p-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the Re/Tc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide.

Introduction: With the goal of developing theranostic agents for application in radiopharmaceutical chemistry, in this work, we studied p-NCS-Bn-NODAGA (1) as a bifunctional chelator for the fac-[M(CO)] core (M = Re, Re, Tc). Specifically, we studied complexes of the formula [M(CO)(L)], where L denotes either Bn-NODAGA-Pyr (2) or Bn-NODAGA-Ser-Ser-RM2 (3).

Methods: The model bioconjugate molecule 2 was synthesized by conjugating pyrrolidine with 1, while 3 was derived from the conjugation of the gastrin-releasing peptide receptor (GRPR)-targeting peptide Ser-Ser-RM2 with 1. Labeling of 2 and 3 was performed with [M(CO)(OH)] (where M = Re, Re, or Tc). The stability of the radioactive complexes was studied against l-histidine and l-cysteine (1 mM in PBS; pH 7.4, 37 °C). GRPR affinity of both peptide 3 and its metallated counterpart, Re-3, were determined with in vitro competitive binding assays in GRPR-expressing PC-3 cells using [I]I-Tyr-BBN as the competitor.

Results: After a thorough radiolabeling optimization process, the [M(CO)(2)] model complexes (M = Re and Tc) were synthesized with 94 ± 2% radiochemical yield (RCY; estimated by radio-HPLC). In stability studies, [Re]Re-2 remained intact through 7 d in l-cysteine and l-histidine. Similarly, stability studies in rat serum at 37 °C showed 99 ± 1% intact [Re]Re-2 through 4 h. Non-specific rat serum protein binding of [Re]Re-2 was found to be 33 ± 4% at 4 h. The [Tc]Tc-2 complex was found to be stable in l-histidine and l-cysteine at 37 °C through 24 h. [Tc]Tc-2 was also stable in rat serum, with 38 ± 3% non-specific protein binding, at 4 h. The [M(CO)(3)] peptide radiometal complex (M = Re and Tc) syntheses were also optimized, resulting in RCYs of 35% for [Re]Re-3 and 47% for [Tc]Tc-3 (estimated by radio-HPLC). [Re]Re-3 showed 98 ± 2% and 84 ± 5% stability in l-histidine and l-cysteine, respectively, through 48 h. Similarly, stability studies in rat serum at 37 °C showed 85 ± 3% intact [Re]Re-3 through 4 h, with 29 ± 7% non-specific protein binding in rat serum. [Tc]Tc-3 was found to be 84 ± 3% and 82 ± 4% stable in l-histidine and l-cysteine at 24 h, respectively. [Tc]Tc-3 in rat serum at 37 °C showed 88 ± 2% stability through 4 h, with 25 ± 2% non-specific protein binding. Both 3 and Re-3 demonstrated high GRPR affinity, with IC values of 3.1 nM and 3.9 nM, respectively.

Conclusions: The low nanomolar IC values obtained for 3 and Re-3 demonstrate high affinity of this novel [M(CO)]-labeled bioconjugate for GRPR. The encouraging stability studies and receptor affinity results demonstrate promise for further development of these metal complexes as a theranostic matched pair for targeting GRPR.