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.