Abstract
2757
Introduction: [18F]AlF-NOTA-MAL-Cys40-Exendin-4 ([18F]AlF-Exendin-4) has been developed and tested for beta cell imaging through labeling of the GLP-1 receptor. However, the reported syntheses of [18F]AlF-Exendin-4 were performed using manual methods(1) with no automation via commercially available synthesis modules which limits the application of the tracer. Our aim was to develop an automated synthesis and produce sufficient radiotracer in GMP quality for clinical trials. Challenges for the automation of this radiolabeled peptide are the amount of peptide precursor and the separation of the unlabeled peptide from the radiolabeled material to obtain desirable product in good yield and molar activity. Here we present a method to label [18F]AlF-Exendin-4 using a NeptisRS module and a quaternary semi-prep HPLC system for gradient purification.
Methods: A NeptisRS module was used for labeling of [18F]AlF-Exendin-4. See Figure 1 for the layout. The synthesis is a two-step one-pot reaction followed by purification of the crude reaction mixture. First, [18F]fluoride is trapped and eluted from an anion exchange cartridge into a reaction vessel charged with a small amount of AlCl3 to form the radiolabeled intermediate [18F]AlF2+. This is followed by the addition of NOTA-MAL-Cys40-Exendin-4. After the radiolabeling (95ºC, 20 min), the mixture was purified by either solid-phase only, or a combination of HPLC and solid-phase method.
Results: Multiple successful runs were performed on a NeptisRS module. Unlike the typical small volume conical reaction vessel used for manual process (1-2 mL vial), larger volume flat reaction vessels (10 mL) on standard cassettes had to be used, which resulted in a higher amount of precursor needed as starting material. We tested different amounts of precursor, 0.1 - 0.5 mg, and the best results were observed by using at least 0.5 mg starting material (yield > 10%). The next step was to investigate the purification method. Similar to 68Ga labeling, we first tried to use only solid-phase purification using a tC18 SepPak cartridge. The resulting samples contained a high amount of cold mass as the precursor was not well-separated from the radiotracer. The next method using an isocratic semi-prep HPLC method for crude product purification also showed to be insufficient due to the challenging elution characteristics of this peptide (early elution of peptide or none at all). Finally, with the use of a gradient method, total mass of the final product has a lower mass with molar activity of at least 550 mCi/mmol, and the yields were 10-12% decay corrected (n = 3). Hence, the use of a gradient semi-prep HPLC method (Phenomenex Gemini, MeCN:0.1M Ammoniumfromate=20:80 to 100:0, 20 min linear gradient) to remove excess precursor from the crude reaction mixture was shown to be crucial. The synthesized [18F]AlF-Exendin-4 was used successfully in preclinical studies.
Conclusions: [18F]AlF-Exendin-4 was successfully synthesized using a commercially available synthesis module. The incorporation of a gradient HPLC method is crucial for purification. Tests to further optimize the purification (different HPLC columns, gradient methods and mobile phase compositions) are currently underway. This should lead to a radiotracer with even better molar activity and will make the tracer suitable for clinical use.