以SCN-Bn-DOTA为螯合基团的PSMA靶向探针^(68)Ga-P214的制备与临床前评估

Preparation and preclinical evaluation of^(68)Ga-P214 PSMA-targeting probe with SCN-Bn-DOTA as chelating group

目的:适当延长三七素类前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)靶向探针的体内循环时间,开发新型^(68)Ga标记的前列腺癌诊断探针。方法:采用固相合成法制备配体P214(以p-SCN-Bn-DOTA为双功能螯合基团),测定其亲和性。将配体加入到含^(68)Ga3+离子的缓冲液中,95℃下反应5 min,纯化后使用放射性薄层色谱法测定其放射化学纯度。评估^(68)Ga-P214的体外稳定性、脂水分配系数和人血清白蛋白结合率,并进行细胞摄取实验。对荷22Rv1瘤小鼠注射^(68)Ga-P214,特定时间进行micro-正电子发射体层成像(positron emission tomography,PET)/计算机体层成像(computed tomography,CT)显像,并与^(68)Ga-P137进行对比显像。尾静脉注射探针30、60和120 min后,处死实验鼠,取感兴趣器官称重、计数、计算每克组织注射剂量活度百分比(%ID/g)。结果:成功合成目标配体P214,其Ki值为0.085 nmol/L,标记产物^(68)Ga-P214放射化学纯度大于95%,在生理盐水和37℃小鼠血清中2 h保持稳定。^(68)Ga-P214的脂水分配系数为-3.17±0.09,对人血清白蛋白的结合率为(88.86±0.51)%,明显高于^(68)Ga-P137的(74.01±1.17)%。细胞实验发现^(68)Ga-P214能够有效被PSMA+细胞22Rv1摄取,温育30、60和120 min后,细胞摄取分别为(0.39±0.14)、(0.55±0.09)、(0.54±0.12)%ID/105细胞,且可被PSMA抑制剂ZJ43所抑制。注射后60 min,^(68)Ga-P214在肿瘤区域内有更高的最大标准摄取值(maximum standard uptake value,SUVmax)(1.40±0.11 vs 0.80±0.04),且随时间而增加。荷瘤小鼠生物分布表明,^(68)Ga-P214在肿瘤的摄取明显高于^(68)Ga-P137[(44.15±6.25)%ID/g vs(19.76±3.56)%ID/g,注射后120 min],^(68)Ga-P214拥有优异的肿瘤/肌肉、肿瘤/血液和肿瘤/肝脏的比值(127.63、33.87和12.68)。结论:^(68)Ga-P214生物分布理想,适当延长了三七素类探针的在体循环时间,在PSMA阳性肿瘤中的摄取显著增加,有望应用于前列腺癌的诊断,有较大的潜力改造为治疗探针。

Objective:To appropriately extend the in vivo circulation time of ginsenoside-like prostate-specific membrane antigen(PSMA)targeting probes and develop a novel^(68)Ga-labeled diagnostic probe for prostate cancer.Methods:The ligand P214(using p-SCN-Bn-DOTA as a bifunctional chelator)was synthesized using solid-phase synthesis,and its affinity was measured.The ligand was added to a buffer containing^(68)Ga3+ions,reacted at 95℃for 5 min,and then purified.The radiochemical purity was determined using radioactive thin-layer chromatography.The in vitro stability,lipophilicity and human serum protein binding rate of^(68)Ga-P214 were evaluated,followed by cell uptake experiments.^(68)Ga-P214 was injected into 22Rv1 tumor-bearing mice,and micro-positron emission tomography(PET)/computed tomography(CT)imaging was performed at specific time points,with comparative imaging using^(68)Ga-P137.Mice were sacrificed at 30,60 and 120 min post-injection via tail vein,and organs of interest were weighed,counted,and the percentage of injected dose per gram of tissue(%ID/g)was calculated.Results:The target ligand P214 was successfully synthesized with a Ki value of 0.085 nmol/L,and the radiochemical purity exceeded 95%.The labeled product^(68)Ga-P214 remained stable in saline and 37℃mouse serum for 2 h.The lipophilicity(partition coefficient)of^(68)Ga-P214 was-3.17±0.09,and the human serum albumin binding rate was(88.86±0.51)%,significantly higher than that of^(68)Ga-P137(74.01±1.17)%.Cell experiments demonstrated that^(68)Ga-P214 was effectively taken up by PSMA+cells(22Rv1),with uptake rates of(0.39±0.14),(0.55±0.09),and(0.54±0.12)%ID/105 cells at 30,60,and 120 min of incubation,respectively,which could be inhibited by the PSMA inhibitor ZJ43.At 60 min post-injection,^(68)Ga-P214 had a higher maximum standard uptake value(SUVmax)in the tumor region(1.40±0.11 vs 0.80±0.04),which increased over time.Biodistribution in tumor-bearing mice showed that^(68)Ga-P214 had significantly higher uptake in tumors compared to^(68)Ga-P137[(44.15±6.25)%ID/g vs(19.76±3.56)%ID/g at 120 min post-injection],with superior tumor/muscle,tumor/blood,and tumor/liver ratios(127.63,33.87,and 12.68,respectively).Conclusion:^(68)Ga-P214 exhibits ideal biodistribution,appropriately extends the in vivo circulation time of ginsenoside-like probes,and significantly increases uptake in PSMA-positive tumors,making it a promising candidate for prostate cancer diagnosis with considerable potential to be developed into a therapeutic probe.