Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.
Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.
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发表时间：
2004
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通讯作者：
Xiaoyuan Chen;Yingping Hou;M. Tohme;R. Park;Vazgen Khankaldyyan;I. Gonzales-Gomez;J. Bading;W. Laug;P. Conti
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作者：
Xiaoyuan Chen;Yingping Hou;M. Tohme;R. Park;Vazgen Khankaldyyan;I. Gonzales-Gomez;J. Bading;W. Laug;P. Conti
未标记的α-整合素是一种细胞黏附分子，在激活的内皮细胞和肿瘤细胞上高表达，而在休眠的内皮细胞或正常细胞上不表达，是肿瘤成像和治疗的一个有吸引力的靶点。我们先前将一个环状Arg-Gly-Asp(RGD)肽c(RGDyK)与1，4，7，10-四氮杂环十二烷-N，N‘，N’‘，N’-四乙酸(DOTA)偶联，并用64Cu(半衰期，12.8h；19%β+)标记RGD-DOTA结合物用于固体肿瘤靶向，具有较高的肿瘤与背景对比度。这种示踪剂快速的肿瘤洗脱率和持久的肝和肾滞留促使我们优化示踪剂以改善药代动力学行为。在这项研究中，我们在DOTA和RGD之间引入了聚乙二醇(PEG3，分子量，3,400)部分，并评价了用于脑肿瘤模型microPET成像的64Cu-DOTA-PEG-RGD示踪剂。方法在微碱性条件下，原位活化DOTA并与RGD-PEGNH_2偶联。用固相受体结合分析法评价125I-ecichatin存在下的alphavbeta3整合素结合亲和力。荷U87 MG胶质母细胞瘤裸鼠皮下注射64CuDOTA-PEGRGD，于注射后30min~4h测定放射性示踪剂的生物分布。用MicroPET(注射后1h静态成像20min)和定量放射自显影对肿瘤进行显示和定量。同样的示踪剂也被用于检测肿瘤的原位U87 MG模型。结果合成的放射性示踪剂具有较高的比活度(14,800~29,600 GBq/mmol[400~800 Ci/mmol])。C(RGDyK)-PEGDOTA配体与α-β3-整合素的结合亲和力中等(50%抑制浓度，67.5+/-7.8nmol/L[Mean+/-SD])。聚乙二醇化的RGD多肽具有较快的血液清除能力(注射后30min注射剂量为0.57+/-0.15%ID/g[Mean+/-SD]，注射后4h注射剂量为0.03+/-0.02%ID/g)。注射后30min，肿瘤内活性积聚迅速且高(2.74+/-0.45%ID/g)，随时间推移有一定的活性丧失(注射后4h为1.62+/-0.18%ID/g)。与()铜-DOTA-RGD相比，该示踪剂的体内动力学有所改善，肝脏摄取显著降低(注射后30min为0.99+/-0.08%ID/g比1.73+/-0.39%ID/g，注射后4 h为0.58+/-0.07%ID/g比2.57+/-0.49%ID/g)。与64Cu-DOTA-RGD相比，聚乙二醇化RGD多肽在感染后30min的早期肾脏蓄积量(3.51+/-0.24%ID/g比2.18+/-0.23%ID/g)更快(在注射后1h为1.82+/-0.29%ID/g比2.01+/-0.25%ID/g)。通过与非放射性标记c(RGDyK)共注射阻断肿瘤摄取，证明了该放射性示踪剂的整合素受体特异性。在U87 MG皮下肿瘤模型中，MicroPET和放射自显影证实了聚乙二醇化RGD多肽示踪剂的高肿瘤器官比(注射后1h：肿瘤与血液的比率为20；肿瘤与肌肉的比率为12；肿瘤与肝脏的比率为2.7；肿瘤与肾脏的比率为1.2)。该示踪剂还能够在将U87 MG细胞植入小鼠前脑的模型中检测到原位脑瘤。尽管原位异种移植的肿瘤摄取量低于皮下移植，但原位肿瘤仍然可见，与正常脑组织形成明显的对比。结论本研究证明了在不影响64CuRGD多肽的肿瘤靶向性和特异性的情况下，聚乙二醇单分子部分适合于改善64CuRGD多肽的体内动力学。系统地研究聚乙二醇的大小和几何形状对肿瘤靶向和体内动力学的影响，将有助于开发适合临床应用的放射性示踪剂，如通过PET可视化和定量αv-整合素的表达。此外，用治疗性放射性核素标记的同一配体可能适用于整合素靶向内放射治疗。
UNLABELLED The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.