Carbon-11 radiolabeling allows small compounds to be used for positron emission tomography or PET without changing the molecule. This is essential for investigating receptor expression levels. The advantage of carbon-11 radiolabeling is that we can use PET to directly evaluate SNAP's bindings kinetics towards the melanin-concentrating hormone receptor in real time.
SNAP as a melanin-concentrating hormone receptor antagonist will contribute to a better understanding of this receptor and its association with metabolic conditions like obesity and diabetes. Efflux transporters expressed at the blood-brain barrier can significantly hamper the use of radiopharmaceuticals for central receptor expression imaging. Therefore it is important to evaluate traces in vitro.
The automated radiosynthesis of SNAP is broadly applicable to radiolabeled low molecular weight compounds with an easily assessable methyl group. Once established this procedure can be adapted for other compounds. Assisting in the demonstration will be senior PhD students Sarah Pfaff and Theresa Balber.
To begin carbon-11 SNAP synthesis set up a clean carbon-11 automated synthesis module with the appropriate reagents and materials. Start the SNAP synthesis process on the module computer which will begin with system checks and conditioning. About 30 to 40 minutes before the module will be ready for C-11 delivery, select the C-11 carbon dioxide target on the cyclotron and start the beam at 65 microamperes.
Once the module is ready for delivery close the inner window and outer door. Confirm that the CO2 trap has cooled to at least 40 degrees Celsius and that the methane trap has started cooling. Once the cyclotron has produced about 120 gigabecquerels of C-11 CO2 click OK in the module software to open the path from the target through the CO2 trap.
At the cyclotron computer start delivering C-11 CO2 to the module. Monitor the activity transfer at the module computer and ensure that it proceeds to the reduction of CO2 to methane after three minutes. Monitor the process as the resulting methane is flushed to the methane trap and converted to methyl iodide by circulation through the iodine oven.
At the end of the conversion sequence wait as byproducts are flushed from the methyl iodide trap to exhaust and then confirm that the reactor is ready to receive activity by clicking OK.Monitor the system as the methyl iodide is released from the trap, flushed through the methyl triflate oven and trapped in the reactor. When the activity in the reactor plateaus confirm that trapping is complete. Wait while the reaction mixture reacts at 75 degrees Celsius for three minutes.
Then monitor the system as the reaction mixture is cooled to below 35 degrees Celsius and quenched with 1.5 milliliters of water. Monitor the system as the reaction mixture is loaded into the HPLC injection loop and is injected into the semi-preparatory HPLC column. Watch the UV and gamma detector traces as chromatography proceeds.
When the product peak starts to appear manually start collecting the product in the round bottom collection flask in the module. Manually end collection when the gamma signal falls below about 400 counts per second. Watch the fluid level in the round bottom flask as the product is loaded onto a solid phase extraction cartridge under helium pressure.
Once the flask is empty and the liquid has passed through the SPE cartridge, click OK to begin washing the products trapped on the cartridge. Then monitor the activity in the products collection vial as the product is first eluted from the cartridge into the vial and then diluted with 0.9%saline solution. Watch as the product solution is transferred to the product vial through sterile filter under helium pressure.
When the transfer is complete close the product output valve. When the radiotracer arrives in the product vial measure the absolute radioactive yield. Then use a lead shielded syringe to transfer 100 microliters of the tracer to a small vial for quality control.
Bring the quality control sample to a shielded quality control working area. Draw up 40 microliters of the QC sample into a gas-tight syringe, load it into a prepared HPLC instrument equipped with an analytical column and inject the product. Perform other quality control measurements during the HPLC run.
Once the HPLC run has finished integrate all peaks in the chromatogram from the radioactivity detector to determine whether the SNAP radio chemical purity is greater than 95%Integrate the peaks in the UV chromatogram to determine the chemical purity of the product. Use the area under the UV product peak to calculate the specific activity. Once the product has been confirmed to meet the requirements for use release the radio tracer to the real-time kinetic experiment lab.
At least 30 minutes before the experiment wash the prepared wild type or transfected cells with DPBS and add two milliliters of serum-free growth medium. To block transfected cells add 0.98 microliters of a 20.4 millimolar solution of racemic verapamil hydrochloride in dimethyl sulfoxide. Incubate the cells on an oblique plane for 30 minutes.
Then turn on the real-time assay instrument and the associated computer. Open the control software and choose one target. Set the number of positions to two, the detection time to three seconds, the detection delay time to two seconds, and the name of the first phase to baseline.
Insert the cell culture dish into the inclined support of the device with the cell pole on the bottom side. Ensure that the cell pole is covered in cell culture medium. Start a 10-minute background measurement and set the filename and path.
Set the time scale to minutes and the nuclide collection to carbon-11. Configure the graph to show the background, target and background subtracted target data. Once the radio tracer has been delivered and approved for use, take a small sample for the assay.
Calculate the needed volume of radio tracer solution and prepare a pipette. In the instrument software pause the run and wait for the dish rotation to stop. Open the lid of the device, turn the support 90 degrees to the left, add the radio tracer with the prepared pipette and return the device to its initial position.
Close the lid of the device and immediately resume the experiment. Let the experiment run for 20 minutes before ending it. Process and analyze the data to evaluate SNAP uptake in the cells.
Now the procedure can be repeated for the DMSO-treated control cell culture dish and wild type cell line. Real-time kinetic assays were performed with non-PGP expressing wild type cells, PGP-expressing cells pre-blocked with verapamil as a PGP inhibitor, and unblocked PGP cells. No significant difference was observed between non-treated cells and vehicle-treated cells.
Carbon-11 SNAP rapidly accumulated in wild type cells while no accumulation was observed in PGP-expressing cells. Pre-blocking the PGP efflux transporter with verapamil resulted in SNAP accumulation in the PGP cells comparable to that of wild type cells. In combination with real-time kinetic measurements this set-up enables a broad understanding of the pharmacokinetics of low molecular weight compounds and is therefore highly important for pre-clinical development of radiopharmaceuticals.
Timing and organization are the main ingredients for a successful carbon-11 radiosynthesis with subsequent real-time kinetic experiments as the half-life of carbon-11 is only 20 minutes. Due a short half-life of carbon-11 many parts of this procedure are performed simultaneously. It is important for everyone involved to understand the scope and timing of their role.
Both product safety and radiation protection are key for the preparation of radiopharmaceuticals. All experiments must follow the as low as reasonably achievable or ALARA Principle to limit the operator's radiation exposure.
