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

Our research focuses on the production of PET radiotracers used for both preclinical and clinical research applications. Our team focuses on the development and optimization of these carbon-11 radiotracers to ensure a safe and optimal dose is administered for a study. Radiolabeling with carbon-11 involves the substitution of a carbon-12 atom with a carbon-11 nuclide, thereby preserving the physical chemical properties of a compound of interest.

Most routine carbon-11 labeling involves alkylation with C-11 methyl iodide or methyl triflate using a reactor. Captive solvent radiolabeling, as explored here, opens the door to innumerable other methods of introducing the carbon-11 label, including previously cumbersome methods such as carbonylation with C-11 carbon dioxide, allowing new classes of chemical structures to be utilized in molecular imaging research. By using the loop method for carbon-11 methylation reactions, our group has been able to increase the overall efficiency for the production of several carbon-11 radiotracers.

This has been demonstrated in-house with the overall time of production, the amount of final activity, and the increase in molar activity at the end of the synthesis. This protocol allows for an additional method to radiolabel carbon-11 radiotracers via carbon-11 methyl iodide or carbon-11 methyl triflate. We have included our replumbing of an automated model to make this type of radiolabeling routine.

Our team has demonstrated that the use of the loop method when comparing to the traditional reaction vessel labeling method is shortening the overall synthesis time, increasing the activity of the radiolabel tracer, as well as an increase in molar activity at the end of the synthesis. By optimizing the known loop method established by Professor Allen Wilson, we hope to inspire other groups to use this method on their existing platforms, as well as think outside of the box to develop new ways in which carbon-11 radiotracers can be routinely produced for research studies. To replumb the module for the loop method production, attach a V-8 valve to a union that connects directly into the HPLC loop to bypass the reaction vessel.

Condition the methane furnace at 350 degrees Celsius for 20 minutes with a hydrogen gas flow of 100 milliliters per minute. Condition the methane trap at 120 degrees Celsius for 20 minutes with a helium gas flow of 50 milliliters per minute. Then condition the methyl iodide trap at 190 degrees Celsius for 20 minutes with a helium gas flow of 50 milliliters per minute.

Load 100 microliters of the prepared mixture into a syringe and inject it through the adapter at position one of the six port valves internal HPLC loop. Next, load the V-4 reservoir with three milliliters of 0.9%sodium chloride, V-5 with one milliliter of 200 proof ethanol, and V-6 with 10 milliliters of sterile deionized water. Add the C-18 cartridge to the synthesis module.

Fill the large receiving flask with 25 milliliters of sterile deionized water. Then fill the formulation flask with six milliliters of 0.9%sodium chloride for injection. Ensure the delivery line is attached to a sterile preassembled final product vial.

Next, prepare the mobile phase solution and condition the semi-preparative HPLC column with four column volumes of mobile phase. Approximately 20 minutes before unloading radioactive C-11 carbon dioxide from the cyclotron to the module, click the start button to begin the validated time list for C-11 ER-176 synthesis. Next, allow the embedded method in the module to proceed with the conversion of C-11 carbon dioxide into C-11 methyl iodide by way of a dry chemistry process.

Convert C-11 carbon dioxide into C-11 methane by reacting it with hydrogen gas at 350 degrees Celsius over a nickel catalyst. Use an Ascarite trap to retain the unconverted C-11 carbon dioxide and formed water. Next, entrap the formed C-11 methane on a carbosphere column at minus 75 degrees Celsius for purification and concentration.

Heat the carbosphere column to 80 degrees Celsius to release the trapped C-11 methane. React the purified C-11 methane with elemental iodine at 720 degrees Celsius to form C-11 methyl iodide by way of the helium recirculation gas pump. Ensure that the hydrogen iodide formed during the process is retained by another Ascarite trap while unconverted C-11 methane returns to the circulation process.

Entrap the formed C-11 methyl iodide at room temperature on the methyl iodide column during the recirculation process. Once the circulation process is complete, release the collected C-11 methyl iodide from the methyl iodide trap by heating it to 190 degrees Celsius under helium flow. Bypass the methy triflate column and guide the C-11 methyl iodide through a check valve into the 1.5 milliliter stainless steel loop containing the preloaded precursor solution.

After C-11 methyl iodide passes through the loop for 180 seconds, inject the reaction mixture onto the semi-preparative HPLC column for purification. Collect the fraction sample into a large receiving flask containing 25 milliliters of sterile deionized water. Load the diluted mixture onto a C-18 light solid phase extraction or SPE cartridge.

Wash the product with an additional 10 milliliters of sterile deionized water. Next, elute the desired product off the C-18 light SPE using one milliliter of 200 proof ethanol. Direct the elution into a formulation flask preloaded with six milliliters of 0.9%sodium chloride for injection.

Further rinse the C-18 light SPE with an additional three milliliters of 0.9%sodium chloride for injection from the V-4 reservoir. Collect the final solution in the formulation flask and pass it through a 0.22 micrometer sterilizing filter into a preassembled sterile apyrogenic USP type one, 50-milliliter glass vial, sealed with a rubber septum and crimped with an aluminum cap. Next, use telemanipulators to transfer a sample from the final product vial into a TB syringe.

Transfer the syringe to the quality control room using a lead-shielded carrier. In a lead-shielded area, expel the sample into a pyrogen-free tube. Dispense the sample into smaller glass vials for HPLC and GC analyses.

Apply a small aliquot to a pH strip to determine the pH of the final product. Analytical HPLC profiles for ER-176 displayed a peak retention time of 6.103 minutes with a concentration of 1.1 micrograms per milliliter. The analytical HPLC profile of radiotracer for C-11 ER-176 exhibited a prominent peak retention time of 6.356 minutes with a radiochemical purity greater than 99%and an average end-of-synthesis radioactivity of 5.4 G becquerel, as well as an average molar activity of 194 G becquerel per micromole.

The overall synthesis time for the production of carbon-11 ER-176 was 36 minutes.