Department of Nuclear Medicine, Second Affiliated Hospital of Zhejiang University School of Medicine, Institute of Nuclear Medicine and Molecular Imaging of Zhejiang University, Center of Excellence in Medical Molecular Imaging of Zhejiang State, Hangzhou, 310009, China.
Bioconjug Chem. 2012 Nov 21;23(11):2300-12. doi: 10.1021/bc300509k. Epub 2012 Nov 8.
Engineering peptide-based targeting agents with residues for site-specific and stable complexation of radionuclides is a highly desirable strategy for producing diagnostic and therapeutic agents for cancer and other diseases. In this report, a model N-S-N(Py) ligand (3) and a cysteine-derived α-melanocyte stimulating hormone (α-MSH) peptide (6) were used as novel demonstrations of a widely applicable chelation strategy for incorporation of the M(I)(CO)(3) (M = Re, (99m)Tc) core into peptide-based molecules for radiopharmaceutical applications. The structural details of the core ligand-metal complexes as model systems were demonstrated by full chemical characterization of fac-Re(I)(CO)(3)(N,S,N(Py)-3) (4) and comparative high-performance liquid chromatography (HPLC) analysis between 4 and (99m)Tc(I)(CO)(3)(N,S,N(Py)-3) (4a). The α-MSH analogue bearing the N-S-N(Py) chelate on a modified cysteine residue (6) was generated and complexed with M(I)(CO)(3) to confirm the chelation strategy's utility when applied in a peptide-based targeting agent. Characterization of the Re(I)(CO)(3)-6 peptide conjugate (7) confirmed the efficient incorporation of the metal center, and the (99m)Tc(I)(CO)(3)-6 analogue (7a) was explored as a potential single photon emission computed tomography (SPECT) compound for imaging the melanocortin 1 receptor (MC1R) in melanoma. Peptide 7a showed excellent radiolabeling yields and in vitro stability during amino acid challenge and serum stability assays. In vitro B16F10 melanoma cell uptake of 7a reached a modest value of 2.3 ± 0.08% of applied activity at 2 h at 37 °C, while this uptake was significantly reduced by coincubation with a nonlabeled α-MSH analogue, NAPamide (3.2 μM) (P < 0.05). In vivo SPECT/X-ray computed tomography (SPECT/CT) imaging and biodistribution of 7a were evaluated in a B16F10 melanoma xenografted mouse model. SPECT/CT imaging clearly visualized the tumor at 1 h post injection (p.i.) with high tumor-to-background contrast. Blocking studies with coinjected NAPamide (10 mg per kg of mouse body weight) confirmed the in vivo specificity of 7a for MC1R-positive tumors. Biodistribution results with 7a yielded a moderate tumor uptake of 1.20 ± 0.09 percentage of the injected radioactive dose per gram of tissue (% ID/g) at 1 h p.i. Relatively high uptake of 7a was also seen in the kidneys and liver at 1 h p.i. (6.55 ± 0.36% ID/g and 4.44 ± 0.17% ID/g, respectively), although reduced kidney uptake was seen at 4 h p.i. (3.20 ± 0.48% ID/g). These results demonstrate the utility of the novel M(I)(CO)(3) chelation strategy when applied in a targeting peptide.
工程肽基靶向剂与用于放射性核素的位点特异性和稳定络合的残基是生产用于癌症和其他疾病的诊断和治疗剂的高度理想策略。在本报告中，使用模型 N-S-N(Py)配体（3）和半胱氨酸衍生的α-促黑素细胞激素（α-MSH）肽（6）作为将M(I)(CO)(3)（M = Re、(99m)Tc）核掺入基于肽的分子中用于放射性药物应用的广泛适用螯合策略的新范例。通过对 fac-Re(I)(CO)(3)(N,S,N(Py)-3)（4）的全化学表征和 4 与 (99m)Tc(I)(CO)(3)(N,S,N(Py)-3)（4a）之间的比较高效液相色谱（HPLC）分析，证明了模型系统中核配体-金属配合物的结构细节。在经修饰的半胱氨酸残基上带有 N-S-N(Py)螯合物的 α-MSH 类似物（6）被生成并与 M(I)(CO)(3)络合，以确认该螯合策略在基于肽的靶向剂中的应用的实用性。Re(I)(CO)(3)-6 肽缀合物（7）的表征证实了金属中心的有效掺入，并且研究了 (99m)Tc(I)(CO)(3)-6 类似物（7a）作为用于成像黑色素皮质素 1 受体（MC1R）的单光子发射计算机断层扫描（SPECT）化合物的潜力在黑色素瘤中。肽 7a 在氨基酸挑战和血清稳定性测定期间显示出优异的放射性标记产率和体外稳定性。在 37°C 下孵育 2 小时时，B16F10 黑色素瘤细胞对 7a 的摄取达到了 2.3±0.08%的应用活性的适度值，而与非标记的 α-MSH 类似物 NAPamide（3.2 μM）共孵育时，这种摄取显著降低（P<0.05）。在 B16F10 黑色素瘤异种移植小鼠模型中评估了 7a 的体内 SPECT/X 射线计算机断层扫描（SPECT/CT）成像和生物分布。SPECT/CT 成像在 1 小时注射后（p.i.）清晰地可视化了肿瘤，具有高肿瘤与背景的对比度。用共注射的 NAPamide（小鼠体重的 10mg）进行的阻断研究证实了 7a 对 MC1R 阳性肿瘤的体内特异性。7a 的生物分布结果在 1 h p.i.时产生了 1.20±0.09%的组织放射性剂量每克组织（% ID/g）的中等肿瘤摄取率。在 1 h p.i.时，7a 在肾脏和肝脏中也有较高的摄取率（分别为 6.55±0.36%ID/g 和 4.44±0.17%ID/g），尽管在 4 h p.i.时肾脏摄取减少（3.20±0.48%ID/g）。这些结果证明了在靶向肽中应用新型M(I)(CO)(3)螯合策略的实用性。
