Prostate-specific membrane antigen (PSMA)–negative neuroendocrine prostate cancer (PCa) is a subtype of PCa likely to be lethal, with limited clinical diagnostic and therapeutic options. High expression of neurotensin receptor subtype 1 (NTR1) is associated with neuroendocrine differentiation of PCa, which makes NTR1 a potential target for neuroendocrine PCa. In this study, the NTR1-targeted tracer ⁶⁸Ga-DOTA-NT-20.3 was synthesized, and its affinity to androgen-dependent (LNCap) and androgen-independent (PC3) xenografts was determined. Methods: ⁶⁸Ga-DOTA-NT-20.3 was labeled using an automated synthesizer module, and its stability, labeling yield, and radiochemical purity were analyzed by radio–high-performance liquid chromatography. Receptor binding affinity was evaluated in NTR1-positive PC3 cells by a competitive binding assay. The biodistribution of ⁶⁸Ga-DOTA-NT-20.3 in vivo was evaluated in PC3 and LNCap xenografts by small-animal PET imaging. NTR1 expression was identified by immunohistochemistry and immunofluorescence evaluation. Results: ⁶⁸Ga-DOTA-NT-20.3 was synthesized successfully, with a yield of 88.07% ± 1.26%, radiochemical purity of at least 99%, and favorable stability. The NTR1 affinity (half-maximal inhibitory concentration) for ⁶⁸Ga-DOTA-NT-20.3 was 7.59 ± 0.41 nM. Small-animal PET/CT of PC3 xenograft animals showed high-contrast images with intense tumor uptake, which revealed specific NTR1 expression. The tumors showed significant radioactivity (4.95 ± 0.67 percentage injected dose per gram of tissue [%ID/g]) at 1 h, which fell to 1.95 ± 0.17 %ID/g (P < 0.01, t = 8.72) after specific blockage by neurotensin. LNCap xenografts had no significant accumulation (0.81 ± 0.06 %ID/g) of ⁶⁸Ga-DOTA-NT-20.3 at 1 h. In contrast, ⁶⁸Ga-PSMA-11 was concentrated mainly in LNCap xenografts (8.60 ± 2.11 %ID/g), with no significant uptake in PC3 tumors (0.53 ± 0.05 %ID/g), consistent with the in vitro immunohistochemistry findings. Biodistribution evaluation showed rapid clearance from the blood and main organs (brain, heart, lung, liver, muscle, and bone), with significantly high tumor-to-liver (4.41 ± 0.73) and tumor-to-muscle (12.34 ± 1.32) ratios at 60 min after injection. Conclusion: ⁶⁸Ga-DOTA-NT-20.3 can be efficiently prepared with a high yield and high radiochemical purity. Its favorable biodistribution and prominent NTR1 affinity make ⁶⁸Ga-DOTA-NT-20.3 a potential radiopharmaceutical for the detection of PSMA-negative PCa and identification of neuroendocrine differentiation.

前列腺特异性膜抗原 (PSMA) 阴性的神经内分泌前列腺癌 (PCa) 是一种可能致命的 PCa 亚型，临床诊断和治疗选择有限。神经降压素受体亚型 1 (NTR1) 的高表达与 PCa 的神经内分泌分化有关，这使得 NTR1 成为神经内分泌 PCa 的潜在靶点。在这项研究中，合成了NTR1 靶向示踪剂^{68 Ga-DOTA-NT-20.3，并确定了其对雄激素依赖性 (LNCap) 和雄激素非依赖性 (PC3) 异种移植物的亲和力。}方法： ⁶⁸使用自动合成器模块标记 Ga-DOTA-NT-20.3，并通过放射高效液相色谱分析其稳定性、标记产量和放射化学纯度。通过竞争性结合测定在 NTR1 阳性 PC3 细胞中评估受体结合亲和力。通过小型动物 PET 成像在 PC3 和 LNCap 异种移植物中评估⁶⁸ Ga-DOTA-NT-20.3 在体内的生物分布。通过免疫组织化学和免疫荧光评估鉴定 NTR1 表达。结果：成功合成了 ⁶⁸ Ga-DOTA-NT-20.3，收率为88.07%±1.26%，放射化学纯度至少为99%，稳定性良好。⁶⁸的 NTR1 亲和力（半数最大抑制浓度）Ga-DOTA-NT-20.3 为 7.59 ± 0.41 nM。PC3 异种移植动物的小动物 PET/CT 显示高对比度图像和强烈的肿瘤吸收，揭示了特定的 NTR1 表达。肿瘤在 1 小时时显示出显着的放射性（每克组织注射剂量百分比为 4.95 ± 0.67 [%ID/g]），在通过特定阻断后降至 1.95 ± 0.17 %ID/g（P < 0.01，t = 8.72）神经降压素。LNCap 异种移植物在 1 小时时没有显着积累 (0.81 ± 0.06 %ID/g) ⁶⁸ Ga-DOTA-NT-20.3。相比之下，⁶⁸Ga-PSMA-11 主要集中在 LNCap 异种移植物中 (8.60 ± 2.11 %ID/g)，在 PC3 肿瘤中没有显着摄取 (0.53 ± 0.05 %ID/g)，这与体外免疫组织化学结果一致。生物分布评估显示血液和主要器官（脑、心脏、肺、肝脏、肌肉和骨骼）的快速清除，具有显着高的肿瘤-肝脏 (4.41 ± 0.73) 和肿瘤-肌肉 (12.34 ± 1.32)注射后 60 分钟的比率。结论： ⁶⁸ Ga-DOTA-NT-20.3可以高效、高收率、高放射化学纯度制备。其良好的生物分布和显着的 NTR1 亲和力使⁶⁸ Ga-DOTA-NT-20.3 成为检测 PSMA 阴性 PCa 和鉴定神经内分泌分化的潜在放射性药物。