Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Aim: An promising therapeutic approach to treat cancer focus on the inhibition of tumourinduced angiogenesis. One target structure involved is integrin alpha(v)beta3. Imaging modalities allowing monitoring of alpha(v)beta3 may provide insights into corresponding molecular process. Labelling techniques becoming more and more interesting for molecular imaging with PET are based on Ga‐68. Especially, due to the straightforward labelling protocols this is an interesting alternative to F‐18 labelling of peptides. Here the imaging properties of c(RGDfK) conjugated to different chelating systems are compared.
Methods: Peptide synthesis follows standard SPPS protocols. The cyclised and selectively deprotected peptides were conjugated with the chelating moieties via the side chain amino function of the lysine. The chelating systems include 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐acetic acid (DOTA), 1,4,7‐triaazacyclononane‐4,7‐acetic acid‐1‐2‐glutaric acid (NODAGA), and a tris(2‐mercaptoethyl)amine derivative (NS3). Labelling was carried out using the fractionated elution method in sodium acetate (DOTA; NODAGA) or phosphate buffer (NS3). In vitro evaluation included log D determination, protein binding assays, plasma stability studies, isolated receptor binding assays, and cell uptake studies. In vivo evaluation was carried out using M21 (alpha(v)beta3 positive) and M21L (alpha(v)beta3 negative) bearing nude mice. For all tracer biodistribution data were collected. For DOTA‐RGD and NODAGA‐RGD also small animal PET imaging was carried out. Results: NODAGA‐RGD and NS3‐RGD could be labelled at room temperature, whereas labelling of DOTA‐RGD has to be carried out at elevated temperature. NODAGA‐RGD and DOTA‐RGD could be labelled in high radiochemical purity without further purification. For NS3‐RGD a subsequent Seppak separation was necessary to obtain the product in high radiochemical purity. The compounds showed comparable log D (‐2.9 ‐ ‐3.9) and IC(50) values (~5 nM) as well as receptor specific uptake. In contrast, great differences were found in the protein binding properties. The found protein bound activity after 1 h incubation was 18.6% (DOTA‐RGD), 42,4% (NS3‐RGD), and 1.4% (NODAGA‐RGD). This performance is reflected in the biodistribution data. Lowest activity concentration in blood (%ID/g 1h p.i.: NODAGA‐RGD=0.12±0.06 ; DOTA‐RGD=0.72±0.07; NS3‐RGD=3.33±1.9) and best tumour/background ratios were found for NODAGA‐RGD. Small animal imaging confirmed these findings and indicated that NODAGA‐RGD might have similar imaging properties as found for F‐18‐Galacto‐RGD. Conclusions: In this series NODAGA‐RGD revealed most promising properties for imaging alpha(v)beta3 expression. Easy radiolabelling at room temperature, low amount of protein bound activity and the resulting lower activity concentration found in blood compared to the other compounds makes it to an attractive alternative to F‐18‐Galacto‐RGD, worth to be tested in clinical studies.