1,4,7-Triazacyclononane-Based Chelators for the Complexation of [186Re]Re- and [99mTc]Tc-Tricarbonyl Cores.

Metal complexes with the general formula [MI(CO)3(k3-L)]+, where M = Re, 186Re, or 99mTc and L = 1,4,7-triazacyclononane (TACN), NOTA, or NODAGA chelators, have previously been conjugated to peptide-based biological targeting vectors and investigated as potential theranostic radiopharmaceuticals. The promising results demonstrated by these bioconjugate complexes prompted our exploration of other TACN-based chelators for suitability for (radio)labeling with the [M(CO)3]+ core. In this work, we investigated the role of the TACN pendant arms in complexation of the [M(CO)3]+ core through (radio)labeling of TACN chelators bearing acid, ester, mixed acid-ester, or no pendant functional groups. The chelators were synthesized from TACN, characterized, and (radio)labeled with nonradioactive Re-, [186Re]Re-, and [99mTc]Tc-tricarbonyl cores. The nonfunctionalized (3), diacid (4), and monoacid monoester (7 and 8) chelators underwent direct labeling, while the diester (M-5 and M-6) complexes required indirect synthesis from M-4. All six chelators demonstrated stable radiometal coordination. The ester-bearing derivatives, which exhibited more lipophilic character than their acid-bearing counterparts, were prone to ester hydrolysis over time, making them less suitable for radiopharmaceutical development. These studies confirmed that the TACN pendant functional groups were key to efficient labeling with the [M(CO)3]+ core, with ionizable pendant arms favored over nonionizable pendant arms.

Pretargeted PET of Osteodestructive Lesions in Dogs.

The last decade has witnessed the creation of a highly effective approach to in vivo pretargeting based on the inverse electron demand Diels-Alder (IEDDA) click ligation between tetrazine (Tz) and trans-cyclooctene (TCO). Despite the steady progression of this technology toward the clinic, concerns have persisted regarding whether this in vivo chemistry will work in humans given their larger size and blood volume. In this work, we describe the use of a 64Cu-labeled Tz radioligand ([64Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted positron emission tomography (PET) imaging of osteodestructive lesions in a large animal model: companion dogs. First, in a small animal pilot study, healthy mice were injected with TCO-BP followed after 1 or 6 h by [64Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [64Cu]Cu-SarAr-Tz, revealing that this approach produced high activity concentrations in the bone (>20 and >15%ID/g in the femur and humerus, respectively, at 24 h post injection) as well as high target-to-background contrast. Subsequently, companion dogs (n = 5) presenting with osteodestructive lesions were administered TCO-BP (5 or 10 mg/kg) followed 1 h later by [64Cu]Cu-SarAr-Tz (2.2-7.3 mCi; 81.4-270.1 MBq). PET scans were collected for each dog 4 h after the administration of the radioligand, and SUV values for the osteodestructive lesions, healthy bones, and kidneys were determined. In these animals, pretargeted PET clearly delineated healthy bone and produced very high activity concentrations in osteodestructive lesions. Low levels of uptake were observed in all healthy organs except for the kidneys and bladder due to the renal excretion of excess radioligand. Ultimately, this work not only illustrates that pretargeted PET with TCO-BP and [64Cu]Cu-SarAr-Tz is an effective tool for the visualization of osteodestructive lesions but also demonstrates for the first time that in vivo pretargeting based on IEDDA click chemistry is feasible in large animals.

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.

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.