This work was supported by the Austrian Science Fund (FWF P26502-B24, M. Mitterhauser). We are grateful for the technical support of T. Zenz and A. Krcal. Furthermore, we thank K. Pallitsch for preparation of the AgOTf and H. Spreitzer for distributing the precursor.

<ol>
	<li>Saito, Y., Maruyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+melanin-concentrating+hormone+receptor+and+its+impact+on+drug+discovery.">Identification of melanin-concentrating hormone receptor and its impact on drug discovery.</a> Journal of Experimental Zoology. 305, 761-768 (2006).</li><li>Philippe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+in+vitro+&#38;+in+vivo+evaluation+of+[11C]SNAP-7941+-+the+first+PET+tracer+for+the+melanin+concentrating+hormone+receptor+1.">Preclinical in vitro &#38; in vivo evaluation of [11C]SNAP-7941 - the first PET tracer for the melanin concentrating hormone receptor 1.</a> Nuclear Medicine and Biology. 40, 919-925 (2013).</li><li>Philippe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosynthesis+of+[+11C]SNAP-7941-the+first+PET-tracer+for+the+melanin+concentrating+hormone+receptor+1+(MCHR1).">Radiosynthesis of [ 11C]SNAP-7941-the first PET-tracer for the melanin concentrating hormone receptor 1 (MCHR1).</a> Applied. Radiation and Isotopes. 70, 2287-2294 (2012).</li><li>Philippe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAPshots+of+the+MCHR1&#8239;:+a+Comparison+Between+the+PET-Tracers+[+18+F+]+FE+@+SNAP+and+[+11+C+]+SNAP-7941.">SNAPshots of the MCHR1&#8239;: a Comparison Between the PET-Tracers [ 18 F ] FE @ SNAP and [ 11 C ] SNAP-7941.</a> Molecular Imaging Biology. , 1-12 (2018).</li><li>Schirmer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheses+of+precursors+and+reference+compounds+of+the+melanin-+concentrating+hormone+receptor+1+(MCHR1)+Tracers+[11C]SNAP-7941+and+[18F]FE@SNAP+for+positron+emission+tomography.">Syntheses of precursors and reference compounds of the melanin- concentrating hormone receptor 1 (MCHR1) Tracers [11C]SNAP-7941 and [18F]FE@SNAP for positron emission tomography.</a> Molecules. 18, 12119-12143 (2013).</li><li>Miller, P. W., Long, N. J., Vilar, R., Gee, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthese+von+11C-,+18F-,+15O-+und+13N-Radiotracern+f&#252;r+die+Positronenemissionstomographie.">Synthese von 11C-, 18F-, 15O- und 13N-Radiotracern f&#252;r die Positronenemissionstomographie.</a> Angewandte Chemie. 120, 9136-9172 (2008).</li><li>Pichler, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+on+PET+radiochemistry:+part+1+-+covalent+labels+-+18+F,+11+C,+and+13+N.">An overview on PET radiochemistry: part 1 - covalent labels - 18 F, 11 C, and 13 N.</a> Journal of Nuclear Medicine. 59, 1350-1354 (2018).</li><li>Pichler, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molar+activity+-+The+keystone+in+11C-radiochemistry:+An+explorative+study+using+the+gas+phase+method.">Molar activity - The keystone in 11C-radiochemistry: An explorative study using the gas phase method.</a> Nuclear Medicine and Biology. 67, 21-26 (2018).</li><li>Larsen, P., Ulin, J., Dahlstr&#248;m, K., Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+[11C]iodomethane+by+iodination+of+[11C]methane.">Synthesis of [11C]iodomethane by iodination of [11C]methane.</a> Applied Radiation and Isotopes. 48, 153-157 (1997).</li><li>Dahl, K., Halldin, C., Schou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+the+preparation+of+carbon-11+labeled+radiopharmaceuticals.">New methodologies for the preparation of carbon-11 labeled radiopharmaceuticals.</a> Clinical and Translational Imaging. 5, 275-289 (2017).</li><li>Raaphorst, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiopharmaceuticals+for+assessing+ABC+transporters+at+the+blood-brain+barrier.">Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier.</a> Clinical. Pharmacollogy &#38; Therapeutics. 97, 362-371 (2015).</li><li>Nics, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speed+matters+to+raise+molar+radioactivity:+Fast+HPLC+shortens+the+quality+control+of+C-11+PET-tracers.">Speed matters to raise molar radioactivity: Fast HPLC shortens the quality control of C-11 PET-tracers.</a> Nuclear Medicine and Biology. 57, 28-33 (2018).</li><li>Zeilinger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+the+reliable+in+vitro+assessment+of+binding+affinity+based+on+high-resolution+real-time+data+acquisition+of+radioligand-receptor+binding+kinetics.">New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.</a> European Journal of Nuclear Medicine and Molecular Imaging (Research). 7, 22 (2017).</li></ol>

We have nothing to disclose.

The radiosynthesis of [11C]SNAP-7941 was established on a commercial synthesis module. Due to the possibility to fully automate the preparation procedure, the radiosynthesis proofed to be reliable, and improvements regarding radiation protection of the operator were achieved. The preparation of the synthesizer has an enormous impact on the quality of the radiotracer, especially in terms of molar activity. Thus, it is essential to constantly work under inert conditions (e.g., helium atmosphere), and to flush all lines located prior to the reaction vessel (target line, [11C]CH3I production cycle and reactor (see Figure 2)). Moreover, heating the respective traps and ovens before the start of the synthesis to remove moisture and atmospheric carbon increases the molar activity advantageously. Especially the AgOTf column, impregnated with graphitized carbon, is extremely sensitive to humidity. Even minor amounts of any source of moisture disturb the conversion of [11C]CH3I to [11C]CH3OTf. Before starting the synthesis, the [11C]CO2 trap and the [11C]CH3I trap have to be cooled down to room temperature again in order to enable subsequent trapping.Furthermore, it is recommended to dissolve the precursor shortly before starting the synthesis and to add the base directly into the precursor solution.
The quality control for carbon-11 radiotracers has to be rationally designed for a continuous and fast workflow. However, the most important parameters for cell culture studies are radiochemical purity and molar activity to obtain valid results. Correct evaluation of the molar activity requires a robust analytical HPLC method and the calibration curve has to cover the concentration range of the final product. The challenging part for radiotracers is to achieve a concentration above the limit of quantification (LOQ) due to small amounts, which are produced during radiosynthesis. Hence, the art is to find the balance between high molar activities to avoid receptor saturation and high enough concentrations to still be able to quantify the non-radioactive signal.
[11C]SNAP-7941 was confirmed to be a potent substrate of the human P-gp transporter, as no accumulation was observed in the untreated or vehicle-treated MDCKII-hMDR1 cells due to rapid efflux. In contrast, both experimental set ups (MDCKII-WT or pre-blocked MDCKII-hMDR1 cells) provided similar results (accumulation of [11C]SNAP-7941), supporting the versatility of this in vitro&#160;assay. MDCKII-hMDR1 cells are highly suitable for LigandTracer experiments due to their stable transfection, fast growth and persistent against shear stress caused by the rotating cell culture dish. The lack of [11C]SNAP-7941 uptake in the rat and mice brain might therefore occur caused by efflux through the P-gp transporter. Owing to the transfection of canine kidney cells with the human multi drug resistance protein 1 (hMDR-1, P-gp), the predictive value of this method for efflux transporter binding in humans is high, which is favorable in terms of a future clinical application. However, so far, the selectivity against other efflux transporter was not verified. Therefore, other cell lines can be used, expressing different prominent efflux transporters as the breast cancer resistance protein (BCRP) or multiple resistance protein-1 (MRP-1), to study interactions towards these transporters. The method is in comparison to classical accumulation or transport assays very simple and gives immediately qualitative results. Moreover, the greatest advantage is that this technology enables evaluation of direct interaction of the PET tracer and the target in real-time, in contrast to the conventional experiment using indirect quantification (mostly displacement). Additionally, the real-time radioassay software provides experimental flexibility (e.g., nuclide decay correction, measuring time and positions, etc.) and therefore, high freedom for users. On the other side, limitations of the method include a low sample throughput, as only one cell dish can be measured at a time. Furthermore, a few other technical and operational issues should be taken into account: the described technology is very sensitive to background radiation; thus, radiation sources should be kept at distance and emphasis should be placed on the background measurement prior to the experiment. Another issue concerning experiments at higher temperatures than room temperature, is the heating of the inclined support: evaporation of the cell culture medium might affect the detector. Instead of heating, the whole device is preferably placed into the incubator. Moreover, the method is limited to adherent cell lines. Through the rotation of the cell culture dish, shear stress sensitive cells might detach from the dish, which can lead to invalid results.
Nevertheless, if the experimenter pays attention to these minor drawbacks the method delivers fast and reliable results for the analysis of the kinetic behavior of preclinical PET-tracers.

[11C]SNAP-7941 was evolved as the first positron emission tomography (PET)-tracer targeting the melanin-concentrating hormone receptor 1 (MCHR1) - a receptor mainly involved in the central regulation of appetite and food intake1. Carbon-11 labelling of SNAP-7941, a well characterised MCHR1 antagonist, yielded the authentic PET-tracer2,3,4,5. However, fully automated radiosynthesis is highly challenging in terms of time efficacy and reproducibility with the short-lived radionuclide carbon-11 offering a half-life of 20 min6. The overall synthesis time should be kept to a minimum, and as a rule of thumb should not exceed 2-3 half-lives (i.e., around 40-60 min for carbon-11)7. Especially, synthesis procedures for radiotracers targeting receptor systems with low expression densities must be extensively optimized to obtain sufficient yields and consequently high molar activity8. The synthetic strategy often follows the radionuclide production within a cyclotron and release of [11C]CO2 to the synthesizer. There, [11C]CO2 is first reduced to [11C]CH4 and subsequently reacted with iodine to yield [11C]CH3I via the gas-phase method9,10. Further treatment with silver triflate yields [11C]CH3OTf directly on-line. Afterwards, this reactive carbon-11 labelled intermediate is introduced into a solution containing the precursor molecule. An automated radiosynthesis additionally involves a purification process with semi-preparative RP-HPLC including subsequent formulation of the product suitable for preclinical and clinical studies.
Regardless of the half-life of the radionuclide and the time effort of the radiosynthesis, the pharmacokinetic of a radiopharmaceutical is the most critical part to be evaluated during PET-tracer development. In terms of neuroimaging, brain entry of the PET-tracer is the main prerequisite. However, the blood brain barrier (BBB), a &#34;security border&#34; of the brain, highly expresses efflux transporters that can unload small molecules (e.g., PET-tracers) and efficiently hamper their applicability.
A huge drawback during preclinical evaluation are unexpected interactions towards these efflux transporters, which are often unrecognized in in vitro experiments and leading to failure of the PET-tracer in vivo, as observed for [11C]SNAP-7941. &#181;PET imaging in rats demonstrated low brain accumulation, which increased dramatically after administration of the P-gp inhibitor tariquidar11. These data suggested that [11C]SNAP-7941 is a substrate of this efflux transporter system impeding ligand binding to central MCHR1. Unfortunately, there is still a lack of adequate in vitro models enabling the prediction of BBB penetration in an early stage of tracer development.
Here, we describe the automated synthesis of [11C]SNAP-7941 using a synthesizer for carbon-11 methylations. The emphasis of this work is to give an overview on how to organize a consecutive experimental approach including the automated synthesis, quality control as well as successive in vitro evaluation with the very short-lived nuclide carbon-11.
First, the key steps for a successful radiosynthesis with minimal time expenditure and maximal yield are described. Then, a reliable quality control procedure is set up making the radiotracer available for potential clinical studies and meeting the criteria of the European Pharmacopoeia12. Quantification of the molar concentration and calculation of the respective molar activity is an essential requirement for the successive kinetic measurements.
Finally, a new and straightforward in vitro method evaluating [11C]SNAP-7941&#39;s interactions towards the efflux transporter, P-gp (hMDR1), is presented. The proposed kinetic model uses an easy-to-handle device that allows an immediate data interpretation and requires minimal cell culture effort13.

<table>
	<tbody>
		<tr>
			<td>Table 1: List of materials and instrumentation of the fully automated radiosynthesis of [11C]SNAP-7941</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni catalyst</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td></td>
			<td>Shimalilte Ni reduced, 80/100 mesh</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>1.04761.0100</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>for DNA synthesis, &#60; 10 ppm H2O</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td></td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>glacial</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>96%</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>B. Braun, Melsungen, Germany</td>
			<td></td>
			<td>0.9%</td>
		</tr>
		<tr>
			<td>Tetrabutylammonium hydroxide</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td></td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>SPE cartridge</td>
			<td>Waters, Milford, MA, USA</td>
			<td></td>
			<td>SepPak C18plus</td>
		</tr>
		<tr>
			<td>Semi-preparative RP-HPLC column</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>Chromolith SemiPrep RP-18e, 100-10 mm</td>
		</tr>
		<tr>
			<td>Precolumn</td>
			<td>Merck, Darmstadt, Germany</td>
			<td></td>
			<td>Chromolith Guard RP-18e, 5-4.6 mm</td>
		</tr>
		<tr>
			<td>Precursor</td>
			<td>University of Vienna, Austria</td>
			<td></td>
			<td>SNAP-acid</td>
		</tr>
		<tr>
			<td>Reference compound</td>
			<td>University of Vienna, Austria</td>
			<td></td>
			<td>SNAP-7941</td>
		</tr>
		<tr>
			<td>Silver trifluoromethanesulfonate</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpa GC</td>
			<td>Alltech, Deerfield, IL, USA</td>
			<td></td>
			<td>80/100 mesh</td>
		</tr>
		<tr>
			<td>PET trace 860 cyclotron</td>
			<td>GE Healthcare, Uppsala, Sweden</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[11C]CO2 high pressure target</td>
			<td>Air Liquide, Vienna, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRACERlabFX2 C</td>
			<td>GE Healthcare, Uppsala, Sweden</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2 + 1% O2</td>
			<td>Air Liquide, Vienna, Austria</td>
			<td></td>
			<td>Target gas</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 2: List of materials and instrumentation of the quality control of [11C]SNAP-7941.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Merck Hitachi LaChrom, L-7100</td>
			<td>Hitachi Vantara Austria GmbH (Vienna, Austria)</td>
			<td></td>
			<td>HPLC pump</td>
		</tr>
		<tr>
			<td>Merck Hitachi, L7400</td>
			<td>Hitachi Vantara Austria GmbH (Tokyo, Japan)</td>
			<td></td>
			<td>UV-detector</td>
		</tr>
		<tr>
			<td>NaI-radiodetector</td>
			<td>Raytest (Straubenhardt, Germany)</td>
			<td></td>
			<td>NaI-radiodetector</td>
		</tr>
		<tr>
			<td>Chromolith Performance RP-18e, 100-4.6 mm</td>
			<td>Merck (Darmstadt, Germany)</td>
			<td></td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>430-GC</td>
			<td>Bruker (Bremen, Germany)</td>
			<td></td>
			<td>Gas chromatograph</td>
		</tr>
		<tr>
			<td>Capillary column ID-BP20; 12 mx0.22 mmx0.25 mm</td>
			<td>SGE Ananlytical Science Pty. Ltd. (Victoria, Australia)</td>
			<td></td>
			<td>Gas capillary</td>
		</tr>
		<tr>
			<td>Wesco, osmometer Vapro 5600</td>
			<td>Sanoya Medical Systems (Vienna, Austria)</td>
			<td></td>
			<td>Osmometer</td>
		</tr>
		<tr>
			<td>g-spectrometer</td>
			<td></td>
			<td></td>
			<td>g-spectrometer</td>
		</tr>
		<tr>
			<td>Gas chromatography controlling software</td>
			<td>VARIAN (Palo Alto, California, U.S.A)</td>
			<td></td>
			<td>Galaxie Version 1.9.302.952</td>
		</tr>
		<tr>
			<td>Gamma spectrometer controlling software</td>
			<td>ORTEC (Oak Ridge, Tenessee, U.S.A.)</td>
			<td></td>
			<td>Maestro for windows Version 6.06</td>
		</tr>
		<tr>
			<td>Gamma spectrum recalling software</td>
			<td>ORTEC (Oak Ridge, Tenessee, U.S.A.)</td>
			<td></td>
			<td>Winplots version 3.21</td>
		</tr>
		<tr>
			<td>HPLC controlling software</td>
			<td>Raytest (Straubenhardt, Germany)</td>
			<td></td>
			<td>Gina Star Version 5.9</td>
		</tr>
		<tr>
			<td>inolab 740</td>
			<td>WTW (Weilheim, Germany)</td>
			<td></td>
			<td>pH meter</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 3: List of materials and instrumentation for the evaluation of the real-time kinetic behaviour of [11C]SNAP-7941.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Madin-Darby Canine Kidney cell line (MDCKII-hMDR1)</td>
			<td>Netherlands Cancer Institute (NKI, Amsterdam, Netherlands)</td>
			<td></td>
			<td>Expressing the human P-glycoprotein (hMDR1)</td>
		</tr>
		<tr>
			<td>Madin-Darby Canine Kidney cell line (MDCKII-WT)</td>
			<td>Netherlands Cancer Institute (NKI, Amsterdam, Netherlands)</td>
			<td></td>
			<td>Wildtype (WT)</td>
		</tr>
		<tr>
			<td>DMEM GlutaMAX</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Gibco 61965-026</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Gibco 10270-106</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Gibco 15140</td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 100 mm x 20 mm, Mfr.No. 664160</td>
		</tr>
		<tr>
			<td>In vitro experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM GlutaMAX</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Gibco 61965-026</td>
		</tr>
		<tr>
			<td>(&#177;)-Verapamil hydrochloride</td>
			<td>Sigma Aldrich (St. Louis, Missouri, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich (St. Louis, Missouri, USA)</td>
			<td></td>
			<td>276855-100 mL</td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 100 mm x 20 mm, Mfr.No. 664160</td>
		</tr>
		<tr>
			<td>Sterile disposable plastic pipettes</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Sterilin, 5 mL &#8211; 25 mL</td>
		</tr>
		<tr>
			<td>Sterile pipette tips</td>
			<td>VWR International GmbH, Vienna, Austria</td>
			<td></td>
			<td>Eppendorf epT.I.P.S. Biopur 20 &#181;L &#8211; 200 &#181;L</td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 250 mL, 75 cm2 red filter screw cap, Mfr.No.658175</td>
		</tr>
		<tr>
			<td>LigandTracer control Version 2.2.2</td>
			<td>Ridgeview Instruments AB, Uppsala, Sweden.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LigandTracer Yellow</td>
			<td>Ridgeview Instruments AB, Uppsala, Sweden.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LigandTracer White</td>
			<td>Ridgeview Instruments AB, Uppsala, Sweden.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 6.0</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld automated Cell Counter</td>
			<td>Millipore Corporation Billerica MA01821</td>
			<td></td>
			<td>Scepter (Cat.No. PHC00000)</td>
		</tr>
		<tr>
			<td>Cell Counter Sensors</td>
			<td>Millipore Corporation Billerica MA01821</td>
			<td></td>
			<td>Scepter Sensor 60 &#181;m (Cat.No. PHCC60050)</td>
		</tr>
	</tbody>
</table>

technical aspect of the automated synthesis and real-time kinetic evaluation of [11c]snap-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

The fully automated radiosynthesis of [11C]SNAP-7941 yielded 5.7 &#177; 2.5 GBq (4.6 &#177; 2.0% at EOB, 14.9 &#177; 5.9% based on [11C]CH3OTf; n = 10) of formulated product. The overall synthesis lasted around 40 min, where 15 min were required for preparation of [11C]CH3OTf via the gas phase method, another 5 min were necessary for radiolabelling of the precursor, followed by 10 min of semi-preparative RP-HPLC purification and 10 min for the C18 cartridge solid phase extraction and formulation. Then, a small aliquot (approx. 100-200 &#181;L) was delivered to the person responsible for the quality control, whereas the original product vial containing the ready-to-use tracer was passed to the experimenter of the real-time kinetic analysis.
Quality control was completed within 10 min after the end of synthesis. The molar activity was in a range of 72 &#177; 41 GBq/&#181;mol (n = 10) and the radiochemical purity was always &#62; 95%. All other parameters (pH, osmolality, residual solvents) fulfilled the release criteria. For the real-time kinetic assay, three different experimental setups were chosen: (A) treated and non-treated (vehicle) MDCKII-WT cells, (B) non-treated or treated with vehicle MDCKII-hMDR1 cells and (C) the latter cell line with blocking prior the real-time kinetic assay of the transporter using (&#177;)-verapamil. Applying [11C]SNAP-7941 to the wildtype cell line MDCKII-WT (P-gp non-expressing) and MDCKII-hMDR1 (P-gp expressing) shows a different kinetic behavior, as there is a fast accumulation to the wildtype cell line, whereas no accumulation was observed for the MDCKII-hMDR1 cells. However, blocking the P-gp efflux transporter inMDCKII-hMDR1 cells with (&#177;)-verapamil led to a comparable real-time kinetic as already seen for the wildtype cell line (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59557/59557fig1.jpg" /> 
Figure 1: Overview of the work-flow. Work-flow of the radiosynthesis, quality control, and performance of the real-time kinetic measurement of [11C]SNAP-7941. The black arrows indicate the transport way of the radioactivity. <a href="https://www.jove.com/files/ftp_upload/59557/59557fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59557/59557fig2.jpg" /> 
Figure 2: Radiosynthetic scheme of the automated synthesizer. Switching circuit of the automated synthesizer, starting with the circulation unit for [11C]CH3I/[11C]CH3OTf production, reactor for introduction of the activity into the precursor solution and SPE purification (SPE = solid phase extraction; PCV = product collection vial). <a href="https://www.jove.com/files/ftp_upload/59557/59557fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59557/59557fig3.jpg" /> 
Figure 3: Representative chromatograms of the fully automated radiosynthesis of [11C]SNAP-7841. The semi-preparative RP-HPLC chromatogram is illustrated at the top and the analytical one after purification on the bottom. <a href="https://www.jove.com/files/ftp_upload/59557/59557fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59557/59557fig4.jpg" /> 
Figure 4: Cell culture dish preparation for real-time experiments. Step 1 includes the seeding of 2.5 x 105 up to 1 x 106 depending on the cell type and their growth rate. Subsequently, the culture dish is placed in an oblique plane (approximately 30-45&#176;). Therefore, the supplied metal apparatus or the lid of a cell culture dish can be used in the incubator to stabilize the slanting position of the dish. The day after (24 h) the dish is adjusted horizontally, and fresh cell culture medium is added to completely cover the cell surface. On the day of experiment, cell viability and confluence are examined. According to the experimental protocol, the cells are washed with DPBS, the medium is replaced by serum-free medium (2 mL), and the culture dish is repositioned to an oblique position till the start of experiments. Henceforth, the cells can be treated with the inhibitor or vehicle. <a href="https://www.jove.com/files/ftp_upload/59557/59557fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59557/59557fig5.jpg" /> 
Figure 5: Real-time kinetic measurements of [11C]SNAP-7941. Representative real-time kinetics of [11C]SNAP-7941 are shown in three different set ups: using MDCKII-WT cells; MDCKII-hMDR1 cells pre-blocked with (&#177;)-verapamil as P-gp inhibitor and MDCKII-hMDR1 cells without blockage. The y-axis illustrates the rate of increase of the pre-blocked MDCKII-hMDR1 and WT cells compared to the results of the untreated or vehicle-treated MDCKII-MDR1 cells, respectively (no uptake, 0%). <a href="https://www.jove.com/files/ftp_upload/59557/59557fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941 at JoVE.com

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

technical-aspect-automated-synthesis-real-time-kinetic-evaluation

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Chemistry

7.4K Views.  Medical University of Vienna. Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Video: Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.