Abstract
1476
Introduction: Glioblastoma (GBM) is a devastating cancer disease affecting 3 of 100000 Europeans. Furthermore, GBM accounts for 25% of all malignant tumors of the central nervous system. Combined approaches, including surgery, chemotherapy and external radiation have shown to only slightly prolong the survival of patients to a maximum of 14 months after first diagnosis. Reasons for this ineffective treatment are the tumor growth pattern, its invasive vascularization and high heterogenicity. Hence, a specific PET radioligand for visualization of GBM biomarkers like EGFR-TKI might help to improve diagnostic and therapy monitoring following anti-angiogenic treatment. Here, we describe the development of a thienopyrimidine based radiotracer from a library of EGFR-TKI from Bugge et al. [1] and its subsequent in vivo characterization and visualization.
Methods: The non-radioactive thienopyrimidine standard 1 and the desmethyl precursor 2 were synthesized according to published methods [1]. Radiosynthesis of [11C]1 was achieved by methylation with [11C]MeI using NaOH as a base in DMSO at 100 °C for 3-5 min in a TRACERlab FX2C module. [11C]1 was purified by preparative HPLC and further passed through a C-18 Sep-Pak cartridge. The eluted ethanolic extract was evaporated to approximately 30 µl and reconstituted to a final volume of 400 µl with 1% polysorbate 80 in isotonic saline for in vivo application. The cell association was evaluated at room temperature by in vitro saturation studies in A431 cells using Erlotinib as a blocking agent. Radiotracer biodistribution in vivo was studied by 1 h dynamic simultaneous PET/MR imaging (DRYMAG 7024/PET, MRsolutions, Guildford, UK) following [11C]1 i.v. injection (5.8±4.3 MBq) in healthy female CD1 mice (31.7±4.2 g, n=4). Initial metabolism was studied in healthy CD1 mice (n=3) administered with 130±45 MBq [11C]1. Animals were sacrificed five minutes after administration and brain, blood and liver were collected and homogenized in ACN/H2O (7:3). Tissue homogenates and plasma were further analyzed by HPLC-RAD-MS. Results: Compounds 1 and 2 were obtained in 29% and 39% overall yields over six steps. Radiomethylation of 2 with [11C]MeI resulted in [11C]1 with 8.9±3.8% (n=12) radiochemical yield (non-decay corrected, EOB) in 45 min total synthesis time. The radiochemical purity was found to be >99% (n=12) and the molar activity achieved was 115±80 GBq/µmol (n=12). In vitro stability in human serum shows >99% intact [11C]1 after 30 minutes. Compound [11C]1 showed uptake in EGFR overexpressing A431 cells which was displaceable with Erlotinib. The radiotracer showed blood-brain-barrier (BBB) penetration and nonsignificant amounts of radiometabolites were detected with the methods applied. However, possible hydroxylated metabolites were found in trace amounts by LC/MS analysis. PET/MR imaging revealed high brain uptake (SUV=1.4 at 5 min p.i.) followed by a wash-out during the investigation time. Conclusion: Our preliminary PET/MR and ex vivo metabolism results indicate that compound [11C]1 crosses the BBB and that non-significant radiometabolite amounts are detected with the analysis methods applied . Cellular uptake in A431 cells renders [11C]1 suitable for subsequent in vivo studies. Hence, further studies in brain tumor bearing animals are planned to show specific binding to GBM tissue and to further develop the radiotracer as a tool for brain tumor imaging with PET. 1. Bugge, S., et al., Structure-activity study leading to identification of a highly active thienopyrimidine based EGFR inhibitor. European Journal of Medicinal Chemistry, 2014. 75: p. 354-374. Acknowledgements: This study is financed by 180°N the Norwegian Nuclear Medicine Consortium