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

The overall goal of this procedure is to produce F-18 labeled 3-fluoro-4-aminophyridine of adequate purity and quality for PET imaging experiments in animal subjects. This method can help answer key questions in the imaging field about the utility of 3F4AP. As a PET tracer for brain imaging to detect demyelinating conditions and other diseases.

The main advantages of this labeling method is, flexibility of the pulse radio labeling hydrogenation. Which could be adapted to the production of other F-18 labeled PET radio tracers. F-18, has a two hour half life and therefore, is produced with a cyclotron before it synthases, to ensure that the product has sufficient activity to be injected.

Given the short half life of fluorine 18, it's strongly recommended to work in groups of two or three, to ensure that the procedure's performed quickly but safely. To begin preparing for the synthesis, use new sterile one millimeter syringes to dispense 400 microliters of tetrabutylammonium bicarbonate and 800 microliters of acetonitrile into a glass vial. Seal the vial with the cream cap.

Add 50 microliters of precursor stock solution and 450 microliters of anhydrous acetonitrile to an oven dried vial and seal the top. Seal 500 microliters of anhydrous acetonitrile in another oven dried vial. Place four milliliters of 0.2%oxalic acid in methanol in another glass vial, and seal the top.

Then, condition a strong anion exchange solid phase extraction cartridge with five milliliters of 8.4%sodium bicarbonate drop wise, followed by five milliliters of ultra pure deionized water. Condition a neutral alumina solid phase extraction cartridge with five milliliters of ultra pure deionized water drop wise, followed by five milliliters of 0.2%oxalic acid in methanol. Load the reagent vials and the extraction cartridges into the integrated fluidic processor.

Turn on the HPLC and condition the column at four milliliters per minute for 30 minutes. During column conditioning, load a 10%palladium carbon catalyst cartridge onto the hydrogenator cartridge holder. Equip the product receiving vial with the vent needle assembly composed of a sterile 20 gauge 1.5 inch needle attached to a 0.22 micron filter.

Connect the vial to the hydrogenator. Open the hydrogen valve, load the HPLC sequence and start the program to initiate the flow of methanol and hydrogen through the cartridge. Load the IFP into the synthesizer, using the software.

Then, start the synthesis module software and perform system checks. Close the hot cell. Open the 3F4AP synthesis sequence, and load the sequence to the synthesis module.

Name the synthesis run and start the sequence. Monitor the instrument and software during the pre-synthesis steps to ensure that the synthesis module is operating normally. Once the reaction vessel reaches 65 degrees celsius, the module is ready for F-18 delivery from the cyclotron.

When the cyclotron has produced the desired amount of F-18 and the synthesis module is ready, deliver the F-18 to the module. Once the activity has been transferred to the receiving vial, continue the synthesis sequence by pressing the green button, resume. Monitor the synthesis as the F-18 is trapped on the strong anion exchange cartridge, convert it to F-18 tetrabutylammonium fluoride and dry it.

Continue monitoring the synthesis as the tetrabutylammonium fluoride is dissolved in acetonitrile. The precursor reacts with the F-18 fluoride for one minute, at which point the reaction is quenched with the oxalic acid solution. It is helpful to take the labeling efficiency with radio HPLC and efficiency of the F-18 trapped in an illusion with activity measurement.

But only, when using very low amount of radioactivity. Monitor the transfer of the quenched reaction mixture through the neutral alumina cartridge to the hydrogenator loop, and the injection of the mixture onto the hydrogenator HPLC. Collect the crude product solution from the hydrogenator, and measure the activity in the vial using a dose calibrator.

The crude product should be injected into a semiprep HPLC system inside a hot cell. After purification, the final product is then collected and dispensed into an aseptic ISO class five laminar air flow hot cell as per USP and FDA regulations. For demonstration, we show extraction of the crude product in that dispensing cell and show the purification step with a small amount of activity.

Place the vial in a shielded container and transport it to the HPLC. Load 100 microliters of the crude product solution onto the HPLC and collect the final product peak with an automated collector. Measure the activity of each fraction and combine those with the highest activity.

Record the end of synthesis time and the volume and activity of the product. Aseptically withdraw about 0.8 milliliters of the product solution for quality control testing from the final product vial. Inspect the dose vial through a leaded glass shield, and verify that the product is clear, colorless and free of particulate metal.

Spot the sample on a TLC plate, next to a reference standard. Develop the plate with the solution of 95%methanol and 5%acetic acid. Visualize the reference with UV light and mark the spot in pencil.

Secure the TLC plate to a radio TLC scanner and record the peak time. Verify that the retention factors of the product and reference are within 5%of each other. Inject 10 microliters of the product onto a HPLC equipped with the radiation detector.

For both the radio HPLC and TLC results, verify that the product peak accounts for at least 95%of the total area under all gamma peaks in that chromatogram. Add 10 microliters of the reference standard to the quality controlled product sample and verify that the single UV peak appears on the HPLC. Inject 10 microliters of the product onto a JC, to verify that the residual solvent content is below the established limits.

Radio TLC is used to ensure the absence of three fluoride, which can provide confounding results during PET imaging. Radio HPLC is used to confirm identity and calculate specific activity. GC is used to analyze the residual solvent content.

Perform a bubble point test, to verify that the filter used in the final product vial, meets or exceeds the manufacturer's specifications for filter integrity. Calculate the specific activity and the half life, verify the radio anaclitic identity and purity. Perform sterility test and measure the endotoxin levels.

Document the results of the quality control tests. Using this method, the radio tracer, 3F4AP, will synthesize by an automated process, in which fluoride exchange was followed by reduction of the anoxide and the nitro groups. The tracer was synthesized with sufficient purity and specific activity, to perform pre-clinical PET imaging studies on animal models of demyelination.

The intermediate product was analyzed during tracer development and was found to have a characteristic UV peak at 313 nanometers. The final product was best visualized by HPLC at 254 nanometers. The observed radio chemical purity in the radio HPLC, was confirmed by radio TLC.

This whole demonstration of this method is critical, as many steps are not commonly employed in chemistry or radiochemistry labs. Generally, individuals new to this method struggle to precisely follow synthesis procedure and to perform the vigorous quality control tests. While developing this procedure, it is important to measure the amount of radioactivity present before and after each step, to determine the reaction efficiency.

But this should only be done with low amounts of radioactivity. Once mastered, this technique can be done in less than two hours, if performed properly. Following animal injection and imaging, procedures such as tissue dissection and blood collection can be performed to answer additional questions about biodistribution and metabolism of the tracer.

After watching this video, you should have a good understanding of the general workflow for synthesizing and performing quality control tests on a PET tracer. Don't forget that working with radioactivity can be extremely hazardous and precautions such as the ALARA principle, As Low As Reasonably Achievable, should always be taken and always work with your radiation safety office.