Abstract
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Objectives: L-Glutamine (Gln) is essential for cell growth and proliferation. In addition to glucose, cancer cells utilize Gln as a carbon source for ATP production, biosynthesis, and as a defense against reactive oxygen species. The utilization of [11C]Gln has been previously reported as a biomarker for tissues with an elevated demand for Gln, however, the previous methodology used for the preparation of [11C]Gln was found to be lacking in several crucial aspects that are important for the transition of a drug product from the preclinical to clinical use. These reports utilized a simple solid phase extraction method to purify the labeled intermediate. However, the process was never designed to remove the unreacted precursor from the labeled intermediate. The presence of the unreacted precursor has the undesired consequence of lowering of the overall chemical purity of the final drug product solution. In addition to the purity concerns, all methodologies reported for the preparation of [11C]Gln have been accomplished using non-commercialized, custom built, reaction platforms. In an effort to allow for fast transfer of technology from production site to production site, development of this product on commercially available platforms is needed. Herein, we report the development and utilization of methodology for the automated production of [11C]Gln that meets criteria for human use.
Methods: The radiosynthesis was carried out using a modified form of a published procedure, using [11C]HCN produced on a GE ProCab C-11 handling unit and the two-reactor capability of either a GE FX2N or an iPhase Flexlab system. Briefly, after trapping of the [11C]HCN with a CsHCO3/18-Crown-6 solution, the [11C]CsCN was reacted with a commercially available precursor. This intermediate was then purified by HPLC, trapped via SPE, eluted, dried under vacuum with inert gas flow, and then partially hydrolyzed and deprotected under acidic conditions. Following passage through an ion-exchange resin, the product was passed through a final filter (0.22 µm) to give the desired [11C]Gln as a sterile injectable. The resulting product was then analyzed for quality assurance.
Results: Automated production by this method provides over 100 mCi of [11C]Gln from ~950 mCi of [11C]CsCN (10-14% ndc, 50-66% dc). Under the HPLC conditions (C18, MeCN/Aqueous Phosphate Buffer), the desired intermediate and precursor are easily separable with product being eluted from 5.5 – 6.5 min. After deprotection and ion-exchange purification, the resulting final drug product has a >99% radiochemical purity, <5% of D-Gln present, no detectable impurities, and has a pH of 5.5-6.0 without the need for pH adjustment. The synthesis is completed within 45 min after the end of bombardment, with efforts underway to further reduce this time as well as quantify the injected mass of glutamine.
Conclusion: A modified automated radiosynthesis was developed using commercially available modules in a rapid time frame. Addition of the HPLC purification of the intermediate allows removal of unreacted precursor as well as any other impurities. This allows production of pure [11C]Gln without fear of contamination with the difficult to detect impurities. Efforts are currently underway to develop a protocol for the quantification of the mass of the final drug product (i.e. specific activity), which is a metric that will be beneficial during the transition to clinical utilization. Research Support: Vanderbilt GI SPORE (5P50CA098131), Vanderbilt Center for Molecular Probes, Vanderbilt Ingram Cancer Center, The Kleberg Foundation