Imaging demyelinated axons after spinal cord injuries with PET tracer [ 18 F]3F4AP.

Spinal cord injuries (SCI) often lead to lifelong disability. Among the various types of injuries, incomplete and discomplete injuries, where some axons remain intact, offer potential for recovery. However, demyelination of these spared axons can worsen disability. Demyelination is a reversible phenomenon, and drugs like 4-aminopyridine (4AP), which target K+ channels in demyelinated axons, show that conduction can be restored. Yet, accurately assessing and monitoring demyelination post-SCI remains challenging due to the lack of suitable imaging methods. In this study, we introduce a novel approach utilizing the positron emission tomography (PET) tracer, [ 18 F]3F4AP, specifically targeting K+ channels in demyelinated axons for SCI imaging. Rats with incomplete contusion injuries were imaged up to one month post-injury, revealing [ 18 F]3F4AP's exceptional sensitivity to injury and its ability to detect temporal changes. Further validation through autoradiography and immunohistochemistry confirmed [ 18 F]3F4AP's targeting of demyelinated axons. In a proof-of-concept study involving human subjects, [ 18 F]3F4AP differentiated between a severe and a largely recovered incomplete injury, indicating axonal loss and demyelination, respectively. Moreover, alterations in tracer delivery were evident on dynamic PET images, suggestive of differences in spinal cord blood flow between the injuries. In conclusion, [ 18 F]3F4AP demonstrates efficacy in detecting incomplete SCI in both animal models and humans. The potential for monitoring post-SCI demyelination changes and response to therapy underscores the utility of [ 18 F]3F4AP in advancing our understanding and management of spinal cord injuries.

Metabolic Stability of the Demyelination Positron Emission Tomography Tracer [18F]3-Fluoro-4-Aminopyridine and Identification of Its Metabolites.

[18F]3-fluoro-4-aminopyridine ([18F]3F4AP) is a positron emission tomography (PET) tracer for imaging demyelination based on the multiple sclerosis drug 4-aminopyridine (4AP, dalfampridine). This radiotracer was found to be stable in rodents and nonhuman primates imaged under isoflurane anesthesia. However, recent findings indicate that its stability is greatly decreased in awake humans and mice. Since both 4AP and isoflurane are metabolized primarily by cytochrome P450 enzymes, particularly cytochrome P450 family 2 subfamily E member 1 (CYP2E1), we postulated that this enzyme may be responsible for the metabolism of 3F4AP. Here, we investigated the metabolism of [18F]3F4AP by CYP2E1 and identified its metabolites. We also investigated whether deuteration, a common approach to increase the stability of drugs, could improve its stability. Our results demonstrate that CYP2E1 readily metabolizes 3F4AP and its deuterated analogs and that the primary metabolites are 5-hydroxy-3F4AP and 3F4AP N-oxide. Although deuteration did not decrease the rate of the CYP2E1-mediated oxidation, our findings explain the diminished in vivo stability of 3F4AP compared with 4AP and further our understanding of when deuteration may improve the metabolic stability of drugs and PET ligands. SIGNIFICANCE STATEMENT: The demyelination tracer [18F]3F4AP was found to undergo rapid metabolism in humans, which could compromise its utility. Understanding the enzymes and metabolic products involved may offer strategies to reduce metabolism. Using a combination of in vitro assays and chemical syntheses, this report shows that cytochrome P450 enzyme CYP2E1 is likely responsible for [18F]3F4AP metabolism, that 4-amino-5-fluoroprydin-3-ol (5-hydroxy-3F4AP, 5OH3F4AP) and 4-amino-3-fluoropyridine 1-oxide (3F4AP N-oxide) are the main metabolites, and that deuteration is unlikely to improve the stability of the tracer in vivo.

Broad-scope Syntheses of [11 C/18 F]Trifluoromethylarenes from Aryl(mesityl)iodonium Salts.

Efficient methods for labeling aryl trifluoromethyl groups to provide novel radiotracers for use in biomedical research with positron emission tomography (PET) are keenly sought. We report a broad-scope method for labeling trifluoromethylarenes with either carbon-11 (t1/2 =20.4 min) or fluorine-18 (t1/2 =109.8 min) from readily accessible aryl(mesityl)iodonium salts. In this method, the aryl(mesityl)iodonium salt is treated rapidly with no-carrier-added [11 C]CuCF3 or [18 F]CuCF3 . The mesityl group acts as a spectator allowing radiolabeled trifluoromethylarenes to be obtained with very high chemoselectivity. Radiochemical yields from aryl(mesityl)iodonium salts bearing either electron-donating or electron-withdrawing groups at meta- or para- position are good to excellent (67-96 %). Ortho-substituted and otherwise sterically hindered trifluoromethylarenes still give good yields (15-34 %). Substituted heteroaryl(mesityl)iodonium salts are also viable substrates. The broad scope of this method was further exemplified by labeling a previously inaccessible target, [11 C]p-trifluoromethylphenyl boronic acid, as a potentially useful labeling synthon. In addition, fluoxetine, leflunomide, and 3-trifluoromethyl-4-aminopyridine, as examples of small drug-like molecules and candidate PET radioligands, were successfully labeled in high yields (69-81 %).

Human biodistribution and radiation dosimetry of the demyelination tracer [18F]3F4AP.

PURPOSE: [18F]3F4AP is a novel PET radiotracer that targets voltage-gated potassium (K+) channels and has shown promise for imaging demyelinated lesions in animal models of neurological diseases. This study aimed to evaluate the biodistribution, safety, and radiation dosimetry of [18F]3F4AP in healthy human volunteers. METHODS: Four healthy volunteers (2 females) underwent a 4-h dynamic PET scan from the cranial vertex to mid-thigh using multiple bed positions after administration of 368 ± 17.9 MBq (9.94 ± 0.48 mCi) of [18F]3F4AP. Volumes of interest for relevant organs were manually drawn guided by the CT, and PET images and time-activity curves (TACs) were extracted. Radiation dosimetry was estimated from the integrated TACs using OLINDA software. Safety assessments included measuring vital signs immediately before and after the scan, monitoring for adverse events, and obtaining a comprehensive metabolic panel and electrocardiogram within 30 days before and after the scan. RESULTS: [18F]3F4AP distributed throughout the body with the highest levels of activity in the kidneys, urinary bladder, stomach, liver, spleen, and brain and with low accumulation in muscle and fat. The tracer cleared quickly from circulation and from most organs. The clearance of the tracer was noticeably faster than previously reported in nonhuman primates (NHPs). The average effective dose (ED) across all subjects was 12.1 ± 2.2 µSv/MBq, which is lower than the estimated ED from the NHP studies (21.6 ± 0.6 µSv/MBq) as well as the ED of other fluorine-18 radiotracers such as [18F]FDG (~ 20 µSv/MBq). No differences in ED between males and females were observed. No substantial changes in safety assessments or adverse events were recorded. CONCLUSION: The biodistribution and radiation dosimetry of [18F]3F4AP in humans are reported for the first time. The average total ED across four subjects was lower than most 18F-labeled PET tracers. The tracer and study procedures were well tolerated, and no adverse events occurred.

Evaluation of the potassium channel tracer [18F]3F4AP in rhesus macaques.

Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of molecular changes involved in demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K+ channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K+ channel PET tracer [18F]3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging. [18F]3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics. [18F]3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of [18F]3F4AP for the focal brain injury was higher than [18F]FDG, [11C]PiB, and [11C]PBR28, and compared favorably to currently used MRI methods.

In-vivo Imaging of Mitochondrial Depolarization of Myocardium With Positron Emission Tomography and a Proton Gradient Uncoupler.

BACKGROUND: We recently reported a method using positron emission tomography (PET) and the tracer 18F-labeled tetraphenylphosphonium (18F-TPP+) for mapping the tissue (i.e., cellular plus mitochondrial) membrane potential (ΔΨT) in the myocardium. The purpose of this work is to provide additional experimental evidence that our methods can be used to observe transient changes in the volume of distribution for 18F-TPP+ and mitochondrial membrane potential (ΔΨm). METHODS: We tested these hypotheses by measuring decreases of 18F-TPP+ concentration elicited when a proton gradient uncoupler, BAM15, is administered by intracoronary infusion during PET scanning. BAM15 is the first proton gradient uncoupler shown to affect the mitochondrial membrane without affecting the cellular membrane potential. Preliminary dose response experiments were conducted in two pigs to determine the concentration of BAM15 infusate necessary to perturb the 18F-TPP+ concentration. More definitive experiments were performed in two additional pigs, in which we administered an intravenous bolus plus infusion of 18F-TPP+ to reach secular equilibrium followed by an intracoronary infusion of BAM15. RESULTS: Intracoronary BAM15 infusion led to a clear decrease in 18F-TPP+ concentration, falling to a lower level, and then recovering. A second BAM15 infusion reduced the 18F-TPP+ level in a similar fashion. We observed a maximum depolarization of 10 mV as a result of the BAM15 infusion. SUMMARY: This work provides evidence that the total membrane potential measured with 18F-TPP+ PET is sensitive to temporal changes in mitochondrial membrane potential.

Structure-activity relationship studies of four novel 4-aminopyridine K+ channel blockers.

4-Aminopyridine (4AP) is a specific blocker of voltage-gated potassium channels (KV1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [18F]3F4AP, a radiofluorinated analog of 4AP, also binds to KV1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate four novel 4AP derivatives containing methyl (-CH3), methoxy (-OCH3) as well as trifluoromethyl (-CF3) in the 2 and 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (basicity and lipophilicity) and analyzed their ability to block Shaker K+ channel under different voltage and pH conditions. Our results demonstrate that three of the four derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP and 3F4AP; 3-methoxy- and 3-trifluoromethyl-4-aminopyridine (3MeO4AP and 3CF34AP) were found to be about 3- to 4-fold less potent than 4AP; and 2-trifluoromethyl-4-AP (2CF34AP) was found to be about 60-fold less active. These results suggest that these novel derivatives are potential candidates for therapy and imaging.

Syntheses of [11C]2- and [11C]3-trifluoromethyl-4-aminopyridine: potential PET radioligands for demyelinating diseases.

Trifluoromethyl groups are of great interest in PET radiopharmaceuticals. Radiolabelled 4-aminopyridine (4AP) derivatives have been proposed for imaging demyelinating diseases. Here, we describe methods for producing 11C-trifluoromethylated derivatives of 4AP and present early imaging results with [11C]3-trifluoromethyl-4AP in a rhesus macaque. This study shows the utility of [11C]CuCF3 for labelling pyridines and provides initial evidence for the potential use of [11C]3-trifluoromethyl-4AP as a PET radioligand.

Tuning the Color Palette of Fluorescent Copper Sensors through Systematic Heteroatom Substitution at Rhodol Cores.

Copper is an essential nutrient for sustaining life, and emerging data have expanded the roles of this metal in biology from its canonical functions as a static enzyme cofactor to dynamic functions as a transition metal signal. At the same time, loosely bound, labile copper pools can trigger oxidative stress and damaging events that are detrimental if misregulated. The signal/stress dichotomy of copper motivates the development of new chemical tools to study its spatial and temporal distributions in native biological contexts such as living cells. Here, we report a family of fluorescent copper sensors built upon carbon-, silicon-, and phosphorus-substituted rhodol dyes that enable systematic tuning of excitation/emission colors from orange to near-infrared. These probes can detect changes in labile copper levels in living cells upon copper supplementation and/or depletion. We demonstrate the ability of the carbon-rhodol based congener, Copper Carbo Fluor 1 (CCF1), to identify elevations in labile copper pools in the Atp7a-/- fibroblast cell model of the genetic copper disorder Menkes disease. Moreover, we showcase the utility of the red-emitting phosphorus-rhodol based dye Copper Phosphorus Fluor 1 (CPF1) in dual-color, dual-analyte imaging experiments with the green-emitting calcium indicator Calcium Green-1 to enable simultaneous detection of fluctuations in copper and calcium pools in living cells. The results provide a starting point for advancing tools to study the contributions of copper to health and disease and for exploiting the rapidly growing palette of heteroatom-substituted xanthene dyes to rationally tune the optical properties of fluorescent indicators for other biologically important analytes.

Stage specific effects of soluble copper and copper oxide nanoparticles during sea urchin embryo development and their relation to intracellular copper uptake.

The effects of exposure to either soluble copper (copper sulfate) or copper oxide nanoparticles (nano-CuO) during specific early developmental stages of sea urchin embryos were analyzed. Soluble copper caused significant malformations in embryos (skeletal malformations, delayed development or gut malformations) when present at any given stage, while cleavage stage was the most sensitive to nano-CuO exposure causing skeletal malformations and decreased total antioxidant capacity. The stage specificity was linked to higher endocytic activity during the first hours of development that leads to higher accumulation of copper in specific cells critical for development. Results indicate that nano-CuO results in higher accumulation of copper inside of embryos and this intracellular copper is more persistent as compared to soluble copper. The possible implications later in development are discussed.

Cell-surface copper transporters and superoxide dismutase 1 are essential for outgrowth during fungal spore germination.

During fungal spore germination, a resting spore returns to a conventional mode of cell division and resumes vegetative growth, but the requirements for spore germination are incompletely understood. Here, we show that copper is essential for spore germination in Schizosaccharomyces pombe Germinating spores develop a single germ tube that emerges from the outer spore wall in a process called outgrowth. Under low-copper conditions, the copper transporters Ctr4 and Ctr5 are maximally expressed at the onset of outgrowth. In the case of Ctr6, its expression is broader, taking place before and during outgrowth. Spores lacking Ctr4, Ctr5, and the copper sensor Cuf1 exhibit complete germination arrest at outgrowth. In contrast, ctr6 deletion only partially interferes with formation of outgrowing spores. At outgrowth, Ctr4-GFP and Ctr5-Cherry first co-localize at the spore contour, followed by re-location to a middle peripheral spore region. Subsequently, they move away from the spore body to occupy the periphery of the nascent cell. After breaking of spore dormancy, Ctr6 localizes to the vacuole membranes that are enriched in the spore body relative to the germ tube. Using a copper-binding tracker, results showed that labile copper is preferentially localized to the spore body. Further analysis showed that Ctr4 and Ctr6 are required for copper-dependent activation of the superoxide dismutase 1 (SOD1) during spore germination. This activation is critical because the loss of SOD1 activity blocked spore germination at outgrowth. Taken together, these results indicate that cell-surface copper transporters and SOD1 are required for completion of the spore germination program.

Thioether Coordination Chemistry for Molecular Imaging of Copper in Biological Systems.

Copper is an essential element in biological systems. Its potent redox activity renders it necessary for life, but at the same time, misregulation of its cellular pools can lead to oxidative stress implicated in aging and various disease states. Copper is commonly thought of as a static cofactor buried in protein active sites; however, evidence of a more loosely bound, labile pool of copper has emerged. To help identify and understand new roles for dynamic copper pools in biology, we have developed selective molecular imaging agents for this metal, drawing inspiration from both biological binding motifs and synthetic model complexes that reveal thioether coordination as a general design strategy for selective and sensitive copper recognition. In this review, we summarize some contributions, primarily from our own laboratory, on fluorescence- and magnetic resonance-based molecular-imaging probes for studying copper in living systems using thioether coordination chemistry.

Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems.

Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed "recognition" and "reactivity". Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the "cuprosome" as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals.

Synthetic fluorescent probes for studying copper in biological systems.

The potent redox activity of copper is required for sustaining life. Mismanagement of its cellular pools, however, can result in oxidative stress and damage connected to aging, neurodegenerative diseases, and metabolic disorders. Therefore, copper homeostasis is tightly regulated by cells and tissues. Whereas copper and other transition metal ions are commonly thought of as static cofactors buried within protein active sites, emerging data points to the presence of additional loosely bound, labile pools that can participate in dynamic signalling pathways. Against this backdrop, we review advances in sensing labile copper pools and understanding their functions using synthetic fluorescent indicators. Following brief introductions to cellular copper homeostasis and considerations in sensor design, we survey available fluorescent copper probes and evaluate their properties in the context of their utility as effective biological screening tools. We emphasize the need for combined chemical and biological evaluation of these reagents, as well as the value of complementing probe data with other techniques for characterizing the different pools of metal ions in biological systems. This holistic approach will maximize the exciting opportunities for these and related chemical technologies in the study and discovery of novel biology of metals.

Copper is an endogenous modulator of neural circuit spontaneous activity.

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.