Preparation and Evaluation of 64Cu-Radiolabled Dual-Ligand Multifunctional Gold Nanoparticles for Tumor Theragnosis.

Gold nanoparticles (AuNPs) are cutting-edge platforms for combined diagnostic and therapeutic approaches due to their exquisite physicochemical and optical properties. Using the AuNPs physically produced by femtosecond pulsed laser ablation of bulk Au in deionized water, with a capping agent-free surface, the conjugation of functional ligands onto the AuNPs can be tunable between 0% and 100% coverage. By taking advantage of this property, AuNPs functionalized by two different types of active targeting ligands with predetermined ratios were fabricated. The quantitatively controllable conjugation to construct a mixed monolayer of multiple biological molecules at a certain ratio onto the surface of AuNPs was achieved and a chelator-free 64Cu-labeling method was developed. We report here the manufacture, radiosynthesis and bioevaluation of three different types of dual-ligand AuNPs functionalized with two distinct ligands selected from glucose, arginine-glycine-aspartate (RGD) peptide, and methotrexate (MTX) for tumor theragnosis. The preclinical evaluation demonstrated that tumor uptakes and retention of two components AuNP conjugates were higher than that of single-component AuNP conjugates. Notably, the glucose/MT- modified dual-ligand AuNP conjugates showed significant improvement in tumor uptake and retention. The novel nanoconjugates prepared in this study make it possible to integrate several modalities with a single AuNP for multimodality imaging and therapy, combining the power of chemo-, thermal- and radiation therapies together.

Development of Fluorinated NP-59: A Revival of Cholesterol Use Imaging with PET.

Imaging of cholesterol use is possible with the 131I scintiscanning/SPECT agent NP-59. This agent provided a noninvasive measure of adrenal function and steroid synthesis. However, iodine isotopes resulted in poor resolution, manufacturing challenges, and high radiation dosimetry to patients that have limited their use and clinical impact. A 18F analog would address these shortcomings while retaining the ability to image cholesterol use. The goal of this study was to prepare and evaluate a 18F analog of NP-59 to serve as a PET imaging agent for functional imaging of the adrenal glands based on cholesterol use. Previous attempts to prepare such an analog of NP-59 have proven elusive. Preclinical and clinical evaluation could be performed once the new fluorine analog of NP-59 production was established. Methods: The recent development of a new reagent for fluorination along with an improved route to the NP-59 precursor allowed for the preparation of a fluorine analog of NP-59, FNP-59. The radiochemistry for the 18F-radiolabeled 18F-FNP-59 is described, and rodent radiation dosimetry studies and in vivo imaging in New Zealand rabbits was performed. After in vivo toxicity studies, an investigational new drug approval was obtained, and the first-in-humans images with dosimetry using the agent were acquired. Results: In vivo toxicity studies demonstrated that FNP-59 is safe for use at the intended dose. Biodistribution studies with 18F-FNP-59 demonstrated a pharmacokinetic profile similar to that of NP-59 but with decreased radiation exposure. In vivo animal images demonstrated expected uptake in tissues that use cholesterol: gallbladder, liver, and adrenal glands. In this first-in-humans study, subjects had no adverse events and images demonstrated accumulation in target tissues (liver and adrenal glands). Manipulation of uptake was also demonstrated with patients who received cosyntropin, resulting in improved uptake. Conclusion: 18F-FNP-59 provided higher resolution images, with lower radiation dose to the subjects. It has the potential to provide a noninvasive test for patients with adrenocortical diseases.

Synthesis and Evaluation of a Fluorine-18 Radioligand for Imaging Huntingtin Aggregates by Positron Emission Tomographic Imaging.

Mutations in the huntingtin gene (HTT) triggers aggregation of huntingtin protein (mHTT), which is the hallmark pathology of neurodegenerative Huntington's disease (HD). Development of a high affinity 18F radiotracer would enable the study of Huntington's disease pathology using a non-invasive imaging modality, positron emission tomography (PET) imaging. Herein, we report the first synthesis of fluorine-18 imaging agent, 6-(5-((5-(2,2-difluoro-2-(fluoro-18F)ethoxy)pyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one ([18F]1), a radioligand for HD and its preclinical evaluation in vitro (autoradiography of post-mortem HD brains) and in vivo (rodent and non-human primate brain PET). [18F]1 was synthesized in a 4.1% RCY (decay corrected) and in an average molar activity of 16.5 ± 12.5 GBq/µmol (445 ± 339 Ci/mmol). [18F]1 penetrated the blood-brain barrier of both rodents and primates, and specific saturable binding in post-mortem brain slices was observed that correlated to mHTT aggregates identified by immunohistochemistry.

Improved Synthesis of [11C]COU and [11C]PHXY, Evaluation of Neurotoxicity, and Imaging of MAOs in Rodent Heart.

The radiotracers [11C]COU and [11C]PHXY are potential PET imaging agents for in vivo studies of monoamine oxidases (MAOs), as previously shown in rodent and primate brain. One-pot, automated methods for the radiosynthesis of [11C]PHXY and [11C]COU were developed to provide reliable and improved radiochemical yields. Although derived from the structure of the neurotoxin MPTP, COU did not exhibit in vivo neurotoxicity to dopaminergic nerve terminals in the mouse brain as assayed by losses of VMAT2 radioligand binding. PET imaging studies in rats demonstrated that both [11C]COU and [11C]PHXY exhibit retention in cardiac tissues that can be blocked by pretreatment with the MAO inhibitors deprenyl (MAO-B) and pargyline (MAO-A and -B). In addition to prior neuroimaging applications, [11C]COU and [11C]PHXY are thus also of interest for studies of MAO enzymatic activity and imaging of sympathetic nerve density in heart.

Fully Automated Radiosynthesis of [11C]Guanidines for Cardiac PET Imaging.

Radiolabeled guanidines such as meta-iodobenzylguanidine (MIBG) find utility in nuclear medicine as both diagnostic imaging agents and radiotherapeutics and, over the years, numerous methods for incorporating radionuclides into guanidines have been developed. In connection with a project developing new positron emission tomography (PET) radiotracers for cardiac sympathetic nerve density, we had cause to prepare [11C]3F-PHPOG. However, it quickly became apparent that radiolabeling of guanidine scaffolds with carbon-11 has remained challenging, and historical methods lack compatibility with modern automated radiochemistry synthesis platforms and current Good Manufacturing Practice (cGMP) requirements. To address this challenge, we report a new automated method for radiolabeling guanidines with carbon-11. The method was used to prepare a series of [11C]guanidines in good radiochemical yield (8-76% by radio-HPLC) and was found to have broad substrate scope and tolerance of unprotected OH and NH functional groups. The method was used to synthesize [11C]3F-PHPOG for preclinical imaging, and suitability of the radiotracer for preclinical use was demonstrated through preliminary cardiac PET in New Zealand white rabbits which revealed good cardiac uptake and expected retention in the heart.

Strategies for PET imaging of the receptor for advanced glycation endproducts (RAGE).

The implication of the receptor for advanced glycation end-products (RAGE) in numerous diseases and neurodegenerative disorders makes it interesting both as a therapeutic target and as an inflammatory biomarker. In the context of investigating RAGE as a biomarker, there is interest in developing radiotracers that will enable quantification of RAGE using positron emission tomography (PET) imaging. We have synthesized potential small molecule radiotracers for both the intracellular ([18F]InRAGER) and extracellular ([18F]RAGER) domains of RAGE. Herein we report preclinical evaluation of both using in vitro (lead panel screens) and in vivo (rodent and nonhuman primate PET imaging) methods. Both radiotracers have high affinity for RAGE and show good brain uptake, but suffer from off-target binding. The source of the off-target PET signal is not attributable to binding to melatonin receptors, but remains unexplained. We have also investigated use of lipopolysaccharide (LPS)-treated mice as a possible animal model with upregulated RAGE for evaluation of new imaging agents. Immunoreactivity of the mouse brain sections revealed increases in RAGE in the male cohorts, but no difference in the female groups. However, it proves challenging to quantify the changes in RAGE due to off-target binding of the radiotracers. Nevertheless, they are appropriate lead scaffolds for future development of 2nd generation RAGE PET radiotracers because of their high affinity for the receptor and good CNS penetration.

Synthesis and Evaluation of 11C- and 18F-Labeled SOAT1 Inhibitors as Macrophage Foam Cell Imaging Agents.

PD-132301, an inhibitor of sterol O-acyltransferase 1 (SOAT1; also known as acyl-coenzyme A:cholesterol acyltransferase-1, ACAT1), is under clinical investigation for numerous adrenal disorders. Radiolabeled SOAT1 inhibitors could support drug discovery and help diagnose SOAT1-related disorders, such as atherosclerosis. We synthesized two radiolabeled SOAT1 inhibitors, [11C]PD-132301 and fluorine analogue [18F]1. Rat biodistribution studies were conducted with both agents and, as the most selective tracer, [11C]PD-132301 was advanced to preclinical positron emission tomography studies in (atherosclerotic) ApoE-/- mice. The uptake of [11C]PD-132301 in SOAT1-rich tissue warrants further investigation into the compound as an atherosclerosis and adrenal imaging agent.

The Effects of Intramuscular Naloxone Dose on Mu Receptor Displacement of Carfentanil in Rhesus Monkeys.

Naloxone (NLX) is a mu receptor antagonist used to treat acute opioid overdoses. Currently approved doses of naloxone to treat opioid overdoses are 4 mg intranasal (IN) and 2 mg intramuscular (IM). However, higher mu receptor occupancy (RO) may be required to treat overdoses due to more potent synthetic opioids such as fentanyl and carfentanil that have entered the illicit drug market recently. To address this need, a higher dose of NLX has been investigated in a 5 mg IM formulation called ZIMHI but, while the effects of intravenous (IV) and IN administration of NLX on the opioid mu receptor occupancy (RO) have been studied, comparatively little is known about RO for IM administration of NLX. The goal of this study was to examine the effect of IM dosing of NLX on mu RO in rhesus macaques using [11C]carfentanil positron emission tomography (PET) imaging. The lowest dose of NLX (0.06 mg/kg) approximated 51% RO. Higher doses of NLX (0.14 mg/kg, 0.28 mg/kg) resulted in higher mu RO of 70% and 75%, respectively. Plasma levels were 4.6 ng/mL, 16.8 ng/mL, and 43.4 ng/mL for the three IM doses, and a significant correlation between percent RO and plasma NLX level was observed (r = 0.80). These results suggest that higher doses of IM NLX result in higher mu RO and could be useful in combating overdoses resulting from potent synthetic opioids.

Strategies for PET imaging of the receptor for advanced glycation endproducts (RAGE) / 药物分析学报

The implication of the receptor for advanced glycation end-products (RAGE) in numerous diseases and neurodegenerative disorders makes it interesting both as a therapeutic target and as an inflammatory biomarker. In the context of investigating RAGE as a biomarker, there is interest in developing radio-tracers that will enable quantification of RAGE using positron emission tomography (PET) imaging. We have synthesized potential small molecule radiotracers for both the intracellular ([18F]InRAGER) and extracellular ([18F]RAGER) domains of RAGE. Herein we report preclinical evaluation of both using in vitro (lead panel screens) and in vivo (rodent and nonhuman primate PET imaging) methods. Both radiotracers have high affinity for RAGE and show good brain uptake, but suffer from off-target binding. The source of the off-target PET signal is not attributable to binding to melatonin receptors, but remains unexplained. We have also investigated use of lipopolysaccharide (LPS)-treated mice as a possible animal model with upregulated RAGE for evaluation of new imaging agents. Immunoreactivity of the mouse brain sections revealed increases in RAGE in the male cohorts, but no difference in the female groups. However, it proves challenging to quantify the changes in RAGE due to off-target binding of the radio-tracers. Nevertheless, they are appropriate lead scaffolds for future development of 2nd generation RAGE PET radiotracers because of their high affinity for the receptor and good CNS penetration.

Structural Basis for Achieving GSK-3ß Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery.

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.

Synthesis and Initial In Vivo Evaluation of [11C]AZ683-A Novel PET Radiotracer for Colony Stimulating Factor 1 Receptor (CSF1R).

Positron emission tomography (PET) imaging of Colony Stimulating Factor 1 Receptor (CSF1R) is a new strategy for quantifying both neuroinflammation and inflammation in the periphery since CSF1R is expressed on microglia and macrophages. AZ683 has high affinity for CSF1R (Ki = 8 nM; IC50 = 6 nM) and >250-fold selectivity over 95 other kinases. In this paper, we report the radiosynthesis of [11C]AZ683 and initial evaluation of its use in CSF1R PET. [11C]AZ683 was synthesized by 11C-methylation of the desmethyl precursor with [11C]MeOTf in 3.0% non-corrected activity yield (based upon [11C]MeOTf), >99% radiochemical purity and high molar activity. Preliminary PET imaging with [11C]AZ683 revealed low brain uptake in rodents and nonhuman primates, suggesting that imaging neuroinflammation could be challenging but that the radiopharmaceutical could still be useful for peripheral imaging of inflammation.

Automated synthesis of [68Ga]oxine, improved preparation of 68Ga-labeled erythrocytes for blood-pool imaging, and preclinical evaluation in rodents.

Radiolabeled erythrocytes have multiple applications in nuclear medicine, including blood pool imaging. Historically they have been labeled with SPECT radionuclides. A PET blood pool imaging agent is highly desirable as it would improve clinical applications with better image quality and resolution, higher sensitivity, and dynamic scanning capabilities. With the coming of age of modern 68Ge/68Ga generator systems, gallium-68 is now widely accessible. In this paper we describe an updated method for the preparation of 68Ga-labeled erythrocytes and their preliminary use in rodent blood pool imaging. A novel automated synthesis of [68Ga]oxine using a 68Ga/68Ge generator and automated synthesis module is reported. [68Ga]Oxine was synthesized in 50 ± 5% (n = 3) non-decay corrected radiochemical yield and >99% radiochemical purity. Rat and human erythrocytes were successfully labeled with the complex in 42% RCY, and the 68Ga-labeled erythrocytes have been shown to clearly image the blood pool in a healthy rat. Human erythrocytes labelled with [68Ga]oxine were shown to be viable up to 2 hours post-labelling, and washout of the radiolabel was minimal up to 1 hour post-labelling. Further optimization of the labeling method to translate for use in human cardiac and oncologic blood pool PET imaging studies, is underway.

Synthesis and pre-clinical evaluation of a potential radiotracer for PET imaging of the dopamine D3 receptor.

There is considerable interest in using positron emission tomography (PET) imaging to understand the function of dopamine D3 receptors. Due to high sequence homology with D2 receptors, development of D3-selective PET radiotracers has been challenging. In an effort to overcome this issue, we report the radiosynthesis of a new selective D3 ligand with carbon-11 ([11C]1 ), and its initial preclincial evaluation as a potential PET radiotracer for in vivo imaging of D3 receptors. [11C]1 was prepared via [11C]CO2 fixation in 0.1% non-corrected radiochemical yield, good radiochemical purity (>95%) and high specific activity (>2000 Ci mmol-1). [11C]1 exhibited specific binding to D3 receptors using ex vivo autoradiography experiments with rat brain, but only 14-fold selectivity over D2 receptors which is lower than the 1400-fold value reported previously for cell studies. Rodent PET imaging revealed reasonable uptake of the radiotracer in areas of the brain known to be rich in D3 receptors.

Development of Positron Emission Tomography Radiotracers for the GABA Transporter 1.

In vivo positron emission tomography (PET) imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[11C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [18F]fluoride was used to prepare the desired candidate radiotracer ( R, E/ Z)-1-(2-((4-fluoro-2-(4-[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid (( R, E/ Z)-[18F]10). PET studies in rats showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of ( R, E/ Z)-[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of ( R, E/ Z)-[18F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 min. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.

Identification of [18F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging.

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first 18F-labeled optimized lead suitable for in vivo imaging of Trk, [18F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity.

[(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease.

OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aß rather than synthetic tau aggregates or Aß fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aß. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aß on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aß plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.

A highly selective and specific PET tracer for imaging of tau pathologies.

Senile plaques and neurofibrillary tangles are prominent neuropathological hallmarks in Alzheimer's disease and are considered to be targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. While there are a number of amyloid-ß positron emission tomography (PET) tracers currently in different stages of clinical development and commercialization, there have been very few reports on imaging agents selectively targeting tau aggregates. In search of [18F]-PET tracers that possess great binding affinity and selectivity toward tau tangles, we tested more than 900 compounds utilizing a unique screening process. A competitive autoradiography assay was set up to test compounds for binding to native tau tangles and amyloid-ß plaques on human brain tissue sections. In our in vitro assays, the 18F labeled compound [18F]-T808 displayed a high level of binding affinity and good selectivity for tau aggregates over amyloid-ß plaques. [18F]-T808 showed rapid uptake and washout in rodent brains. Our in vitro and preclinical in vivo studies suggest that [18F]-T808 possesses suitable properties and characteristics to be a specific and selective PET probe for imaging of paired helical filament tau in human brains.