Quantifying cardiac sympathetic denervation: first studies of 18F-fluorohydroxyphenethylguanidines in cardiomyopathy patients.

PURPOSE: 4-18F-Fluoro-m-hydroxyphenethylguanidine (18F-4F-MHPG) and 3-18F-fluoro-p-hydroxyphenethylguanidine (18F-3F-PHPG) were developed for quantifying regional cardiac sympathetic nerve density using tracer kinetic analysis. The aim of this study was to evaluate their performance in cardiomyopathy patients. METHODS: Eight cardiomyopathy patients were scanned with 18F-4F-MHPG and 18F-3F-PHPG. Also, regional resting perfusion was assessed with 13N-ammonia. 18F-4F-MHPG and 18F-3F-PHPG kinetics were analyzed using the Patlak graphical method to obtain Patlak slopes Kp (mL/min/g) as measures of regional nerve density. Patlak slope polar maps were used to evaluate the pattern and extent of cardiac denervation. For comparison, "retention index" (RI) values (mL blood/min/mL tissue) were also calculated and used to assess denervation. Perfusion polar maps were used to estimate the extent of hypoperfusion. RESULTS: Patlak analysis of 18F-4F-MHPG and 18F-3F-PHPG kinetics was successful in all subjects, demonstrating the robustness of this approach in cardiomyopathy patients. Substantial regional denervation was observed in all subjects, ranging from 25 to 74% of the left ventricle. Denervation zones were equal to or larger than the size of corresponding areas of hypoperfusion. The two tracers provided comparable metrics of regional nerve density and the extent of left ventricular denervation. 18F-4F-MHPG exhibited faster liver clearance than 18F-3F-PHPG, reducing spillover from the liver into the inferior wall. 18F-4F-MHPG was also metabolized more consistently in plasma, which may allow application of population-averaged metabolite corrections. CONCLUSION: The advantages of 18F-4F-MHPG (more rapid liver clearance, more consistent metabolism in plasma) make it the better imaging agent to carry forward into future clinical studies in patients with cardiomyopathy. TRIAL REGISTRATION: Registered at the ClinicalTrials.gov website (NCT02669563). URL: https://clinicaltrials.gov/ct2/show/NCT02669563.

Improved synthesis of 4-[18 F]fluoro-m-hydroxyphenethylguanidine using an iodonium ylide precursor.

Fluorine-18 labeled hydroxyphenethylguanidines were recently developed in our laboratory as a new class of PET radiopharmaceuticals for quantifying regional cardiac sympathetic nerve density in heart disease patients. Studies of 4-[18 F]fluoro-m-hydroxyphenethylguanidine ([18 F]4F-MHPG) and 3-[18 F]fluoro-p-hydroxyphenethylguanidine ([18 F]3F-PHPG) in human subjects have shown that these radiotracers can be used to generate high-resolution maps of regional sympathetic nerve density using the Patlak graphical method. Previously, these compounds were synthesized using iodonium salt precursors, which provided sufficient radiochemical yields for on-site clinical PET studies. However, we were interested in exploring new methods that could offer significantly higher radiochemical yields. Spirocyclic iodonium ylide precursors have recently been established as an attractive new approach to radiofluorination of electron-rich aromatic compounds, offering several advantages over iodonium salt precursors. The goal of this study was to prepare a spirocyclic iodonium ylide precursor for synthesizing [18 F]4F-MHPG and evaluate its efficacy in production of this radiopharmaceutical. Under optimized automated reaction conditions, the iodonium ylide precursor provided radiochemical yields averaging 7.8% ± 1.4% (n = 8, EOS, not decay corrected), around threefold higher than those achieved previously using an iodonium salt precursor. With further optimization and scale-up, this approach could potentially support commercial distribution of [18 F]4F-MHPG to PET centers without on-site radiochemistry facilities.

First-in-Human Studies of [18F] Fluorohydroxyphenethylguanidines.

BACKGROUND: Disease-induced damage to cardiac autonomic nerve populations is associated with an increased risk of sudden cardiac death. The extent of cardiac sympathetic denervation, assessed using planar scintigraphy or positron emission tomography, has been shown to predict the risk of arrhythmic events in heart failure patients staged for implantable cardioverter defibrillator therapy. The goal of this study was to perform first-in-human evaluations of 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine, 2 new positron emission tomography radiotracers developed for quantifying regional cardiac sympathetic nerve density. METHODS AND RESULTS: Cardiac positron emission tomography studies with 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine were performed in normal subjects (n=4 each) to assess their imaging properties and organ kinetics. Patlak graphical analysis of their myocardial kinetics was evaluated as a technique for generating nerve density metrics. Whole-body biodistribution studies (n=4 each) were acquired and used to calculate human radiation dosimetry estimates. Patlak analysis proved to be an effective approach for quantifying regional nerve density. Using 960 left ventricular volumes of interest, across-subject Patlak slopes averaged 0.107±0.010 mL/min per gram for 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 0.116±0.010 mL/min per gram for 3-[18F]fluoro-para-hydroxyphenethylguanidine. Tracer uptake was highest in heart, liver, kidneys, and salivary glands. Urinary excretion was the main elimination pathway. CONCLUSIONS: 4-[18F]fluoro-meta-hydroxyphenethylguanidine and 3-[18F]fluoro-para-hydroxyphenethylguanidine each produce high-quality positron emission tomography images of the distribution of sympathetic nerves in human heart. Patlak analysis provides reproducible measurements of regional cardiac sympathetic nerve density at high spatial resolution. Further studies of these tracers in heart failure patients will be performed to identify the best agent for clinical development. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT02385877.

[18F]Fluoro-Hydroxyphenethylguanidines: Efficient Synthesis and Comparison of Two Structural Isomers as Radiotracers of Cardiac Sympathetic Innervation.

Fluorine-18 labeled phenethylguanidines are currently under development in our laboratory as radiotracers for quantifying regional cardiac sympathetic nerve density using PET imaging techniques. In this study, we report an efficient synthesis of 18F-hydroxyphenethylguanidines consisting of nucleophilic aromatic [18F]fluorination of a protected diaryliodonium salt precursor followed by a single deprotection step to afford the desired radiolabeled compound. This approach has been shown to reliably produce 4-[18F]fluoro-m-hydroxyphenethylguanidine ([18F]4F-MHPG, [18F]1) and its structural isomer 3-[18F]fluoro-p-hydroxyphenethylguanidine ([18F]3F-PHPG, [18F]2) with good radiochemical yields. Preclinical evaluations of [18F]2 in nonhuman primates were performed to compare its imaging properties, metabolism, and myocardial kinetics with those obtained previously with [18F]1. The results of these studies have demonstrated that [18F]2 exhibits imaging properties comparable to those of [18F]1. Myocardial tracer kinetic analysis of each tracer provides quantitative metrics of cardiac sympathetic nerve density. Based on these findings, first-in-human PET studies with [18F]1 and [18F]2 are currently in progress to assess their ability to accurately measure regional cardiac sympathetic denervation in patients with heart disease, with the ultimate goal of selecting a lead compound for further clinical development.

4-[18F]Fluoro-m-hydroxyphenethylguanidine: a radiopharmaceutical for quantifying regional cardiac sympathetic nerve density with positron emission tomography.

4-[(18)F]Fluoro-m-hydroxyphenethylguanidine ([(18)F]4F-MHPG, [(18)F]1) is a new cardiac sympathetic nerve radiotracer with kinetic properties favorable for quantifying regional nerve density with PET and tracer kinetic analysis. An automated synthesis of [(18)F]1 was developed in which the intermediate 4-[(18)F]fluoro-m-tyramine ([(18)F]16) was prepared using a diaryliodonium salt precursor for nucleophilic aromatic [(18)F]fluorination. In PET imaging studies in rhesus macaque monkeys, [(18)F]1 demonstrated high quality cardiac images with low uptake in lungs and the liver. Compartmental modeling of [(18)F]1 kinetics provided net uptake rate constants Ki (mL/min/g wet), and Patlak graphical analysis of [(18)F]1 kinetics provided Patlak slopes Kp (mL/min/g). In pharmacological blocking studies with the norepinephrine transporter inhibitor desipramine (DMI), each of these quantitative measures declined in a dose-dependent manner with increasing DMI doses. These initial results strongly suggest that [(18)F]1 can provide quantitative measures of regional cardiac sympathetic nerve density in human hearts using PET.

Quantification of cardiac sympathetic nerve density with N-11C-guanyl-meta-octopamine and tracer kinetic analysis.

UNLABELLED: Most cardiac sympathetic nerve radiotracers are substrates of the norepinephrine transporter (NET). Existing tracers such as (123)I-metaiodobenzylguanidine ((123)I-MIBG) and (11)C-(-)-meta-hydroxyephedrine ((11)C-HED) are flow-limited tracers because of their rapid NET transport rates. This prevents successful application of kinetic analysis techniques and causes semiquantitative measures of tracer retention to be insensitive to mild-to-moderate nerve losses. N-(11)C-guanyl-(-)-meta-octopamine ((11)C-GMO) has a much slower NET transport rate and is trapped in storage vesicles. The goal of this study was to determine whether analyses of (11)C-GMO kinetics could provide robust and sensitive measures of regional cardiac sympathetic nerve densities. METHODS: PET studies were performed in a rhesus macaque monkey under control conditions or after intravenous infusion of the NET inhibitor desipramine (DMI). Five desipramine dose levels were used to establish a range of available cardiac NET levels. Compartmental modeling of (11)C-GMO kinetics yielded estimates of the rate constants K1 (mL/min/g), k2 (min(-1)), and k3 (min(-1)). These values were used to calculate a net uptake rate constant K(i) (mL/min/g) = (K1k3)/(k2 + k3). In addition, Patlak graphical analyses of (11)C-GMO kinetics yielded Patlak slopes K(p) (mL/min/g), which represent alternative measurements of the net uptake rate constant K(i). (11)C-GMO kinetics in isolated rat hearts were also measured for comparison with other tracers. RESULTS: In isolated rat hearts, the neuronal uptake rate of (11)C-GMO was 8 times slower than (11)C-HED and 12 times slower than (11)C-MIBG. (11)C-GMO also had a long neuronal retention time (>200 h). Compartmental modeling of (11)C-GMO kinetics in the monkey heart proved stable under all conditions. Calculated net uptake rate constants K(i) tracked desipramine-induced reductions of available NET in a dose-dependent manner, with a half maximal inhibitory concentration (IC50) of 0.087 ± 0.012 mg of desipramine per kilogram. Patlak analysis provided highly linear Patlak plots, and the Patlak slopes Kp also declined in a dose-dependent manner (IC50 = 0.068 ± 0.010 mg of desipramine per kilogram). CONCLUSION: Compartmental modeling and Patlak analysis of (11)C-GMO kinetics each provided quantitative parameters that accurately tracked changes in cardiac NET levels. These results strongly suggest that PET studies with (11)C-GMO can provide robust and sensitive quantitative measures of regional cardiac sympathetic nerve densities in human hearts.

Synthesis and bioevaluation of [(18)F]4-fluoro-m-hydroxyphenethylguanidine ([(18)F]4F-MHPG): a novel radiotracer for quantitative PET studies of cardiac sympathetic innervation.

A new cardiac sympathetic nerve imaging agent, [(18)F]4-fluoro-m-hydroxyphenethylguanidine ([(18)F]4F-MHPG), was synthesized and evaluated. The radiosynthetic intermediate [(18)F]4-fluoro-m-tyramine ([(18)F]4F-MTA) was prepared and then sequentially reacted with cyanogen bromide and NH4Br/NH4OH to afford [(18)F]4F-MHPG. Initial bioevaluations of [(18)F]4F-MHPG (biodistribution studies in rats and kinetic studies in the isolated rat heart) were similar to results previously reported for the carbon-11 labeled analog [(11)C]4F-MHPG. The neuronal uptake rate of [(18)F]4F-MHPG into the isolated rat heart was 0.68ml/min/g wet and its retention time in sympathetic neurons was very long (T1/2 >13h). A PET imaging study in a nonhuman primate with [(18)F]4F-MHPG provided high quality images of the heart, with heart-to-blood ratios at 80-90min after injection of 5-to-1. These initial kinetic and imaging studies of [(18)F]4F-MHPG suggest that this radiotracer may allow for more accurate quantification of regional cardiac sympathetic nerve density than is currently possible with existing neuronal imaging agents.

Radiotracers for cardiac sympathetic innervation: transport kinetics and binding affinities for the human norepinephrine transporter.

INTRODUCTION: Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [(11)C]-(-)-meta-hydroxyephedrine, [(11)C]-(-)-epinephrine, and a series of [(11)C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [(3)H]-labeled biogenic amines were also determined. METHODS: Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [(3)H]mazindol provided estimates of binding affinities (KI) for NET. RESULTS: Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for 'tracer concentrations' of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r(2)=0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. CONCLUSION: The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.

Drosophila ninaB and ninaD act outside of retina to produce rhodopsin chromophore.

The Drosophila ninaB gene encodes a beta,beta-carotene-15,15'-oxygenase responsible for the centric cleavage of beta-carotene that produces the retinal chromophore of rhodopsin. The ninaD gene encodes a membrane receptor required for efficient use of beta-carotene. Despite their importance to the synthesis of visual pigment, we show that these genes are not active in the retina. Mosaic analysis shows that ninaB and ninaD are not required in the retina, and exclusive retinal expression of either gene, or both genes simultaneously, does not support rhodopsin biogenesis. In contrast, neuron-specific expression of ninaB and ninaD allows for rhodopsin biogenesis. Additional directed expression studies failed to identify other tissues supporting ninaB activity in rhodopsin biogenesis. These results show that nonretinal sites of NinaB beta,beta-carotene-15,15'-oxygenase activity, likely neurons of the central nervous system, are essential for production of the visual chromophore. Retinal or another C(20) retinoid, not members of the beta-carotene family of C(40) carotenoids, are supplied to photoreceptors for rhodopsin biogenesis.