Evaluation of Re/99mTc-labeled somatostatin receptor-targeting peptide complexes synthesized via direct metal cyclization

With interest in the development of somatostatin receptor (SSTR) targeting agents for potential application in diagnostic SPECT imaging (Tc-99m) or Peptide Radionuclide Receptor Therapy (PRRT, Re-186 or Re-188) of neuroendocrine tumors, we present herein Tc-99m/Re (radio)complexes synthesized by the integrated (radio)labeling approach of peptide cyclization via metal complexation. In particular, we utilized the potent SSTR2 peptide antagonist sequence DOTA-4-NO2-Phe-c((D)Cys-Tyr-(D)Trp-Lys-Thr-Cys)-(D)Tyr-NH2 (DOTA-sst(2)-ANT) and report the syntheses and in vitro evaluations of its respective [Tc-99m]Tc/Re-cyclized peptides ([Tc-99m]Tc/Re-cyc-DOTA-sst(2)-ANT). The Re-cyc-DOTA-sst(2)-ANT complex was synthesized via an on-resin Re(V)-cyclization reaction using the ReOCl3(PPh3)(2) precursor and consisted of three isomers characterized by LC-ESI-MS. The [Tc-99m]Tc-cyclized analogue was prepared via a ligand exchange reaction of the [Tc-99m][TcO](3+) core through a [Tc-99m]Tc-glucoheptonate intermediate with linear DOTA-sst(2)-ANT and was characterized by comparative HPLC studies against Re-cyc-DOTA-sst(2)-ANT. Good in vitro binding affinity was demonstrated in SSTR-expressing cells (AR42J) by the Re-cyc-DOTA-sst(2)-ANT major isomer, similar to the potent binder Lu-DOTA-sst(2)-ANT, in which the Lu metal was complexed by the bifunctional chelator DOTA versus via peptide cyclization. [Tc-99m]Tc-cyc-DOTA-sst(2)-ANT was obtained in high radiochemical yield, also with an elution pattern of three isomers observed by HPLC analysis, which were comparable yet not identical to those of Re-cyc-DOTA-sst(2)-ANT. The [Tc-99m]Tc-tracer complex was shown to be hydrophilic, and stability studies at 4 h demonstrated that it remained intact in both PBS and in rat serum, with low non-specific rat serum protein binding, while exhibiting more moderate stability in 1 mM cysteine. These findings demonstrate that direct Re/[Tc-99m]Tc-cyclization of DOTA-sst(2)-ANT is feasible and may be used as an alternative approach to the bifunctional chelate labeling strategy. However, given that the non-radioactive (Re) and radiotracer (Tc-99m) analogues are not identical and both form isomeric products in equilibrium, additional design modifications will be necessary prior to in vivo application of [Tc-99m]Tc/Re-cyc-DOTA-sst(2)-ANT.

Automated summary

This study synthesized Tc-99m/Re (radio)complexes for potential application in diagnostic imaging and therapy of neuroendocrine tumors. The compounds showed high binding affinity and good stability in vitro.