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111In-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: August 29, 2012.

Chemical name:111In-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4Image RAFTRGD.jpg
Abbreviated name:111In-DOTA-RAFT-RGD
Agent Category:Peptides
Target:Integrin αvβ3
Target Category:Receptors
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal / contrast:111In
  • Checkbox In vitro
  • Checkbox Rodents
Structure of 111In-DOTA-RAFT-RGD (1).



111In-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-regioselectively addressable functionalized template (RAFT)-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4, abbreviated as 111In-DOTA-RAFT-RGD, is a tetrameric Arg-Gly-Asp (RGD)-based peptide conjugate synthesized by Ahmadi et al. for tumor imaging by targeting integrin αvβ3 (1).

Angiogenesis is a process of neovascular development and growth from pre-existing vessels. Different from the normal blood vessel system, the neovasculature is chaotic and irregular in tumors with abnormal expression of diverse vascular surface biomarkers. These biomarkers have fewer kinetic compartments that must be crossed for intravenously administered agents to reach them (2, 3). Integrin αvβ3 is one of these biomarkers and has been intensively investigated as a target for imaging and antiangiogenic therapy. Integrin αvβ3 is minimally expressed in normal blood vessels but is significantly overexpressed in newly sprouting vasculature in tumors (4, 5). Small peptides are one group of ligands frequently used for targeting purposes, such as RGD, Asp-Gly-Arg, His-Gly-Phe, and Arg-Arg-Leu (3). The RGD tripeptide sequence is an adhesive protein recognition site, presenting in the extracellular matrix and blood. Integrin αvβ3 binds extracellular matrix proteins through the exposed RGD tripeptide sequence. In general, RGD peptides are less selective (binding with 8 of the 24 integrins), degrade rapidly in vivo, and have a relatively low binding affinity (3, 6).

Development of multivalent RGD peptides is one approach to improve their properties. Boturyn et al. synthesized a RAFT-RGD platform based on a cyclic decapeptide scaffold that incorporates and presents in a spatially controlled manner two independent functional domains: a clustered ligand domain for integrin recognition and cell targeting, and a labeling domain for detection and characterization of this binding (7). This platform has been labeled with Cy5, 99mTc, and 64Cu and has been shown to efficiently accumulate in tumors in animal models (8-11). Although the results are promising, approximately 30%–50% of the total dose of the labeled RAFT-RGD has been found to remain trapped in the kidneys several days after injection, which is at least partially due to the tetramerization of RGD peptides (1, 8, 12). Although peptides can be reabsorbed, endocytosed, and degraded by the lysosomes of proximal tubular cells, radiometal-chelated amino acids may be retained in the tubular cells, leading to radiation-induced toxicity and a weak imaging contrast for tumors surrounding the kidneys.

To reduce the renal accumulation of fluorescent and radiolabeled RAFT-RGD, Briat et al. evaluated the effect of Gelofusine on the renal reabsorption of these agents (13). Gelofusine is a gelatin-based plasma expander that consists of succinylated bovine gelatine molecules, a mixture of collagen-derived peptides. Infusion of Gelofusine has been reported to increase the renal excretion of megalin ligands and decrease the retention of radiolabeled compounds in the cortical proximal tubules (14-16). The results by Briat et al. showed that Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while it increased the ratio of tumor to healthy tissue (13). This chapter summarizes the data obtained with 111In-DOTA-RAFT-RGD and Gelofusine.



Boturyn et al. described the three-step synthesis of DOTA-RAFT-RGD (7). Ahmadi et al. described the radiolabeling of DOTA-RAFT-RGD with [111In]InCl3 (1). The optimized labeling reaction was determined to be the addition of 55 MBq (1.49 mCi) [111In]InCl3 to 100 µg DOTA-RAFT-RGD in an ammonium acetate buffer (pH 4.8), heated for 30 min at 70°C for 30 min. DOTA-RAFT-RGD was labeled at a high radiochemical yield (> 97%) and purity (> 97%) in this condition (1). The specific activity was not reported.

In Vitro Studies: Testing in Cells and Tissues


Ahmadi et al. analyzed the in vitro stability of 111In-DOTA-RAFT-RGD after incubation with human serum for 30 min to 24 h (1). 111In-DOTA-RAFT-RGD remained stable (95.6% intact) at 24 h, and only 2.5% of nonbound 111In was detected in human serum at 24 h, indicating a weak exchange reaction with plasma proteins and a high stability of the labeled complex.

Animal Studies



Ahmadi et al. evaluated the in vivo metabolic stability of 111In-DOTA-RAFT-RGD in mice at 1, 4, and 24 h after injection of 2 MBq (0.054 mCi) of the tracer (n = 3 mice/time point, except 24 h, where n = 2) (1). Tissue homogenates were extracted, and the extraction efficiency for all tissues was between 77% and 99%. More than 90% of injected radioactivity was recovered from the purification column. Analysis of urine and liver extracts at 5 min and 25 min showed two peaks, which corresponded respectively to nonbound 111In and 111In-DOTA-RAFT-RGD. The radiochemical purity of 111In-DOTA-RAFT-RGD at 4 h after injection was 90% and 85% in the urine and liver, respectively. In the kidneys, 54.3% of the radioactivity corresponded to the intact tracer and ~25% to the nonbound 111In. Blood radioactivity was undetectable at 1 h after the tracer injection.

Ahmadi et al. further investigated the biodistribution of 111In-DOTA-RAFT-RGD in αvβ3-positive TS/A-pc (murine mammary carcinoma cells) tumor-bearing mice (n = 8) (Table 1) (1). After injection of 111In-DOTA-RAFT-RGD (3.7 MBq (0.1 mCi)) through the tail vein, 15-min whole-body planar imaging was performed at 1, 4, 24, and 48 h, respectively. Animals were then euthanized. At 1 h after injection, 111In-DOTA-RAFT-RGD uptake in tumors was 1.63% injected dose per gram (ID/g), which decreased over time and stabilized between 24 h and 48 h. Clearance of the radioactivity from the blood circulation was fast, with the kidneys as the main route. Radioactivity in the kidneys was persistent with >30% ID/g. The tumor/blood and tumor/muscle ratios rapidly reached values above 1, and indicated that the imaging contrast was better at 24 h or 48 h after injection. Scintigraphic imaging confirmed these results and allowed the visualization of all tumors between 1 h and 48 h.

Table 1: Biodistribution data of 111In-DOTA-RAFT-RGD in TS/A-pc tumor-bearing mice.*

Time after injection
Tissue (% ID/g)1 h4 h24 h48 h
Liver2.16 ± 0.811.30 ± 0.101.15 ± 0.081.29 ± 0.61
Kidney32.08 ± 14.5952.73 ± 9.4432.18 ± 1.5832.69 ± 5.56
Blood2.77 ± 1.700.12 ± 0.050.03 ± 0.0020.03 ± 0.01
Tumor1.63 ± 0.671.26 ± 0.180.77 ± 0.070.71 ± 0.17

*Data were obtained by Ahmadi et al. (1).

Briat et al. studied the effect of Gelofusine on the biodistribution of 111In-DOTA-RAFT-RGD in mice bearing TS/A-pc tumors (n = 6 mice/group) (Table 2) (13). Each mouse received a tail vein injection of 12 MBq (0.32 mCi) 111In-DOTA-RAFT-RGD. Gelofusine (4%) or phosphate-buffered saline (PBS) was either pre-injected (5 min before tracer injection) or co-injected with the tracer. Similar to the data obtained by Ahmadi et al., 111In-DOTA-RAFT-RGD was primarily and rapidly cleared through the renal route. The amount of 111In-DOTA-RAFT-RGD was <1% ID/g in most organs at 1 h after injection except in the tumors and kidneys. Pre-injection and co-injection of Gelofusine strongly reduced the radioactivity in the kidneys by 49.2% and 47.9%, respectively (P = 0.002 and P < 0.0001). The radioactivity in the blood was comparable in the presence or absence of Gelofusine. Muscle uptake of 111In-DOTA-RAFT-RGD decreased by 10% in the presence of Gelofusine, whereas tumor uptake was not significantly affected (P > 0.05).

Briat et al. performed SPECT-CT imaging at 1 h and 24 h after injection of 111In-DOTA-RAFT-RGD (13). PBS or Gelofusine was pre-injected 5 min before the tracer (Table 2). Images showed that 111In-DOTA-RAFT-RGD was mainly localized in the renal cortex. In the presence of Gelofusine, this renal capture was also localized in the cortex but was greatly reduced. At 24 h after injection, the tracer uptake in the kidneys with Gelofusine preinjection was reduced by 49% and 66% as measured in the dissected kidneys and from the images, respectively, compared with PBS. The tumor/kidney but not the tumor/muscle ratio was significantly different between mice with PBS and those with Gelofusine injection (P < 0.05).

Table 2: Effect of Gelofusine on the biodistribution of 111In-DOTA-RAFT-RGD.

Ratio1 h24 hDissected*1 h24 hDissected*
Kidney13.35 ± 1.555.83 ± 0.5242.30 ± 9.304.95 ± 0.951.97 ± 0.2321.50 ± 9.30
T/M4.24 ± 0.444.50 ± 0.906.42 ± 2.303.63 ± 0.665.21 ± 0.767.03 ± 1.62
T/K0.06 ± 0.010.05 ± 0.010.03 ± 0.010.16 ± 0.020.16 ± 0.010.06 ± 0.01

*Data were obtained from dissected organs at 24 h after injection. Other data in the table were obtained directly from the images.

T/M = tumor/muscle ratio; T/K = tumor/kidney ratio.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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