Evaluation of Re/${}^{99m}$Tc-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 (${}^{99m}$Tc) or Peptide Radionuclide Receptor Therapy (PRRT, ${}^{186}$Re or ${}^{188}$Re) of neuroendocrine tumors, we present herein ${}^{99m}$Tc/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-NO${}_2$-Phe-c(${}_D$Cys-Tyr-${}_D$Trp-Lys-Thr-Cys)-${}_D$Tyr-NH${}_2$ (DOTA-sst${}_2$-ANT) and report the syntheses and in vitro evaluations of its respective [${}^{99m}$Tc]Tc/Re-cyclized peptides ([${}^{99m}$Tc]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 ReOCl${}_3$(PPh${}_3$)${}_2$ precursor and consisted of three isomers characterized by LC-ESI-MS. The [${}^{99m}$Tc]Tc-cyclized analogue was prepared via a ligand exchange reaction of the [${}^{99m}$Tc][TcO]${}^{3+}$ core through a [${}^{99m}$Tc]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. [${}^{99m}$Tc]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 [${}^{99m}$Tc]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/[${}^{99m}$Tc]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 (${}^{99m}$Tc) analogues are not identical and both form isomeric products in equilibrium, additional design modifications will be necessary prior to in vivo application of [${}^{99m}$Tc]Tc/Re-cyc-DOTA-sst${}_2$-ANT.