Abstract View

Background: Glioblastoma (GBM) remains a deadly cancer, with no significant improvement in its clinical intervention in more than 20 years, and a 5-year overall survival rate of 5%. Amino acid derivatives have had some success as diagnostic radiopharmaceuticals for GBM, with [¹⁸F]FDOPA, [¹⁸F]fluoroethyl tyrosine, and [¹¹C]methionine, which are taken up by the LAT1 system. Recently, system X_C^- has gained interest as a target for GBM targeting, with [¹⁸F]FSPG explored clinically¹, and [¹⁸F]hGTS13 as a second generation X_C^- tracer, with reduced uptake in inflammatory cells². We present the development of a third generation X_C^- targeting tracer, hED1, amenable to labeling with both diagnostic and therapeutic radiohalogens.

Methods: The precursor and non-radioactive standards were synthesized from bis-tert-butoxy protected homoglutamate and analyzed by NMR, HPLC, and high-resolution MS. The precursor was labelled with fluorine-18, bromine-77, and iodine-131 in copper mediated processes (Figure 1A). The radiofluorination process was automated with a Trasis ALLINONE mini synthesizer. The resulting radiohalogenated hED1s were purified by HPLC and formulated in phosphate buffered saline for use in vitro and in vivo.

System X_C^- mediated cellular uptake of [¹⁸F]F-hED1 and [¹³¹I]I-hED1 were investigated in the presence and absence of 1 mM glutamate, phenylalanine, or aspartic acid to evaluate amino acid transporter specificity. Orthotopic murine GL261 and human derived GBM43 mouse models of GBM were assessed using T1-weighted contrast enhanced magnetic resonance imaging (T1 CE MRI) and [¹⁸F]F-hED1 for positron emission tomography (PET).

Results: The radiohalogenation precursor was synthesized in 4 steps in 13% overall yield, and the non-radioactive standards ^natF-hED1, ^natBr-hED1, and ^natI-hED1 synthesized in 3-6% overall yields. The precursor was labelled with fluorine-18, bromine-77, and iodine-131 using Cu(py)₄(OTf)₂. This gave [¹⁸F]F-hED1, [⁷⁷Br]Br-hED1, and [¹³¹I]I-hED1 with non-decay-corrected radiochemical yields (n.d.c. RCY) of 10% ± 5% (n = 6), 62% (n = 1), and 70% ± 6% (n = 3), respectively. Automated [¹⁸F]F-hED1 synthesis gave n.d.c. RCY of 9% ± 4% in 186 ± 16 min (n = 9).

In vitro cell uptake studies on GL261 cells (n = 4–6 technical, n = 2 biological) show uptake of 1.03 ± 0.2 percent activity per 100,000 cells (%A/100k) and 1.11 ± 0.14 %A/100k for [¹⁸F]F-hED1and [¹³¹I]I-hED1, respectively. Pre-incubation with glutamate reduced uptake to 0.45 ± 0.12 %A/100k for [¹⁸F]F-hED1, but no significant change for phenylalanine and aspartic acid (Figure 1B). In vivo PET imaging with [¹⁸F]F-hED1 shows tumor accumulation with low background brain uptake, with a tumor-to-brain ratio of 9, and high kidney uptake, indicative of renal clearance (Figure 1C).

Conclusions: [¹⁸F]F-hED1/[¹³¹I]I-hED1 is a promising theranostic candidate for system X_C- targeting of GBM in murine cancer models. We are investigating how diastereomeric purity affects the uptake in cells and tumor models. The same precursor may also be extended to astatine-211 labeling.

Acknowledgements: We gratefully acknowledge the University of Wisconsin Cyclotron group for providing [¹⁸F]fluoride and a Pilot Award from the University of Wisconsin Institute for Clinical and Translational Research and Department of Radiology.

References:

1.        Baek S, et al. Clinical Cancer Research. 2012;18(19):5427-5437.

2.        Moses A, et al. Theranostics. 2025;15(3):836-849.