Click Chemistry in the Design and Production of Hybrid Tracers.

Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For αβ-integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers and were obtained in fewer steps than and had partition coefficients of log  = -2.55 ± 0.10, -1.45 ± 0.03, and -2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of and were, respectively, 23 × 10 and 40 × 10 M cm; lower than that of the reference tracer (50 × 10 M cm). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for and , respectively; 36 ± 5% bound for ;  > 0.05). ( = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound ( = 30.3 ± 5.7; < 0.0001), but had a statistically significantly lower affinity ( = 76.5 ± 18.3 nM; < 0.0001). Both and enabled in vivo visualization of the 4T1 tumor via fluorescence and single-photon emission computed tomography (SPECT) imaging. Biodistribution data (% ID/g) revealed a significant increase in nonspecific uptake in the kidney, liver, and muscle for both and . As a result of the higher background activity, the tumor-to-background ratio of the click-labeled RGD analogues was twofold lower compared to the end-labeled reference compound. The use of click chemistry labeling did not yield a pronounced negative effect on serum protein binding, in vitro stability, and receptor affinity; and tumors could still be visualized using SPECT and fluorescence imaging. However, quantitative in vivo biodistribution data suggest that the triazole and strained cyclooctyne moieties associated with this type of click chemistry negatively influence the pharmacokinetics of RGD peptides. Nevertheless, the design might still hold promise for other targets/targeting moieties.