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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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111In-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid-Glu{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)}-{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)} (PEG4 = 15 amino-4,7,10,13-tetraoxapentadecanoic acid)

, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: March 22, 2012.

Chemical name:111In-Labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid-Glu{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)}-{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)} (PEG4 = 15 amino-4,7,10,13-tetraoxapentadecanoic acid)Image In1112P4GRGD4.jpg
Abbreviated name:111In(DOTA-2P4G-RGD4)
Agent Category:Peptides
Target:Integrin alphavbeta3 (αvβ3)
Target Category:Receptors
Method of detection:Single-photon emission computed tomography (SPECT) or planar imaging
Source of signal / contrast:111In
  • Checkbox In vitro
  • Checkbox Rodents
Structures of 111In(DOTA-2P4G-RGD4) by Shi et al. (1).



The 111In-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA)-Glu{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)}-{PEG4-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-d-Phe)}, abbreviated as 111In(DOTA-2P4G-RGD4) or 111In-DOTA-E{PEG4-E[Gly3-c(RGDfK)]2}2, was synthesized by Shi et al. as an agent for molecular imaging of tumor angiogenesis by targeting integrin alphavbeta3 (αvβ3) (1). Here, P = PEG4 = 15 amino-4,710,13-tetraoxapentadecanoic acid = a linker between two c(RGDfK) motifs; c(RGDfK) = cyclo(Lys-Arg-Gly-Asp-d-Phe); RGD4 = four motifs of c(RGDfK); E = Glu; and (G = G3 = Gly-Gly-Gly).

Integrin αvβ3 is a receptor that is overexpressed on the activated endothelial cells of tumors (2). Because the integrin αvβ3 binds with extracellular matrix proteins (e.g., vitronectin, fibronectin) through the exposed Arg-Gly-Asp tripeptide sequence, RGD-containing peptides have been intensively studied in the past decade as a vector for imaging αvβ3 expression (3, 4). Although significant progress has been made, improving the binding affinity and pharmacokinetics of RGD peptides remains the major consideration in the design of agents, and various strategies have been developed for this improvement, such as the use of RGD multimers, the introduction of sugar amino acids or d-amino acids into the RGD peptides, and the conjugation of the RGD peptides with chelators or polyethylene glycol (PEG) chains (5, 6).

The concept of multimerization has been developed to address the question of multimeric binding by "poly-potent" ligands. Multimers are formed by bridging monomeric units through linker(s) (2, 7). The multimer effect has been demonstrated in different studies, showing that the binding affinity of RGD peptides increases in the order of monomer < dimer < tetramer < octamer. For example, the cyclo(-RGDfE-)-monomer, dimer, and tetramer containing heptaethylene glycol spacer units have been shown to exhibit αvβ3-binding affinities that increase by a factor of ten with each duplication of binding units (8). The integrin affinity of the DOTA-RGD octamer has been shown to be three-fold higher than that of the DOTA-RGD tetramer (7).

Recently, investigators from Purdue University suggested a concept of "bivalency" for the αvβ3-binding affinity of RGD-containing peptides (1, 6, 9). The main point of this concept is that the multimeric cyclic RGD peptides are likely bivalent rather than multivalent in binding to integrin αvβ3, and enough distance between two RGD motifs is the key for bivalency (1, 5, 10). For example, the dimeric peptide E[c(RGDfK)]2 (RGD2) is monovalent, whereas the dimeric peptides PEG4-E[PEG4-c(RGDfK)]2 (3P-RGD2) and G3-E[G3-c(RGDfK)]2 (3G-RGD2) (G = G3 = Gly-Gly-Gly linker) are bivalent because of the increased distance between the two cyclic RGD motifs for simultaneous integrin αvβ3 binding in the latter two peptides (5). The cyclic RGD tetramers, such as DOTA-RGD4 and DOTA-E{G3-E[G3-c(RGDfK)]2}2 (DOTA-6G-RGD4), are also likely bivalent in binding to integrin αvβ3 even though they contain four identical RGD motifs (5, 10). Studies with three other tetramers (DOTA-2P-RGD4, DOTA-2P4G-RGD4, and DOTA-6P-RGD4) have further shown that the three tetramers are not tetravalent, although the tetramers exhibited a higher binding affinity to αvβ3 than monomers and dimers, and the "locally enriched" RGD concentration has been considered to contribute to the higher binding affinity (1, 6). The linkers between RGD motifs may have a significant impact on the integrin αvβ3-targeting capability, biodistribution, excretion kinetics, and metabolic stability of cyclic RGD peptides (1, 11, 12).

This chapter summarizes the data obtained with 111In(DOTA-2P4G-RGD4). Other chapters summarize the data obtained with 111In(DOTA-2P-RGD4) and 111In(DOTA-6P-RGD4), respectively.



The RGD-containing peptides were all custom-made, including P-RGD, P2G-RGD2, 3P-RGD2, and 3P-RGK2 (RGK = cyclo(Arg-Gly-Lys-d-Phe-Asp)). The tetramers were synthesized by the reaction of Boc-E(OSu)2 and their corresponding dimers (1). Conjugation of the RGD peptides with DOTA-OSu resulted in their corresponding conjugates (DOTA-P-RGD, DOTA-P-RGD2, DOTA-3P-RGD2, DOTA-2P-RGD4, DOTA-2P4G-RGD4, and DOTA-6P-RGD4, respectively). DOTA-6P-RGK4 was prepared using a procedure identical to that for DOTA-6P-RGD4. DOTA-6P-RGK4 has the identical amino acids, but its sequence is scrambled to demonstrate the RGD-specificity of DOTA-6P-RGD4. The identities for all RGD conjugates were confirmed, and their purities were all >95% before being used for 111In labeling and determination of their integrin αvβ3 binding affinity. Table 1 lists the molecular weights and yields of each DOTA-conjugated peptide (1).

Table 1: The physicochemical characteristics of the RGD peptides.


*The data for DOTA-3P-RGD2 were obtained from Shi et al. (5).

All 111In-labeled peptides were prepared by reacting 111InCl3 with the respective DOTA conjugates in NH4OAc buffer (100 mM, pH = 5.5). Radiolabeling was completed by heating the reaction mixture at 100°C for ~15 min. After purification, the radiochemical purity was >95% and the specific activity was >40 mCi/μmol (1.48 GBq/µmol) for all 111In-labeled RGD peptides (1).

In Vitro Studies: Testing in Cells and Tissues


The integrin binding affinity and specificity of the RGD peptides were assessed in U87MG human glioma whole cells, with 125I-c(RGDyK) as the integrin-specific radioligand (1). The αvβ3 binding affinity of the peptides followed the order of DOTA-6P-RGD4 ~ DOTA-2P4G-RGD4 ~ DOTA-2P-RGD4 > DOTA-P-RGD2 > DOTA-P-RGD > RGD (IC50 = 49.9 ± 5.5) (Table 1). The binding affinity of DOTA-3P-RGD2 was significantly higher than that of DOTA-P-RGD and DOTA-P-RGD2 (P < 0.01). However, the investigators found that the integrin αvβ3 binding affinities of DOTA-2P-RGD4, DOTA-2P4G-RGD4, and DOTA-6P-RGD4 were only marginally higher than that of DOTA-3P-RGD2, suggesting that they might share the same bivalency in binding to the integrin αvβ3.

Shi et al. also determined the water-octanol partition coefficients and stability of the 111In-labeled peptides (1). The log P values are listed in Table 2. The 111In-labeled peptides remained stable for >72 h after purification in the presence of 3 mM EDTA.

Table 2: The IC50 and log P values of the 111In-labeled cyclic RGD peptides.

IC50 (nM)
Log P-3.48-3.22-4.20-3.87-3.93-3.68-3.61

IC50: half-maximal inhibitory concentration.

Animal Studies



Biodistribution, imaging, and metabolic studies were performed in athymic nude mice bearing U87MG human glioma xenografts in both left and right upper flanks (1).

For the biodistribution studies, each mouse was administered ~3 μCi (~0.111 MBq) of the agent via tail vein injection (1). Mice were then euthanized at 0.5, 1, 4, 24, and 72 h after injection (n = 5 mice/time point). The organ uptake was calculated as the percentage of injected dose per gram of organ mass (% ID/g). The tumor and intestinal uptakes of 111In(DOTA-2P-RGD4) were high and close to those of 111In(DOTA-6P-RGD4) over the 72-h period. 111In(DOTA-2P-RGD4) also had a rapid clearance from normal organs, such as the blood, kidneys, and liver. Therefore, the tumor/liver and tumor/kidney ratios of 111In(DOTA-2P-RGD4) were comparable to those obtained for 111In(DOTA-6P-RGD4) and 111In(DOTA-6G-RGD4.

Table 3: Selected biodistribution data of 111In(DOTA-2P-RGD4).

% ID/g0.5 h1 h24 h72 h
Blood0.84 ± 0.320.13 ± 0.000.02 ± 0.000.03 ± 0.01
Kidney10.72 ± 2.135.57 ± 0.631.98 ± 0.310.85 ± 0.09
Liver3.62 ± 0.852.36 ± 0.270.91 ± 0.080.25 ± 0.06
U87MG11.43 ± 2.9210.18 ± 1.615.79 ± 0.672.66 ± 0.81
Tumor/Blood13.58 ± 3.4780.74 ± 10.67266.19 ± 59.0293.96 ± 22.32
Tumor/Bone5.27 ± 1.355.80 ± 0.657.02 ± 1.396.76 ± 2.74
Tumor/Kidney1.07 ± 0.271.84 ± 0.262.96 ± 0.363.14 ± 0.84
Tumor/Liver3.15 ± 0.814.39 ± 1.136.46 ± 1.3310.71 ± 1.45
Tumor/Lungs2.68 ± 0.694.38 ± 1.285.59 ± 0.638.73 ± 1.70
Tumor/Muscle6.99 ± 1.7910.43 ± 1.7524.04 ± 14.2410.16 ± 2.72

No blocking experiment was performed with 111In(DOTA-2P4G-RGD4). The data obtained from blocking experiments with 111In(DOTA-6P-RGD4) are summarized in the chapter on 111In(DOTA-6P-RGD4) in MICAD (1).

The imaging studies were performed after tail vein administration of ~100 μCi (3.7 MBq) 111In(DOTA-2P4G-RGD4) (n = 3 mice) (1). The whole-body images were acquired at 0.5, 1, 4, 24, and 72 h after injection. The tumors were clearly visualized with excellent tumor/background contrast as early as 1 h after injection. A long tumor retention time was observed, which was consistent with the biodistribution results. The time to half-maximal radioactivity in tumors was >30 h for 111In(DOTA-2P-RGD4), 111In(DOTA-2P4G-RGD4), and 111In(DOTA-6P-RGD4) (1).

For the metabolism studies, each mouse was given ~100 μCi (3.7 MBq) 111In(DOTA-6P-RGD4) via tail vein injection (1). The urine samples were collected at 30 min and 120 min after injection, and the feces samples were collected at 120 min after injection. The percentage of radioactivity recovery was >95% (by γ-counting) for both urine and feces. There was very limited radioactivity accumulation in the liver and kidneys. There was little metabolism detected in the urine or feces samples over the 2-h study period for 111In(DOTA-2P4G-RGD4).

Shi et al. comparatively analyzed the biodistribution data for 111In(DOTA-2P-RGD4), 111In(DOTA-2P4G-RGD4), 111In(DOTA-6P-RGD4), 111In(DOTA-6G-RGD4), 111In(DOTA-3P-RGD2), 111In(DOTA-P-RGD2), and 111In(DOTA-P-RGD) (1). Over the first 24 h, the tumor uptake difference between the tetramers and dimers was not significant (P > 0.05). The tumor uptake at 72 h (% ID/g) followed the general order of 111In(DOTA-6P-RGD4) (3.04) > 111In(DOTA-2P-RGD4) (2.87) ~ 111In(DOTA-2P4G-RGD4) (2.66) > 111In(DOTA-3P-RGD2) (2.18), which was similar to the trend observed with integrin αvβ3 binding affinity (IC50, nM) of DOTA-6P-RGD4 (0.3 ± 0.1) ~ DOTA-2P4G-RGD4 (0.2 ± 0.1) ~ DOTA-2P-RGD4 (0.5 ± 0.1) > DOTA-3P-RGD2 (1.5 ± 0.2). Among the 111In-labeled radiotracers, 111In(DOTA-6G-RGD4) had the highest uptake in the tumor and intestine at 72 h after injection. The half-life of 111In(DOTA-6G-RGD4) in the tumor was estimated to be 60 h. In contrast, 111In(DOTA-3P-RGD2) and 111In(DOTA-2P4G-RGD4) had low uptake in the intestine, kidneys, and liver over the 72-h period. As a result, 111In(DOTA-3P-RGD2) and 111In(DOTA-2P4G-RGD4) had tumor/kidney and tumor/liver ratios that were significantly better (P < 0.05) than those of 111In(DOTA-6G-RGD4) and 111In(DOTA-6P-RGD4) during that period of time. As expected, 111In(DOTA-P-RGD) had the lowest tumor uptake (3.7% ID/g and 0.7% ID/g at 0.5 h and 72 h after injection, respectively). 111In(DOTA-P-RGD2) had a relatively high tumor uptake (6.1 at 0.5 h after injection), but it had a significant washout from the tumor, with uptake values being 4.9, 4.8, 3.1, and 1.6% ID/g at 1, 4, 24, and 72 h after injection, respectively. The tumor uptake values follow the trend of 111In(DOTA-3P-RGD2) > 111In(DOTA-P-RGD2) > 111In(DOTA-P-RGD), which was consistent with the order of their αvβ3 binding affinities (IC50 value): DOTA-3P-RGD2 < DOTA-P-RGD2 < DOTA-P-RGD.

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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Dijkgraaf I., Beer A.J., Wester H.J. Application of RGD-containing peptides as imaging probes for alphavbeta3 expression. Front Biosci. 2009;14:887–99. [PubMed: 19273106]
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