Name: Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of 11C SNAP-7941 (Video) | JoVE Journal
Uploaded: 2019-04-28T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Learn automated synthesis of [11C]SNAP-7941 for real-time PET imaging of receptor binding kinetics. This method evaluates efflux transporter effects and enables preclinical radiopharmaceutical development.

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5 minutes

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/pubmed?term=((Identification+of+melanin-concentrating+hormone+receptor+and+its+impact+on+drug+discovery%5BTitle%5D)+AND+%22Journal+of+Experimental+Zoology%22%5BJournal%5D)">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/pubmed?term=((Preclinical+in+vitro+%26+in+vivo+evaluation+of+%5B11C%5DSNAP-7941+-+the+first+PET+tracer+for+the+melanin+concentrating+hormone+receptor+1%5BTitle%5D)+AND+%22Nuclear+Medicine+and+Biology%22%5BJournal%5D)">Preclinical in vitro & 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/pubmed?term=((Radiosynthesis+of+%5B+11C%5DSNAP-7941-the+first+PET-tracer+for+the+melanin+concentrating+hormone+receptor+1+%28MCHR1%29%5BTitle%5D)+AND+%22Applied.+Radiation+and+Isotopes%22%5BJournal%5D)">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/pubmed?term=((SNAPshots+of+the+MCHR1%E2%80%AF%3A+a+Comparison+Between+the+PET-Tracers+%5B+18+F+%5D+FE+%40+SNAP+and+%5B+11+C+%5D+SNAP-7941%5BTitle%5D)+AND+%22Molecular+Imaging+Biology%22%5BJournal%5D)">SNAPshots of the MCHR1 : 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/pubmed?term=((Syntheses+of+precursors+and+reference+compounds+of+the+melanin-+concentrating+hormone+receptor+1+%28MCHR1%29+Tracers+%5B11C%5DSNAP-7941+and+%5B18F%5DFE%40SNAP+for+positron+emission+tomography%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">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/pubmed?term=((Synthese+von+11C-%2C+18F-%2C+15O-+und+13N-Radiotracern+f%C3%BCr+die+Positronenemissionstomographie%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">Synthese von 11C-, 18F-, 15O- und 13N-Radiotracern für 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/pubmed?term=((An+overview+on+PET+radiochemistry%3A+part+1+-+covalent+labels+-+18+F%2C+11+C%2C+and+13+N%5BTitle%5D)+AND+%22Journal+of+Nuclear+Medicine%22%5BJournal%5D)">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/pubmed?term=((Molar+activity+-+The+keystone+in+11C-radiochemistry%3A+An+explorative+study+using+the+gas+phase+method%5BTitle%5D)+AND+%22Nuclear+Medicine+and+Biology%22%5BJournal%5D)">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., Dahlstrøm, K., Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+of+%5B11C%5Diodomethane+by+iodination+of+%5B11C%5Dmethane%5BTitle%5D)+AND+%22Applied+Radiation+and+Isotopes%22%5BJournal%5D)">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/pubmed?term=((New+methodologies+for+the+preparation+of+carbon-11+labeled+radiopharmaceuticals%5BTitle%5D)+AND+%22Clinical+and+Translational+Imaging%22%5BJournal%5D)">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/pubmed?term=((Radiopharmaceuticals+for+assessing+ABC+transporters+at+the+blood-brain+barrier%5BTitle%5D)+AND+%22Clinical.+Pharmacollogy+%26+Therapeutics%22%5BJournal%5D)">Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier.</a> Clinical. Pharmacollogy & Therapeutics. 97, 362-371 (2015).</li>
<li>Nics, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Speed+matters+to+raise+molar+radioactivity%3A+Fast+HPLC+shortens+the+quality+control+of+C-11+PET-tracers%5BTitle%5D)+AND+%22Nuclear+Medicine+and+Biology%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22European+Journal+of+Nuclear+Medicine+and+Molecular+Imaging+%28Research%29%22%5BJournal%5D)">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>&lt;strong&gt;Table 1: List of materials and instrumentation of the fully automated radiosynthesis of [11C]SNAP-7941&lt;/strong&gt;</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, &amp;lt; 10 ppm H&lt;sub&gt;2&lt;/sub&gt;O</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>[&lt;sup&gt;11&lt;/sup&gt;C]CO&lt;sub&gt;2&lt;/sub&gt; 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>N&lt;sub&gt;2&lt;/sub&gt; + 1% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Air Liquide, Vienna, Austria</td><td></td><td>Target gas</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 2: List of materials and instrumentation of the quality control of [&lt;sup&gt;11&lt;/sup&gt;C]SNAP-7941.&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 3: List of materials and instrumentation for the evaluation of the real-time kinetic behaviour of [&lt;sup&gt;11&lt;/sup&gt;C]SNAP-7941.&lt;/strong&gt;</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>&lt;em&gt;In vitro&lt;/em&gt; 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>(&amp;plusmn;)-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 &amp;ndash; 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 &amp;micro;L &amp;ndash; 200 &amp;micro;L</td></tr><tr><td>Cell culture flasks</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>Cellstar 250 mL, 75 cm&lt;sup&gt;2 &lt;/sup&gt;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 &amp;micro;m (Cat.No. PHCC60050)</td></tr></tbody></table>

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

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 ± 2.5 GBq (4.6 ± 2.0% at EOB, 14.9 ± 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 µL) 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 ± 41 GBq/µmol (n = 10) and the radiochemical purity was always &gt; 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 (±)-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 (±)-verapamil led to a comparable real-time kinetic as already seen for the wildtype cell line (Figure 5).
<img alt="Radiosynthesis process diagram; shows synthesizer preparation, irradiation, in vitro evaluation steps." class="xfigimg" src="/files/ftp_upload/59557/59557fig1.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
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="Tracerlab FX2 C schematic; radiolabelling synthesis with HPLC column and SPE cartridge setup." class="xfigimg" src="/files/ftp_upload/59557/59557fig2.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
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="HPLC chromatogram showing SNAP-7941 detection peaks; UV and gamma detectors; compound analysis." class="xfigimg" src="/files/ftp_upload/59557/59557fig3.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
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="Cell culture method, diagram of angled seeding and media change; incubation process illustration." class="xfigimg" src="/files/ftp_upload/59557/59557fig4.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
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°). 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="MDCKII cell growth graph; time vs. fold increase; WT, hMDR1, pre-blocked; scientific analysis." class="xfigimg" src="/files/ftp_upload/59557/59557fig5.jpg" data-altupdatedat="1788282455000" data-alt="Figure 5"> 
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 (±)-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>

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.

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

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Research

JoVE Journal

Chemistry

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature.   In this video,  we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent...

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Here, we describe a bioassay using 3-(4′,5′-dimethylthiazol-2′-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Optimization of Radiochemical Reactions using Droplet Arrays

Here, we describe a protocol for the optimization and parameterization of amino acid residues modified with reactive carbonyl species, adaptable to protein systems. The protocol steps include structure design and optimization, charge assignments, parameter construction, and preparation of protein systems.

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the “loop method” [11C]methylation. We describe the procedure to synthesize [11C]ER-176,  with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

Learn automated synthesis of [11C]SNAP-7941 for real-time PET imaging of receptor binding kinetics. This method evaluates efflux transporter effects and enables preclinical radiopharmaceutical development.

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

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