Increasing evidence indicated that targeting tumor angiogenesis for specific drug transfer into tumor masses and metastasis is a promising approach. On the one hand, integrin poorly expressed on quiescent vessels and selectively overexpressed on activated endothelial cells of growing vessels, on the other hand, integrin αvβ3 frequently overexpresses on tumor cells, as observed in lung cancers, or breast cancers, and other tumors. Especially, many studies have indicated that the internalization of monomeric RGD-ligands is independent of its αvβ3 receptor and occurs via a fluid-phase endocytic pathway. Accordingly, in the blog, we would like to introduce a potent and selective inhibitor of the αvβ3 integrin, Cyclo(-RGDfK). The peptide exhibits an IC50 of 0.94 nM for αvβ3.

Cyclo(-RGDfK) TFA potently targets tumor microvasculature and cancer cells through the specific binding to the αvβ3 integrin on the cell surface.

Indeed, integrins exposed on the surface of HEK293(β3) cells, and the cells were incubated in the presence of 0–1 µmol/l RAFT-RGD or 0-4 µmol/l Cyclo(-RGDfK) for 10 minutes. The control group established the normal endocytosis of integrin αvβ3. As a result, in the presence of RAFT-RGD, internalization increased in a dose-dependent manner and reached 21 ± 2% at 1 µmol/L, corresponding to an increase of 79% versus control. In contrast, increasing doses of Cyclo(-RGDfK) (from 0.1 to 4 µmol/l) did not affect integrin internalization at all, which remained similar to that of the control.

What is more, [66Ga]DOTA-E-[c(RGDfK)]2 can be prepared with high radiochemical purity (>97%), specific activity (36-67GBq/μM), in vitro stability, and moderate protein binding. MicroPET imaging up to 24 post-injection showed contrasting tumors reflecting αvβ3-targeted tracer accumulation.

What’s more, in vivo, uptake of [66Ga]DOTA-E-[c(RGDfK)]2 by the C6 tumor xenograft indicated stable retention of the tracer with minimal wash-out up to 24 h. And not only that, tumor-to-muscle ratios (% ID/g) were 1.40 ± 0.97 and 1.78 ± 0.57 at 0.5 h and 24 h p.i., respectively. While tumor-to-blood ratio was 0.20 ± 0.09 at 0.5 h, increasing to 1.97 ± 0.37 at 24 h p.i.

To conclude, Cyclo (-RGDfK) is a potent and selective inhibitor of the αvβ3 integrin.


Lucie Sancey, et al. Clustering and internalization of integrin alphavbeta3 with a tetrameric RGD-synthetic peptide. Mol Ther. 2009 May;17(5):837-43.

V Lopez-Rodriguez, et al. Preparation and preclinical evaluation of (66)Ga-DOTA-E(c(RGDfK))2 as a potential theranostic radiopharmaceutical. Nucl Med Biol. 2015 Feb;42(2):109-14.