18F-fluorobenzoylcystine - A novel radiotracer for tracking apoptosis

Objectives Induction of apoptosis is often associated with an oxidative stress response. N-(4-[18F]fluorobenzoyl)cystine (18F-FBC), a potential substrate of the cystine-glutamate exchanger (xCT) which is known to be upregulated under oxidative stress conditions, could serve as novel apoptosis imaging agent.

Methods Radiosynthesis of 18F-FBC was achieved by coupling 18F-SFB with L-cystine di-tert-butyl ester followed by deprotection. Apoptosis was induced in EL4 and Jurkat cells with etoposide and staurosporine, respectively. Cell uptake of 18F-FBC in treated and non-treated cells and mixtures thereof was investigated. Apoptotic level was measured by Caspase-Glo assay. The distribution of 18F-FBC in EL4 tumor-bearing Balb/C mice 24h after i.p. administration of etoposide and cyclophosphamide or without treatment was assessed via PET/CT and gamma counting of excised organs. In vitro and ex vivo tracer stability was examined by HPLC analysis of mouse plasma and urine.

Results Radiochemical yields of 18F-FBC of 5.9±4.4% (n=8, decay-corrected to end of synthesis of 18F-SFB) were achieved. Higher uptake of 18F-FBC was observed in cells induced for apoptosis than in non-treated cells (up to 5.6±1.2-fold for late apoptotic Jurkat). A strong correlation of uptake of 18F-FBC with apoptotic level was found for all cell types (R2=0.88-1). Uptake of 18F-FBC in late apoptotic EL4 could be partially blocked (57%) by co-incubation with 0.5mM L-cystine. Ex vivo biodistribution studies at 60min p.i. showed that 18F-FBC does not accumulate in EL4 tumors neither without (0.21±0.14 %ID/g, n=3) or with treatment (0.17±0.04 %ID/g, n=3). As confirmed by PET/CT imaging, it is instead excreted almost entirely by the kidneys (112-142 %ID/g). This is not due to complete degradation of 18F-FBC, as 60min p.i. 29% and 18% of intact tracer were still found in blood and urine, respectively.

Conclusions Although uptake of 18F-FBC correlates well with apoptotic level in vitro, its potential as apoptosis imaging agent is limited because no in vivo tumor uptake was observed, at least in the model used in this study.

Research Support We thank the Radiochemistry Facility and the Small Animal Imaging Center at Stanford for supporting this study.