p-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the 186Re/99mTc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide

Abstract

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)₃]⁺ core (M = ^natRe, ¹⁸⁶Re, ^99mTc). Specifically, we studied complexes of the formula [M(CO)₃(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)₃(OH₂)₃]⁺ (where M = ^natRe, ¹⁸⁶Re, or ^99mTc). 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 [¹²⁵I]I-Tyr⁴-BBN as the competitor.

Results: After a thorough radiolabeling optimization process, the [M(CO)₃(2)]⁺ model complexes (M = ¹⁸⁶Re and ^99mTc) were synthesized with 94 ± 2% radiochemical yield (RCY; estimated by radio-HPLC). In stability studies, [¹⁸⁶Re]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 [¹⁸⁶Re]Re-2 through 4 h. Non-specific rat serum protein binding of [¹⁸⁶Re]Re-2 was found to be 33 ± 4% at 4 h. The [^99mTc]Tc-2 complex was found to be stable in l-histidine and l-cysteine at 37 °C through 24 h. [^99mTc]Tc-2 was also stable in rat serum, with 38 ± 3% non-specific protein binding, at 4 h. The [M(CO)₃(3)]⁺ peptide radiometal complex (M = ¹⁸⁶Re and ^99mTc) syntheses were also optimized, resulting in RCYs of 35% for [¹⁸⁶Re]Re-3 and 47% for [^99mTc]Tc-3 (estimated by radio-HPLC). [¹⁸⁶Re]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 [¹⁸⁶Re]Re-3 through 4 h, with 29 ± 7% non-specific protein binding in rat serum. [^99mTc]Tc-3 was found to be 84 ± 3% and 82 ± 4% stable in l-histidine and l-cysteine at 24 h, respectively. [^99mTc]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 IC₅₀ values of 3.1 nM and 3.9 nM, respectively.

Conclusions: The low nanomolar IC₅₀ values obtained for 3 and Re-3 demonstrate high affinity of this novel [M(CO)₃]-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.

Keywords: Gastrin-releasing peptide receptor; Rhenium-186; Technetium-99m; Theranostics; Tricarbonyl metal complexes; p-NCS-Bn-NODAGA.