Synthesis and initial characterization of [¹²⁵I]-5-iodotubercidin, a radioligand for imaging adenosine kinase.

Objectives: Adenosine kinase (ADK) has been found to be overexpressed in the epileptogenic focus of both rodents and humans secondary to local astrogliosis. Hence, ADK may serve as an imaging biomarker for localizing seizure foci in patients with epilepsy. Here we report the radiosynthesis and initial evaluation of [¹²⁵I]-5-iodotubercidin, a well-known potent inhibitor of ADK (K_i = 9nM), as a prospective radioligand for monitoring ADK expression.

Methods: [¹²⁵I]-5-iodotubercidin was prepared from 5-bromotubercidin by treatment with [¹²⁵I]NaI, CuSO4 and SnSO4 in DMSO at 130°C, and the radiolabeled product was isolated by HPLC. [¹²⁵I]-5-iodotubercidin was evaluated in vitro by cell uptake studies using SKOV-3 cells, which are known to have robust ADK expression; blocking studies were performed with adenosine and equilibrative nucleoside transporter (ENT) inhibitors dipyridamole and nitrobenzylthioinosine (NBMPR). Uptake and blocking experiments were also performed with commercially available [³H]adenosine, for comparison. Biodistribution studies were carried out in C57BL/6 mice following intravenous administration of [¹²⁵I]-5-iodotubercidin; tissues and blood samples were collected and assayed for radioactivity after euthanizing mice at various time points. In vitro autoradiography and immunohistochemistry (IHC) were conducted on the brains of wild-type C57BL/6 mice and transgenic (fb-Adk-def) mice with regional alterations of ADK expression in the brain, and blocking experiments were performed with adenosine.

Results: [¹²⁵I]-5-Iodotubercidin was produced via halogen exchange reaction in 30-39% radiochemical yield and >99% radiochemical purity. Uptake of [¹²⁵I]-5-iodotubercidin in SKOV-3 cells peaked at 30 min incubation, and was blocked by adenosine in a dose-dependent manner with minimal nonspecific binding. Treatment of SKOV-3 cells with NBMPR and dipyridamole resulted in potent inhibition of [³H]adenosine uptake at nanomolar concentrations, whereas inhibition of [¹²⁵I]-5-iodotubercidin uptake required much higher (>1000x) concentrations. Moreover, [¹²⁵I]-5-iodotubercidin uptake was reduced by more than 50% in Na+ free buffer, while [³H]adenosine uptake was unaffected. These findings suggest that [¹²⁵I]-5-iodotubercidin uptake in SKOV-3 cells is mediated by both ENT and sodium-dependent mechanisms, unlike adenosine uptake which is mediated primarily via an ENT mechanism. Biodistribution studies with [¹²⁵I]-5-iodotubercidin revealed that peak brain activity occurred at 60 min post injection, and measured 1.53 %ID/g. However, >85% of the radiotracer remained in the blood pool at 60 min, and further analysis revealed that the radiotracer was being trapped in RBCs, with >99% of the activity in the cell pellet (compared to plasma) at both 5 min and 60 min post injection. In vitro autoradiography demonstrated increased radiotracer uptake in the striatum of transgenic mice compared to wild-type mice, in concordance with IHC analysis.

Conclusion: A high-affinity first-in-class ADK-specific radiotracer, [¹²⁵I]-5-iodotubercidin, was successfully prepared and its potential for measuring changes in ADK expression was demonstrated in vitro. However, in vivo imaging was not feasible due to trapping of radiotracer within RBCs. The synthesis of other nonnucleoside and nucleoside-based ADK-specific radiotracers, which have the potential for more rapid blood pool clearance and increased brain uptake, is currently underway. Research Support: Ralph Schlaeger Charitable Foundation Research Fellow Grant #RF1214 (RSNA), Penn University Research Foundation Award.