Novel ^99mTc '4 + 1' peptide conjugates: Tuning the biodistribution by variation of coligands

A sophisticated coligand strategy is presented for peptide-derived radioconjugates based on ^99mTc '4 + 1' mixed-ligand complexes. The new pharmacologically active coligands are assessed for ^99mTc-labeling of the RGD-peptide cyclo(Arg-Gly-Asp-D-Tyr-Lys) which is an established vehicle to target a_vß₃ integrins playing a crucial part in tumor pathogenesis.
Complexes of the general formula [^99mTc(NS₃R)X] were synthesized and evaluated, in which Tc(III) is coordinated by NS₃R, a derivative of the tetradentate chelator 2,2´,2´´-nitrilotriethanethiol (NS₃), and by X, a monodentate binding isocyanide bearing the biomolecule. The novel tetradentate chelators (NS₃R = NS₃crown, NS₃en, NS₃(COOH)₃) constitute NS₃ with a crown ether, an amine or a tricarboxylic acid as pharmacological modifiers. The isocyanides (X = L2-RGD, L2-Lys) contained the linker isocyanobutanoic acid (L2) coupled to N⁶-Lys of the RGD-peptide and additionally to a single Lys.
The lipophilicity (distribution coefficient log D_O/W, pH = 7.4) of the RGD-containing radiotracers decreased in the order of the coligands NS₃crown (-1.7 +/- 0.1), NS₃en (-2.7 +/- 0.1) and NS₃(COOH)₃ (-3.3 +/- 0.1). In the same order of the coligands, the biodistribution of the series [^99mTc(NS₃R)(L2-RGD)] in normal rats showed a decrease of hepatobiliary and an increase of urinary excretion.
The ratio of specifically to unspecifically uptaken activity (sum of surface bound and internalized activity) in a_vß₃ integrin-expressing M21 cells was in the range of approximately 4-5 and comparable for all [^99mTc(NS₃R)(L2-RGD)] tracers. The biodistribution of [^99mTc(NS₃en)(L2-RGD)] in v/v mice bearing M21 and M21L (control) tumor xenografts exhibited a specific tumor uptake with a low target-background ratio.
The metabolic stability of the [^99mTc(NS₃R)(L2-RGD)] tracers in normal rats was high, since 75-87% of the radioactivity in the plasma extract was assigned to the injected radiotracers 60 min after intravenous
application in a rat. The hypothetical metabolites [^99mTc(NS₃R)(L2-Lys)] were not found.
These results demonstrate a considerable improvement of in vivo properties of ^99mTc '4 + 1' peptide conjugates and open up the possibility of applying the labeling approach for further radiodiagnostic peptides.