ABSTRACT
INTRODUCTION: Recently, [(18)F]FE@SUPPY and [(18)F]FE@SUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A(3) receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A(3) receptor PET tracers. METHODS: In vitro carboxylesterase (CES) experiments were conducted using incubation mixtures containing different concentrations of the two substrates, porcine CES and phosphate-buffered saline. Enzymatic reactions were stopped by adding acetonitrile/methanol (10:1) after various time points and analyzed by a high-performance liquid chromatography (HPLC) standard protocol. In vivo experiments were conducted in male wild-type rats; tracers were injected through a tail vein. Rats were sacrificed after various time points (n=3), and blood and brain samples were collected. Sample cleanup was performed by an HPLC standard protocol. RESULTS: The rate of enzymatic hydrolysis by CES demonstrated Michaelis-Menten constants in a micromolar range (FE@SUPPY, 20.15 microM, and FE@SUPPY:2, 13.11 microM) and limiting velocities of 0.035 and 0.015 microM/min for FE@SUPPY and FE@SUPPY:2, respectively. Degree of metabolism in blood showed the following: 15 min pi 47.7% of [(18)F]FE@SUPPY was intact compared to 33.1% of [(18)F]FE@SUPPY:2; 30 min pi 30.3% intact [(18)F]FE@SUPPY was found compared to 15.6% [(18)F]FE@SUPPY:2. In brain, [(18)F]FE@SUPPY:2 formed an early hydrophilic metabolite, whereas metabolism of [(18)F]FE@SUPPY was not observed before 30 min pi CONCLUSION: Knowing that metabolism in rats is several times faster than in human, we conclude that [(18)F]FE@SUPPY should be stable for the typical time span of a clinical investigation. As a consequence, from a metabolic point of view, one would tend to decide in favor of [(18)F]FE@SUPPY.
Subject(s)
Nicotinic Acids/metabolism , Animals , Carboxylesterase/metabolism , Drug Stability , Fluorine Radioisotopes , Male , Nicotinic Acids/chemistry , Positron-Emission Tomography , Radioactive Tracers , Radiochemistry , RatsABSTRACT
PURPOSE: Since the late 1980s, cocaine analogues based on the phenyltropane structure, such as [(11)C]CFT and [(123)I]beta-CIT have been used for the imaging of the dopamine transporter. FE@CIT (fluoropropyl ester) and FP-CIT (N-fluoropropyl derivative) are further analogues. The aim of this study was to (1) evaluate and compare the metabolic stability of beta-CIT, FP-CIT and FE@CIT against carboxyl esterases and (2) evaluate selectivity of [(18)F]FE@CIT compared to [(123)I]beta-CIT and [(123)I]FP-CIT using autoradiography. METHODS: In vitro enzymatic hydrolysis assays were performed using different concentrations of beta-CIT, FE@CIT and FP-CIT with constant concentrations of carboxyl esterase. Autoradiography was performed on coronal 20-microm rat brain sections incubated with different radioactivity concentrations of [(123)I]beta-CIT, [(123)I]FP-CIT or [(18)F]FE@CIT and, additionally, with 3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile [serotonin transporter (SERT)] and nisoxetine [norepinephrine transporter (NET)] for blocking experiments. RESULTS: In vitro assays showed Michaelis-Menten constants of 175 micromol (beta-CIT), 183 micromol (FE@CIT) and 521 micromol (FP-CIT). Limiting velocities were 0.1005 micromol/min (beta-CIT), 0.1418 micromol/min (FE@CIT) and 0.1308 micromol/min (FP-CIT). This indicates a significantly increased stability of FP-CIT, whereas carboxyl esterase stability of beta-CIT and FE@CIT showed no significant difference. Autoradiographic analyses revealed a good correlation between dopamine transporter (DAT)-rich regions and the uptake pattern of FE@CIT. Blocking experiments showed a higher DAT selectivity for [(18)F]FE@CIT than for the other two tracers. CONCLUSION: We found that (1) the metabolic stability of FE@CIT was comparable to that of beta-CIT, whereas FP-CIT showed higher resistance to enzymatic hydrolysis; and (2) the overall uptake pattern of [(18)F]FE@CIT on brain slices was comparable to that of [(123)I]beta-CIT and [(123)I]FPCIT. After blocking of NET and SERT binding, a significantly higher DAT selectivity was observed for [(18)F]FE@CIT. Hence, [(18)F]FE@CIT may be of interest for further clinical application.
Subject(s)
Cocaine/analogs & derivatives , Nortropanes/metabolism , Tropanes/metabolism , Aniline Compounds/pharmacology , Animals , Autoradiography , Binding, Competitive , Carboxylesterase/pharmacology , Cocaine/metabolism , Dopamine Plasma Membrane Transport Proteins/drug effects , Dopamine Plasma Membrane Transport Proteins/metabolism , Drug Stability , Fluoxetine/analogs & derivatives , Fluoxetine/pharmacology , Kinetics , Male , Norepinephrine Plasma Membrane Transport Proteins/antagonists & inhibitors , Rats , Rats, Wistar , Selective Serotonin Reuptake Inhibitors/pharmacology , Sulfides/pharmacologyABSTRACT
INTRODUCTION: Changes of the adenosine A(3) receptor subtype (A3AR) expression have been shown in a variety of pathologies, especially neurological and affective disorders, cardiac diseases and oncological and inflammation processes. Recently, 5-(2-fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate (FE@SUPPY) was presented as a high-affinity ligand for the A3AR with good selectivity. Our aims were the development of a suitable labeling precursor, the establishment of a reliable radiosynthesis for the fluorine-18-labeled analogue [(18)F]FE@SUPPY and a first evaluation of [(18)F]FE@SUPPY in rats. METHODS: [(18)F]FE@SUPPY was prepared in a feasible and reliable manner by radiofluorination of the corresponding tosylated precursor. Biodistribution was carried out in rats, and organs were removed and counted. Autoradiography was performed on rat brain slices in the presence or absence of 2-Cl-IB-MECA. RESULTS: Overall yields and radiochemical purity were sufficient for further preclinical and clinical applications. The uptake pattern of [(18)F]FE@SUPPY found in rats mainly followed the described mRNA distribution pattern of the A3AR. Specific uptake in brain was demonstrated by blocking with a selective A3AR agonist. CONCLUSION: We conclude that [(18)F]FE@SUPPY has the potential to serve as the first positron emission tomography tracer for the A3AR.
Subject(s)
Fluorine Radioisotopes , Nicotinic Acids/chemical synthesis , Positron-Emission Tomography , Radiopharmaceuticals/chemical synthesis , Receptor, Adenosine A3/metabolism , Animals , Autoradiography , Male , Nicotinic Acids/metabolism , Radiopharmaceuticals/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Tissue DistributionABSTRACT
INTRODUCTION: The translation of 11C-labeled compounds into their respective 18F-labeled derivatives is an important tool in the rapid development of positron emission tomography (PET) tracers. Thus, our aim was the development of a general method for the preparation of 18F-fluoroethylated compounds that (a) is applicable to a variety of precursors, (b) can be performed in a fully automated commercially available synthesizer and (c) enables this rapid translation of 11C-methylated tracers into their 18F-fluoroethylated analogs sharing the same precursor molecules. METHODS: Ten methods for the preparation and purification of different 18F-fluoroethylating agents were compared. Subsequently, five 18F-labeled PET tracers were synthesized under fully automated conditions. RESULTS: Radiochemical yields ranged from 34.4% to 60.8%, and time consumption ranged from 20 to 55 min for all methods. Use of 1-bromo-2-[18F]fluoroethane and distillation evinced as the method of choice. CONCLUSIONS: We were able to develop a general method for the preparation of a variety of 18F-fluoroethylated molecules. The provided tool is solely based on commercially available resources and has the potential to simplify and accelerate innovative PET tracer development in the future.
