Synthesis and in vivo evaluation of [18F]UCB-J for PET imaging of synaptic vesicle glycoprotein 2A (SV2A).

PURPOSE: Synaptic abnormalities have been implicated in a variety of neuropsychiatric disorders, including epilepsy, Alzheimer's disease, and schizophrenia. Hence, PET imaging of the synaptic vesicle glycoprotein 2A (SV2A) may be a valuable in vivo biomarker for neurologic and psychiatric diseases. We previously developed [11C]UCB-J, a PET radiotracer with high affinity and selectivity toward SV2A; however, the short radioactive half-life (20 min for 11C) places some limitations on its broader application. Herein, we report the first synthesis of the longer-lived 18F-labeled counterpart (half-life: 110 min), [18F]UCB-J, and its evaluation in nonhuman primates. METHODS: [18F]UCB-J was synthesized from the iodonium precursors. PET imaging experiments with [18F]UCB-J were conducted in rhesus monkeys to assess the pharmacokinetic and in vivo binding properties. Arterial samples were taken for analysis of radioactive metabolites and generation of input functions. Regional time-activity curves were analyzed using the one-tissue compartment model to derive regional distribution volumes and binding potentials for comparison with [11C]UCB-J. RESULTS: [18F]UCB-J was prepared in high radiochemical and enantiomeric purity, but low radiochemical yield. Evaluation in nonhuman primates indicated that the radiotracer displayed pharmacokinetic and imaging characteristics similar to those of [11C]UCB-J, with moderate metabolism rate, high brain uptake, fast and reversible binding kinetics, and high specific binding signals. CONCLUSION: We have accomplished the first synthesis of the novel SV2A radiotracer [18F]UCB-J. [18F]UCB-J is demonstrated to be an excellent imaging agent and may prove to be useful for imaging and quantification of SV2A expression, and synaptic density, in humans.

Novel Kappa Opioid Receptor Agonist as Improved PET Radiotracer: Development and in Vivo Evaluation.

The kappa opioid receptor (KOR) is involved in depression, alcoholism, and drug abuse. The current agonist radiotracer 11C-GR103545 is not ideal for imaging KOR due to its slow tissue kinetics in human. The aim of our project was to develop novel KOR agonist radiotracers with improved imaging properties. A novel compound FEKAP ((( R))-4-(2-(3,4-dichlorophenyl)acetyl)-3-((ethyl(2-fluoroethyl)amino)methyl) piperazine-1-carboxylate) was designed, synthesized, and assayed for in vitro binding affinities. It was then radiolabeled and evaluated in rhesus monkeys. Baseline and blocking scans were conducted on a Focus-220 scanner to assess binding specificity and selectivity. Metabolite-corrected arterial activities over time were measured and used as input functions to analyze the brain regional time-activity curves and derive kinetic and binding parameters with kinetic modeling. FEKAP displayed high KOR binding affinity ( Ki = 0.43 nM) and selectivity (17-fold over mu opioid receptor and 323-fold over delta opioid receptor) in vitro. 11C-FEKAP was prepared in high molar activity (mean of 718 GBq/µmol, n = 19) and >99% radiochemical purity. In monkeys, 11C-FEKAP metabolized fairly fast, with ∼31% of intact parent fraction at 30 min post-injection. In the brain, it exhibited fast and reversible kinetics with good uptake. Pretreatment with the nonselective opioid receptor antagonist naloxone (1 mg/kg) decreased uptake in high binding regions to the level in the cerebellum, and the selective KOR antagonist LY2456302 (0.02 and 0.1 mg/kg) reduced 11C-FEKAP specific binding in a dose-dependent manner. As a measure of specific binding signals, the mean binding potential ( BPND) values of 11C-FEKAP derived from the multilinear analysis-1 (MA1) method were greater than 0.5 for all regions, except for the thalamus. The novel KOR agonist tracer 11C-FEKAP demonstrated binding specificity and selectivity in vivo and exhibited attractive properties of fast tissue kinetics and high specific binding.

The Search for a Subtype-Selective PET Imaging Agent for the GABAA Receptor Complex: Evaluation of the Radiotracer [11C]ADO in Nonhuman Primates.

The myriad physiological functions of γ-amino butyric acid (GABA) are mediated by the GABA-benzodiazepine receptor complex comprising of the GABAA, GABAB, and GABAC groups. The various GABAA subunits with region-specific distributions in the brain subserve different functional and physiological roles. For example, the sedative and anticonvulsive effects of classical benzodiazepines are attributed to the α1 subunit, and the α2 and α3 subunits mediate the anxiolytic effect. To optimize pharmacotherapies with improved efficacy and devoid of undesirable side effects for the treatment of anxiety disorders, subtype-selective imaging radiotracers are required to assess target engagement at GABA sites and determine the dose-receptor occupancy relationships. The goal of this work was to characterize, in nonhuman primates, the in vivo binding profile of a novel positron emission tomography (PET) radiotracer, [11C]ADO, which has been indicated to have functional selectivity for the GABAA α2/α3 subunits. High specific activity [11C]ADO was administrated to 3 rhesus monkeys, and PET scans of 120-minute duration were performed on the Focus-220 scanner. In the blood, [11C]ADO metabolized at a fairly rapid rate, with ∼36% of the parent tracer remaining at 30 minutes postinjection. Uptake levels of [11C]ADO in the brain were high (peak standardized uptake value of ∼3.0) and consistent with GABAA distribution, with highest activity levels in cortical areas, intermediate levels in cerebellum and thalamus, and lowest uptake in striatal regions and amygdala. Tissue kinetics was fast, with peak uptake in all brain regions within 20 minutes of tracer injection. The one-tissue compartment model provided good fits to regional time-activity curves and reliable measurement of kinetic parameters. The absolute test-retest variability of regional distribution volumes ( VT) was low, ranging from 4.5% to 8.7%. Pretreatment with flumazenil (a subtype nonselective ligand, 0.2 mg/kg, intravenous [IV], n = 1), Ro15-4513 (an α5-selective ligand, 0.03 mg/kg, IV, n = 2), and zolpidem (an α1-selective ligand, 1.7 mg/kg, IV, n = 1) led to blockade of [11C]ADO binding by 96.5%, 52.5%, and 76.5%, respectively, indicating the in vivo binding specificity of the radiotracer. Using the nondisplaceable volume of distribution ( VND) determined from the blocking studies, specific binding signals, as measured by values of regional binding potential ( BPND), ranged from 0.6 to 4.4, which are comparable to those of [11C]flumazenil. In conclusion, [11C]ADO was demonstrated to be a specific radiotracer for the GABAA receptors with several favorable properties: high brain uptake, fast tissue kinetics, and high levels of specific binding in nonhuman primates. However, subtype selectivity in vivo is not obvious for the radiotracer, and thus, the search for subtype-selective GABAA radiotracers continues.

Synthesis of a 2-aryl-3-aroyl indole salt (OXi8007) resembling combretastatin A-4 with application as a vascular disrupting agent.

The natural products colchicine and combretastatin A-4 are potent inhibitors of tubulin assembly, and they have inspired the design and synthesis of a large number of small-molecule, potential anticancer agents. The indole-based molecular scaffold is prominent among these SAR modifications, leading to a rapidly increasing number of agents. The water-soluble phosphate prodrug 33 (OXi8007) of 2-aryl-3-aroylindole-based phenol 8 (OXi8006) was prepared by chemical synthesis and found to be strongly cytotoxic against selected human cancer cell lines (GI50 = 36 nM against DU-145 cells, for example). The free phenol, 8 (OXi8006), was a strong inhibitor (IC50 = 1.1 µM) of tubulin assembly. The corresponding phosphate prodrug 33 (OXi8007) also demonstrated pronounced interference with tumor vasculature in a preliminary in vivo study utilizing a SCID mouse model bearing an orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent that may prove useful for the treatment of cancer.

Inhibition of NETosis by a Nuclear-Penetrating Anti-DNA Autoantibody.

Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase-dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Positron Emission Tomography Imaging Evaluation of a Novel 18F-Labeled Sigma-1 Receptor Radioligand in Cynomolgus Monkeys.

We report a convenient radiosynthesis and the first positron emission tomography (PET) imaging evaluation of [18F]FBFP as a potent sigma-1 (σ1) receptor radioligand with advantageous characteristics. [18F]FBFP was synthesized in one step from an iodonium ylide precursor. In cynomolgus monkeys, [18F]FBFP displayed high brain uptake and suitable tissue kinetics for quantitative analysis. It exhibited heterogeneous distribution with higher regional volume of distribution (VT) values in the amygdala, hippocampus, insula, and frontal cortex. Pretreatment with the σ1 receptor agonist SA4503 (0.5 mg/kg) significantly reduced radioligand uptake in the monkey brain (>95%), indicating high binding specificity of [18F]FBFP in vivo. Compared with (S)-[18F]fluspidine, [18F]FBFP possessed higher regional nondisplaceable binding potential (BPND) values across the brain regions. These findings demonstrate that [18F]FBFP is a highly promising PET radioligand for imaging and quantification of σ1 receptors in humans.

PET Imaging of Pancreatic Dopamine D2 and D3 Receptor Density with 11C-(+)-PHNO in Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) has traditionally been characterized by a complete destruction of ß-cell mass (BCM); however, there is growing evidence of possible residual BCM present in T1DM. Given the absence of in vivo tools to measure BCM, routine clinical measures of ß-cell function (e.g., C-peptide release) may not reflect BCM. We previously demonstrated the potential utility of PET imaging with the dopamine D2 and D3 receptor agonist 3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol (11C-(+)-PHNO) to differentiate between healthy control (HC) and T1DM individuals. Methods: Sixteen individuals participated (10 men, 6 women; 9 HCs, 7 T1DMs). The average duration of diabetes was 18 ± 6 y (range, 14-30 y). Individuals underwent PET/CT scanning with a 120-min dynamic PET scan centered on the pancreas. One- and 2-tissue-compartment models were used to estimate pancreas and spleen distribution volume. Reference region approaches (spleen as reference) were also investigated. Quantitative PET measures were correlated with clinical outcome measures. Immunohistochemistry was performed to examine colocalization of dopamine receptors with endocrine hormones in HC and T1DM pancreatic tissue. Results: C-peptide release was not detectable in any T1DM individuals, whereas proinsulin was detectable in 3 of 5 T1DM individuals. Pancreas SUV ratio minus 1 (SUVR-1) (20-30 min; spleen as reference region) demonstrated a statistically significant reduction (-36.2%) in radioligand binding (HCs, 5.6; T1DMs, 3.6; P = 0.03). Age at diagnosis correlated significantly with pancreas SUVR-1 (20-30 min) (R2 = 0.67, P = 0.025). Duration of diabetes did not significantly correlate with pancreas SUVR-1 (20-30 min) (R2 = 0.36, P = 0.16). Mean acute C-peptide response to arginine at maximal glycemic potentiation did not significantly correlate with SUVR-1 (20-30 min) (R2 = 0.57, P = 0.05), nor did mean baseline proinsulin (R2 = 0.45, P = 0.10). Immunohistochemistry demonstrated colocalization of dopamine D3 receptor and dopamine D2 receptor in HCs. No colocalization of the dopamine D3 receptor or dopamine D2 receptor was seen with somatostatin, glucagon, or polypeptide Y. In a separate T1DM individual, no immunostaining was seen with dopamine D3 receptor, dopamine D2 receptor, or insulin antibodies, suggesting that loss of endocrine dopamine D3 receptor and dopamine D2 receptor expression accompanies loss of ß-cell functional insulin secretory capacity. Conclusion: Thirty-minute scan durations and SUVR-1 provide quantitative outcome measures for 11C-(+)-PHNO, a dopamine D3 receptor-preferring agonist PET radioligand, to differentiate BCM in T1DM and HCs.

Synthesis and Preclinical Evaluation of an 18F-Labeled Synaptic Vesicle Glycoprotein 2A PET Imaging Probe: [18F]SynVesT-2.

Synaptic vesicle glycoprotein 2A (SV2A) is a 12-pass transmembrane glycoprotein ubiquitously expressed in presynaptic vesicles. In vivo imaging of SV2A using PET has potential applications in the diagnosis and prognosis of a variety of neuropsychiatric diseases, e.g., Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, autism, epilepsy, stroke, traumatic brain injury, post-traumatic stress disorder, depression, etc. Herein, we report the synthesis and evaluation of a new 18F-labeled SV2A PET imaging probe, [18F]SynVesT-2, which possesses fast in vivo binding kinetics and high specific binding signals in non-human primate brain.

Development of synthetic methodology suitable for the radiosynthesis of combretastatin A-1 (CA1) and its corresponding prodrug CA1P.

Synthetic methodology has been established suitable for the preparation of combretastatin A-1 (CA1) and its corresponding phosphate prodrug salt (CA1P) in high specific activity radiolabeled form. Judicious selection of appropriate phenolic protecting groups to distinguish positions on the A-ring from the B-ring of the stilbenoid was paramount for the success of this project. Methylation of the C-4' phenolic moiety by removal of the tert-butyldimethylsilyl protecting group in the presence of methyl iodide was accomplished in excellent yield without significant Z to E isomerization. This step (carried out with (12)C-methyl iodide as proof of concept in this study) represents the process in which a (14)C radioisotope could be incorporated in an actual radiosynthesis. CA1 is a natural product isolated from the African bush willow tree (Combretum caffrum) that has important medicinal value due, in part, to its ability to inhibit tubulin assembly. As a prodrug, CA1P (OXi4503) is in human clinical trials as a vascular disrupting agent.

Evaluation of 11C-LSN3172176 as a Novel PET Tracer for Imaging M1 Muscarinic Acetylcholine Receptors in Nonhuman Primates.

The M1 muscarinic acetylcholine receptor (mAChR) plays an important role in learning and memory, and therefore is a target for development of drugs for treatment of cognitive impairments in Alzheimer disease and schizophrenia. The availability of M1-selective radiotracers for PET will help in developing therapeutic agents by providing an imaging tool for assessment of drug dose-receptor occupancy relationship. Here we report the synthesis and evaluation of 11C-LSN3172176 (ethyl 4-(6-(methyl-11C)-2-oxoindolin-1-yl)-[1,4'-bipiperidine]-1'-carboxylate) in nonhuman primates. Methods:11C-LSN3172176 was radiolabeled via the Suzuki-Miyaura cross-coupling method. PET scans in rhesus macaques were acquired for 2 h with arterial blood sampling and metabolite analysis to measure the input function. Blocking scans with scopolamine (50 µg/kg) and the M1-selective agent AZD6088 (0.67 and 2 mg/kg) were obtained to assess tracer binding specificity and selectivity. Regional brain time-activity curves were analyzed with the 1-tissue-compartment model and the multilinear analysis method (MA1) to calculate regional distribution volume. Nondisplaceable binding potential values were calculated using the cerebellum as a reference region. Results:11C-LSN3172176 was synthesized with greater than 99% radiochemical purity and high molar activity. In rhesus monkeys, 11C-LSN3172176 metabolized rapidly (29% ± 6% parent remaining at 15 min) and displayed fast kinetics and extremely high uptake in the brain. Imaging data were modeled well with the 1-tissue-compartment model and MA1 methods. MA1-derived distribution volume values were high (range, 10-81 mL/cm3) in all known M1 mAChR-rich brain regions. Pretreatment with scopolamine and AZD6088 significantly reduced the brain uptake of 11C-LSN3172176, thus demonstrating its binding specificity and selectivity in vivo. The cerebellum appeared to be a suitable reference region for derivation of nondisplaceable binding potential, which ranged from 2.42 in the globus pallidus to 8.48 in the nucleus accumbens. Conclusion:11C-LSN3172176 exhibits excellent in vivo binding and imaging characteristics in nonhuman primates and appears to be the first appropriate radiotracer for PET imaging of human M1 AChR.

Imaging the Enzyme 11ß-Hydroxysteroid Dehydrogenase Type 1 with PET: Evaluation of the Novel Radiotracer 11C-AS2471907 in Human Brain.

The 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) enzyme converts cortisone to cortisol and participates in the regulation of glucocorticoid levels in tissues. 11ß-HSD1 is expressed in the liver, kidney, adipose tissue, placenta, and brain. 11ß-HSD1 is a target for treatment of depression, anxiety, posttraumatic stress disorder, and also against age-related cognitive function and memory loss. In this study, we evaluated the radiotracer 11C-AS2471907 (3-(2-chlorophenyl)-4-(methyl-11C)-5-[2-[2,4,6-trifluorophenoxy]propan-2-yl]-4H-1,2,4-triazole) to image 11ß-HSD1 availability in the human brain with PET. Methods: Fifteen subjects were included in the study. All subjects underwent one 2-h scan after a bolus administration of 11C-AS2471907. Two subjects underwent an additional scan after blockade with the selective and high-affinity 11ß-HSD1 inhibitor ASP3662 to evaluate 11C-AS2471907 nondisplaceable distribution volume. Five subjects also underwent an additional scan to evaluate the within-day test-retest variability of 11C-AS2471907 volumes of distribution (VT). Results:11C-AS2471907 time-activity curves were best fitted by the 2-tissue-compartment (2TC) model. 11C-AS2471907 exhibited a regionally varying pattern of uptake throughout the brain. The VT of 11C-AS2471907 ranged from 3.7 ± 1.5 mL/cm3 in the caudate nucleus to 14.5 ± 5.3 mL/cm3 in the occipital cortex, with intermediate values in the amygdala, white matter, cingulum, insula, frontal cortex, putamen, temporal and parietal cortices, cerebellum, and thalamus (from lowest to highest VT). From the blocking scans, nondisplaceable distribution volume was determined to be 0.16 ± 0.04 mL/cm3 for 11C-AS2471907. Thus, nearly all uptake was specific and the binding potential ranged from 22 in the caudate to 90 in the occipital cortex. Test-retest variability of 2TC VT values was less than 10% in most large cortical regions (14% in parietal cortex) and ranged from 14% (cerebellum) to 51% (amygdala) in other regions. The intraclass correlation coefficient of 2TC VT values ranged from 0.55 in the white matter to 0.98 in the cerebellum. Conclusion:11C-AS2471907 has a high fraction of specific binding in vivo in humans and reasonable within-day reproducibility of binding parameters.

Fluorine-18-Labeled Antagonist for PET Imaging of Kappa Opioid Receptors.

Kappa opioid receptor (KOR) antagonists are potential drug candidates for diseases such as treatment-refractory depression, anxiety, and addictive disorders. PET imaging radiotracers for KOR can be used in occupancy study to facilitate drug development, and to investigate the roles of KOR in health and diseases. We have previously developed two 11C-labeled antagonist radiotracers with high affinity and selectivity toward KOR. What is limiting their wide applications is the short half-life of 11C. Herein, we report the synthesis of a first 18F-labeled KOR antagonist radiotracer and the initial PET imaging study in a nonhuman primate.

2-(3-tert-Butyldimethylsiloxy-4-methoxyphenyl)-6-methoxy-3-(3,4,5-trimethoxybenzoyl)indole.

In the crystal structure of the title compound, C(32)H(39)NO(7)Si, all geometric parameters fall within experimental error of expected values. The analysis of molecular-packing plots reveals an infinite two-dimensional linear array running parallel to the b axis, formed by one N[bond]H...O intermolecular hydrogen-bonding interaction. Several potential C[bond]H...O interactions are also present.