Name: solid phase 11c-methylation, purification and formulation for the production of pet tracers
Uploaded: 2019-10-24T14:30:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers at JoVE.com

This study was partially supported by a grant 18-05 from the Alzheimer&#8217;s Society of Canada (for A. K.) and Brain Canada Foundation with support from Health Canada. The authors would like to acknowledge the McGill University Faculty of Medicine, Montreal Neurological Institute and McConnell Brain Imaging Centre for support of this work. We also thank Mrs. Monica Lacatus-Samoila for help with quality control procedures and Drs. Jean-Paul Soucy and Gassan Massarweh for access to radioisotopes and the radiochemistry facility.

1. Preparation of buffers and eluents
<ol>
	<li>Dissolve 2.72 g of sodium acetate trihydrate in 100 mL of water to prepare 0.2 M sodium acetate solution (solution A).</li>
	<li>Dissolve 11.4 mL of glacial acetic acid in 1 L of water to prepare 0.2 M acetic acid solution (solution B).</li>
	<li>Combine 50 mL of solution A with 450 mL of solution B to prepare the acetate buffer at pH 3.7 (buffer 1) according to the buffer reference center15. Verify the pH of the buffer with pH strips or a pH meter.</li>
	<li>Combine 12.5 mL of absolute ethanol with 87.5 mL of buffer 1 to make 12.5% aqueous EtOH solution (wash 1) in a 100 mL bottle.</li>
	<li>Combine 15 mL of absolute ethanol with 85 mL of buffer 1 to make 15% aqueous EtOH solution (wash 2) in a 100 mL bottle.</li>
	<li>Combine 5 mL of absolute ethanol with 5 mL of buffer 1 to make 50% aqueous EtOH solution (final eluent) and draw 2.5 mL of this solution into a 10 mL syringe.</li>
</ol>
2. Application of the precursor to the cartridge
<ol>
	<li>Pass 10 mL of water followed by 5 mL of acetone through the tC18 cartridge to precondition it.</li>
	<li>Dry the cartridge with a stream of nitrogen at 50 mL/min for 1 min.</li>
	<li>Dissolve 2 mg of the precursor 6-OH-BTA-0 in 1 mL of anhydrous acetone.</li>
	<li>Holding a Luer-tip 250 &#181;L precision glass syringe downwards, withdraw 100 &#181;L of the precursor solution and 50 &#181;L of air cushion on top of the liquid. Remove the needle and apply the precursor solution on the tC18 cartridge from the female end by slowly pushing the plunger all the way down. Do not push the solution any further!</li>
</ol>
3. Setting up the manifold for automated synthesis
<ol>
	<li>Secure the standard 5-port disposable manifold on the synthesis module and assemble it according to the Figure 2 and steps 3.2 - 3.5 below. 
	NOTE: We recommend using acetone-resistant manifolds (see Table of Materials).</li>
	<li>Port 1 has two positions. Connect the horizontal inlet to the automated dispenser fitted with a 20 mL syringe. Connect the vertical inlet to the bottle with wash 1.</li>
	<li>Connect the output of the module which produces [11C]CH3OTf to port 2 of the manifold.</li>
	<li>Install the tC18 cartridge loaded with precursor 6-OH-BTA-0 between ports 3 and 4.</li>
	<li>Port 5 has two positions. Connect the horizontal outlet to the waste bottle which must hold at least 200 mL. Connect the vertical outlet to the sterile vial for tracer collection via the sterile filter.</li>
</ol>
4. Radiosynthesis of [11C]PiB
CAUTION: All manipulations involving radioactive isotopes must be performed in a lead-shielded hot cell by personnel with adequate training to work with radioactive materials. 
NOTE: This protocol does not cover the details of production of [11C]CO2 in the cyclotron and its conversion into [11C]CH3OTf using the radiochemistry module. These procedures will depend on the individual equipment of the radiochemistry lab and are outside the scope of this protocol. Our PET centre is equipped with an IBA cyclotron, which produces carbon-11 in the chemical form of [11C]CO2 via the 14N(p,&#945;)11C nuclear reaction with a N2/O2 gas mixture (99.5:0.5) in the gas target, and a commercially available module for production of [11C]CH3I via the &#34;dry method&#34; (catalytic reduction to [11C]CH4 followed by radical iodination). [11C]CH3OTf is produced by passing [11C]CH3I over a silver triflate column heated to 175 &#176;C at 20 mL/min.
<ol>
	<li>Deliver [11C]CH3OTf into the manifold through port 2 and pass it through the loaded tC18 cartridge at 20 mL/min output flow regulated by the [11C]CH3OTf module, via ports 3 and 4 and into the waste bottle as shown on Figure 2A.</li>
	<li>Once all the radioactivity has been transferred and trapped on the tC18 cartridge as monitored by the radioactivity detector behind the cartridge holder, stop the flow of gas by closing port 2. Let the cartridge sit for 2 min to complete the reaction.</li>
	<li>Withdraw 19 mL of wash 1 solution (see step 1.4) from the 100 mL bottle into the dispenser syringe through port 1 at 100 mL/min as shown on Figure 2B.</li>
	<li>Dispense 18.5 mL of wash 1 solution from the dispenser through the tC18 cartridge via ports 3 and 4 and into the waste bottle at 50 mL/min as shown on Figure 2C. Ensure the absence of air bubbles in the manifold as they might diminish the separation efficiency.</li>
	<li>Repeat steps 4.3 and 4.4 four times, withdrawing and dispensing 18.5 mL of wash 1 solution each time. The total volume of wash 1 solution passed through tC18 is 92.5 mL; however, it can vary within the 90 - 100 mL range depending on the particular synthesis module used.</li>
	<li>Switch the input line on port 1 from wash 1 to wash 2 solution (see step 1.5).</li>
	<li>Repeat steps 4.3 and 4.4 three times, withdrawing and dispensing 18.5 mL of wash 2 solution each time. The total volume of wash 2 solution passed through tC18 is 55.5 mL. However, it can vary within the 50 - 60 mL range depending on the particular synthesis module used.</li>
	<li>Toggle valve 5 towards the final vial as shown on Figure 2D. Disconnect the line from the dispenser and connect it to the 10 mL syringe containing 2.5 mL of the final eluent solution (50% aqueous EtOH, see step 1.6) and 7.5 mL of air.</li>
	<li>Holding the syringe downwards, manually push the final eluent solution (2.5 mL) followed by air (7.5 mL) through the tC18 cartridge via ports 3 and 4 and into the sterile vial for tracer collection via the sterile filter as shown on Figure 2D.</li>
	<li>Disconnect the empty syringe, connect the 10 mL syringe containing 10 mL of the sterile phosphate buffer (recipe not included as it may vary) and push the entire volume through the tC18 cartridge into the sterile vial as described above (Figure 2D). Disconnect the syringe and flush the line with 10 mL of air using the same syringe.</li>
	<li>Withdraw 0.7 mL of the final tracer formulation and collect samples for quality control procedures (0.1 mL), bacterial endotoxin test (0.1 mL) and sterility (0.5 mL).</li>
</ol>
5. Quality control procedures
CAUTION: Each batch of the radiotracer must be subjected to the appropriate quality control procedures (QC) prior to release to the PET imaging site for administration into human or animal subjects. The authors of this manuscript are not responsible for the compliance of the radiotracer produced at other centers with local health authority regulations.
<ol>
	<li>Perform pre-release QC procedures, which must include tests for radiochemical identity (RCI), radiochemical purity (RCP), chemical purity and molar activity of the tracer as well as residual solvent content and pH of the formulation.</li>
	<li>Determine the RCI, RCP, chemical purity and molar activity by means of analytical HPLC system equipped with UV (monitoring at 350 nm) and radioactivity detectors, and a reversed-phase column. Determine the retention times of 6-OH-BTA-0 and 6-OH-BTA-1 and calibrate the instrument to quantify the content of each compound.</li>
	<li>Determine the residual solvent content by means of analytical gas chromatography system equipped with a capillary column. Determine the retention times of acetone and ethanol and calibrate the instrument to quantify the content of each solvent.</li>
	<li>Perform the bacterial endotoxins test using a cartridge reader equipped with suitable cartridges.</li>
	<li>Perform the sterility analysis of the sample at least 14 day after the synthesis to ensure the absence of bacterial growth or send the sterility sample to a laboratory accredited by the local health authority.</li>
</ol>

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The authors declare that they have no competing financial interests.

Despite the recent emergence and FDA approval of several 18F-labeled PET tracers, such as florbetapir, florbetaben and flutemetamol, [11C]PiB remains a gold standard tracer for amyloid imaging due to the fast brain uptake and low non-specific binding. Currently this tracer is synthesized via either wet chemistry16 or using a &#34;dry loop&#34; approach4,17. Both methods require HPLC purification followed by r...

Positron emission tomography (PET) is a molecular imaging modality which relies on detecting the radioactive decay of an isotope attached to a biologically active molecule to enable the in vivo visualization of biochemical processes, signals and transformations. Carbon-11 (t1/2 = 20.3 min) is one of the most commonly used radioisotopes in PET because of its abundance in organic molecules and short half-life which allows for multiple tracer administrations on the same day to the same human or animal subject and reduces the radiation burden on the patients. Many tracers labeled with this isotope are used in clinical studies and in basic health research for in vivo PET imaging of classical and emerging biologically relevant targets - [11C]raclopride for D2/D3 receptors, [11C]PiB for amyloid plaques, [11C]PBR28 for translocator protein - to name just a few.
Carbon-11 labeled PET tracers are predominantly produced via 11C-methylation of non-radioactive precursors containing -OH (alcohol, phenol and carboxylic acid), -NH (amine and amide) or -SH (thiol) groups. Briefly, the isotope is generated in the gas target of a cyclotron via a 14N(p,&#945;)11C nuclear reaction in the chemical form of [11C]CO2. The latter is then converted into [11C]methyl iodide ([11C]CH3I) via either wet chemistry (reduction to [11C]CH3OH with LiAlH4 followed by quenching with HI)1 or dry chemistry (catalytic reduction to [11C]CH4 followed by radical iodination with molecular I2)2. [11C]CH3I can then be further converted to the more reactive 11C-methyl triflate ([11C]CH3OTf) by passing it over a silver triflate column3. The 11C-methylation is then performed by either bubbling the radioactive gas into a solution of non-radioactive precursor in organic solvent or via the more elegant captive solvent &#34;loop&#34; method4,5. The 11C-tracer is then purified by means of HPLC, reformulated in a biocompatible solvent, and passed through a sterile filter before being administered to human subjects. All of these manipulations must be fast and reliable given the short half-life of carbon-11. However, the use of an HPLC system significantly increases the losses of the tracer and production time, often necessitates the use of toxic solvents, complicates automation and occasionally leads to failed syntheses. Furthermore, the required cleaning of the reactors and HPLC column prolongs delays between the syntheses of subsequent tracer batches and increases the exposure of personnel to radiation.
The radiochemistry of fluorine-18 (t1/2 = 109.7 min), the other widely used PET isotope, has been recently advanced via the development of cassette-based kits that obviate the need for HPLC purification. By employing solid phase extraction (SPE) cartridges, these fully disposable kits allow for the reliable routine production of 18F-tracers, including [18F]FDG, [18F]FMISO, [18F]FMC and others, with shorter synthesis times, reduced personnel involvement and minimal maintenance of the equipment. One of the reasons carbon-11 remains a less popular isotope in PET imaging is a lack of similar kits for the routine production of 11C-tracers. Their development would significantly improve synthetic reliability, increase radiochemical yields and simplify automation and preventive maintenance of the production modules.
Currently available production kits take advantage of inexpensive, disposable, SPE cartridges instead of HPLC columns for the separation of the radiotracer from unreacted radioactive isotope, precursor and other radioactive and non-radioactive by-products. Ideally, the radiolabeling reaction also proceeds on the same cartridge; for example, the [18F]fluoromethylation of dimethylaminoethanol with gaseous [18F]CH2BrF in the production of prostate cancer imaging PET tracer [18F]fluoromethylcholine occurs on a cation-exchange resin cartridge6. Although similar procedures for the radiolabeling of several 11C-tracers on cartridges have been reported7,8 and became especially powerful for the radiosynthesis of [11C]choline9 and [11C]methionine10, these examples remain limited to oncological PET tracers where the separation from the precursor is often not required. We recently reported the development of &#34;[11C]kits&#34; for the production of [11C]CH3I11 and subsequent 11C-methylation, as well as solid phase-supported synthesis12 in our endeavours to simplify the routine production of 11C-tracers. Here, we wish to demonstrate our progress using the example of the solid phase supported radiosynthesis of [11C]PiB, a radiotracer for A&#946; imaging which revolutionized the field of Alzheimer&#39;s disease (AD) imaging when it was first developed in 2003 (Figure 1)13,14. In this method, volatile [11C]CH3OTf (bp 100 &#176;C) is passed over 6-OH-BTA-0 precursor deposited on the resin of a disposable cartridge. PET tracer [11C]PiB is then separated from the precursor and radioactive impurities by elution from the cartridge with biocompatible aqueous ethanol. Further, we automated this method of [11C]PiB radiosynthesis using a remotely operated radiochemistry synthesis module and disposable cassette kits. Specifically, we implemented this radiosynthesis on a 20-valve radiochemistry module, equipped with syringe drive (dispenser) which fits standard 20 mL disposable plastic syringe, gas flow controller, vacuum pump and gauge. Due to the simplicity of this method, we are confident that it can be modified to most commercially available automated synthesizers, either cassette-based or those equipped with stationary valves. This solid phase supported technique facilitates [11C]PiB production compliant with Good Manufacturing Practice (GMP) regulations and improves synthesis reliability. The technique described here also reduces the amount of precursor required for radiosynthesis, uses only &#34;green&#34; biocompatible solvents and decreases the time between consecutive production batches.

<table><tbody><tr><td>6-OH-BTA-0</td><td>ABX advanced biochemical compounds</td><td>5101</td><td>Non-radioactive precursor of [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>6-OH-BTA-1</td><td>ABX advanced biochemical compounds</td><td>5140</td><td>Non-radioactive standard of [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Agilent 1200 HPLC system</td><td>Agilent</td><td>Agilent 1200</td><td>Analytical HPLC system</td></tr><tr><td>Ethanol absolute</td><td>Commercial alcohols</td><td>432526</td><td></td></tr><tr><td>Hamilton syringe (luer-tip, 250 &amp;micro;L)</td><td>Hamilton</td><td>HAM80701</td><td></td></tr><tr><td>MZ Analytical PerfectSil 120</td><td>MZ-Analysentechik GmbH</td><td>MZ1440-100040</td><td>Analytical HPLC column</td></tr><tr><td>Perkin Elmer Clarus 480 GC system</td><td>Perkin Elmer</td><td>Clarus 480</td><td>Gas chromotograph</td></tr><tr><td>polycarbonate manifold</td><td>Scintomics</td><td>ACC-101</td><td>Synthesis manifold</td></tr><tr><td>Restek MTX-Wax column (30 m, 0.53 mm)</td><td>Restek</td><td>70625-273</td><td>Analytical GC column</td></tr><tr><td>Scintomics GRP module</td><td>Scintomics</td><td>Scintomics GRP</td><td>Automated synthesis unit</td></tr><tr><td>Sep-Pak tC18 Plus</td><td>Waters</td><td>WAT020515</td><td>Solid phase extraction cartridge</td></tr><tr><td>solvent-resistant manifold</td><td>Scintomics</td><td>ACC-201</td><td>Synthesis manifold</td></tr><tr><td>Spinal needle</td><td>BD</td><td>405181</td><td></td></tr><tr><td>Sterile extension line</td><td>B. Braun</td><td>8255059</td><td></td></tr><tr><td>Sterile filter</td><td>Millipore</td><td>SLLG013SL</td><td></td></tr><tr><td>Sterile vial (20mL)</td><td>Huayi</td><td>SVV-20A</td><td></td></tr><tr><td>Sterile water</td><td>Baxter</td><td>JF7623</td><td></td></tr><tr><td>Synthra MeIplus Research</td><td>Synthra</td><td>MeIplus Research</td><td>[&lt;sup&gt;11&lt;/sup&gt;C]CH&lt;sub&gt;3&lt;/sub&gt;I/[&lt;sup&gt;11&lt;/sup&gt;C]CH&lt;sub&gt;3&lt;/sub&gt;OTf module</td></tr><tr><td>Syringe (10 mL)</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Syringe (1mL)</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Syringe (20 mL)</td><td>B. Braun</td><td>4617207V</td><td>Dispenser syringe</td></tr><tr><td>Vent filter</td><td>Millipore</td><td>TEFG02525</td><td></td></tr></tbody></table>

solid phase 11c-methylation, purification and formulation for the production of pet tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

To summarize a typical radiosynthesis of [11C]PiB, gaseous [11C]CH3OTf is first passed through a tC18 cartridge preloaded with a solution of precursor (Figure 1). Separation of the reaction mixture is then achieved by successive elution with aqueous ethanol solutions as follows. First, 12.5% EtOH elutes the majority of unreacted [11C]CH3OTf and 6-OH-BTA-0, then 15% EtOH washes out the residual impurities,...

Watch this Scientific Journal Video about Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers at JoVE.com

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

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

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

solid-phase-11c-methylation-purification-formulation-for-production

Research

JoVE Journal

Chemistry

6.6K Views.  McGill University. 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.

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

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

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

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

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

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.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

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).

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.

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

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Cancer Research

18F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor (SiFA) for Positron Emission Tomography

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the radiochemist&#39;s toolkit for incorporating radioactive [18F]fluoride into tracers for use in positron emission tomography. In comparison to conventional radiolabeling strategies, isotopic exchange of fluorine-19 from SiFA with [18F]fluoride is carried out at room temperature and requires minimal reaction participants. The formation of by-products is thus negligible, and purification is greatly simplified. However, while the precursor molecule used for labeling and the final radiolabeled product are isotopically discrete, they are chemically identical and are thus inseparable during purification procedures. The SiFA tag is also susceptible to degradation under the basic conditions arising from the processing and drying of [18F]fluoride. The &#39;4 drop method&#39;, wherein only the first 4 drops of eluted [18F]fluoride are used from the solid-phase extraction, reduces the amount of base in the reaction, facilitates lower molar amounts of precursor, and reduces degradation.

18F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor SiFA for Positron Emission Tomography

The synthesis of fluorine-18 (18F) labeled radiopharmaceuticals for positron emission tomography typically requires months of experience. When incorporated into a radiotracer, the silicon-fluoride acceptor (SiFA) motif enables a simple 18F-labeling protocol that is independent of costly equipment and preparatory training, while reducing precursor quantity needed and utilizing milder reaction conditions.

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the ...

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, &#945;-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models.
This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 &#177; 5% (decay corrected at the end of synthesis, n &#62; 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol

Here, we describe the radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-tryptophan, a positron emission tomography imaging agent for studying tryptophan metabolism, using a one-pot, two-step strategy in a radiochemistry synthesis system with good radiochemical yields, high enantiomeric excess, and high reliability.

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

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

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;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.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

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

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

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.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

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

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

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

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

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

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

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