Radiosynthesis and biological evaluation of [¹ ⁸F]AG-120 as imaging agent for the detection of the mutant isocitrate dehydrogenase 1 in glioma by PET

Introduction: Glioma are clinically challenging tumors due to their location and invasiveness, limiting their complete surgical resection. Mutated isocitrate dehydrogenase enzymes (IDH) are frequent alterations - the most common being the IDH1R132H - expressed only in glioma but not in healthy brain, correlating with prognosis. Hence, the evaluation of the IDH mutation status has become essential in clinical patient stratification. Currently, the IDH mutations are determined either directly by invasive biopsies or indirectly by magnetic resonance spectroscopy. Here, we propose a transdisciplinary approach to develop an 18F-labeled ligand to detect the IDH1R132H protein directly and non-invasively by positron emission tomography (PET) imaging. Such an imaging tool could improve the selection of cancer patients who are most likely to benefit from precision medicine. In the present study, we performed the radiofluorination of AG-120 (Ivosidenib), an FDA-approved small molecule inhibitor of mutant-IDH.

Methods: The stannyl precursor (HL174) was synthetized according to the published synthesis of AG-120 [1] with minor modifications. The radiosynthesis was performed by copper-mediated radiofluorination of HL174 (Fig. 1) using a TRACERlab FX2 N synthesis module. The inhibitory potency of AG-120 was measured with a diaphorase/resazurin coupled assay using recombinant IDH1R132H or IDH1 (n=2). Internalization of [18F]AG-120 were determined in vitro using U251 human glioblastoma cells stably transfected with IDH1 or IDH1R132H [2]. In vivo metabolism was investigated in healthy CD-1 mice (n=3) by radio-chromatographic analyses of plasma and brain tissue. Dynamic PET (Mediso, nanoScan® PET/CT) imaging studies were performed in nude rats bearing U251-IDH1 (n=2) or U251-IDH1R132H (n=2) glioblastoma.

Results: For the first time, diastereomerically pure [18F]AG-120 was prepared by an automated copper-mediated radiolabelling approach without azeotropic drying starting from the stannyl precursor HL174 (Fig.1). AG-120 shows a high inhibitory potency toward the IDH1R132H (IC50 IDH1R132H=5.11 nM). Internalization studies revealed a significantly higher uptake (0.422 vs. 0.014% AD/μg protein at 120min) of [18F]AG-120 in U251-IDH1R132H cells in comparison to U251-IDH1 cells, suppressible by self-blocking (0.422 vs. 0.009% AD/μg protein at 120min). [18F]AG-120 is metabolized relatively slow with parent fractions of 85% and 91% in plasma and brain, respectively, at 30min p.i.. Dynamic PET studies show a limited blood-brain barrier (BBB) permeation of [18F]AG-120 along with a low uptake in the brain tumor (TAC peak value ~0.4 SUV at 0.6min), which did not differ significantly between IDH1R132H- and IDH1-tumors (tumor-to-blood ratio[40-60min]: ~1.7 vs. ~1.3).

Conclusions: We successfully established an automated radiosynthesis of [18F]AG-120. The preliminary preclinical evaluation revealed a target-specific internalization in vitro and a high metabolic stability in vivo. Despite a generally low BBB penetration of [18F]AG-120, a slightly higher accumulation of activity in IDH1R132H-glioblastoma in comparison to the IDH1 glioblastoma could be observed. Altogether, these results encourage the evaluation of [18F]AG-120 in models of peripheral cancers such as chondrosarcoma.

Acknowledgements: We thank the European-Regional-Development-Fund and the Sächsische-Aufbaubank for the financial support (project no. 100364142); Dr. Kessler, Prof. Vordermark for the cells and Mrs. Barth for the PET experiments.

References:

[1] Popovici-Muller et al., 2018. ACS Med Chem Lett, 9: 300-305.
[2] Kessler et al., 2015. Radiother Oncol, 116: 381-387.