前列腺癌是全球高发的恶性肿瘤，严重威胁中老年男性生命健康。放射性诊疗药物对前列腺癌的早期诊断、治疗及疗效评估具有重要意义。前列腺特异性膜抗原（Prostate-specific membrane antigen，PSMA）在大多数前列腺癌中特异性高表达，是前列腺癌诊断与治疗的理想靶点。PSMA靶向放射性诊断药物[68Ga]Ga-PSMA-11和治疗药物[177Lu]Lu-PSMA-617已被美国食品药品监督管理局（Food and drug administration，FDA）批准用于前列腺癌的诊断和治疗。神经内分泌性前列腺癌（Neuroendocrine prostate cancer，NEPC）是前列腺癌的一种侵袭性变体，不同病灶PSMA表达水平各异，临床用PSMA靶向显像剂诊断NEPC时，病灶漏诊率较高。生长抑素受体2（Somatostatin receptor 2，SSTR2）是NEPC诊疗的另一潜在靶点，其表达水平与NEPC的疾病进展呈正相关，SSTR2靶向放射性药物可以辅助NEPC的诊疗。因而，开发双靶向PSMA和SSTR2的放射性药物，成为改善NEPC诊断和治疗效果的新思路。
本文以谷氨酸-脲-赖氨酸为PSMA靶向基团，JR11为SSTR2靶向基团，HBED-CC、DOTAGA和DOTA(GA)2为双功能螯合剂，甘氨酸-酪氨酸-苯基丙氨酸和PEG链为连接基团，以68Ga和177Lu分别作为诊断和治疗核素，设计合成了5个68Ga/177Lu标记的双靶化合物，并进行了一系列体内外生物评价实验。具体内容如下：
1.    PSMA和SSTR2双靶向化合物的设计合成
设计合成了3个前体化合物21、26、28，3个natGa配合物标准品，2个natLu配合物标准品，其中涉及27个中间体。所有化合物经核磁和质谱表征，确证其化学结构。
2.    68Ga/177Lu放射性标记和稳定性研究
对影响放射标记产率和标记产物放射化学纯度的主要因素（前体量、pH、温度和反应时间）进行条件优化，建立68Ga/177Lu放射性标记方法。结果表明，10 - 20 nmol前体在50 - 95 ℃下反应10 - 20 min即可得到高放射化学纯度（> 95%）的标记产物（[68Ga]Ga/[177Lu]Lu-21、[68Ga]Ga-26和[68Ga]Ga/[177Lu]Lu-28）。在磷酸缓冲液和小鼠血清中进一步评价了5个68Ga/177Lu标记的双靶化合物的体外稳定性，结果表明，3个68Ga标记的双靶化合物在磷酸缓冲液和小鼠血清中孵育120 min后，放射化学纯度仍高于95%；2个177Lu标记的双靶化合物在磷酸缓冲液和小鼠血清中孵育3 d后，放射化学纯度仍高于95%，5个68Ga/177Lu标记的双靶化合物均具有良好的体外稳定性，可进一步用于生物评价实验。
3.    体外细胞摄取与阻断实验以及竞争结合实验
采用22Rv1-FoLH1-oe细胞（PSMA阳性）和HEK293-SSTR2细胞（SSTR2阳性）对5个68Ga/177Lu标记的双靶化合物进行了体外细胞摄取与阻断实验以及竞争结合实验，结果显示，[68Ga]Ga-26和[68Ga]Ga/[177Lu]Lu-28在两种细胞中均表现出高特异性摄取，竞争结合实验测得化合物26和28的半抑制浓度为57.0 - 94.0 nM，表明化合物26和28及对应的68Ga/177Lu标记产物与PSMA和SSTR2均具有高亲和力和靶向特异性。
4.    荷瘤小鼠体内PET显像和生物分布实验
分别在22Rv1（PSMA阳性）和H69（SSTR2阳性）肿瘤小鼠体内进行了PET显像和生物分布实验。PET显像结果表明，[68Ga]Ga-26和[68Ga]Ga-28在22Rv1和H69肿瘤中显示较高的特异性摄取，其中，[68Ga]Ga-28的非靶器官摄取更低，因此PET图像对比度更高，可视化肿瘤的效果更好。生物分布实验结果进一步表明，[68Ga]Ga/[177Lu]Lu-28特异性靶向22Rv1和H69肿瘤，非靶器官摄取较低，靶与非靶比高，主要经肾脏系统代谢且清除快速。
综上所述，[68Ga]Ga/[177Lu]Lu-28显示出良好的PSMA和SSTR2靶向亲和性和特异性，在PSMA阳性和SSTR2阳性肿瘤中摄取高，药代动力学性质优良，可以作为NEPC放射性诊疗候选药物展开更深入的研究。

Prostate cancer is a highly prevalent malignant tumor which is often detected in middle-aged and elderly men. Because of its highly expressed in prostate cancer, prostate-specific membrane antigen (PSMA) has emerged as an ideal target for the diagnosis and treatment of prostate cancer. PSMA-targeted radiopharmaceuticals [68Ga]Ga-PSMA-11 for diagnosis and [177Lu]Lu-PSMA-617 for radiotherapy have been approved by the U.S. Food and Drug Administration (FDA) for prostate cancer. Neuroendocrine prostate cancer (NEPC) is an invasive variant of prostate cancer, with varying levels of PSMA expression. Somatostatin receptor 2 (SSTR2), which is associated with NEPC progression, represents an alternative potential target for NEPC diagnosis and treatment. SSTR2-targeted radiopharmaceuticals can be used for diagnosis and treatment of NEPC. Therefore, the development of dual-targeted radiopharmaceuticals targeting both PSMA and SSTR2 may a potential approach to improve the diagnosis and treatment of NEPC.
In this study，five dual-targeted ligands were designed and synthesized by a combination of glutamic acid-urea-lysine as the PSMA targeting moiety, JR11 as the SSTR2-targeting moiety, HBED-CC, DOTAGA and DOTA(GA)2 as bifunctional chelators，and glycine-tyrosine-phenylalanine and PEG linkers. Dual-targeting ligands were labeled with 68Ga/177Lu, respectively. A series of in vitro and in vivo biological evaluations demonstrating their biological properties were conducted, as follows:
1.    Design and synthesis of dual-targeted ligands
Three dual-targeting precursor ligands (21, 26, and 28) and five natGa/natLu standard complexes involving 27 intermediates were synthesized. The chemical structures of all compounds were confirmed by nuclear magnetic resonance and mass spectrometry.
2.    Radiolabeling with 68Ga/177Lu and stability studies 
Radiolabeling of these new dual-targeting ligands (21, 26, and 28) with 68Ga/177Lu was evaluated. Key factors affecting the radiolabeling yield and radiochemical purity (precursor concentration, pH, temperature, and reaction time) were optimized to establish the radiolabeling methods. The results showed that high radiochemical purity (RCP > 95%) of the labeled products ([68Ga]Ga/[177Lu]Lu-21, [68Ga]Ga-26, and [68Ga]Ga/[177Lu]Lu-28) were obtained by reacting 10 - 20 nmol of the precursors at temperatures ranging from 50 to 95°C for 10 - 20 minutes. The in vitro stability of the five complexes were further evaluated in phosphate buffer (PBS) and mouse serum, demonstrating that three 68Ga-labeled complexes retained high RCP (> 95%) after incubation for 120 minutes, while two 177Lu-labeled complexes retained high RCP (> 95%) after 3 days. All five radiolabeled complexes exhibiting excellent stability underwent further biological evaluation experiments.
3.    In vitro cell uptake experiments and competitive binding assays
To investigate the PSMA and SSTR2 binding of five complexes, cell uptake experiments and competitive binding assays were performed using 22Rv1-FoLH1-oe cells (PSMA-positive) and HEK293-SSTR2 cells (SSTR2-positive). Results showed that [68Ga]Ga-26 and [68Ga]Ga/[177Lu]Lu-28 exhibited high specific uptake in both cell types, and the half-inhibition concentrations (IC50) of compounds 26 and 28 (IC50: 57.0 - 94.0 nM) were determined by competitive binding assays, indicating high affinity and target specificity of compounds 26 and 28 for PSMA and SSTR2 binding sites, respectively.
4.    In vivo PET imaging and biodistribution in tumor-bearing mice
PET imaging and biodistribution experiments were conducted in 22Rv1 (PSMA-positive) and H69 (SSTR2-positive) tumor-bearing mice. PET imaging showed specific accumulation of [68Ga]Ga-26 and [68Ga]Ga-28 in 22Rv1 and H69 tumor-bearing mice models, respectively, indicating the specific high tumor uptake. Additionally, [68Ga]Ga-28 exhibited lower uptake in non-target organs, resulting in enhanced contrast in PET images and improved tumor visualization. The biodistribution experiments further confirmed the specific targeting of [68Ga]Ga/[177Lu]Lu-28 to 22Rv1 and H69 tumor-bearing mice models with lower uptake in non-target organs and rapid renal metabolism and clearance.
In conclusion, dual-targeting agent, [68Ga]Ga/[177Lu]Lu-28, exhibits excellent affinity and specifically accumulated in PSMA-positive and SSTR2-positive tumor. [68Ga]Ga/[177Lu]Lu-28 showed a desirable high tumor uptake and favorable pharmacokinetic properties in tumor-bearing mice models, indicating that it may be potentially useful for theranostic applications in neuroendocrine prostate cancer (NEPC) patients.