally, there is an insufficient number of preclinical studies monitoring these therapies with high spatiotemporal resolution. To address these limitations, we report the first longitudinal fluorescence-based intravital microscopy imaging of CAR T-cells within an orthotopic GBM preclinical model to illustrate the necessity of RT for complete therapeutic response. Additionally, we detail the first usage of murine-derived CAR T-cells targeting the disialoganglioside GD2 in an immunocompetent tumor model. Cell culture assays demonstrated substantial GD2 CAR T-cell-mediated killing of murine GBM cell lines SB28 and GL26 induced to overexpress GD2. Complete antitumor response in advanced syngeneic orthotopic models of GBM was achieved only when a single intravenous dose of GD2 CAR T-cells was following either sub-lethal whole-body irradiation or focal RT. Intravital microscopy imaging successfully visualized CAR T-cell homing and T-cell mediated apoptosis of tumor cells in real-time within the tumor stroma. Findings indicate that RT allows for rapid CAR T-cell extravasation from the vasculature and expansion within the tumor microenvironment, leading to a more robust and lasting immunologic response. These exciting results highlight potential opportunities to improve intravenous adoptive T-cell administration in the treatment of GBM through concurrent RT. Additionally, they emphasize the need for advancements in immunotherapeutic homing to and extravasation through the tumor microenvironment., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is found in high levels in glioblastoma (GBM) cells with marginal expression within healthy brain tissue, rendering it a key biomarker of GBM metabolic re-programming. Our group has reported the development of a novel radiotracer, 1-((2-fluoro- 6-[ 18 F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([ 18 F]DASA- 23), to non-invasively detect PKM2 levels with positron emission tomography (PET)., Procedure: U87 human GBM cells were treated with the IC50 concentration of various agents used in the treatment of GBM, including alkylating agents (temozolomide, carmustine, lomustine, procarbazine), inhibitor of topoisomerase I (irinotecan), vascular endothelial and epidermal growth factor receptor inhibitors (cediranib and erlotinib, respectively) anti-metabolite (5-fluorouracil), microtubule inhibitor (vincristine), and metabolic agents (dichloroacetate and IDH1 inhibitor ivosidenib). Following drug exposure for three or 6 days (n = 6 replicates per condition), the radiotracer uptake of [ 18 F]DASA-23 and 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) was assessed. Changes in PKM2 protein levels were determined via Western blot and correlated to radiotracer uptake., Results: Significant interactions were found between the treatment agent (n = 12 conditions total comprised 11 drugs and vehicle) and the duration of treatment (3- or 6-day exposure to each drug) on the cellular uptake of [ 18 F]DASA-23 (p = 0.0001). The greatest change in the cellular uptake of [ 18 F]DASA-23 was found after exposure to alkylating agents (p < 0. 0001) followed by irinotecan (p = 0. 0012), erlotinib (p = 0. 02), and 5-fluorouracil (p = 0. 005). Correlation of PKM2 protein levels and [ 18 F]DASA-23 cellular uptake revealed a moderate correlation (r = 0.44, p = 0.15)., Conclusions: These proof of principle studies emphasize the superiority of [ 18 F]DASA-23 to [ 18 F]FDG in detecting the glycolytic response of GBM to multiple classes of anti-neoplastic drugs in cell culture. A clinical trial evaluating the diagnostic utility of [ 18 F]DASA-23 PET in GBM patients (NCT03539731) is ongoing.

The aim of this study was development of an improved PET radiotracer for measuring x C - activity with increased tumor uptake and reduced uptake in inflammatory cells compared with ( S )-4-(3- 18 F-fluoropropyl)-l-glutamate ( 18 F-FSPG). Methods: A racemic glutamate derivative, 18 F-hGTS13, was evaluated in cell culture and animal tumor models. 18 F-hGTS13 was separated into C5 epimers, and the corresponding 18 F-hGTS13-isomer1 and 18 F-hGTS13-isomer2 were evaluated in H460 tumor-bearing rats. Preliminary studies investigated the cellular uptake of 18 F-hGTS13-isomer2 in multiple immune cell populations and states. Results: 18 F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor-associated radioactivity was significantly higher for 18 F-hGTS13 (7.5 ± 0.9 percentage injected dose [%ID]/g, n = 3) than for 18 F-FSPG (4.6 ± 0.7 %ID/g, n = 3, P = 0.01). 18 F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3 ± 1.1 %ID/g, n = 3) and significantly reduced uptake in multiple immune cell populations relative to 18 F-FSPG. 18 F-hGTS13-isomer2 exhibited increased liver uptake relative to 18 F-FSPG (4.6 ± 0.8 vs. 0.7 ± 0.01 %ID/g), limiting its application in hepatocellular carcinoma. Conclusion: 18 F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of x C - activity that may provide information on tumor oxidation states. 18 F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax because of the low background signal in healthy tissue., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

There is a growing need for monitoring or imaging gene therapy in the central nervous system (CNS). This can be achieved with a positron emission tomography (PET) reporter gene strategy. Here we report the development of a PET reporter gene system using the PKM2 gene with its associated radiotracer [ 18 F]DASA-23. The PKM2 reporter gene was delivered to the brains of mice by adeno-associated virus (AAV9) via stereotactic injection. Serial PET imaging was carried out over 8 wk to assess PKM2 expression. After 8 wk, the brains were excised for further mRNA and protein analysis. PET imaging at 8 wk post-AAV delivery showed an increase in [ 18 F]DASA-23 brain uptake in the transduced site of mice injected with the AAV mice over all controls. We believe PKM2 shows great promise as a PET reporter gene and to date is the only example that can be used in all areas of the CNS without breaking the blood-brain barrier, to monitor gene and cell therapy., Competing Interests: The authors declare no conflict of interest.

Endogenous biomarkers remain at the forefront of early disease detection efforts, but many lack the sensitivities and specificities necessary to influence disease management. Here, we describe a cell-based in vivo sensor for highly sensitive early cancer detection. We engineer macrophages to produce a synthetic reporter on adopting an M2 tumor-associated metabolic profile by coupling luciferase expression to activation of the arginase-1 promoter. After adoptive transfer in colorectal and breast mouse tumor models, the engineered macrophages migrated to the tumors and activated arginase-1 so that they could be detected by bioluminescence imaging and luciferase measured in the blood. The macrophage sensor detected tumors as small as 25-50 mm 3 by blood luciferase measurements, even in the presence of concomitant inflammation, and was more sensitive than clinically used protein and nucleic acid cancer biomarkers. Macrophage sensors also effectively tracked the immunological response in muscle and lung models of inflammation, suggesting the potential utility of this approach in disease states other than cancer.

Purpose: There is a strong, unmet need for superior positron emission tomography (PET) imaging agents that are able to measure biochemical processes specific to prostate cancer. Pyruvate kinase M2 (PKM2) catalyzes the concluding step in glycolysis and is a key regulator of tumor growth and metabolism. Elevation of PKM2 expression was detected in Gleason 8-10 tumors compared to Gleason 6-7 carcinomas, indicating that PKM2 may potentially be a marker of aggressive prostate cancer. We have recently reported the development of a PKM2-specific radiopharmaceutical [ 18 F]DASA-23 and herein describe its evaluation in cell culture and preclinical models of prostate cancer., Procedure: The cellular uptake of [ 18 F]DASA-23 was evaluated in a panel of prostate cancer cell lines and compared to that of [ 18 F]FDG. The specificity of [ 18 F]DASA-23 to measure PKM2 levels in cell culture was additionally confirmed through the use of PKM2-specific siRNA. PET imaging studies were then completed utilizing subcutaneous prostate cancer xenografts using either PC3 or DU145 cells in mice., Results: [ 18 F]DASA-23 uptake values over 60-min incubation period in PC3, LnCAP, and DU145 respectively were 23.4 ± 4.5, 18.0 ± 2.1, and 53.1 ± 4.6 % tracer/mg protein. Transient reduction in PKM2 protein expression with siRNA resulted in a 50.1 % reduction in radiotracer uptake in DU145 cells. Small animal PET imaging revealed 0.86 ± 0.13 and 1.6 ± 0.2 % ID/g at 30 min post injection of radioactivity in DU145 and PC3 subcutaneous tumor bearing mice respectively., Conclusion: Herein, we evaluated a F-18-labeled PKM2-specific radiotracer, [ 18 F]DASA-23, for the molecular imaging of prostate cancer with PET. [ 18 F]DASA-23 revealed rapid and extensive uptake levels in cellular uptake studies of prostate cancer cells; however, there was only modest tumor uptake when evaluated in mouse subcutaneous tumor models.

The aim of this study was to develop a positron emission tomography (PET) tracer to visualize and monitor therapeutic response to bacterial infections. In our continued efforts to find maltose based PET tracers that can image bacterial infections, we have designed and prepared 6''-[ 18 F]fluoromaltotriose as a second generation PET imaging tracer targeting the maltodextrin transporter of bacteria. We have developed methods to synthesize 6''-deoxy-6''-[ 18 F]fluoro-α-D-glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4)-O-D-glucopyranose (6''-[ 18 F]-fluoromaltotriose) as a bacterial infection PET imaging agent. 6''-[ 18 F]fluoromaltotriose was prepared from precursor, 2'',3'',4''-tri-O-acetyl-6''-O-nosyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose (per-O-acetyl-6''-O-nosyl-maltotriose 4). This method utilizes the reaction between precursor 4 and anhydrous [ 18 F]KF/Kryptofix 2.2.2 in dimethylformamide (DMF) at 85°C for 10 minutes to yield per-O-acetyl-6''-deoxy-6-'' [ 18 F]-fluoromaltotriose (7). Successive acidic and basic hydrolysis of the acetyl protecting groups in 7 produced 6''-[ 18 F]fluoromaltotriose (8). Also, cold 6''- [ 19 F]fluoromaltotriose was prepared from per-O-acetyl-6''-hydroxymaltotriose via a diethylaminosulfur trifluoride reaction followed by a basic hydrolysis. A successful synthesis of 6''-[ 18 F]-fluoromaltotriose has been accomplished in 8 ± 1.2% radiochemical yield (decay corrected). Total synthesis time was 120 minutes. Serum stability of 6''-[ 18 F]fluoromaltotriose at 37°C indicated that 6''-[ 18 F]-fluoromaltotriose remained intact up to 2 hours. In conclusion, we have successfully synthesized 6''-[ 18 F]-fluoromaltotriose via direct fluorination of an appropriate precursor of a protected maltotriose., (Copyright © 2018 John Wiley & Sons, Ltd.)

Purpose: A hallmark of cancer is metabolic reprogramming, which is exploited by cancer cells to ensure rapid growth and survival. Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key step in tumor metabolism and growth. Recently, we reported the radiosynthesis of the first positron emission tomography tracer for visualizing PKM2 in vivo-i.e., [ 11 C]DASA-23. Due to the highly promising imaging results obtained with [ 11 C]DASA-23 in rodent model glioblastoma, we set out to generate an F-18-labeled version of this tracer, with the end goal of clinical translation in mind. Herein, we report the radiosynthesis of 1-((2-fluoro-6-[ 18 F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([ 18 F]DASA-23) and our initial investigation of its binding properties in cancer cells., Procedure: We synthesized [ 18 F]DASA-23 via fluorination of 1-((2-fluoro-6-nitrophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine (10) with K[ 18 F]F/K2.2.2 in N,N-dimethylformamide at 110 °C for 20 min. Subsequently, we evaluated uptake of [ 18 F]DASA-23 in HeLa cervical adenocarcinoma cells and in vitro stability in human and mouse serum., Results: We successfully prepared [ 18 F]DASA-23 in 2.61 ± 1.54 % radiochemical yield (n = 10, non-decay corrected at end of synthesis) with a specific activity of 2.59 ± 0.44 Ci/μmol. Preliminary cell uptake experiments revealed high uptake in HeLa cells, which was effectively blocked by pretreating cells with the structurally distinct PKM2 activator, TEPP-46. [ 18 F]DASA-23 remained intact in human and mouse serum up to 120 min., Conclusion: Herein, we have identified a F-18-labeled PKM2 specific radiotracer which shows potential for in vivo imaging. The promising cell uptake results reported herein warrant the further evaluation of [ 18 F]DASA-23 for its ability to detect and monitor cancer noninvasively.

Background: The availability of GABA A receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [ 18 F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [ 18 F]flumazenil to GABA A receptors in mice., Methods: Brain and whole blood radioactivity concentrations were measured ex vivo by scintillation counting or in vivo by PET in four groups of mice following administration of [ 18 F]flumazenil: awake mice and mice anesthetized with isoflurane, dexmedetomidine, or ketamine/dexmedetomidine. Dynamic PET recordings were obtained for 60 min in mice anesthetized by either isoflurane or ketamine/dexmedetomidine. Static PET recordings were obtained at 25 or 55 min after [ 18 F]flumazenil injection in awake or dexmedetomidine-treated mice acutely anesthetized with isoflurane. The apparent distribution volume (V T *) was calculated for the hippocampus and frontal cortex from either the full dynamic PET scans using an image-derived input function or from a series of ex vivo experiments using whole blood as the input function., Results: PET images showed persistence of high [ 18 F]flumazenil uptake (up to 20 % ID/g) in the brains of mice scanned under isoflurane or ketamine/dexmedetomidine anesthesia, whereas uptake was almost indiscernible in late samples or static scans from awake or dexmedetomidine-treated animals. The steady-state V T * was twofold higher in hippocampus of isoflurane-treated mice and dexmedetomidine-treated mice than in awake mice., Conclusions: Anesthesia has pronounced effects on the binding and blood-brain distribution of [ 18 F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABA A receptors involving the use of anesthesia.

Homomeric α7 nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation of cognitive processes such as memory and attention and have potential as therapeutic targets for the treatment of the cognitive deficits associated with schizophrenia. Though numerous α7 nAChR agonists have been developed, and several have progressed to clinical trials, these are derived from few common chemotypes. Consequently, many of these α7 nAChR clinical candidates share unfavorable side-effect profile. SEN12333 represents a novel chemotype for the development of α7 nAChR agonists, and exploration of this scaffold has produced structurally diverse ligands with promising pharmacological properties. This review will summarize structure-affinity and -activity relationships surrounding analogs of SEN12333.

Synthetic cannabinoid (SC) designer drugs based on indole and indazole scaffolds and featuring l-valinamide or l-tert-leucinamide side chains are encountered with increasing frequency by forensic researchers and law enforcement agencies and are associated with serious adverse health effects. However, many of these novel SCs are unprecedented in the scientific literature at the time of their discovery, and little is known of their pharmacology. Here, we report the synthesis and pharmacological characterization of AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, 5F-ADBICA, and several analogues. All synthesized SCs acted as high potency agonists of CB1 (EC50 = 0.24-21 nM) and CB2 (EC50 = 0.88-15 nM) receptors in a fluorometric assay of membrane potential, with 5F-ADB-PINACA showing the greatest potency at CB1 receptors. The cannabimimetic activities of AB-FUBINACA and AB-PINACA in vivo were evaluated in rats using biotelemetry. AB-FUBINACA and AB-PINACA dose-dependently induced hypothermia and bradycardia at doses of 0.3-3 mg/kg, and hypothermia was reversed by pretreatment with a CB1 (but not CB2) antagonist, indicating that these SCs are cannabimimetic in vivo, consistent with anecdotal reports of psychoactivity in humans.

Synthetic cannabinoid (SC) designer drugs featuring bioisosteric fluorine substitution are identified by forensic chemists and toxicologists with increasing frequency. Although terminal fluorination of N-pentyl indole SCs is sometimes known to improve cannabinoid type 1 (CB1) receptor binding affinity, little is known of the effects of fluorination on functional activity of SCs. This study explores the in vitro functional activities of SC designer drugs JWH-018, UR-144, PB-22, and APICA, and their respective terminally fluorinated analogues AM-2201, XLR-11, 5F-PB-22, and STS-135 at human CB1 and CB2 receptors using a FLIPR membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 2.8-1959 nM) and CB2 (EC50 = 6.5-206 nM) receptors, with the fluorinated analogues generally showing increased CB1 receptor potency (∼2-5 times). Additionally, the cannabimimetic activities and relative potencies of JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135 in vivo were evaluated in rats using biotelemetry. All SCs dose-dependently induced hypothermia and reduced heart rate at doses of 0.3-10 mg/kg. There was no consistent trend for increased potency of fluorinated SCs over the corresponding des-fluoro SCs in vivo. Based on magnitude and duration of hypothermia, the SCs were ranked for potency (PB-22 > 5F-PB-22 = JWH-018 > AM-2201 > APICA = STS-135 = XLR-11 > UR-144).

Homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs) have implications in the regulation of cognitive processes such as memory and attention, and have shown promise as a therapeutic target for the treatment of the cognitive deficits associated with schizophrenia. Multiple α7 nAChR agonists have entered human trials; however, unfavorable side effects and pharmacokinetic issues have hindered the development of a clinical α7 nAChR agonist. Currently, EVP-6124 is in phase III clinical trials, and several other α7 nAChR agonists (GTS-21 and AQW051) are in earlier stages of development. This review will summarize the recent advances and failures of α7 nAChR agonists in clinical trials for the treatment of the aforementioned pathology.

Alpha7 nicotinic acetylcholine receptors (nAChRs) have implications in the regulation of cognitive processes such as memory and attention and have been identified as a promising therapeutic target for the treatment of the cognitive deficits associated with schizophrenia and Alzheimer's disease (AD). Structure affinity relationship studies of the previously described α7 agonist SEN12333 (8), have resulted in the identification of compound 45, a potent and selective agonist of the α7 nAChR with enhanced affinity and improved physicochemical properties over the parent compound (SEN12333, 8)., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Sixteen new phenyl alkyl ether derivatives (12, 14-28) of the 5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-ylacetamide (DPA) class were synthesized and evaluated in a competition binding assay against [(3)H]PK11195 using 18 kDa translocator protein (TSPO) derived from rat kidney mitochondrial fractions. All analogues showed superior binding affinities for TSPO compared to DPA-713 (5) and DPA-714 (6). Picomolar affinities were observed for this class of TSPO ligands in this assay for the first time, with phenethyl ether 28 showing the greatest affinity (Ki = 0.13 nM). Additionally, all analogues increased pregnenolone biosynthesis (134-331% above baseline) in a rat C6 glioma cell steroidogenesis assay., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Several lines of experimental evidence support the involvement of the α7 nAChR in schizophrenia and Alzheimer's disease. Modulators of the α7 nAChR have been extensively reviewed for the treatment of the cognitive deficits associated with these pathologies. SEN12333 represents a novel α7 nAChR agonist chemotype with potential for reduced side effects but requiring further SAR exploration. The present work investigates the amide bond of SEN12333, specifically its connectivity and replacement with the tetrazole functionality, a known cis amide isostere. The results reveal the original amide bond connectivity of SEN12333 to be favorable for binding affinity and agonist activity at α7 nAChRs. The use of a tetrazole isostere completely abolishes affinity and functional activity and suggests that SEN12333 binds in a linear conformation. Results reported herein also suggest the pyridine nitrogen within the terminal aromatic ring of SEN12333 is not essential for binding affinity or functional activity. Further SAR investigations involving manipulation of other moieties contained within SEN12333 are warranted., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

GluN2B subtype-selective NMDA antagonists represent promising therapeutic targets for the symptomatic treatment of multiple CNS pathologies. A series of N-benzyl substituted benzamidines were synthesised and the benzyl ring was further replaced with various polycyclic moieties. Compounds were evaluated for activity at GluN2B containing NMDA receptors where analogues 9, 12, 16 and 18 were the most potent of the series, replacement of the benzyl ring with polycycles resulted in a complete loss of activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of ligands based on SEN12333, containing either contracted or elongated alkyl chains, were synthesized and evaluated in molecular docking studies against a homology model of the α7 nicotinic acetylcholine receptor (nAChR) subtype. The predicted binding of all ligands was highly similar, with the exception of the analog containing a 5 methylene unit spacer. However, in vitro competition binding assays revealed that the ligands possessed dissimilar binding affinities, with a K(i) range of more than an order of magnitude (K(i)=0.50 to >10 μM), and only SEN12333 itself exhibited functional activity at the α7 nAChR., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In our continued exploration of trishomocubane derivatives with central nervous system (CNS) activity, N-arylalkyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (10-13) displaying affinity for the sigma (σ) receptor were also found, in several cases, to interact with the dopamine transporter (DAT). Compound 12 was identified as the first trishomocubane-derived high affinity DAT ligand (K(i) = 1.2 nM), with greater than 8300-fold selectivity over the monoamine transporters NET and SERT, and only low to moderate affinity for σ(1) and σ(2) receptors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A series of N-(benzofuran-2-ylmethyl)-N'-benzylpiperazines bearing alkyl or fluoroalkyl aryl ethers were synthesized and evaluated at various central nervous system receptors. Examination of in vitro sigma1 {[3H]+-pentazocine} and sigma2 ([3H]DTG) receptor binding profiles of piperazines 11-13 and 25-36 revealed several highly potent and sigma1 selective ligands, notably, N-(benzofuran-2-ylmethyl)-N'-(4'-methoxybenzyl)piperazine (13, Ki=2.7 nM, sigma2/sigma1=38) and N-(benzofuran-2-ylmethyl)-N'-(4'-(2''-fluoroethoxy)benzyl)piperazine (30, Ki=2.6 nM, sigma2/sigma1=187). Structural features for optimal sigma1 receptor affinity and selectivity over the sigma2 receptor were identified. On the basis of its favorable log D value, 13 was selected as a candidate for the development of a sigma1 receptor positron emission tomography radiotracer. [11C]13 showed high uptake in the brain and other sigma receptor-rich organs of a Papio hamadryas baboon. The in vivo evaluation of [11C]13 indicates that this radiotracer is a suitable candidate for imaging the sigma1 receptor in neurodegenerative processes.