正电子发射断层显像（Positron Emission Tomography，PET）是先进的分子成像技术，用于癌症、心血管疾病和神经系统疾病等重大疾病的诊断，正电子药物一直是PET发展的关键和瓶颈，研发性质优良的PET显像药物具有重要意义。正电子核素Ga-68可通过Ge-68/Ga-68发生器获得，标记方法简单高效，广泛用于临床PET显像药物制备。双功能螯合剂HBED-CC（N，N'-双[2-羟基-5-（羧乙基）苄基]-乙二胺-N，N'-二乙酸）与[⁶⁸Ga]Ga³⁺在室温下快速形成稳定的金属螯合物，美国食品药品监督局（U.S. Food and Drug Administration，FDA）于2020年批准的首个前列腺癌显像剂 [⁶⁸Ga]Ga-HBED-CC-PSMA-11即为Ga-HBED-CC类标记物。

胰岛β细胞上高度表达的胰高血糖素样肽-1受体（The Glucagon-Like Peptide-1 Receptor，GLP-1R）是检测β细胞质量（β cell mass，BCM）的理想靶点，可用于糖尿病的诊断。针对已报道的Ga-68标记Exendin-4（GLP-1R激动剂）显像剂存在肾脏摄取高的缺点，本论文以HBED-CC为螯合剂设计合成3种新型Exendin-4标记前体和三种Ga-68标记物，评价筛选靶向性高，肾脏摄取低、具有临床应用潜力的显像剂；

前列腺癌是严重威胁男性健康的癌症，前列腺癌细胞表面过表达前列腺特异性膜抗原（Prostate-Specific Membrane Antigen，PSMA），靶向PSMA的放射性药物广泛用于前列腺癌的临床诊疗。前列腺癌也是具有乏氧特性的实体瘤，2-硝基咪唑是乏氧组织靶向基团，本论文设计研究了2种含有2-硝基咪唑结构的新型靶向PSMA化合物，以期筛选出肿瘤摄取更高和滞留时间更长的前列腺癌诊断药物。具体研究如下：

1. 新型Ga-68标记靶向GLP-1R显像药物的设计、合成和体内外生物评价

化学合成：在本课题组前期[⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4研究基础上，引入白蛋白结合基团——长链烷基、4-(4-碘苯基)丁酸和4-(对甲苯基)丁酸，设计合成了3个新型Ga-68标记前体C₁₂-HBED-CC-MAL-Cys³⁹-Exendin-4（20）、p-I-ABM-HBED-CC-MAL-Cys³⁹-Exendin-4（21）和p-Me-ABM-HBED-CC-MAL-Cys³⁹-Exendin-4（22），并与[^natGa]Ga³⁺螯合得到3个标准品[^natGa]Ga-20、[^natGa]Ga-21和[^natGa]Ga-22，通过结构表征，纯度均大于95%。

放射性标记：通过对标记前体浓度优化，得到3种前体的最佳标记条件：在较低前体浓度（8.7 μM）、温和标记条件下（pH 4, 50℃）加热10 min即可得到放射化学纯度（Radiochemical Purity，RCP）大于95%的标记产物 [⁶⁸Ga]Ga-20、[⁶⁸Ga]Ga-21和[⁶⁸Ga]Ga-22。放射性标记物在PBS（磷酸缓冲液）和37 ℃大鼠血清内孵育2 h，RCP仍大于95%，稳定性满足体内外生物评价要求。

细胞摄取实验：体外INS-1细胞（高表达GLP-1R的大鼠胰岛细胞瘤细胞）实验结果显示，[⁶⁸Ga]Ga-20、[⁶⁸Ga]Ga-21和[⁶⁸Ga]Ga-22均被INS-1细胞摄取（60 min摄取值分别为：22.03 ± 0.12 、5.09 ± 0.19和6.14 ± 0.86 %ID/10⁶ cells），且可被过量的Cys³⁹-Exendin-4阻断，表明3种Ga-68标记物与GLP-1R特异性结合，其中[⁶⁸Ga]Ga-20的INS-1细胞摄取值显著高于其它2种Ga-68标记物。

体内生物评价：正常大鼠离体PET显像初步结果显示，[⁶⁸Ga]Ga-20、[⁶⁸Ga]Ga-21和[⁶⁸Ga]Ga-22在注射15 min后的胰腺摄取值SUV_max分别为：1.55、0.85 和 0.55，均高于本课题组已报道的[⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4（SUV_max = 0.33），[⁶⁸Ga]Ga-20的肾脏摄取显著低于[⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4（SUV_max：0.91 vs 2.42）。生物分布结果显示，正常大鼠和糖尿病大鼠分别注射[⁶⁸Ga]Ga-20后60 min，二者的胰腺摄取值具有显著差异（正常大鼠1.01 ± 0.07 %ID/g、糖尿病大鼠0.30 ± 0.04 %ID/g），值得进一步深入研究。

2. 具有2-硝基咪唑结构的新型Ga-68标记靶向PSMA显像药物研究

化学合成：设计合成了2个具有2-硝基咪唑结构的靶向PSMA标记前体NI-HBED-CC-PSMA-11（40）和NI-HBED-CC-PSMA-093（42），与[^natGa]Ga³⁺螯合得到2个标准品，[^natGa]Ga-40和[^natGa]Ga-42，均通过结构表征，纯度均大于95%。

放射性标记：2种标记前体在较低前体浓度（28 μM）、温和标记条件下（pH 4, 50℃）加热10 min即可得到RCP大于98%的标记产物 [⁶⁸Ga]Ga-40和[⁶⁸Ga]42，在PBS和37 ℃裸鼠血清内孵育2 h，RCP仍大于95%，满足体内外生物评价要求。

细胞摄取实验：体外22RV1-FOLH1-oe细胞（转染PSMA的人前列腺癌细胞）实验结果显示，[⁶⁸Ga]Ga-40和[⁶⁸Ga]Ga-42均能被22RV1-FOLH1-oe细胞摄取（60 min摄取值为：10.69 ± 0.14和21.51 ± 0.37 %ID/2×10⁵ cells），并被过量PSMA-11阻断，表明2种Ga-68标记物与PSMA特异性结合。与对照物（[⁶⁸Ga]Ga-HBED-CC-PSMA-11和[⁶⁸Ga]Ga-HBED-CC-PSMA-093的细胞摄取无显著差异，进一步的体外乏氧细胞结合和荷瘤小鼠生物分布实验正在进行中。

Positron radiopharmaceuticals play a crucial role in PET imaging, which is a non-invasive medical imaging technique for diagnosis and monitoring various diseases, including cancer, cardiovascular disease, and neurological disorders. Ga-68 is a positron emitting isotope that has gained significant attention in recent years due to its convenient availability from Ge-68/Ga-68 generators and the simplicity and high efficiency of the ⁶⁸Ga-labelled method. HBED-CC is a bifunctional chelator that forms a stable complex with Ga-68 rapidly. This chelator has become widely used in the development of clinical PET radiopharmaceuticals due to its high stability and good pharmacokinetics. [⁶⁸Ga]Ga-HBED-CC-PSMA-11 is a PET radiopharmaceutical based on the HBED-CC chelator that was approved by the U.S. FDA in 2020. It is used to detect prostate-specific membrane antigen (PSMA) expression in patients with prostate cancer and has shown promising results in clinical trials.

Previously, Several radiolabeled exendin-4 agents targeting GLP-1R, which are highly and specifically expressed on the surface of pancreatic β-cells, have been investigated for imaging insulinomas and diabetes. They showed modest uptake in target tissues, but high accumulation in kidneys prevented its full clinical potential. To improve in vivo a series of novel ⁶⁸Ga-labeled HBED-CC labeled GLP-1R targeted exendin-4 analogs were designed, synthesized and evaluated. It was hypothesized that adding HBED-CC as the chelate, resulting ⁶⁸Ga-labeled exendin-4 may showed a lower renal uptake and improved clinical potential.

Imaging agents targeting PSMA that is overexpressed on the surface of prostate cancer cells are currently being widely used clinically. Additionally, one of the common characteristics of many solid tumors, such as prostate cancer, is hypoxia. Imaging hypoxia using 2-nitroimidazole (NI) derivatives has been extensively reported. It is hypothesized that adding PSMA binding group and using 2-nitroimidazole (NI) for targeting hypoxia may produce bivalent targeting agents with higher tumor uptake and better detection. Several novel PSMA-targeting compounds containing 2-nitroimidazole, a chemical structure for hypoxia tissue, were designed and synthesized. In vitro preliminary evaluation was performed.

The specific research contents are as follows:

1. Design, synthesis and evaluation novel ⁶⁸Ga-labeled GLP-1R targeting imaging agent.

Chemical synthesis：To improved blood circulating time derivatives of 4-(4-iodophenyl)butanoic acid and 4-(p-tolyl)butanoic acid were introduced in reported [⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4 analogs. Three new radiolabeled precursors ABM-HBED-CC-MAL-Cys³⁹-Exendin-4 (20, 21 and 22) were designed and synthesized and they are chelated with [^natGa]Ga³⁺ to give three ^natGa-complexes ([^natGa]Ga-20, [^natGa]Ga-21 and [^natGa]Ga-22) as corresponding standard compound. Chemical purity of all above compounds are more than 95%

Radiochemistry：Three precursors were labeled with ⁶⁸Ga to give three corresponding new ⁶⁸Ga-complexes [⁶⁸Ga]Ga-20, [⁶⁸Ga]Ga-21 and [⁶⁸Ga]Ga-22, respectively. Excellent radiochemical purity (RCP, > 98%) of ⁶⁸Ga-complexes were obtained by a simple one-step reaction (pH 4, 50 °C, 5 min) without any purification. It was found that ⁶⁸Ga-complexes were stable in PBS and rat serum at 37℃ for more than 2 h. They are suitable for biological evaluation in vitro and in vivo.

In vitro cell uptake studies: The specific binding for GLP-1R was first examined by in vitro uptake and blocking studies in INS-1 cells (rat islet cell tumor cells with high expression of GLP-1R). All ⁶⁸Ga-labeled compounds showed a rapid uptake by INS-1 cells and the uptake was significantly inhibited when an excess amount of Cys³⁹-exendin-4 peptide was added as the blocking agent. It was indicated that the ⁶⁸Ga-labeled compounds selectively bound to GLP-1R. The cell uptake of [⁶⁸Ga]Ga-C₁₂-HBED-CC-MAL-Cys³⁹-Exendin-4 in INS-1 cells was significantly higher than that of the other three ⁶⁸Ga-labeled compounds. Results suggested that addition of albumin binding moiety did not preclude the GLP-1R binding.

 PET imaging ex vivo and biodistribution: To examine the in vivo biological distribution properties of these new [⁶⁸Ga]Ga-C₁₂-HBED-CC-MAL-Cys³⁹-Exendin-4 analogs, a series of microPET imaging studies were carried out using rats with special emphasis on pancreas tissue where GLP-1R are located. The results of preliminary PET imaging in normal rats showed that pancreas uptake of the three novel [⁶⁸Ga]Ga-ABM-HBED-CC-MAL-Cys³⁹-Exendin-4 was higher than that of [⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4. The results of biodistribution in normal rats and diabetic rats showed that [⁶⁸Ga]Ga-20 can be uptake specifically by the pancreas; while the renal uptake was significantly lower than that of [⁶⁸Ga]Ga-HBED-CC-MAL-Cys³⁹-Exendin-4. Therefore, [⁶⁸Ga]Ga-20 is potentially an insulinoma/diabetes imaging agent.

2. Design, synthesis and biological in vitro evaluation of novel bivalent targeting agents: ⁶⁸Ga-labeled PSMA and 2-nitroimidazole (NI) hypoxia agents

Chemical synthesis：Two bivalent targeting precursors with 2-nitroimidazole (NI), NI-HBED-CC-PSMA-11 (40) and NI-HBED-CC-PSMA-093 (42), were designed and synthesized. and they are chelated with [^natGa]Ga³⁺ to give two ^natGa-complexes ([^natGa]Ga-40 and [^natGa]Ga-42) as corresponding standard compound. Chemical purity of all above compounds are more than 95%

Radiochemistry：[⁶⁸Ga]Ga-40 and [⁶⁸Ga]Ga-42 were successfully prepared by a simple one-step reaction (pH 4, 50 °C, 10 min) without any purification (RCP > 98%). Two radiolabeled compounds were stable in PBS and 37℃ nude mouse serum for more than 2 h. They are suitable for in vitro and in vivo biological evaluation studies.

In vitro cell uptake studies: To examine the biological properties of these bivalent targeted agents were evaluated by in vitro cell uptake studies. Results of in vitro uptake and blocking studies in 22RV1-FOLH1-oe cells (human prostate cancer cells with high expression of PSMA) showed that both ⁶⁸Ga-labeled bivalent (PSMA and hypoxia) agents were uptake by 22RV1-FOLH1-oe cells rapidly, and the uptake was significantly blocked by excessive "cold" PSMA-11. Cell uptake and blocking studies results of [⁶⁸Ga]Ga-NI-HBED-CC-PSMA-11 and [⁶⁸Ga]Ga-NI-HBED-CC-PSMA-093 in normoxic conditions showed similar specific uptake as that of corresponding standard controls, while in vitro cellular uptake of the [⁶⁸Ga]Ga-NI-HBED-CC-PSMA-11 and [⁶⁸Ga]Ga-NI-HBED-CC-PSMA-093 was not affected by the introduction of nitroimidazole (NI) group. Specific PSMA binding for these bivalent agents was clearly demonstrated adding extra 2-nitroimidazole (NI) group has no impact on the binding. Further evaluations, i.e., cell binding studies in hypoxic condition and in vivo biodistribution in tumor bearing mice models will be investigated.