Prostate-specific membrane antigen (PSMA)-binding tracers have been shown to be promising agents for the specific targeting of prostate tumors. On labeling with the short-lived isotopes 18F and 68Ga, excellent molecular imaging performance is achieved. This potential could be further exploited using long-lived isotopes. Because of the favorable half-life of 64Cu, tracers labeled with this PET nuclide could solve logistic problems. Moreover, this isotope provides a theranostic pair with the therapeutic copper isotope 67Cu. Hence, 9 novel tracers that combine dedicated copper chelators with the PSMA-specific urea-based binding motif were developed. Methods: The precursors were obtained by solid-phase synthesis. The purity and molecular weight of the PSMA ligands were confirmed by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The compounds were labeled with 64Cu, with a radiolabeling yield of more than 99%. Competitive cell binding assays and internalization assays were performed with C4-2 cells, a subline of the PSMA-positive cell line LNCaP (human lymph node carcinoma of the prostate). In vitro serum stability, the stability of 64Cu-CA003 in blood, and the in vivo fate of neat 64Cu-chloride or 64Cu-CA003 were determined to prove whether the stability of the radiolabeled compounds is sufficient to ensure no significant loss of copper during the targeting process. For PET imaging and biodistribution studies, a C4-2 tumor-bearing mouse model was used. Results: The radiolabeled 64Cu-PSMA ligands showed high serum stability. All PSMA ligands showed high inhibition potencies, with equilibrium inhibition constants in the low nanomolar range. 64Cu-CA003 and 64Cu-CA005 showed high internalization ratios (34.6% ± 2.8 and 18.6% ± 4.4, respectively). Both the in vitro serum stability determination and the in vivo characterization of the main radiolabeled compounds confirmed that, except for 64Cu-PSMA-617, all compounds showed high serum stability within the observation period of 24 h. Small-animal PET imaging demonstrated high tumor uptake within 20 min. Organ distribution studies confirmed high specific uptake in the tumor, with 30.8 ± 12.6 percentage injected dose (%ID)/g at 1 h after injection. Rapid clearance from the kidneys was observed-a decrease from 67.0 ± 20.9 %ID/g at 1 h after injection to 7.5 ± 8.51 %ID/g at 24 h after injection (in the case of CA003). The performance of CA003, the compound with the best preclinical properties, was assessed in a first patient. In line with its preclinical data, PET imaging resulted in clear visualization of the cancer lesions, with high contrast. Conclusion: The 64Cu-labeled PSMA ligands are promising agents to target PSMA and visualize PSMA-positive tumor lesions as shown in preclinical evaluation by small-animal PET studies, organ distribution, and a patient application. Most importantly, the images obtained at 20 h enabled delineation of unclear lesions, showing that the compounds fulfill the prerequisite for dosimetry in the course of therapy planning with 67Cu. Thus, we suggest clinical use of copper-labeled CA003 for diagnostics and radiotherapy of prostate cancer.

中文翻译：

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用于成像和铜同位素治疗的新型PSMA配体的开发。

前列腺特异性膜抗原（PSMA）结合示踪剂已被证明是特异性靶向前列腺肿瘤的有前途的药物。用短寿命同位素18F和68Ga标记时，可获得出色的分子成像性能。使用长寿命同位素可以进一步开发这种潜力。由于64Cu具有良好的半衰期，因此用这种PET核素标记的示踪剂可以解决逻辑问题。此外，该同位素与治疗性铜同位素67Cu提供了一个治疗对。因此，开发了9种新颖的示踪剂，将专用的铜螯合剂与PSMA特异性的脲基结合基序结合在一起。方法：通过固相合成获得前驱体。通过高效液相色谱和液相色谱-质谱法确定了PSMA配体的纯度和分子量。化合物用64Cu标记，放射标记产率超过99％。竞争性细胞结合测定和内在化测定是使用C4-2细胞（PSMA阳性细胞系LNCaP（前列腺的人淋巴结癌）的子系）进行的。测定体外血清稳定性，血液中64Cu-CA003的稳定性以及纯净的64Cu-氯化物或64Cu-CA003的体内命运，以证明放射性标记化合物的稳定性是否足以确保在检测过程中铜没有明显损失。定位过程。对于PET成像和生物分布研究，使用了C4-2荷瘤小鼠模型。结果：放射性标记的64Cu-PSMA配体具有较高的血清稳定性。所有PSMA配体均显示出高抑制能力，且平衡抑制常数在低纳摩尔范围内。64Cu-CA003和64Cu-CA005显示出较高的内在化比率（分别为34.6％±2.8和18.6％±4.4）。体外血清稳定性测定和主要放射性标记化合物的体内表征均证实，除64Cu-PSMA-617外，所有化合物在24小时的观察期内均显示出较高的血清稳定性。小动物PET成像显示20分钟内肿瘤吸收率很高。器官分布研究证实了在肿瘤中的高特异性摄取，注射后1 h的注射剂量（％ID）/ g为30.8±12.6％。观察到从肾脏的快速清除-从注射后1小时的67.0±20.9％ID / g降至7.5±8。注射后24小时（对于CA003）为51％ID / g。在第一例患者中评估了具有最佳临床前特性的化合物CA003的性能。根据其临床前数据，PET成像可清晰显示癌症病变，并具有高对比度。结论：64Cu标记的PSMA配体是靶向PSMA并可视化PSMA阳性肿瘤病变的有前途的药物，如通过小动物PET研究，器官分布和患者应用的临床前评估所示。最重要的是，在20小时时获得的图像能够勾画出不清楚的病灶，表明这些化合物在67Cu的治疗计划过程中满足了剂量测定的先决条件。因此，我们建议铜标记的CA003在前列腺癌的诊断和放射治疗中的临床应用。