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.

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.

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.

Discovery of a CSF-1R inhibitor and PET tracer for imaging of microglia and macrophages in the brain.

Colony stimulating factor 1 receptor (CSF-1R) is a kinase expressed on macrophages and microglia in the brain. It has been recognized as a potential drug and imaging target in treatment of neuroinflammatory diseases and glioblastoma. Despite several attempts, no validated CSF-1R PET tracer is currently available. The aim of this work was to develop a brain permeable CSF-1R PET tracer for non-invasive imaging of CSF-1R in vivo. Based on fragments of two potent and selective CSF-1R inhibitors, novel hybrid molecules were designed and synthesized. Affinity for human recombinant CSF-1R and selectivity over c-KIT and PDGFR-ß was determined using a FRET based in vitro assay. Radiosynthesis was performed by fully automated [11C]CH3I methylation of the corresponding des-methyl precursor. PET imaging was performed at baseline, efflux transporter blocking and CSF-1R blocking conditions. Moreover, tracer distribution and blood and plasma radiometabolites were determined following injection in healthy mice. The most promising CSF-1R inhibitor, compound 4, showed high selectivity and high affinity for CSF-1R (IC50: 12 ± 3 nM) and no affinity for kinase family members c-KIT and PDGFR-beta. [11C]4 was obtained in good yield (15 ± 0.2 % decay corrected yield, (2.0 ± 0.26 GBq at end of synthesis) and excellent purity. The compound demonstrated high brain penetration and good metabolic stability (>2 %ID/g at 60 min post injection and 79 ± 8 % intact [11C]4 in brain at 60 min post injection) and no strong efflux transporter substrate behavior. Blocking CSF-1R prior to imaging with [11C]4 resulted in significant decrease in brain uptake. In conclusion, [11C]4 shows good potential as a novel PET tracer for imaging of CSF-1R in the CNS and future experiments in relevant animal models are warranted.

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.

A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2-Induced Glycolytic Reprogramming in Glioblastoma.

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-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM. EXPERIMENTAL DESIGN: [18F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/µmol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma. RESULTS: In mouse imaging studies, [18F]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, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [18F]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 [18F]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 [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

Effects of the sigma-1 receptor agonist blarcamesine in a murine model of fragile X syndrome: neurobehavioral phenotypes and receptor occupancy.

Fragile X syndrome (FXS), a disorder of synaptic development and function, is the most prevalent genetic form of intellectual disability and autism spectrum disorder. FXS mouse models display clinically-relevant phenotypes, such as increased anxiety and hyperactivity. Despite their availability, so far advances in drug development have not yielded new treatments. Therefore, testing novel drugs that can ameliorate FXS' cognitive and behavioral impairments is imperative. ANAVEX2-73 (blarcamesine) is a sigma-1 receptor (S1R) agonist with a strong safety record and preliminary efficacy evidence in patients with Alzheimer's disease and Rett syndrome, other synaptic neurodegenerative and neurodevelopmental disorders. S1R's role in calcium homeostasis and mitochondrial function, cellular functions related to synaptic function, makes blarcamesine a potential drug candidate for FXS. Administration of blarcamesine in 2-month-old FXS and wild type mice for 2 weeks led to normalization in two key neurobehavioral phenotypes: open field test (hyperactivity) and contextual fear conditioning (associative learning). Furthermore, there was improvement in marble-burying (anxiety, perseverative behavior). It also restored levels of BDNF, a converging point of many synaptic regulators, in the hippocampus. Positron emission tomography (PET) and ex vivo autoradiographic studies, using the highly selective S1R PET ligand [18F]FTC-146, demonstrated the drug's dose-dependent receptor occupancy. Subsequent analyses also showed a wide but variable brain regional distribution of S1Rs, which was preserved in FXS mice. Altogether, these neurobehavioral, biochemical, and imaging data demonstrates doses that yield measurable receptor occupancy are effective for improving the synaptic and behavioral phenotype in FXS mice. The present findings support the viability of S1R as a therapeutic target in FXS, and the clinical potential of blarcamesine in FXS and other neurodevelopmental disorders.

BLZ945 derivatives for PET imaging of colony stimulating factor-1 receptors in the brain.

BACKGROUND: The kinase colony stimulating factor-1 receptor (CSF-1R) has recently been identified as a novel therapeutic target for decreasing tumor associated macrophages and microglia load in cancer treatment. In glioblastoma multiforme (GBM), a high-grade cancer in the brain with extremely poor prognosis, macrophages and microglia can make up to 50% of the total tumor mass. Currently, no non-invasive methods are available for measuring CSF-1R expression in vivo. The aim of this work is to develop a PET tracer for imaging of CSF-1R receptor expression in the brain for future GBM patient selection and treatment monitoring. METHODS: BLZ945 and a derivative that potentially allows for fluorine-18 labeling were synthesized and evaluated in vitro to determine their affinity towards CSF-1R. BLZ945 was radiolabeled with carbon-11 by N-methylation of des-methyl-BLZ945 using [11C]CH3I. Following administration to healthy mice, metabolic stability of [11C]BLZ945 in blood and brain and activity distribution were determined ex vivo. PET scanning was performed at baseline, efflux transporter blocking, and CSF-1R blocking conditions. Finally, [11C]BLZ945 binding was evaluated in vitro by autoradiography on mouse brain sections. RESULTS: BLZ945 was the most potent compound in our series with an IC50 value of 6.9 ± 1.4 nM. BLZ945 was radiolabeled with carbon-11 in 20.7 ± 1.1% decay corrected radiochemical yield in a 60 min synthesis procedure with a radiochemical purity of >95% and a molar activity of 153 ± 34 GBq·µmol-1. Ex vivo biodistribution showed moderate brain uptake and slow wash-out, in addition to slow blood clearance. The stability of BLZ945 in blood plasma and brain was >99% at 60 min post injection. PET scanning demonstrated BLZ945 to be a substrate for efflux transporters. High brain uptake was observed, which was shown to be mostly non-specific. In accordance, in vitro autoradiography on brain sections revealed high non-specific binding. CONCLUSIONS: [11C]BLZ945, a CSF-1R PET tracer, was synthesized in high yield and purity. The tracer has high potency for the target, however, future studies are warranted to address non-specific binding and tracer efflux before BLZ945 or derivatives could be translated into humans for brain imaging.

Evaluation of carbon-11 labeled 5-(1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)nicotinamide as PET tracer for imaging of CSF-1R expression in the brain.

Pharmacological targeting of tumor associated macrophages and microglia in the tumor microenvironment is a novel therapeutic strategy in the treatment of glioblastoma multiforme. As such, the colony stimulating factor-1 receptor (CSF-1R) has been identified as a druggable target. However, no validated companion diagnostic marker for these therapies exists to date. Towards development of a CSF-1R PET tracer, a set of six compounds based on recently reported CSF-1R inhibitor 5-(1-methyl-1H-pyrazol-4-yl)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)nicotinamide (Compound 5) was designed, synthesized and evaluated in vitro for potency and selectivity. The highest affinity for CSF-1R was found for compound 5 (IC50: 2.7 nM). Subsequent radiosynthesis of [11C]5 was achieved in 2.0 ± 0.2% yield (decay corrected to start of synthesis) by carbon-11 carbon monoxide aminocarbonylation in 40 min after end of bombardment. In vitro autoradiography with [11C]5 on rat brain sections demonstrated high specific binding, but also strong off-target binding. Ex vivo, only intact tracer was observed in blood plasma at 90 min post injection in healthy rats. PET scanning results demonstrated negligible brain uptake under baseline conditions and this brain uptake did not increase by blocking of efflux transporters using Tariquidar. To conclude, [11C]5 was successfully synthesized and evaluated in healthy rats. However, the inability of [11C]5 to cross the blood-brain-barrier excludes its use for imaging of CSF-1R expression in the brain.

GABA Measurement in a Neonatal Fragile X Syndrome Mouse Model Using 1H-Magnetic Resonance Spectroscopy and Mass Spectrometry.

Fragile X syndrome (FXS) is the leading monogenetic cause of autism spectrum disorder and inherited cause of intellectual disability that affects approximately one in 7,000 males and one in 11,000 females. In FXS, the Fmr1 gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABAA subunits. Therefore, FMRP loss adversely impacts the neuronal firing of the GABAergic system which creates an imbalance in the excitatory/inhibitory ratio within the brain. Current FXS treatment strategies focus on curing symptoms, such as anxiety or decreased social function. While treating symptoms can be helpful, incorporating non-invasive imaging to evaluate how treatments change the brain's biology may explain what molecular aberrations are associated with disease pathology. Thus, the GABAergic system is suitable to explore developing novel therapeutic strategies for FXS. To understand how the GABAergic system may be affected by this loss-of-function mutation, GABA concentrations were examined within the frontal cortex and thalamus of 5-day-old wild type and Fmr1 knockout mice using both 1H magnetic resonance imaging (1H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice in vivo and evaluate whether 1H-MRS is suitable to capture regional GABA concentration differences at the front end of the critical cortical period where abnormal neurodevelopment occurs due to FMRP loss is first detected. 1H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and Fmr1 knockout mice. To substantiate the results of our 1H-MRS studies, in vitro LC-MS/MS was also performed on brain homogenates from age-matched mice. We found significant changes in GABA concentration between the frontal cortex and thalamus within each mouse from both wild type and Fmr1 knockout mice using 1H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and Fmr1 knockout mice by LC-MS/MS, validating that FMRP loss directly affects the GABAergic system. Thus, these new findings support the need to develop an effective non-invasive imaging method to monitor novel GABAergic strategies aimed at treating patients with FXS.

Visualizing Nerve Injury in a Neuropathic Pain Model with [18F]FTC-146 PET/MRI.

The ability to locate nerve injury and ensuing neuroinflammation would have tremendous clinical value for improving both the diagnosis and subsequent management of patients suffering from pain, weakness, and other neurologic phenomena associated with peripheral nerve injury. Although several non-invasive techniques exist for assessing the clinical manifestations and morphological aspects of nerve injury, they often fail to provide accurate diagnoses due to limited specificity and/or sensitivity. Herein, we describe a new imaging strategy for visualizing a molecular biomarker of nerve injury/neuroinflammation, i.e., the sigma-1 receptor (S1R), in a rat model of nerve injury and neuropathic pain. The two-fold higher increase of S1Rs was shown in the injured compared to the uninjured nerve by Western blotting analyses. With our novel S1R-selective radioligand, [18F]FTC-146 (6-(3-[18F]fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one), and positron emission tomography-magnetic resonance imaging (PET/MRI), we could accurately locate the site of nerve injury created in the rat model. We verified the accuracy of this technique by ex vivo autoradiography and immunostaining, which demonstrated a strong correlation between accumulation of [18F]FTC-146 and S1R staining. Finally, pain relief could also be achieved by blocking S1Rs in the neuroma with local administration of non-radioactive [19F]FTC-146. In summary, [18F]FTC-146 S1R PET/MR imaging has the potential to impact how we diagnose, manage and treat patients with nerve injury, and thus warrants further investigation.