Development of Amino Ester Prodrug Tracers for Imaging Glioblastoma

Introduction: Current neuroimaging techniques for high grade gliomas including glioblastoma (GBM) are limited, resulting in many equivocal imaging results, delays in treating recurrences, overtreatment of false positives, and ineffective therapy. Glioblastoma is the most common adult malignant primary brain tumor with a median survival time of 14.6 months. Tumor locations with an intact tumor-brain barrier do not enhance with conventional CT or MRI contrast agents and present major challenges to GBM imaging and treatment. We have developed a novel ¹⁸F-labeled amino ester prodrug imaging method to address the urgent demand for improved GBM imaging agents. Here we report the initial development and evaluation of novel amino esters based on two ¹⁸F-labeled AAs, MeFAMP and FAMPe, that produce high-to-brain contrast but do not cross the BBB in their underivatized forms. MeFAMP is a system A transport substrate while FAMPe is transported primarily by non-system A neutral amino acid transporters. Ethyl (Et) and Butyl (Bu) esters were used for the target amino esters.

Methods: The synthesis of radiolabeling precursors and non-radioactive analytic standards for (R)-[¹⁸F]MeFAMP, Et-[¹⁸F]MeFAMP, Bu-[¹⁸F]MeFAMP, (S)-[¹⁸F]FAMPe, Et-[¹⁸F]FAMPe, and Bu-[¹⁸F]MeFAMPe was performed using commercially available starting materials. Labeling with [¹⁸F]fluoride was accomplished using standard S_N2 nucleophilic substitution reactions. For the synthesis of novel amino ester prodrugs as single enantiomers, a new reproducible radiosynthesis route was established. Ester prodrug hydrolysis was investigated in rat brain homogenate, plasma, and buffer. We compared amino ester PET brain uptake to free AA PET tracers in dynamic PET/CT scans of normal rats from 0 to 30 mins. Using the cluster tray method and assay conditions that included medium with and without AA transport inhibitors, in vitro AA uptake assays with novel ¹⁸F-labeled amino esters and free AAs were performed in glioma cell lines to demonstrate their transport mechanisms. The AA transporter inhibitors used were MeAIB, a mixture of alanine, serine, cysteine, and L-glutamine.

Results: Our synthetic and radiolabeling methods produced free amino acids and target amino esters in sufficient yield and radiochemical purity for preclinical experiments. Ester prodrugs incubated in rat brain homogenate, plasma, and buffer demonstrated varying rates of hydrolysis based on the size of the ester and the structure of the parent amino acid. In rat brain homogenate, we observed conversion rates of up to 91% of Et-MeFAMP to MeFAMP, in contrast to the 54% conversion rate seen in blood plasma. Brain homogenate for the amino ester prodrug Bu-MeFAMP contained 0% Bu-MeFAMP and 100% MeFAMP after 5 mins, whereas plasma contained 48.85% Bu-MeFAMP, 25% MeFAMP, 25% unknown radioactive product. Overall, the conversion rates for Et-[¹⁸F]MeFAMP, Bu-[¹⁸F]MeFAMP, Et-[¹⁸F]FAMPe, and Bu-[¹⁸F]FAMPe into brain homogenate following enzyme hydrolysis of amino ester prodrugs were 91%, 100%, 35%, and 5%, respectively, at the time of 60 mins.Et- and Bu-MeFAMP did not undergo hydrolysis after 45 min. Bu-[¹⁸F] MeFAMP hydrolyzes faster. Lipophilic amino ester prodrugs penetrated the BBB better than free amino acids at 0-4 min after injection, as predicted by dynamic PET. Bu-[¹⁸F] MeFAMP has the highest brain uptake (mean SUV up to 50)and retains a small amount for 30 mins. Bu-MeFAMP, Et-MeFAMP, and MeFAMP had mean SUVs of 11.71, 4.44, and 3.76 in the rat brain 5 minutes after injection. Et-[¹⁸F] amino ester prodrugs MeFAMP and Bu-[¹⁸F] MeFAMP inhibits MeAIB without system AA. Amino ester prodrugs like Et-[¹⁸F] FAMPe and Bu-[¹⁸F] FAMPe blocked neither MeAIB nor ACS inhibitors.

Conclusions: We have shown that ¹⁸F-labeled amino ester prodrugs can cross the intact BBB followed by rapid hydrolysis to free amino acids by esterases. Studies on cellular uptake indicate that amino ester prodrugs do not cross cell membranes via the same amino acid transporter as parent free amino acids.