Age, sex, and apolipoprotein E isoform alter contextual fear learning, neuronal activation, and baseline DNA damage in the hippocampus.

Age, female sex, and apolipoprotein E4 (E4) are risk factors to develop Alzheimer's disease (AD). There are three major human apoE isoforms: E2, E3, and E4. Compared to E3, E4 increases while E2 decreases AD risk. However, E2 is associated with increased risk and severity of post-traumatic stress disorder (PTSD). In cognitively healthy adults, E4 carriers have greater brain activation during learning and memory tasks in the absence of behavioral differences. Human apoE targeted replacement (TR) mice display differences in fear extinction that parallel human data: E2 mice show impaired extinction, mirroring heightened PTSD symptoms in E2 combat veterans. Recently, an adaptive role of DNA double strand breaks (DSBs) in immediate early gene expression (IEG) has been described. Age and disease synergistically increase DNA damage and decrease DNA repair. As the mechanisms underlying the relative risks of apoE, sex, and their interactions in aging are unclear, we used young (3 months) and middle-aged (12 months) male and female TR mice to investigate the influence of these factors on DSBs and IEGs at baseline and following contextual fear conditioning. We assessed brain-wide changes in neural activation following fear conditioning using whole-brain cFos imaging in young female TR mice. E4 mice froze more during fear conditioning and had lower cFos immunoreactivity across regions important for somatosensation and contextual encoding compared to E2 mice. E4 mice also showed altered co-activation compared to E3 mice, corresponding to human MRI and cognitive data, and indicating that there are differences in brain activity and connectivity at young ages independent of fear learning. There were increased DSB markers in middle-aged animals and alterations to cFos levels dependent on sex and isoform, as well. The increase in hippocampal DSB markers in middle-aged animals and female E4 mice may play a role in the risk for developing AD.

Development of [18F]DASA-10 for enhanced imaging of pyruvate kinase M2.

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 [18F]DASA-23. EXPERIMENTAL DESIGN: First, we synthesized [18F]DASA-10 and tested its uptake and retention compared to [18F]DASA-23 in human and mouse glioma cell lines. We then confirmed the specificity of [18F]DASA-10 by transiently modulating the expression of PKM2 in DU145 and HeLa cells. Next, we determined [18F]DASA-10 pharmacokinetics in healthy nude mice using PET imaging and subsequently assessed the ability of [18F]DASA-10 versus [18F]DASA-23 to enable in vivo detection of intracranial gliomas in syngeneic C6 rat models of glioma. RESULTS: [18F]DASA-10 demonstrated excellent cellular uptake and retention with values significantly higher than [18F]DASA-23 in all cell lines and timepoints investigated. [18F]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. [18F]DASA-10 showed favorable biodistribution and pharmacokinetic properties and a significantly improved tumor-to-brain ratio in rat C6 glioma models relative to [18F]DASA-23 (3.2 ± 0.8 versus 1.6 ± 0.3, p = 0.01). CONCLUSION: [18F]DASA-10 is a new PET radiotracer for molecular imaging of PKM2 with potential to overcome the prior limitations observed with [18F]DASA-23. [18F]DASA-10 shows promise for clinical translation to enable imaging of brain malignancies owing to its low background signal in the healthy brain.

Development and Initial Assessment of [18F]OP-801: a Novel Hydroxyl Dendrimer PET Tracer for Preclinical Imaging of Innate Immune Activation in the Whole Body and Brain.

PURPOSE: Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [18F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge. PROCEDURES: OP-801 was radiolabeled in two steps: [18F]fluorination of a tosyl precursor to yield [18F]3-fluoropropylazide, followed by a copper-catalyzed click reaction. After purification and stability testing, [18F]OP-801 (150-250 µCi) was intravenously injected into female C57BL/6 mice 24 h after intraperitoneal administration of LPS (10 mg/kg, n=14) or saline (n=6). Upon completing dynamic PET/CT imaging, mice were perfused, and radioactivity was measured in tissues of interest via gamma counting or autoradiography. RESULTS: [18F]OP-801 was produced with >95% radiochemical purity, 12-52 µCi/µg specific activity, and 4.3±1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n=4) demonstrated high stability in mice (97±3% intact tracer >120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including the liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: the liver (p=0.009), lung (p=0.030), and spleen (p=0.004). Brain PET/CT images (summed 50-60 min) revealed linearly increasing [18F]OP-801 uptake in the whole brain that significantly correlated with murine sepsis score (r=0.85, p<0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p<0.05). CONCLUSION: [18F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation.

Clinical Radiosynthesis and Translation of [18F]OP-801: A Novel Radiotracer for Imaging Reactive Microglia and Macrophages.

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 ([18F]OP-801), which is selectively taken up by reactive microglia and macrophages. Here, we describe further important characterization of [18F]OP-801 in addition to optimization and validation of a two-step clinical radiosynthesis. [18F]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 [18F]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 [18F]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.

PET imaging of TREM1 identifies CNS-infiltrating myeloid cells in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) that causes substantial morbidity and diminished quality of life. Evidence highlights the central role of myeloid lineage cells in the initiation and progression of MS. However, existing imaging strategies for detecting myeloid cells in the CNS cannot distinguish between beneficial and harmful immune responses. Thus, imaging strategies that specifically identify myeloid cells and their activation states are critical for MS disease staging and monitoring of therapeutic responses. We hypothesized that positron emission tomography (PET) imaging of triggering receptor expressed on myeloid cells 1 (TREM1) could be used to monitor deleterious innate immune responses and disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We first validated TREM1 as a specific marker of proinflammatory, CNS-infiltrating, peripheral myeloid cells in mice with EAE. We show that the 64Cu-radiolabeled TREM1 antibody-based PET tracer monitored active disease with 14- to 17-fold higher sensitivity than translocator protein 18 kDa (TSPO)-PET imaging, the established approach for detecting neuroinflammation in vivo. We illustrate the therapeutic potential of attenuating TREM1 signaling both genetically and pharmacologically in the EAE mice and show that TREM1-PET imaging detected responses to an FDA-approved MS therapy with siponimod (BAF312) in these animals. Last, we observed TREM1+ cells in clinical brain biopsy samples from two treatment-naïve patients with MS but not in healthy control brain tissue. Thus, TREM1-PET imaging has potential for aiding in the diagnosis of MS and monitoring of therapeutic responses to drug treatment.

PET Imaging of Innate Immune Activation Using 11C Radiotracers Targeting GPR84.

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11C-MGX-10S and 11C-MGX-11Svia carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro. In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11C-MGX-10S compared to 11C-MGX-11S. Subsequent use of 11C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies-11C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology.

Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer.

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 [11C]AZD1283 with radioactive precursor [11C]NH4CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [11C]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 [11C]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 [11C]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 [11C]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 [11C]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.

Amifostine (WR-2721) Mitigates Cognitive Injury Induced by Heavy Ion Radiation in Male Mice and Alters Behavior and Brain Connectivity.

The deep space environment contains many risks to astronauts during space missions, such as galactic cosmic rays (GCRs) comprised of naturally occurring heavy ions. Heavy ion radiation is increasingly being used in cancer therapy, including novel regimens involving carbon therapy. Previous investigations involving simulated space radiation have indicated a host of detrimental cognitive and behavioral effects. Therefore, there is an increasing need to counteract these deleterious effects of heavy ion radiation. Here, we assessed the ability of amifostine to mitigate cognitive injury induced by simulated GCRs in C57Bl/6J male and female mice. Six-month-old mice received an intraperitoneal injection of saline, 107 mg/kg, or 214 mg/kg of amifostine 1 h prior to exposure to a simplified five-ion radiation (protons, 28Si, 4He, 16O, and 56Fe) at 500 mGy or sham radiation. Mice were behaviorally tested 2-3 months later. Male mice that received saline and radiation exposure failed to show novel object recognition, which was reversed by both doses of amifostine. Conversely, female mice that received saline and radiation exposure displayed intact object recognition, but those that received amifostine prior to radiation did not. Amifostine and radiation also had distinct effects on males and females in the open field, with amifostine affecting distance moved over time in both sexes, and radiation affecting time spent in the center in females only. Whole-brain analysis of cFos immunoreactivity in male mice indicated that amifostine and radiation altered regional connectivity in areas involved in novel object recognition. These data support that amifostine has potential as a countermeasure against cognitive injury following proton and heavy ion irradiation in males.