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Abstract: Introduction: Our aim was the optimization of radiochemical parameters for the microfluidic preparation of [18F]altanserin. The four main parameters evaluated were (1) precursor concentration, (2) reaction temperature, (3) bolus flow rate through the microreactor and (4) bolus volume. Methods: For the determination of optimal reaction conditions within a flow-through microreactor synthesizer, 5–400 μL of precursor and dried [18F]fluoride solution were simultaneously pushed through the temperature-controlled reactor (180–220°C) with defined bolus flow rates of 10–60 μL/min. Radiochemical incorporation yields (RCIYs) were examined using a thin layer chromatography (TLC) set-up and radio- high-performance liquid chromatography (HPLC). Results: Optimum reaction parameters for the microfluidic set-up were determined as following: 220°C, 5–10 μL/min pump rate per reactant (10–20 μL/min reaction overall flow rate) and 2mg/mL precursor concentration in the reaction mixture. Applying these optimized conditions, RCIYs of 53.7±7.9 were observed for scaled-up preparations. A positive “bolus effect” was observed: applying higher reaction volume resulted in increased RCIYs. Conclusion: This study proved that the reaction bolus volume is an essential parameter influencing the RCIY of [18F]altanserin. A possible explanation is the inhomogeneous distribution within the reaction volume probably caused by diffusion at the bolus interface. This important finding should be considered an important variable for the evaluation of all novel radiotracers labeled using a flow-through reactor device. [Copyright &y& Elsevier]

Abstract: Introduction: Recently, first applications of microfluidic principles for radiosyntheses of positron emission tomography compounds were presented, but direct comparisons with conventional methods were still missing. Therefore, our aims were (1) the set-up of a microfluidic procedure for the preparation of the recently developed adenosine A3-receptor tracers [18F]FE@SUPPY [5-(2-[18F]fluoroethyl)2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] and [18F]FE@SUPPY:2 [5-ethyl-2,4-diethyl-3-((2-[18F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] and (2) the direct comparison of reaction conditions and radiochemical yields of the no-carrier-added nucleophilic substitution with [18F]fluoride between microfluidic and conventional methods. Methods: For the determination of optimal reaction conditions within an Advion NanoTek synthesizer, 5–50 μl of precursor and dried [18F]fluoride solution were simultaneously pushed through the temperature-controlled reactor (26°C–180°C) with defined reactant bolus flow rates (10–50 μl/min). Radiochemical incorporation yields (RCIYs) and overall radiochemical yields for large-scale preparations were compared with data from conventional batch-mode syntheses. Results: Optimal reaction parameters for the microfluidic set-up were determined as follows: 170°C, 30-μl/min pump rate per reactant (reaction overall flow rate of 60 μl/min) and 5-mg/ml precursor concentration in the reaction mixture. Applying these optimized conditions, we observed a significant increase in RCIY from 88.2% to 94.1% (P<.0001, n≥11) for [18F]FE@SUPPY and that from 42.5% to 95.5% (P<.0001, n≥5) for [18F]FE@SUPPY:2 using microfluidic instead of conventional heating. Precursor consumption was decreased from 7.5 and 10 mg to 1 mg per large-scale synthesis for both title compounds, respectively. Conclusion: The direct comparison of radiosyntheses data applying a conventional method and a microfluidic approach revealed a significant increase of RCIY using the microfluidic approach. [Copyright &y& Elsevier]

Abstract: Introduction: Carboxylesterases (CES) play a very important role in the hydrophilic biotransformation of a huge number of structurally diverse drugs and especially play a leading part in the catabolic pathway of carboxylesters or thioesters. Hence, the aim of the present study was the comparison of the in vitro stability of methyl- and ethylesters with fluoroethylesters. Methods: We incubated methyl 3β-(4-iodophenyl)tropane-2β-carboxylate (β-CIT)/2-fluoroethyl 3β-(4-iodophenyl)tropane-2β-carboxylate (FE@CIT), methyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MTO)/ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (ETO)/2-fluoroethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (FETO), ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4]diazepine-3-carboxylate (FMZ)/2-fluoroethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo-[f]imidazo[1,5-a]-[1,4]diazepine-3-carboxylate (FFMZ), methyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate (CFN)/2-fluoroethyl 1-phenylethyl-4-(N-propanoylanilino)piperidine-4-carboxylate (FE@CFN) and methyl 2,4-diethyl-3-methylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate [(Me)2@SUPPY]/2-fluorethyl 2,4-diethyl-3-ethylsulfanylcarbonyl-6-phenylpyridine-5-carboxylate (FE@SUPPY) under physiological conditions. The enzymatic reactions were stopped at different time points and analyzed by a standard protocol. Results: The Michaelis–Menten constants (K M) and limiting velocities (V max) are comparable. The statistical K M values were as follows: β-CIT/FE@CIT, P>.05; MTO/FETO, P>.06; ETO/FETO, P>.09; FMZ/FFMZ, P>.05; CFN/FE@CFN, P>.9; (Me)2@SUPPY/FE@SUPPY, P>.07. Conclusion: We found no statistical difference in stability against CES in vitro. These findings support the strategy to translate C-11-methyl-/ethylesters into their longer-lived F-18-fluoroethyl analogues. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Recently, [18F]FE@SUPPY and [18F]FE@SUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A3 receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A3 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 μM, and FE@SUPPY:2, 13.11 μM) and limiting velocities of 0.035 and 0.015 μM/min for FE@SUPPY and FE@SUPPY:2, respectively. Degree of metabolism in blood showed the following: 15 min pi 47.7% of [18F]FE@SUPPY was intact compared to 33.1% of [18F]FE@SUPPY:2; 30 min pi 30.3% intact [18F]FE@SUPPY was found compared to 15.6% [18F]FE@SUPPY:2. In brain, [18F]FE@SUPPY:2 formed an early hydrophilic metabolite, whereas metabolism of [18F]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 [18F]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 [18F]FE@SUPPY. [Copyright &y& Elsevier]

Abstract: Purpose: Since the late 1980s, cocaine analogues based on the phenyltropane structure, such as [11C]CFT and [123I]β-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 β-CIT, FP-CIT and FE@CIT against carboxyl esterases and (2) evaluate selectivity of [18F]FE@CIT compared to [123I]β-CIT and [123I]FP-CIT using autoradiography. Methods: In vitro enzymatic hydrolysis assays were performed using different concentrations of β-CIT, FE@CIT and FP-CIT with constant concentrations of carboxyl esterase. Autoradiography was performed on coronal 20-μm rat brain sections incubated with different radioactivity concentrations of [123I]β-CIT, [123I]FP-CIT or [18F]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 μmol (β-CIT), 183 μmol (FE@CIT) and 521 μmol (FP-CIT). Limiting velocities were 0.1005 μmol/min (β-CIT), 0.1418 μmol/min (FE@CIT) and 0.1308 μmol/min (FP-CIT). This indicates a significantly increased stability of FP-CIT, whereas carboxyl esterase stability of β-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 [18F]FE@CIT than for the other two tracers. Conclusion: We found that (1) the metabolic stability of FE@CIT was comparable to that of β-CIT, whereas FP-CIT showed higher resistance to enzymatic hydrolysis; and (2) the overall uptake pattern of [18F]FE@CIT on brain slices was comparable to that of [123I]β-CIT and [123I]FPCIT. After blocking of NET and SERT binding, a significantly higher DAT selectivity was observed for [18F]FE@CIT. Hence, [18F]FE@CIT may be of interest for further clinical application. [Copyright &y& Elsevier]

Abstract: Introduction: Changes of the adenosine A3 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 [18F]FE@SUPPY and a first evaluation of [18F]FE@SUPPY in rats. Methods: [18F]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 [18F]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 [18F]FE@SUPPY has the potential to serve as the first positron emission tomography tracer for the A3AR. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Introduction: The objectives of this study were to develop a simple preparation method for [68Ga]-EDTMP and to evaluate the applicability of [68Ga]-EDTMP as a potential positron emission tomography (PET) bone imaging agent using pre vivo, ex vivo and in vivo models. Methods: [68Ga]-EDTMP was prepared using [68Ga]-gallium chloride eluted from the 68Ge/68Ga generator and commercially available Multibone kits. Binding affinity to bone compartments was evaluated using a recently established pre vivo model. In vivo (microPET) and ex vivo experiments were performed in mice, and the results of which were compared with those obtained with [18F]-fluoride. Results: [68Ga]-EDTMP was accessible via simple kit preparation and predominantly accumulated in bone tissue in vivo, ex vivo and pre vivo. Binding to mineral bone was irreversible, and low binding was observed in organic bone. In vivo microPET evaluation revealed predominant uptake in bone with renal excretion. Compared with [18F]-fluoride, the uptake was lower and the PET image quality was reduced. Conclusions: From the present evaluation, apart from the autonomy for PET centers without an onsite cyclotron, the advantage of [68Ga]-EDTMP over [18F]-fluoride is not apparent and the future clinical prospect of [68Ga]-EDTMP remains speculative. [Copyright &y& Elsevier]

Abstract: The present study focused on the preparation of novel bone tracers containing yttrium as radionuclide or carrier. Moreover, these preparations were comparatively evaluated in vitro on the basis of a recently presented pre vivo model comprising binding studies on synthetic and human bone powder. It was shown that among the therapeutic radionuclides, no carrier added [90Y]-EDTMP exceeded [188Re]-EDTMP while yielding lower binding values than [153Sm]-EDTMP. Furthermore, the authors investigated the influence of “foreign” carriers added to [90Y]-EDTMP, [99mTc]-EDTMP and [111In]-EDTMP by the method of cross-complexation. The findings reveal a new paradigm: a carrier more foreign to the complexed radionuclide causes a higher binding increase on human bone matrices in vitro than a more “related” carrier. [Copyright &y& Elsevier]

[11C]Flumazenil, a highly selective benzodiazepine antagonist is the most extensively used GABAA ligand for PET so far. To overcome half life disadvantages of 11C a [18F]-labeled flumazenil derivative, 2′-[18F]fluoroflumazenil (FFMZ) was developed and biologically evaluated with respect to the GABAA receptor. Organ with the highest uptake was the pituitary gland. Brain uptake was high and followed the order cortex>thalamus>cerebellum>rest brain. Fluoroflumazenil displaced [3H]flumazenil binding from membrane GABAA receptors with an IC50value (3.5 nM) comparable to that of Flumazenil (2.8 nM). The presented data confirm the potential of [18F]FFMZ for PET imaging of the GABA-ergic system. [Copyright &y& Elsevier]