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.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects. This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

89Zr-panitumumab Combined With 18F-FDG PET Improves Detection and Staging of Head and Neck Squamous Cell Carcinoma.

PURPOSE: Determine the safety and specificity of a tumor-targeted radiotracer (89Zr-pan) in combination with 18F-FDG PET/CT to improve diagnostic accuracy in head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Adult patients with biopsy-proven HNSCC scheduled for standard-of-care surgery were enrolled in a clinical trial and underwent systemic administration of 89Zirconium-panitumumab and panitumumab-IRDye800 followed by preoperative 89Zr-pan PET/CT and intraoperative fluorescence imaging. The sensitivity, specificity, and AUC were evaluated. RESULTS: A total of fourteen patients were enrolled and completed the study. Four patients (28.5%) had areas of high 18F-FDG uptake outside the head and neck region with maximum standardized uptake values (SUVmax) greater than 2.0 that were not detected on 89Zr-pan PET/CT. These four patients with incidental findings underwent further workup and had no evidence of cancer on biopsy or clinical follow-up. Forty-eight lesions (primary tumor, LNs, incidental findings) with SUVmax ranging 2.0-23.6 were visualized on 18F-FDG PET/CT; 34 lesions on 89Zr-pan PET/CT with SUVmax ranging 0.9-10.5. The combined ability of 18F-FDG PET/CT and 89Zr-pan PET/CT to detect HNSCC in the whole body was improved with higher specificity of 96.3% [confidence interval (CI), 89.2%-100%] compared to 18F-FDG PET/CT alone with specificity of 74.1% (CI, 74.1%-90.6%). One possibly related grade 1 adverse event of prolonged QTc (460 ms) was reported but resolved in follow-up. CONCLUSIONS: 89Zr-pan PET/CT imaging is safe and may be valuable in discriminating incidental findings identified on 18F-FDG PET/CT from true positive lesions and in localizing metastatic LNs.

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.

Tracking Innate Immune Activation in a Mouse Model of Parkinson's Disease Using TREM1 and TSPO PET Tracers.

Parkinson's disease (PD) is associated with aberrant innate immune responses, including microglial activation and infiltration of peripheral myeloid cells into the central nervous system (CNS). Methods to investigate innate immune activation in PD are limited and have not yet elucidated key interactions between neuroinflammation and peripheral inflammation. Translocator protein 18 kDa (TSPO) PET is a widely evaluated imaging approach for studying activated microglia and peripheral myeloid lineage cells in vivo but has yet to be fully explored in PD. Here, we investigate the utility of TSPO PET in addition to PET imaging of triggering receptor expressed on myeloid cells 1 (TREM1)-a novel biomarker of proinflammatory innate immune cells-for detecting innate immune responses in the 6-hydroxydopamine mouse model of dopaminergic neuron degeneration. Methods: C57/BL6J and TREM1 knockout mice were stereotactically injected with 6-hydroxydopamine in the left striatum; control mice were injected with saline. At day 7 or 14 after surgery, mice were administered 18F-GE-180, 64Cu-TREM1 monoclonal antibody (mAb), or 64Cu-isotype control mAb and imaged by PET/CT. Ex vivo autoradiography was performed to obtain high-resolution images of tracer binding within the brain. Immunohistochemistry was conducted to verify myeloid cell activation and dopaminergic cell death, and quantitative polymerase chain reaction and flow cytometry were completed to assess levels of target in the brain. Results: PET/CT images of both tracers showed elevated signal within the striatum of 6-hydroxydopamine-injected mice compared with those injected with saline. Autoradiography afforded higher-resolution brain images and revealed significant TSPO and TREM1 tracer binding within the ipsilateral striatum of 6-hydroxydopamine mice compared with saline mice at both 7 and 14 d after toxin. Interestingly, 18F-GE-180 enabled detection of inflammation in the brain and peripheral tissues (blood and spleen) of 6-hydroxydopamine mice, whereas 64Cu-TREM1 mAb appeared to be more sensitive and specific for detecting neuroinflammation, in particular infiltrating myeloid cells, in these mice, as demonstrated by flow cytometry findings and higher tracer binding signal-to-background ratios in brain. Conclusion: TSPO and TREM1 PET tracers are promising tools for investigating different cell types involved in innate immune activation in the context of dopaminergic neurodegeneration, thus warranting further investigation in other PD rodent models and human postmortem tissue to assess their clinical potential.