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.

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.

[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.

Imaging the Bacterial Cell Wall Using N-Acetyl Muramic Acid-Derived Positron Emission Tomography Radiotracers.

Imaging infections in patients is challenging using conventional methods, motivating the development of positron emission tomography (PET) radiotracers targeting bacteria-specific metabolic pathways. Numerous techniques have focused on the bacterial cell wall, although peptidoglycan-targeted PET tracers have been generally limited to the short-lived carbon-11 radioisotope (t1/2 = 20.4 min). In this article, we developed and tested new tools for infection imaging using an amino sugar component of peptidoglycan, namely, derivatives of N-acetyl muramic acid (NAM) labeled with the longer-lived fluorine-18 (t1/2 = 109.6 min) radioisotope. Muramic acid was reacted directly with 4-nitrophenyl 2-[18F]fluoropropionate ([18F]NFP) to afford the enantiomeric NAM derivatives (S)-[18F]FMA and (R)-[18F]FMA. Both diastereomers were easily isolated and showed robust accumulation by human pathogens in vitro and in vivo, including Staphylococcus aureus. These results form the basis for future clinical studies using fluorine-18-labeled NAM-derived PET radiotracers.

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.

Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from [ 18 F]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-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG), the most common tracer used in clinical imaging, to form [ 18 F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [ 18 F]FDG was reacted with ß-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[ 18 F]-fluoro-maltose ([ 18 F]FDM) and 2-deoxy-2-[ 18 F]-fluoro-sakebiose ([ 18 F]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-[ 18 F]fluoro-trehalose ([ 18 F]FDT), 2-deoxy-2-[ 18 F]fluoro-laminaribiose ([ 18 F]FDL), and 2-deoxy-2-[ 18 F]fluoro-cellobiose ([ 18 F]FDC). We subsequently tested [ 18 F]FDM and [ 18 F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [ 18 F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [ 18 F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [ 18 F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Organ-specific fuel rewiring in acute and chronic hypoxia redistributes glucose and fatty acid metabolism.

Oxygen deprivation can be detrimental. However, chronic hypoxia is also associated with decreased incidence of metabolic syndrome and cardiovascular disease in high-altitude populations. Previously, hypoxic fuel rewiring has primarily been studied in immortalized cells. Here, we describe how systemic hypoxia rewires fuel metabolism to optimize whole-body adaptation. Acclimatization to hypoxia coincided with dramatically lower blood glucose and adiposity. Using in vivo fuel uptake and flux measurements, we found that organs partitioned fuels differently during hypoxia adaption. Acutely, most organs increased glucose uptake and suppressed aerobic glucose oxidation, consistent with previous in vitro investigations. In contrast, brown adipose tissue and skeletal muscle became "glucose savers," suppressing glucose uptake by 3-5-fold. Interestingly, chronic hypoxia produced distinct patterns: the heart relied increasingly on glucose oxidation, and unexpectedly, the brain, kidney, and liver increased fatty acid uptake and oxidation. Hypoxia-induced metabolic plasticity carries therapeutic implications for chronic metabolic diseases and acute hypoxic injuries.

Antigen-Dependent Inducible T-Cell Reporter System for PET Imaging of Breast Cancer and Glioblastoma.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

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.

Longitudinal Imaging of T Cells and Inflammatory Demyelination in a Preclinical Model of Multiple Sclerosis Using 18F-FAraG PET and MRI.

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2'-deoxy-2'-18F-fluoro-9-ß-d-arabinofuranosylguanine (18F-FAraG) PET imaging to noninvasively assess infiltrating T cells and to provide, in combination with MRI, a novel tool to determine lesion types. Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis to reproducibly induce 2 brain inflammatory lesion types, differentiated by their T cell content. 18F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed before disease induction, during demyelination with high levels of innate immune cells, and after T cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T cells, microglia/macrophages, myelin, and blood-brain barrier (BBB) integrity were performed to validate the in vivo findings. Results:18F-FAraG signal was significantly increased in the brain and spinal cord at the time point of T cell infiltration. 18F-FAraG signal from white matter (corpus callosum) and gray matter (cortex, hippocampus) further correlated with T cell density. T2-weighted MRI detected white matter lesions independently of T cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T cell infiltration. Fingolimod treatment prevented motor deficits and decreased T cell and microglia/macrophage levels. In agreement, 18F-FAraG signal was decreased in the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and 18F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses.

Cyclic 68Ga-Labeled Peptides for Specific Detection of Human Angiotensin-Converting Enzyme 2.

In this study, we developed angiotensin-converting enzyme 2 (ACE2)-specific, peptide-derived 68Ga-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. Methods: A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga to generate peptide radiotracers (68Ga-NOTA-PEP). The aminocaproate-derived radiotracer 68Ga-NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. Results: Cyclic DX-600-derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga, and the biodistribution of 68Ga-NOTA-PEP4 was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of 68Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. Conclusion: NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, 68Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine-organs known to be affected in SARS-CoV-2 infection. These results suggest that 68Ga-NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2-infected murine models and COVID-19 patients.

68Ga-PSMA-11 NDA Approval: A Novel and Successful Academic Partnership.

The University of California Los Angeles (UCLA) and University of California San Francisco (UCSF) codeveloped 68Ga-PSMA-11 by conducting a bicentric pivotal phase 3 clinical trial for PET imaging for prostate cancer. On December 1, 2020, 2 separate new drug applications (NDAs) submitted by each institution (NDA 212642 for UCLA and NDA 212643 for UCSF) were approved by the Food and Drug Administration as the first drug for PET imaging of prostate-specific membrane antigen (PSMA)-positive lesions in men with prostate cancer. This article briefly describes the background, clinical development, regulatory approach, and regulatory process for NDA filing and approval. In the second part of this article, key chemistry, manufacturing, and controls (CMC) information is provided to facilitate abbreviated new drug application (ANDA) submission.

The Synthesis and Structural Requirements for Measuring Glucocorticoid Receptor Expression In Vivo with (±)-11C-YJH08 PET.

Noninvasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to elaborate the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-11C-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]-quinoline ((±)-11C-YJH08), a radioligand for PET that engages the ligand binding domain on GR. Methods: (±)-11C-YJH08 was synthesized by reacting the phenol precursor with 11C-methyl iodide. The biodistribution was studied in vivo. Specific binding was tested in vivo with adrenalectomy and ligand competition. A library of analogs was synthesized and studied in vitro and in vivo to understand the (±)-11C-YJH08 structure-activity relationship. Rodent dosimetry studies were performed to estimate the human-equivalent doses of (±)-11C-YJH08. Results: (±)-11C-YJH08 was synthesized by reaction of the phenolic precursor with 11C-methyl iodide, giving a radiochemical yield of 51.7% ± 4.7% (decay-corrected to starting 11C-methyl iodide). Specific binding was observed in many tissues, including the brain. An analysis of the (±)-YJH08 structure-activity relationship showed that (R)- and (S)-enantiomers are equally avid for GR by occupying discrete binding modes. A focused chemical screen revealed that the aryl fluoride motif on YJH08 is essential for high-affinity GR binding in vitro, high tissue uptake in vivo, and efficient passage across the blood-brain barrier. Lastly, we performed dosimetry studies on rodents, from which we estimated the human-equivalent doses of (±)-11C-YJH08 to be commensurate with the widely used 11C and 18F tracers. Conclusion: These studies reveal the molecular determinants of a high-affinity and high-selectivity ligand-receptor interaction and support the use of (±)-11C-YJH08 PET to make the first measurements of GR expression in human subjects.

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.

Positron emission tomography evaluation of oxime countermeasures in live rats using the tracer O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate [18 F]-VXS.

Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [18 F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [18 F]-VXS was evaluated after an LD50 dose (250 µg/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes. Rats given [18 F]-VXS tracer alone had significantly higher radioactivity (two- to threefold) in the heart and lung than rats given LD50 POX at 20 or 60 min prior to [18 F]-VXS. When rats were given LD50 POX followed by 2-PAM (cationic), RS194b (ionizable), or monoisonitrosoacetone (MINA) (neutral), central nervous system (CNS) radioactivity returned to levels at or above untreated naive rats (no POX), whereas CNS radioactivity did not increase in rats given the dication oximes HI-6 or MMB-4. MINA showed a significant, pairwise increase in CNS brain radioactivity compared with POX-treated rats. This new in vivo dynamic platform using [18 F]-VXS tracer measures and quantifies peripheral and CNS relative changes in AChE availability after POX exposure and is suitable for comparing oxime delivery and AChE reactivation in rats.

A Novel Radioligand Reveals Tissue Specific Pharmacological Modulation of Glucocorticoid Receptor Expression with Positron Emission Tomography.

The complexity of glucocorticoid receptor (GR) signaling cannot be measured with direct tissue analysis in living subjects, which has stifled our understanding of GR's role in human physiology or disease and impeded the development of selective GR modulators. Herein, we report 18F-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]quinoline (18F-YJH08), a radioligand that enables noninvasive measurements of tissue autonomous GR expression levels in vivo with positron emission tomography (PET). YJH08 potently binds GR (Ki ∼ 0.4 nM) with ∼100-fold selectivity compared to nuclear hormone receptors in the same subfamily. 18F-YJH08 was prepared via Cu(OTf)2(py)4-mediated radiofluorination of an arylboronic acid pinacol ester with ∼12% decay corrected radiochemical yield from the starting 18F-fluoride ion. We applied treatment with the tissue-wide GR agonist dexamethasone and adrenalectomy and generated an adipocyte specific GR knockout mouse to show that 18F-YJH08 specifically binds GR in normal mouse tissues, including those for which aberrant GR expression is thought to drive severe diseases (e.g., brain, adipose tissue, kidneys). Remarkably, 18F-YJH08 PET also revealed that JG231, a potent and bioavailable HSP70 inhibitor, selectively degrades GR only in the adipose tissue of mice, a finding that foreshadows how GR targeted PET might be integrated into drug discovery to screen for selective GR modulation at the tissue level, beyond the historical screening that was performed at the transcriptional level. In summary, 18F-YJH08 enables a quantitative assessment of GR expression levels in real time among multiple tissues simultaneously, and this technology is a first step toward unraveling the daunting complexity of GR signaling and rationally engineering tissue specific therapeutic modulators in vivo.