imaging-cd19-b-cells-an-experimental-autoimmune-encephalomyelitis

Imaging CD19<sup>+</sup> 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 ...

Stanford University

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, , 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, [F]FTC-146 (6-(3-[F]fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[]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  autoradiography and immunostaining, which demonstrated a strong correlation between accumulation of [F]FTC-146 and S1R staining. Finally, pain relief could also be achieved by blocking S1Rs in the neuroma with local administration of non-radioactive [F]FTC-146. In summary, [F]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.

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

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  gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABA 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  knockout mice using both H magnetic resonance imaging (H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice  and evaluate whether H-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. H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and  knockout mice. To substantiate the results of our H-MRS studies,  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  knockout mice using H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and  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.

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

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 (IC: 2.7 nM). Subsequent radiosynthesis of [C]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 [C]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, [C]5 was successfully synthesized and evaluated in healthy rats. However, the inability of [C]5 to cross the blood-brain-barrier excludes its use for imaging of CSF-1R expression in the brain.

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.

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.

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

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 [F]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.

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

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-[F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM.

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

Brain cell signaling abnormalities are detected in blood in a murine model of Fragile X syndrome and corrected by Sigma-1 receptor agonist Blarcamesine.

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.

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

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.

Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs.

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-β was determined using a FRET based in vitro assay. Radiosynthesis was performed by fully automated [C]CHI 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 (IC: 12 ± 3 nM) and no affinity for kinase family members c-KIT and PDGFR-beta. [C]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 [C]4 in brain at 60 min post injection) and no strong efflux transporter substrate behavior. Blocking CSF-1R prior to imaging with [C]4 resulted in significant decrease in brain uptake. In conclusion, [C]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.

Samantha T. Reyes

Department of Radiology

Stanford University

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

Samantha T. Reyes

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Radiology

Stanford University

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

Department of Radiology

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