Real-time pharmacodynamic evaluation of a therapeutic system X_C- inhibitor using [¹⁸F]FSPG-PET

Purpose: PET/CT offers the unique potential to noninvasively evaluate therapeutic target engagement. Our overall goal is to develop novel drugs that target the System X_C^- antiporter, which has been shown to be overexpressed in many malignancies, including GBM and pancreatic cancer. System X_C^- is composed of SLC7A11 and SLC3A2, and functions to exchange intracellular glutamate with extracellular cystine. The downstream effects of this exchange include protein and glutathione synthesis and it plays a critical role in maintaining extracellular redox balance. Our objective here is to develop a non-invasive means of monitoring System X_C^- target engagement by a novel class of System X_C^- inhibitors.

Methods: We examined the cell uptake of [¹⁸F]FSPG ((4S)-4-(3-[18F]-fluoropropyl)-L-glutamate) in GBM and pancreatic cancer cell lines without and with IKE (imidazole ketone erastin; 10 uM mg/ml), a selective inhibitor of system X_C^-. For cell uptake studies,100,000 cells were plated in 24-well cell culture plates overnight and treated with tracer for 60 minutes followed by 4 washes with cold PBS. Cells were then lysed with 0.1M NaOH and counted using a HIDEX well counter. For tumor slice studies, fresh slices of tumor were extracted immediately after mice were euthanized and placed in specialized media for 4 hours. Slices were treated with [¹⁸F]FSPG with or without pretreatment of IKE, washed and imaged for 60 minutes on an Inveon microPET scanner. Results. [¹⁸F]FSPG was taken up robustly by GBM and pancreatic cancer cells. Uptake was significantly blocked in the presence of IKE (>80% blockade), confirming that IKE suppresses system X_C^- activity. Similarly, [¹⁸F]FSPG was taken up in the fresh tumor sections, which was also significantly blocked with excess IKE. Conclusions. We have established that [¹⁸F]FSPG can be used to monitor pharmacodynamic effects of IKE, a System X_C^-selective inhibitor and ferroptosis inducer. These findings suggest that we can utilize [¹⁸F]FSPG PET as a means to identify target engagement in vivo to personalize therapeutic selection as well as develop second generation compounds with higher affinity or better pharmacodynamics and pharmacokinetics. Support: NIH/NCI 5P01CA207206. The PET early drug development program is supported by the Columbia Dept. of Radiology and Irving Institute CTSA Translational Therapeutics Accelerator (UL1TR001873).