[11C]Paraoxon: Radiosynthesis, Biodistribution and In Vivo Positron Emission Tomography Imaging in Rat.

Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([11C]POX) and profiling in live rats is reported. Naïve rats intravenously injected with [11C]POX showed a rapid decrease in parent tracer to ∼1%, with an increase in radiolabeled serum proteins to 87% and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 minutes, reflecting covalent modification of proteins by [11C]POX. Ex vivo biodistribution and imaging profiles in naïve rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection and persisted over the 60-minute experiment. This showed for the first time that even low POX exposures (∼200 ng tracer) can rapidly enter brain. Rats given an LD50 dose of nonradioactive paraoxon at the LD50 20 or 60 minutes prior to [11C]POX tracer revealed that protein pools were blocked. Blood radioactivity at 20 minutes was markedly lower than naïve levels due to rapid protein modification by nonradioactive POX; however, by 60 minutes the blood radioactivity returned to near naïve levels. Live rat tissue imaging-derived radioactivity values were 10%-37% of naïve levels in nonradioactive POX pretreated rats at 20 minutes, but by 60 minutes the area under the curve (AUC) values had recovered to 25%-80% of naïve. The live rat imaging supported blockade by nonradioactive POX pretreatment at 20 minutes and recovery of proteins by 60 minutes. SIGNIFICANCE STATEMENT: Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX, etc. To study the distribution and penetration of POX into the central nervous system (CNS) and other tissues, a positron emission tomography (PET) tracer analog, carbon-11-labeled paraoxon ([11C]POX), was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.

Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

BACKGROUND: Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. METHODS: We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. RESULTS: In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. CONCLUSIONS: Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Biodistribution of a Mitochondrial Metabolic Tracer, [18F]F-AraG, in Healthy Volunteers.

Purpose: [18F]F-AraG is a radiolabeled nucleoside analog that shows relative specificity for activated T cells. The aim of this study was to investigate the biodistribution of [18F]F-AraG in healthy volunteers and assess the preliminary safety and radiation dosimetry. Methods: Six healthy subjects (three female and three male) between the ages of 24 and 60 participated in the study. Each subject received a bolus venous injection of [18F]F-AraG (dose range: 244.2-329.3 MBq) prior to four consecutive PET/MR whole-body scans. Blood samples were collected at regular intervals and vital signs monitored before and after tracer administration. Regions of interest were delineated for multiple organs, and the area under the time-activity curves was calculated for each organ and used to derive time-integrated activity coefficient (TIAC). TIACs were input for absorbed dose and effective dose calculations using OLINDA. Results: PET/MR examination was well tolerated, and no adverse effects to the administration of [18F]F-AraG were noted by the study participants. The biodistribution was generally reflective of the expression and activity profiles of the enzymes involved in [18F]F-AraG's cellular accumulation, mitochondrial kinase dGK, and SAMHD1. The highest uptake was observed in the kidneys and liver, while the brain, lung, bone marrow, and muscle showed low tracer uptake. The estimated effective dose for [18F]F-AraG was 0.0162 mSv/MBq (0.0167 mSv/MBq for females and 0.0157 mSv/MBq for males). Conclusion: Biodistribution of [18F]F-AraG in healthy volunteers was consistent with its association with mitochondrial metabolism. PET/MR [18F]F-AraG imaging was well tolerated, with a radiation dosimetry profile similar to other commonly used [18F]-labeled tracers. [18F]F-AraG's connection with mitochondrial biogenesis and favorable biodistribution characteristics make it an attractive tracer with a variety of potential applications.

Imaging joint infections using D-methyl-11C-methionine PET/MRI: initial experience in humans.

PURPOSE: Non-invasive imaging is a key clinical tool for detection and treatment monitoring of infections. Existing clinical imaging techniques are frequently unable to distinguish infection from tumors or sterile inflammation. This challenge is well-illustrated by prosthetic joint infections that often complicate joint replacements. D-methyl-11C-methionine (D-11C-Met) is a new bacteria-specific PET radiotracer, based on an amino acid D-enantiomer, that is rapidly incorporated into the bacterial cell wall. In this manuscript, we describe the biodistribution, radiation dosimetry, and initial human experience using D-11C-Met in patients with suspected prosthetic joint infections. METHODS: 614.5 ± 100.2 MBq of D-11C-Met was synthesized using an automated in-loop radiosynthesis method and administered to six healthy volunteers and five patients with suspected prosthetic joint infection, who were studied by PET/MRI. Time-activity curves were used to calculate residence times for each source organ. Absorbed doses to each organ and body effective doses were calculated using OLINDA/EXM 1.1 with both ICRP 60 and ICRP 103 tissue weighting factors. SUVmax and SUVpeak were calculated for volumes of interest (VOIs) in joints with suspected infection, the unaffected contralateral joint, blood pool, and soft tissue background. A two-tissue compartment model was used for kinetic modeling. RESULTS: D-11C-Met was well tolerated in all subjects. The tracer showed clearance from both urinary (rapid) and hepatobiliary (slow) pathways as well as low effective doses. Moreover, minimal background was observed in both organs with resident micro-flora and target organs, such as the spine and musculoskeletal system. Additionally, D-11C-Met showed increased focal uptake in areas of suspected infection, demonstrated by a significantly higher SUVmax and SUVpeak calculated from VOIs of joints with suspected infections compared to the contralateral joints, blood pool, and background (P < 0.01). Furthermore, higher distribution volume and binding potential were observed in suspected infections compared to the unaffected joints. CONCLUSION: D-11C-Met has a favorable radiation profile, minimal background uptake, and fast urinary extraction. Furthermore, D-11C-Met showed increased uptake in areas of suspected infection, making this a promising approach. Validation in larger clinical trials with a rigorous gold standard is still required.

Evaluating Radioactive Analogs of Doxorubicin to Quantify ChemoFilter Binding and Whole-Body Positron Emission Tomography/Magnetic Resonance Imaging for Drug Biodistribution.

PURPOSE: To evaluate radiolabeled doxorubicin (Dox) analogs as tracers of baseline Dox biodistribution in vivo during hepatic intra-arterial chemotherapy and to assess the efficacy of ChemoFilter devices to bind Dox in vitro. MATERIALS AND METHODS: In an in vitro static experiment, [fluorine-18]N-succinimidyl 4-fluorobenzoate ([18F]SFB) and [fluorine-18]fluorobenzoyl-doxorubicin ([18F]FB-Dox) were added to a beaker containing a filter material (Dowex cation exchange resin, single-stranded DNA (ssDNA) resin, or sulfonated polymer coated mesh). In an in vitro flow model, [18F]FB-Dox was added into a Dox solution in phosphate-buffered saline, and the solution flowed via a syringe column containing the filter materials. In an in vitro flow experiment, using micro-positron emission tomography (PET), images were taken as [18F]SFB and [18F]FB-Dox moved through a phantom. For in vivo biodistribution testing, a catheter was placed into the common hepatic artery of a swine, and [18F]FB-Dox was infused over 30 seconds. A 10-minute dynamic image and three 20-minute static images were acquired using 3T PET/MR imaging. RESULTS: In the in vitro static experiment, [18F]FB-Dox demonstrated 76.7%, 88.0%, and 52.4% binding to the Dowex resin, ssDNA resin, and coated mesh, respectively. In the in vitro flow model, the first-pass binding of [18F]FB-Dox to the Dowex resin, ssDNA resin, and coated mesh was 76.7%, 74.2%, and 76.2%, respectively, and the total bound fraction was 80.9%, 84.6%, and 79.9%, respectively. In the in vitro flow experiment using micro-PET, the phantom demonstrated a greater amount of [18F]FB-Dox bound to both filter cartridges than of the control [18F]SFB. In in vivo biodistribution testing, the first 10 minutes depicted [18F]FB-Dox moving through the right upper quadrant of the abdomen. A region-of-interest analysis showed that the relative amount increased by 2.97 times in the gallbladder and 1.08 times in the kidney. The amount decreased by 0.74 times in the brain and 0.57 times in the heart. CONCLUSIONS: [18F]FB-Dox can be used to assess Dox binding to ChemoFilters as well as in vivo biodistribution. This sets the stage for the evaluation of ChemoFilter effectiveness in reducing systemic toxicity from intra-arterial chemotherapy.

Biological Distribution and Metabolic Profiles of Carbon-11 and Fluorine-18 Tracers of VX- and Sarin-Analogs in Sprague-Dawley Rats.

Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [18F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.

Arabinofuranose-derived positron-emission tomography radiotracers for detection of pathogenic microorganisms.

PURPOSE: Detection of bacteria-specific metabolism via positron emission tomography (PET) is an emerging strategy to image human pathogens, with dramatic implications for clinical practice. In silico and in vitro screening tools have recently been applied to this problem, with several monosaccharides including l-arabinose showing rapid accumulation in Escherichia coli and other organisms. Our goal for this study was to evaluate several synthetically viable arabinofuranose-derived 18 F analogs for their incorporation into pathogenic bacteria. PROCEDURES: We synthesized four radiolabeled arabinofuranose-derived sugars: 2-deoxy-2-[18 F]fluoro-arabinofuranoses (d-2-18 F-AF and l-2-18 F-AF) and 5-deoxy-5-[18 F]fluoro-arabinofuranoses (d-5-18 F-AF and l-5-18 F-AF). The arabinofuranoses were synthesized from 18 F- via triflated, peracetylated precursors analogous to the most common radiosynthesis of 2-deoxy-2-[18 F]fluoro-d-glucose ([18 F]FDG). These radiotracers were screened for their uptake into E. coli and Staphylococcus aureus. Subsequently, the sensitivity of d-2-18 F-AF and l-2-18 F-AF to key human pathogens was investigated in vitro. RESULTS: All 18 F radiotracer targets were synthesized in high radiochemical purity. In the screening study, d-2-18 F-AF and l-2-18 F-AF showed greater accumulation in E. coli than in S. aureus. When evaluated in a panel of pathologic microorganisms, both d-2-18 F-AF and l-2-18 F-AF demonstrated sensitivity to most gram-positive and gram-negative bacteria. CONCLUSIONS: Arabinofuranose-derived 18 F PET radiotracers can be synthesized with high radiochemical purity. Our study showed absence of bacterial accumulation for 5-substitued analogs, a finding that may have mechanistic implications for related tracers. Both d-2-18 F-AF and l-2-18 F-AF showed sensitivity to most gram-negative and gram-positive organisms. Future in vivo studies will evaluate the diagnostic accuracy of these radiotracers in animal models of infection.

Synthesis and Initial Biological Evaluation of Boron-Containing Prostate-Specific Membrane Antigen Ligands for Treatment of Prostate Cancer Using Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate-specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed a strong binding affinity to PSMA in a competition radioligand binding assay (IC50 from 555.7 to 20.3 nM). Three selected compounds 1a, 1d, and 1f were administered to mice, and their in vivo blocking of 68Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4-7 µg/g in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-l-phenylalanine (BPA). Taken together, these data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

Radiosynthesis of O-(1-[18 F]fluoropropan-2-yl)-O-(4-nitrophenyl)methylphosphonate: A novel PET tracer surrogate of sarin.

O-(1-Fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate is a reactive organophosphate ester (OP) developed as a surrogate of the chemical warfare agent sarin that forms a similar covalent adduct at the active site serine of acetylcholinesterase. The radiolabeled O-(1-[18 F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate ([18 F] fluorosarin surrogate) has not been previously prepared. In this paper, we report the first radiosynthesis of this tracer from the reaction of bis-(4-nitrophenyl) methylphosphonate with 1-[18 F]fluoro-2-propanol in the presence of DBU. The 1-[18 F]fluoro-2-propanol was prepared by reaction of propylene sulfite with Kryptofix 2.2.2 and [18 F] fluoride ion. The desired tracer O-(1-[18 F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate was obtained in a >98% radiochemical purity with a 2.4% ± 0.6% yield (n = 5, 65 minutes from start of synthesis) based on starting [18 F] fluoride ion and a molar activity of 49.9 GBq/µmol (1.349 ± 0.329 Ci/µmol, n = 3). This new facile radiosynthesis routinely affords sufficient quantities of [18 F] fluorosarin surrogate in high radiochemical purity, which will further enable the tracer development as a novel radiolabeled OP acetylcholinesterase inhibitor for assessment of OP modes of action with PET imaging in vivo.

In Vivo PET Imaging of the Activated Immune Environment in a Small Animal Model of Inflammatory Arthritis.

BACKGROUND: Evolving immune-mediated therapeutic strategies for rheumatoid arthritis (RA) may benefit from an improved understanding of the complex role that T-cell activation plays in RA. This study assessed the potential of fluorine-18-labeled 9-ß-d-arabinofuranosylguanine ([18F]F-AraG) positron emission tomography (PET) imaging to report immune activation in vivo in an adjuvant-induced arthritis (AIA) small animal model. METHODS: Using positron emission tomography-computed tomography imaging, uptake of [18F]F-AraG in the paws of mice affected by arthritis at 6 (acute) and 20 (chronic) days following AIA induction in a single paw was assessed and compared to uptake in contralateral control paws. Fractions of T cells and B cells demonstrating markers of activation at the 2 time points were determined by flow cytometry. RESULTS: Differential uptake of [18F]F-AraG was demonstrated on imaging of the affected joint when compared to control at both acute and chronic time points with corresponding changes in markers of T-cell activation observed on flow cytometry. CONCLUSION: [18F]F-AraG may serve as an imaging biomarker of T-cell activation in inflammatory arthritis. Further development of this technique is warranted and could offer a tool to explore the temporal link between activated T cells and RA as well as to monitor immune-mediated therapies for RA in clinical trials.

Ictal lack of binding to brain parenchyma suggests integrity of the blood-brain barrier for 11C-dihydroergotamine during glyceryl trinitrate-induced migraine.

SEE DREIER DOI 101093/AWW112 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: For many decades a breakdown of the blood-brain barrier has been postulated to occur in migraine. Hypothetically this would facilitate access of medications, such as dihydroergotamine or triptans, to the brain despite physical properties otherwise restricting their entry. We studied the permeability of the blood-brain barrier in six migraineurs and six control subjects at rest and during acute glyceryl trinitrate-induced migraine attacks using positron emission tomography with the novel radioligand (11)C-dihydroergotamine, which is chemically identical to pharmacologically active dihydroergotamine. The influx rate constant Ki, average dynamic image and time activity curve were assessed using arterial blood sampling and served as measures for receptor binding and thus blood-brain barrier penetration. At rest, there was binding of (11)C-dihydroergotamine in the choroid plexus, pituitary gland, and venous sinuses as expected from the pharmacology of dihydroergotamine. However, there was no binding to the brain parenchyma, including the hippocampus, the area with the highest density of the highest-affinity dihydroergotamine receptors, and the raphe nuclei, a postulated brainstem site of action during migraine, suggesting that dihydroergotamine is not able to cross the blood-brain barrier. This binding pattern was identical in migraineurs during glyceryl trinitrate-induced migraine attacks as well as in matched control subjects. We conclude that (11)C-dihydroergotamine is unable to cross the blood-brain barrier interictally or ictally demonstrating that the blood-brain barrier remains tight for dihydroergotamine during acute glyceryl trinitrate-induced migraine attacks.

An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer.

O-(2-Fluoroethyl)-O-(p-nitrophenyl) methylphosphonate 1 is an organophosphate cholinesterase inhibitor that creates a phosphonyl-serine covalent adduct at the enzyme active site blocking cholinesterase activity in vivo. The corresponding radiolabeled O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [18 F]1, has been previously prepared and found to be an excellent positron emission tomography imaging tracer for assessment of cholinesterases in live brain, peripheral tissues, and blood. However, the previously reported [18 F]1 tracer synthesis was slow even with microwave acceleration, required high-performance liquid chromatography separation of the tracer from impurities, and gave less optimal radiochemical yields. In this paper, we report a new synthetic approach to circumvent these shortcomings that is reliant on the facile reactivity of bis-(O,O-p-nitrophenyl) methylphosphonate, 2, with 2-fluoroethanol in the presence of DBU. The cold synthesis was successfully translated to provide a more robust radiosynthesis. Using this new strategy, the desired tracer, [18 F]1, was obtained in a non-decay-corrected radiochemical yield of 8 ± 2% (n = 7) in >99% radiochemical and >95% chemical purity with a specific activity of 3174 ± 345 Ci/mmol (EOS). This new facile radiosynthesis routinely affords highly pure quantities of [18 F]1, which will further enable tracer development of OP cholinesterase inhibitors and their evaluation in vivo.

An updated synthesis of [11 C]carfentanil for positron emission tomography (PET) imaging of the µ-opioid receptor.

[11 C]Carfentanil ([11 C]CFN) is a selective radiotracer for in vivo positron emission tomography imaging studies of the µ-opioid system that, in our laboratories, is synthesized by methylation of the corresponding carboxylate precursor with [11 C]MeOTf, and purified using a C2 solid-phase extraction cartridge. Changes in the commercial availability of common C2 cartridges have necessitated future proofing the synthesis of [11 C]CFN to maintain reliable delivery of the radiotracer for clinical imaging studies. An updated synthesis of [11 C]CFN is reported that replaces a now obsolete purification cartridge with a new commercially available version and also substitutes the organic solvents used in traditional production methods with ethanol.

Caged [(18)F]FDG Glycosylamines for Imaging Acidic Tumor Microenvironments Using Positron Emission Tomography.

Solid tumors are hypoxic with altered metabolism, resulting in secretion of acids into the extracellular matrix and lower relative pH, a feature associated with local invasion and metastasis. Therapeutic and diagnostic agents responsive to this microenvironment may improve tumor-specific delivery. Therefore, we pursued a general strategy whereby caged small-molecule drugs or imaging agents liberate their parent compounds in regions of low interstitial pH. In this manuscript, we present a new acid-labile prodrug method based on the glycosylamine linkage, and its application to a class of positron emission tomography (PET) imaging tracers, termed [(18)F]FDG amines. [(18)F]FDG amines operate via a proposed two-step mechanism, in which an acid-labile precursor decomposes to form the common radiotracer 2-deoxy-2-[(18)F]fluoro-d-glucose, which is subsequently accumulated by glucose avid cells. The rate of decomposition of [(18)F]FDG amines is tunable in a systematic fashion, tracking the pKa of the parent amine. In vivo, a 4-phenylbenzylamine [(18)F]FDG amine congener showed greater relative accumulation in tumors over benign tissue, which could be attenuated upon tumor alkalinization using previously validated models, including sodium bicarbonate treatment, or overexpression of carbonic anhydrase. This new class of PET tracer represents a viable approach for imaging acidic interstitial pH with potential for clinical translation.

A boronate-caged [¹8F]FLT probe for hydrogen peroxide detection using positron emission tomography.

Reactive oxygen species (ROS) play important roles in the development and progression of cancer and other diseases, motivating the development of translatable technologies for biological ROS imaging. Here we report Peroxy-Caged-[(18)F]Fluorodeoxy thymidine-1 (PC-FLT-1), an oxidatively immolative positron emission tomography (PET) probe for H2O2 detection. PC-FLT-1 reacts with H2O2 to generate [(18)F]FLT, allowing its peroxide-dependent uptake and retention in proliferating cells. The relative uptake of PC-FLT-1 was evaluated using H2O2-treated UOK262 renal carcinoma cells and a paraquat-induced oxidative stress cell model, demonstrating ROS-dependent tracer accumulation. The data suggest that PC-FLT-1 possesses promising characteristics for translatable ROS detection and provide a general approach to PET imaging that can be expanded to the in vivo study of other biologically relevant analytes.

A Feasibility Study of [18F]F-AraG Positron Emission Tomography (PET) for Cardiac Imaging-Myocardial Viability in Ischemia-Reperfusion Injury Model.

PURPOSE: Myocardial infarction (MI) with subsequent inflammation is one of the most common heart conditions leading to progressive tissue damage. A reliable imaging marker to assess tissue viability after MI would help determine the risks and benefits of any intervention. In this study, we investigate whether a new mitochondria-targeted imaging agent, 18F-labeled 2'-deoxy-2'-18F-fluoro-9-ß-d-arabinofuranosylguanine ([18F]F-AraG), a positron emission tomography (PET) agent developed for imaging activated T cells, is suitable for cardiac imaging and to test the myocardial viability after MI. PROCEDURE: To test whether the myocardial [18F]-F-AraG signal is coming from cardiomyocytes or immune infiltrates, we compared cardiac signal in wild-type (WT) mice with that of T cell deficient Rag1 knockout (Rag1 KO) mice. We assessed the effect of dietary nucleotides on myocardial [18F]F-AraG uptake in normal heart by comparing [18F]F-AraG signals between mice fed with purified diet and those fed with purified diet supplemented with nucleotides. The myocardial viability was investigated in rodent model by imaging rat with [18F]F-AraG and 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) before and after MI. All PET signals were quantified in terms of the percent injected dose per cc (%ID/cc). We also explored [18F]FDG signal variability and potential T cell infiltration into fibrotic area in the affected myocardium with H&E analysis. RESULTS: The difference in %ID/cc for Rag1 KO and WT mice was not significant (p = ns) indicating that the [18F]F-AraG signal in the myocardium was primarily coming from cardiomyocytes. No difference in myocardial uptake was observed between [18F]F-AraG signals in mice fed with purified diet and with purified diet supplemented with nucleotides (p = ns). The [18F]FDG signals showed wider variability at different time points. Noticeable [18F]F-AraG signals were observed in the affected MI regions. There were T cells in the fibrotic area in the H&E analysis, but they did not constitute the predominant infiltrates. CONCLUSIONS: Our preliminary preclinical data show that [18F]F-AraG accumulates in cardiomyocytes indicating that it may be suitable for cardiac imaging and to evaluate the myocardial viability after MI.

A feasibility study of [18F]F-AraG positron emission tomography (PET) for cardiac imaging - myocardial viability in ischemia-reperfusion injury model.

Purpose: Myocardial infarction (MI) with subsequent inflammation is one of the most common heart conditions leading to progressive tissue damage. A reliable imaging marker to assess tissue viability after MI would help determine the risks and benefits of any intervention. In this study, we investigate whether a new mitochondria-targeted imaging agent, 18F-labeled 2'-deoxy-2'-18F-fluoro-9-ß-d-arabinofuranosylguanine ([18F]F-AraG), a positron emission tomography (PET) agent developed for imaging activated T cells, is suitable for cardiac imaging and to test the myocardial viability after MI. Procedure: To test whether the myocardial [18F]-F-AraG signal is coming from cardiomyocytes or immune infiltrates, we compared cardiac signal in wild-type (WT) mice with that of T cell deficient Rag1 knockout (Rag1 KO) mice. We assessed the effect of dietary nucleotides on myocardial [18F]F-AraG uptake in normal heart by comparing [18F]F-AraG signals between mice fed with purified diet and those fed with purified diet supplemented with nucleotides. The myocardial viability was investigated in rodent model by imaging rat with [18F]F-AraG and 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) before and after MI. All PET signals were quantified in terms of the percent injected dose per cc (%ID/cc). We also explored [18F]FDG signal variability and potential T cell infiltration into fibrotic area in the affected myocardium with H&E analysis. Results: The difference in %ID/cc for Rag1 KO and WT mice was not significant (p = ns) indicating that the [18F]F-AraG signal in the myocardium was primarily coming from cardiomyocytes. No difference in myocardial uptake was observed between [18F]F-AraG signals in mice fed with purified diet and with purified diet supplemented with nucleotides (p = ns). The [18F]FDG signals showed wider variability at different time points. Noticeable [18F]F-AraG signals were observed in the affected MI regions. There were T cells in the fibrotic area in the H&E analysis, but they did not constitute the predominant infiltrates. Conclusions: Our preliminary preclinical data show that [18F]F-AraG accumulates in cardiomyocytes indicating that it may be suitable for cardiac imaging and to evaluate the myocardial viability after MI.

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.