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99mTc-Labeled, PEGylated (NαHis)Ac-β3hLys-βAla-βAla-Gln7-Trp8-Ala9-Val10-Gly11-His12-Cha13-Nle14-NH2

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

Created: ; Last Update: April 11, 2012.

Chemical name:99mTc-Labeled, PEGylated (NαHis)Ac-β3hLys-βAla-βAla-Gln7-Trp8-Ala9-Val10-Gly11-His12-Cha13-Nle14-NH2
Abbreviated name:99mTc-PEGx-Lys-BN (x = 5, 10, and 20 kDa)
Synonym:99mTc-PEG5-Lys-BN, 99mTc-PEG10-Lys-BN, and 99mTc-PEG20-Lys-BN
Agent Category:Peptides
Target:Gastrin-releasing peptide receptors (GRPR)
Target Category:Receptors
Method of detection:Single-photon emission computed tomography (SPECT) or planar imaging
Source of signal / contrast:99mTc
  • Checkbox In vitro
  • Checkbox Rodents
No structure available



The 99mTc-labeled, PEGylated (NαHis)Ac-β3hLys-βAla-βAla-Gln7-Trp8-Ala9-Val10-Gly11-His12-Cha13-Nle14-NH2 (Lys-BN), abbreviated as 99mTc-PEGx-Lys-BN (x = 5, 10, and 20 kDa), are PEGylated bombesin (BN) analogs that were synthesized by Dapp et al. for imaging of tumors that overexpress gastrin-releasing peptide receptors (GRPR) (1).

Engineered proteins and peptides are widely applied in the development of molecular imaging agents; however, they exhibit some unfavorable pharmacokinetic properties when used in vivo, such as rapid clearance, immunogenicity, and poor stability (e.g., aggregation, degradation, deamination, oxidation, etc.) (2, 3). As a technique to overcome these limits of proteins and peptides, PEGylation has been extensively studied recently, and the number of agents newly developed with PEGylation is increasing continuously (3, 4). PEGylation is defined as the covalent attachment of poly(ethylene glycol) (PEG) chains to bioactive substances (3). PEG possesses three key properties: great flexibility due to the absence of bulky substituents along the chain; high hydration of the polymeric backbone; and a high degree of safety, with toxicity only at very high doses (1, 3, 4). Furthermore, PEG can be coupled with virtually any exposed surface and even some buried amino acids in a protein, and this coupling can be achieved at the N- or C-terminus, the cysteines located far from the receptor-binding site, or the incorporated unnatural amino acids. PEG increases the blood circulation of a given protein by increasing its hydrodynamic volume, prevents its immunogenicity, reduces its aggregation, and increases its thermal stability. However, a reduction in biological potency is common after PEGylation because of the steric entanglement of polymer chains during the protein/receptor recognition process (3). This reduction is also related to the PEGylation methods and PEG selected. The properties of PEG vary significantly with molecular weight and concentration.

Radiolabeled BN analogs are promising radiotracers for tumor imaging and therapy by targeting GRPR (5-7). However, the low in vivo stability of BN analogs limits their clinical application (8, 9). Dapp et al. prepared a series of PEGylated BN(7-14) analogs and evaluated their properties in vitro and in vivo (1). PEGylation was performed with linear PEG molecules of various sizes (5 kDa (PEG5), 10 kDa (PEG10), and 20 kDa (PEG20)) through the ɛ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the (NαHis)Ac chelator. In vitro results showed that PEGylation did not affect the binding affinity of BN analogs, but it did slow their binding kinetics (1). In vivo results showed that PEGylation increased the stability of the analogs, improved their pharmacokinetics, and enhanced the tumor retention.



The BN peptide Lys-BN was synthesized with peptide solid-phase synthesis (1). Methoxypolyethylene glycol succinimidyl esters (MeO-PEG-NHS; 5, 10, and 20 kDa) were commercially available. To PEGylate the BN peptides, MeO-PEG-NHS was mixed with each BN analog and incubated for 1 h at room temperature. More MeO-PEG-NHS was added after 30 min and again after 45 min. The solvents were removed by evaporation, and the final product was dissolved in water before labeling with 99mTc. The yields of PEGx-Lys-BN analogs were 85%, 74%, and 46% for PEG5, PEG10, and PEG20, respectively. The molecular weight was 1,441.9 for Lys-BN, 6,335.1 for PEG5-Lys-BN, 10,945.4 for PEG10-Lys-BN, and 22,709.5 for PEG20-Lys-BN.

To label 99mTc, a solution of Na[99mTcO4], sodium boranocarbonate, borax, potassium sodium tartrate tetrahydrate, and sodium carbonate was heated for 40 s at 150°C. After adjusting the pH to 6.5, the solution was mixed with the PEGylation analogs and heated for 2 min at 90°C. The non-PEGylated Lys-BN was similarly labeled with 99mTc. High-performance liquid chromatography was performed to separate the non-PEGylated from the PEGylated 99mTc-labeled peptides, and the 99mTc-labeled from the unlabeled peptides on the basis of the difference of their retention times. The radiochemical purities and specific radioactivities for both 99mTc-Lys-BN and 99mTc-PEG-Lys-BN analogs were >95% and 5.02 TBq/μmol (135.7 Ci/μmol), respectively (1). The radiochemical yields for the analogs were not reported.

In Vitro Studies: Testing in Cells and Tissues


The octanol/phosphate-buffered saline partition coefficients (log D) were determined at pH 7.4, and the log D values were −0.74 ± 0.06, −1.61 ± 0.30, −1.05 ± 0.16, and −1.08 ± 0.25 for 99mTc-Lys-BN, 99mTc-PEG5-Lys-BN, 99mTc-PEG10-Lys-BN, and 99mTc-PEG20-Lys-BN, respectively, showing that PEGylation resulted in increased hydrophilicity of the analogs (1).

The in vitro stability was evaluated after 24 h of incubation with human plasma (1). The analog 99mTc-Lys-BN was rapidly degraded, with only 16.6 ± 2.3% intact at 24 h. In contrast, all PEGylated BN analogs remained intact. The half-life of 99mTc-Lys-BN was 11.4 h, whereas the half-lives of the PEGylated BN analogs were >24 h. The cell binding assay confirmed these results (1). With the analogs obtained after incubation with human plasma for 24 h, the specific binding of the 99mTc-Lys-BN to PC-3 cells was reduced to 17.2 ± 1.9% in comparison to the specific binding without incubation in human plasma. The percentages of the PEGylated BN analogs after 24 h of incubation with human plasma were still 115.5 ± 18.3%, 96.0 ± 3.2%, and 99.9 ± 4.9% for 99mTc-PEG5-Lys-BN, 99mTc-PEG10-Lys-BN, and 99mTc-PEG20-Lys-BN, respectively.

Saturation binding assays were performed with PC-3 cells to evaluate the binding kinetics of the 99mTc-labeled analogs (1). The dissociation constants (Kd) for 99mTc-PEG-Lys-BN analogs were 0.89 ± 0.32, 0.53 ± 0.04, and 0.63 ± 0.20 nM for PEG5, PEG10, and PEG20, respectively. These values were in the same range as the Kd value of Lys-BN, which was 0.65 ± 0.35 nM. Compared to the non-PEGylated 99mTc-Lys-BN, the PEGylated forms had slower binding kinetics.

On cell internalization, all analogs showed specific, time-dependent cell uptake (1). 99mTc-Lys-BN internalized rapidly, reached its maximum within the first 30 min of incubation (~30% per 106 cells), and remained virtually constant for about 2 h. All PEGylated analogs showed lower internalization into PC-3 cells, and longer incubation was required to reach the plateau (between 4 h and 24 h). The size of the PEG entity had an influence on the amount of internalized fraction. After incubation for 4 h, the internalization rates were 2.9 ± 0.9%, 1.7 ± 1.4%, and 1.2 ± 1.2% for 99mTc-PEG5-Lys-BN, 99mTc-PEG10-Lys-BN, and 99mTc-PEG20-Lys-BN, respectively.

On cell externalization, 99mTc-Lys-BN was externalized quickly, with 73.37 ± 1.30% of the internalized activity released within the first 2.5 h, and only 21.44 ± 2.09% of the internalized fraction remained in the cells after 24 h. In contrast, the externalization of PEGylated analogs was slower, with 80.2 ± 3.7% (99mTc-PEG5-Lys-BN), 42.8 ± 0.9% (99mTc-PEG10-Lys-BN), and 54.2 ± 12.1% (99mTc-PEG20-Lys-BN) of the internalized fraction remaining in the cells after 24 h (1).

Animal Studies



The effect of PEGylation on in vivo stability was tested in Balb/c mice after injection of the radiolabeled BN analogs (10–20 MBq (0.27–0.54 mCi)) (n = 2 mice/time point) (1). The non-PEGylated 99mTc-Lys-BN was rapidly metabolized in mice, with only 5.28 ± 0.06% intact at 5 min and no intact 99mTc-Lys-BN at 30 min after injection in blood. 99mTc-PEG5-Lys-BN showed higher stability in vivo than 99mTc-Lys-BN, with 13.20 ± 1.59% intact at 5 min and 4.38 ± 0.60% intact at 30 min after injection. No data were reported for 99mTc-PEG10-Lys-BN and 99mTc-PEG20-Lys-BN.

The effect of PEGylation on biodistribution was tested in mice bearing PC-3 tumor xenografts after injection of 0.5–3.5 MBq (13.51–94.59 µCi) of the analogs (Table 1) (1). Mice (n = 3–6/group) were euthanized at 1, 4, and 24 h after injection. The radioactivity in each tissue was determined and expressed as a percentage of injected dose per gram of tissue (% ID/g).

The blood clearance of 99mTc-Lys-BN was fast. Conjugation with PEG5 did not affect the blood clearance of the BN analog, whereas conjugation with PEG10 and especially PEG20 led to longer blood circulation times and slower clearance rates (P < 0.01) (1).

The highest tumor uptake values for 99mTc-Lys-BN, 99mTc-PEG10-Lys-BN, and 99mTc-PEG5-Lys-BN were found 1 h after injection (2.80%, 1.79%, and 3.91% ID/g, respectively), with the uptake of 99mTc-PEG5-Lys-BN being significantly higher than that of 99mTc-Lys-BN (P < 0.05). The highest tumor uptake for 99mTc-PEG20-Lys-BN, however, was found 4 h after injection (4.86% ID/g). The tumor washout was slower for the PEGylated analogs; only 0.53% ID/g of the 99mTc-Lys-BN remained in the tumor, whereas 1.73%, 1.14%, and 4.12% ID/g were found at 24 h after injection for the PEG5, PEG10 and PEG20 analogs, respectively.

99mTc-PEG5-Lys-BN showed the highest tumor/nontarget ratios at all time points, especially at 24 h after injection. Compared to the non-PEGylated 99mTc-Lys-BN, PEG10 did not lead to improved tumor/nontarget ratios, while PEG20 improved tumor/kidney ratios (2- and 4-fold increase at 4 h and 24 h after injection, respectively) and tumor/liver ratios (2- and 3-fold increase at the same time points, respectively).

In the GRPR-expressing pancreas, all analogs showed highest uptake at 1 h after injection, but there was no statistical difference in uptake between PEGylated and non-PEGylated analogs. In the GRPR-expressing colon, the uptake was different for the PEGylated and non-PEGylated analogs. 99mTc-PEG5-Lys-BN and 99mTc-PEG10-Lys-BN showed significantly lower colon uptake, while 99mTc-PEG20-Lys-BN and 99mTc-Lys-BN showed comparable uptake.

In the liver, the highest uptake was observed with 99mTc-Lys-BN at 1 h after injection (3.92% ID/g); however, its clearance was relatively fast, with only 0.56% ID/g of the radioactivity remaining at 24 h after injection. 99mTc-PEG5-Lys-BN exhibited a significantly lower liver uptake at all time points (<1% ID/g). 99mTc-PEG10-Lys-BN showed lower liver uptake at early time points but slow clearance. There was no difference in the liver uptake between 99mTc-PEG20-Lys-BN and 99mTc-Lys-BN.

Accumulation in the kidney and washout from the kidneys were similar for both 99mTc-Lys-BN and 99mTc-PEG5-Lys-BN. Despite a slightly lower accumulation of 99mTc-PEG10-Lys-BN in the kidneys (4.10% ID/g at 1 h after injection), the accumulated radioactivity was cleared slowly, with 2.16% ID/g found at 24 h after injection. 99mTc-PEG20-Lys-BN showed the highest kidney uptake at all time points and the slowest clearance from kidney.

Table 1: Influence of PEGylation on the biodistribution of BN analogs

Blood0.96 ± 0.301.00 ± 0.083.70 ± 0.2614.46 ± 1.27
Kidneys5.79 ± 0.955.09 ± 1.834.10 ± 0.267.73 ± 0.48
Pancreas13.75 ± 2.3212.92 ± 0.539.43 ± 1.9412.42 ± 1.23
Colon4.94 ± 0.652.04 ± 0.682.27 ± 0.274.35 ± 0.86
Liver3.92 ± 0.390.64 ± 0.062.03 ± 0.223.61 ± 0.36
Tumor2.80 ±0.283.91 ± 0.441.79 ± 0.392.79 ± 0.34

*Data were obtained at 1 h after injection of analogs (n = 3-4)

Blocking studies were performed at 1 h after co-injection of unlabeled BN(1-14) (100 μg) and radiolabeled analogs (n = 3 mice) (1). The uptake in the GRPR-expressing tissues, such as the pancreas, colon, and tumor, was markedly reduced. The uptake of the non-PEGylated BN analog was inhibited the most, while the inhibition was less effective for the PEGylated analogs in a size-dependent manner. The pancreas and colon uptake values were reduced by 54%–95% and 50%–86%, respectively, and no inhibition was found for 99mTc-PEG20-Lys-BN. The tumor uptake was significantly inhibited for 99mTc-Lys-BN and 99mTc-PEG5-Lys-BN (70% and 58% reduction, respectively). However, no significant differences were found in the tumor uptake of 99mTc-PEG10-Lys-BN and 99mTc-PEG20-Lys-BN for the blocked and the unblocked groups.

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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