Research Reactor Center, University of Missouri, Columbia, MO 65211, USA; Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, Mianyang, Sichuan 621900, PR China.
Research Reactor Center, University of Missouri, Columbia, MO 65211, USA.
Nucl Med Biol. 2021 Mar-Apr;94-95:46-52. doi: 10.1016/j.nucmedbio.2020.12.007. Epub 2021 Jan 15.
With the long-term goal of developing a diagnostic (Tc) and therapeutic (Re) agent pair for targeting somatostatin receptor (SSTR)-positive neuroendocrine tumors (NETs), we developed novel metal-cyclized peptides through direct labeling of the potent SSTR2 antagonist Ac-4-NO-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH (1) with Re (in Re-1), Tc (in [Tc]Tc-1) and Re (in [Re]Re-1).
Re-1 was characterized by LC-ESI-MS and HR-ESI-MS and was tested for receptor affinity in SSTR-expressing cells (AR42J). Radiolabeling of the peptide was achieved via ligand exchange from Tc-labeled glucoheptonate or [Re]ReOCl(PPh), yielding [Tc]Tc-1 or [Re]Re-1, respectively. In vitro stability of [Tc]Tc-1/[Re]Re-1 in PBS (10 mM) at pH 7.4 and 37 °C was determined by HPLC analysis. Moreover, [Tc]Tc-1 stability was tested in cysteine (1 mM) and rat serum under the same conditions.
Re-1 consisted of two isomers, confirmed by LC-ESI-MS, with good SSTR2 affinity (IC = 43 ± 6 nM). Optimization of the Tc labeling through varying reaction parameters such as pH, reaction time, and Sn and ligand concentrations resulted in high radiochemical yield (RCY ≥92%). Similarly, [Re]Re-1 was prepared in reasonable RCY (≥50%). Both Tc/Re-tracers consisted of two product isomers as identified by HPLC co-injection with Re-1. While [Tc]Tc-1 was sufficiently stable in vitro (≥71% intact through 4 h in PBS, cysteine and rat serum), [Re]Re-1 exhibited more moderate in vitro stability (58% intact after 1 h in PBS).
Novel Tc/Re-cyclized SSTR2 antagonist peptides were synthesized and characterized using the Re-cyclized analogue as a reference. Due to the nanomolar SSTR2 affinity of Re-1 and good in vitro stability of [Tc]Tc-1, the latter shows early promise for development as a radiodiagnostic agent for SSTR-expressing NETs.
The Tc-cyclized complex showed promising in vitro properties, and future in vivo studies will determine the potential for translating such a design into the human clinic.
我们的长期目标是开发一种用于靶向生长抑素受体（SSTR）阳性神经内分泌肿瘤（NET）的诊断（Tc）和治疗（Re）药物对，为此我们通过直接标记有效的 SSTR2 拮抗剂 Ac-4-NO-Phe-c（DCys-Tyr-DTrp-Lys-Thr-Cys）-DTyr-NH（1），开发了新型金属环化肽，用于 Re（在 Re-1 中）、Tc（在[Tc]Tc-1 中）和 Re（在[Re]Re-1 中）。
通过 LC-ESI-MS 和 HR-ESI-MS 对 Re-1 进行了表征，并在表达 SSTR 的细胞（AR42J）中测试了该配体的受体亲和力。通过从 Tc 标记的葡庚糖酸盐或[Re]ReOCl（PPh）进行配体交换，实现了肽的放射性标记，分别得到[Tc]Tc-1 或[Re]Re-1。通过 HPLC 分析，在 pH 值为 7.4 和 37°C 的 PBS（10 mM）中确定[Tc]Tc-1/[Re]Re-1 的体外稳定性。此外，在相同条件下，还在半胱氨酸（1 mM）和大鼠血清中测试了[Tc]Tc-1 的稳定性。
Re-1 由两种异构体组成，通过 LC-ESI-MS 得到证实，对 SSTR2 具有良好的亲和力（IC=43±6 nM）。通过改变反应参数（例如 pH、反应时间、Sn 和配体浓度）对 Tc 标记进行优化，得到了高放射性化学产率（RCY≥92%）。同样，[Re]Re-1 也以合理的 RCY（≥50%）制备。通过与 Re-1 进行 HPLC 共注射，确定两种 Tc/Re 示踪剂均由两种产物异构体组成。尽管[Tc]Tc-1 在体外稳定性良好（在 PBS、半胱氨酸和大鼠血清中 4 小时内≥71%保持完整），但[Re]Re-1 的体外稳定性中等（在 PBS 中 1 小时后 58%保持完整）。
使用 Re 环化类似物作为参考，合成并表征了新型 Tc/Re 环化 SSTR2 拮抗剂肽。由于 Re-1 具有纳摩尔级的 SSTR2 亲和力和[Tc]Tc-1 的良好体外稳定性，后者在开发用于表达 SSTR 的 NET 的放射性诊断剂方面显示出早期的潜力。
Tc 环化复合物表现出有希望的体外特性，未来的体内研究将确定将这种设计转化为人类临床应用的潜力。
