Systematic alteration of PEGylation grade of 68Ga- and 64Cu-labeled L-oligonucleotides for tailored pharmacokinetics as prerequisite for tumor pretargeting technologies

Systematic alteration of PEGylation grade of 68Ga- and 64Cu-labeled L-oligonucleotides for tailored pharmacokinetics as prerequisite for tumor pretargeting technologies

Förster, C.; Schubert, M.; Bergmann, R.; Walther, M.; Pietzsch, J.; Pietzsch, H.-J.; Steinbach, J.


The favorable properties of non-natural, L-configured oligonucleotides (L ON) such as rapid hybridization, the lack of natural hybridization targets in vivo as well as their high metabolic stability [1] award L-ONs as a promising tool for tumor pretargeting technologies. As a result of their oligoanionic nature, 17mer-L-ONs are correlated with very fast blood clearance and high kidney retention [1]. Therefore, improvement of these two parameters was indispensable. To minimize the kidney accumulation and to increase the bioavailability by increasing of the area under the blood curve (AUC), polyethylene glycol (PEG) units of increasing size 2000-20000 Da were conjugated to the 17mer lead structure. Beside effect of PEGylation, the influences of different chelators and labeling ions on the biodistribution behavior of 64Cu- and 68Ga-labeled 17mer-L-ON-PEGs were investigated.
17mer-L-DNA (HO-C6H12-S-S-C6H12-5’GCGGCTGTG-CGGTGCGG3’-amino modifier) was 3’-modified with different PEG substituents. By using the mercaptohexyl spacer at 5’-position, new developed NOTA-maleimide derivatives as well as a commercially available DOTA-maleimide were conjugated. After radiolabeling with 64Cu and 68Ga and intravenously administration in healthy Wistar rats, biodistribution data by organ and tissue sampling were determined 5 min p.i. as well as 60 min p.i. Blood clearance half lifes were calculated using of dynamic PET studies. The influence of PEGylation on hybridization was investigated in vitro by gel-electrophoresis.
The increasing molecular mass of the conjugated PEG units led to significantly decreased kidney retention at 60 min from 56.3±4.1%ID (64Cu; 2 kDa PEG) and 52.4±6.1%ID (68Ga; 2 kDa PEG) to 6.5±0.4%ID (64Cu; 20 kDa PEG) and 6.9±0.6% (68Ga; 20 kDa PEG), respectively. However, the liver uptake was slightly increased at 60 min p.i. from 4.8±0.2%ID (64Cu; 2 kDa PEG) and 2.6±0.4%ID (68Ga; 2 kDa PEG) to 13.3±2.1%ID (64Cu; 20 kDa PEG) and 10.7±2.7%ID (68Ga; 20 kDa PEG). The activity concentrations in all other measured organs, except the blood, were at 60 min p.i. negligible (< 0.5%ID/g or <1 SUV). The biological half-lives in the blood were 10.8 min (2 kDa PEG), 9.6 min (5 kDa PEG), 27.7 min (10 kDa PEG), and 39.4 min (20 kDa PEG) for 68Ga-labeled compounds. Based on gel-electrophoresis investigations, no PEG-induced inhibition of hybridization was found. In vitro hybridization was completed in less than 30 s, whereas the optimal ratio of complementary L DNA single strands was between 1:1 (2-5 kDa) to 1:1,25 (10-20 kDa).
PEGylation of our 17mer-L-ON resulted in significantly reduced kidney retention, favorable blood circulation half-life, and very low unspecific tissue accumulations. Low kidney and liver accumulations were found for 5 kDa and 10 kDa PEG derivatives. Increasing size of PEG units shifted the hybridization equilibrium to slightly higher DNA ratios without clear hybridization inhibition. The introduction of NOTA or DOTA as chelating moiety enables radiolabeling with various metallic radionuclides for diagnostics and therapy. These results emphasize the high-grade potential of L-ON as a complementary tool for tumor pretargeting technologies.
Schlesinger, J. et al., (2008), Lett. Drug Des. Discovery, 3, 330-335

  • Lecture (Conference)
    19th Internatzional Symposium on Radiopharmaceutical Sciences, 28.08.-02.09.2011, Amsterdam, Niederlande
  • Abstract in refereed journal
    Journal of Labelled Compounds and Radiopharmaceuticals 54(2011), S62
    ISSN: 0362-4803

Publ.-Id: 16255