Planar chromatographic analysis and quantification of short-lived radioactive metabolites from microdialysis fractions.

A sensitive radiochromatographic method for the quantitative determination of compounds labelled with short-lived beta-emitting radionuclides in microdialysates is described. The method is well suited for microdialysis (MD) samples, which have small volumes and low concentrations of compounds. An 18F-labelled (beta+; T(1/2)=109.8 min) radiopharmaceutical, (1R,2S)-4-[18F]fluorometaraminol (FMR), was injected intravenously into rats, and microdialysis fractions were then collected from the blood at 15 min intervals. Fractions were analyzed for FMR and its radioactive metabolites by planar chromatography combined with digital photostimulated luminescence autoradiography. The lowest detectable 18F-radioactivity was 0.24 Bq/application and the limit of quantification was 0.31 Bq/application with 4-16 h exposure. The method was found to be highly sensitive and linear in the range of 0.1 Bq-2 kBq. This method thus allows the quantification of beta-emitting radiopharmaceuticals in sequential microdialysis fractions with good time-resolution.

A novel electrophilic synthesis and evaluation of medium specific radioactivity (1R,2S)-4-[18F]fluorometaraminol, a tracer for the assessment of cardiac sympathetic nerve integrity with PET.

(1R,2S)-4-[18F]fluorometaraminol (4-[18F]FMR), a tracer for cardiac sympathetic innervation, was synthesized by electrophilic aromatic substitution. A trimethylstannyl precursor, protected with tert-butoxycarbonyl protecting groups, was radiofluorinated with high specific radioactivity [18F]F2. Specific radioactivity of 4-[18F]FMR, in average 11.8 +/-3.3 GBq/micromol, was improved 40-800-fold in comparison to the previous electrophilic fluorinations. The biodistribution of 4-[18F]FMR in rat was in accordance with the known distribution of sympathetic innervation. 4-[18F]FMR showed no metabolic degradation in left ventricle of rat heart, where the uptake was high, rapid and specific.

Relative uptake, metabolism, and beta-receptor binding of (1R,2S)-4-(18)F-fluorometaraminol and (123)I-MIBG in normotensive and spontaneously hypertensive rats.

UNLABELLED: The objective of the study was to compare relative uptake, metabolism, and beta-receptor affinity of the new positron-emitting uptake-1 tracer (1R,2S)-4-(18)F-fluorometaraminol (4-FM) with those of the SPECT pharmaceutical meta-(123)I-iodobenzylguanidine (MIBG) in Wistar Kyoto (WKY) rats and spontaneously hypertensive (SHR) rats. METHODS: No-carrier-added 4-(18)F-FM was applied to SHR and WKY rats in vivo and to retrogradely perfused hearts in vitro. Cardiac and extracardiac distribution was assessed, and metabolite formation was determined by thin-layer chromatography. The in vivo experiments were repeated with no-carrier-added (123)I-MIBG. By means of autoradiography, the beta-receptor affinity of 4-FM was compared with that of MIBG and propranolol (10 micromol/L) through displacement of (125)I-iodocyanopindolol (1.5 pmol/L) in slices of heart and spleen. RESULTS: Cardiomyopathic hearts showed heterogeneous 4-(18)F-FM uptake with gradients up to 3.6 in vivo and in vitro between different regions of the heart. Control hearts showed such gradients in 4-(18)F-FM uptake only in vitro. (123)I-MIBG exhibited a less heterogeneous in vivo distribution in SHR hearts. Extracardiac differences between WKY and SHR were found for uptake of 4-(18)F-FM in the spleen (63.3% plus minus 4% vs. 38.8% plus minus 5.7% of cardiac activity) and for renal uptake of (123)I-MIBG (373% plus minus 27% vs. 81.4% plus minus 17% of cardiac activity). Metabolites of 4-(18)F-FM were found only in the liver and those of (123)I-MIBG were found in the liver and kidney with a nearly equal relative fraction in both types of animals of about 20%, 60%, and 30%, respectively. 4-FM suppressed cardiac-specific beta-receptor binding of (125)I-iodocyanopindolol in heart and spleen of both types of animals significantly, whereas MIBG had almost no effect. CONCLUSION: The more heterogeneous cardiac distribution of 4-(18)F-FM suggests that it reflects alterations in uptake-1 better than (123)I-MIBG in addition to the possibility of quantification and higher spatial resolution by PET compared with SPECT. Altered biotransformation in cardiomyopathic diseases may also impair the evaluation of (123)I-MIBG-SPECT data. The beta-receptor binding of 4-(18)F-FM must be further elucidated.

Synthesis of high-specific-radioactivity 4- and 6-[18F]fluorometaraminol-PET tracers for the adrenergic nervous system of the heart.

Fluorine-18- (t(1/2) 109.8 min) and carbon-11 (t(1/2) 20.4 min)-labeled norepinephrine analogues have been found previously to be useful positron-emission-tomography (PET) radioligands to map adrenergic nerve terminals of the heart. Metaraminol ((1R,2S)-2-amino-1-(3-hydroxyphenyl)-1-propanol) is a metabolically stable structural analogue of norepinephrine and possesses high affinity towards the norepinephrine transporter and the vesicular monoamine transporter. This paper presents the radiosynthesis of new positron-emission-tomography halogeno analogues of metaraminol labeled with high specific radioactivity. Firstly, fluorine-18-labeled 4-fluorometaraminol (4-[18F]FMR or (1R,2S)-2-amino-1-(4-[18F]fluoro-3-hydroxyphenyl)-1-propanol) and its three other stereoisomers were prepared based on the following key steps: (a) condensation of the corresponding no-carrier-added labeled fluorobenzaldehyde with nitroethane, and (b) HPLC (C18 and chiral) resolution of the diastereomeric product mixture into the four individual enantiomers. Secondly, the corresponding 6-fluoro analogues, fluorine-18-labeled 6-fluorometaraminol (6-[18F]FMR or (1R,2S)-2-amino-1-(2-[18F]fluoro-5-hydroxyphenyl)-1-propanol) and its three other enantiomers, were prepared in an analogous way. Typically, 0.48-0.55 GBq of 4-[18F]FMR and 0.14-0.15 GBq of 6-[18F]FMR could be obtained after 120-160 min total synthesis time, with a specific radioactivity of 56-106 GBq/micromol. Furthermore, the synthesis of racemic 4-fluorometaraminol and 6-fluorometaraminol as reference compounds was performed. as well as independent chiral syntheses of the optically active (1R,2S) enantiomers. For the chiral syntheses, the key step was an electrophilic fluorination with acetyl hypofluorite of (1R,2S)-configurated organometallic derivatives of metaraminol. Tissue distribution studies in rats suggested that both 4-[18F]FMR and 6-[18F]FMR display similar affinity towards the presynaptic adrenergic nerve terminal in the heart. From a practical point of view, 4-[18F]FMR appeared to be the more attractive candidate for future PET investigations, due to higher radiochemical yields.

High specific radioactivity (1R,2S)-4-[(18)F]fluorometaraminol: a PET radiotracer for mapping sympathetic nerves of the heart.

The radiolabeled catecholamine analogue (1R, 2S)-6-[(18)F]fluorometaraminol (6-[(18)F]FMR) is a substrate for the neuronal norepinephrine transporter. It has been used as a positron emission tomography (PET) ligand to map sympathetic nerves in dog heart. 6-[(18)F]FMR could be only synthesized with low specific radioactivity, which precluded its use in human subjects. We have recently prepared (1R,2S)-4-[(18)F]fluorometaraminol (4-[(18)F]FMR), a new fluoro-analogue of metaraminol, with high specific radioactivity (56-106 GBq/micromol). In the present study, we demonstrate in rats that 4-[(18)F]FMR possesses similar affinity toward myocardial norepinephrine transport mechanisms as 6-[(18)F]FMR. When compared with control animals, an 80% and 76% reduction in myocardial uptake was observed in animals pretreated with desipramine (an inhibitor of the neuronal norepinephrine transporter) and with reserpine (a blocker of the vesicular storage of monoamines), respectively. The entire radioactivity in rat myocardium represented unmetabolized parent tracer as determined by high performance liquid chromatography analysis of tissue extracts. In dogs, myocardial kinetics of 4-[(18)F]FMR were assessed using PET. A rapid and high uptake was observed, followed by prolonged cardiac retention. A heart-to-lung ratio of 15 was reached 10 min after injection of the radiotracer. Pretreatment with desipramine reduced the heart half-life of 4-[(18)F]FMR by 90% compared with control. Moreover, an infusion of tyramine caused a rapid decline of radioactivity in the heart. This demonstrates that 4-[(18)F]FMR specifically visualizes sympathetic neurons in dog heart. High specific radioactivity 4-[(18)F]FMR is a promising alternative to 6-[(18)F]FMR for myocardial neuronal mapping with PET in humans.

Effect of regional myocardial ischemia on sympathetic nervous system as assessed by fluorine-18-metaraminol.

With the introduction of radiolabeled catecholamine analogues, the noninvasive evaluation of the cardiac sympathetic nervous system has become possible. This study evaluated the effect of regional ischemia on myocardial retention of the new norepinephrine analogue 6-[18F] fluorometaraminol (FMR) in the open chest dog model. Six dogs were injected intravenously with FMR following 30-min occlusion of the left anterior descending artery. Six sham animals served as control group. Regional myocardial blood flow as determined by microspheres decreased 87% during ischemia (p less than 0.01), but was not significantly different from control myocardium following reperfusion. Regional myocardial 18F activity as determined postmortem was significantly reduced in reperfused myocardium (-34%), which paralleled an 18% reduction of tissue norepinephrine concentration. Thus, short time periods of coronary occlusion affect neuronal function indicating the sensitivity of the sympathetic nerve terminals to ischemia. FMR provides a new tracer approach for the characterization of neuronal integrity in postischemic myocardium.

Neuronal mapping of the heart with 6-[18F]fluorometaraminol.

The false neurotransmitter metaraminol labeled with fluorine-18 has been used to noninvasively assess regional adrenergic nerve density in the canine heart. Intravenous administration of 6-[18F]fluorometaraminol (FMR) results in high, selective accumulation of radioactivity in the heart; drug blocking studies with desipramine and reserpine confirm the neuronal locus of FMR. Iodine-125 labeled metaraminol, however, shows no selective accumulation in the canine heart. Positron emission tomography (PET) analyses with FMR of closed-chest dogs bearing left ventricular neuronal defects clearly delineate the region of neuronal impairment; blood perfusion in the left ventricle wall was homogeneous as determined by [13N]NH3 tomograms. The accumulation of FMR in regionally denervated dog heart correlates closely (r = 0.88) with endogenous norepinephrine concentrations. PET-generated 18F time-activity curves demonstrate marked kinetic differences between normal and denervated myocardium. FMR/PET analysis could be used to assess the heterogeneity of sympathetic innervation in human heart disease contingent on the development of FMR with sufficiently high specific activity to clearly avoid pressor activity.

The heritage of radiotracers for positron emission tomography.

The history of PET research clearly demonstrates that it is advances in chemistry coupled with a detailed examination of the biochemistry of new radiotracers that have allowed the PET method to be applied to new areas of biology and medicine. Radiotracers whose regional distribution reflects glucose metabolism, neurotransmitter activity and enzyme activity have all required the development of rapid synthetic methods for the radiotracers themselves and the characterization of their biochemical behavior. This article traces some of the advances in the production of labeled precursors and in radiotracer synthesis and evaluation which have shaped the rapidly expanding application of PET to problems in the neurosciences, in cardiology and in oncology.

Metabolic fate of the heart agent [18F]6-fluorometaraminol.

Studies were performed to determine whether [18F]6-fluorometaraminol (18F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with 18F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25-90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of 18F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (less than or equal to 0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that 18F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.

6-[18F]fluorometaraminol: a radiotracer for in vivo mapping of adrenergic nerves of the heart.

The false neurotransmitter metaraminol has been 18F labeled and evaluated as a possible heart imaging agent on the basis of its selective accumulation in adrenergic nerves. Reaction of 6-(acetoxymercurio)-N-t-BOC-metaraminol with acetyl hypofluorite followed by removal of the BOC group provides a regiospecific synthesis of 6-fluorometaraminol (4). Use of acetyl hypo[18F]fluorite gives [18F]-4 in 60 min in 20-42% radiochemical yield. Systemic blockade of the neuronal uptake-1 carrier with desmethylimipramine or systemic destruction of the adrenergic nerves with 6-hydroxydopamine lowers [18F]-4 accumulation greater than or equal to 85% in all four regions of the rat heart. These preliminary findings suggest that [18F]-4 could be used to assess neuronal damage in various heart diseases by positron emission tomography.