Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis and thrombus. The hexapeptide PGDLSR was described as a selective and high affinity ligand for PS. In this work, we synthesized and evaluated a gallium labelled-PGDLSR peptide as a potential and selective radiopharmaceutical for nuclear imaging. PGDLSR-β-alanine-NODAGA (P04087) was prepared using Fmoc-based synthesis and then chelated with cold gallium, 68Ga and 67Ga. The affinity of Ga-P04087 for PS was evaluated by a competitive binding assay using biotinylated AnnexinV. The in vitro stability of the radiotracer was checked at room temperature and after incubation in human serum at 37 °C with and without a metalloprotease inhibitor. The in vivo binding of 67Ga-P04087 to phosphatidylserine was evaluated in a rat model of infective endocarditis. PGDLSR was successfully prepared with a yield of 31%. P04087 was obtained with a yield of 28% and in high chemical purity (>95%). The radiochemical purities of 67Ga-P04087 and 68Ga-P04087 exceeded 98% in all cases. IC50 of P04087 and Ga-P04087 were in the same order of magnitude (10−7M). The radiolabelled product was stable for 24 h at room temperature, but was very rapidly degraded in human serum in the absence of a protease inhibitor, which had a stabilizing effect. No focal uptake could be detected visually in the cardiac area on SPECT images. On autoradiography however, a focal uptake of 67Ga-P04087 in the valve area was present and histological slices demonstrated localization of peptide binding at the peripheral layer of vegetations. In spite of the preservation of the peptide affinity to the PS after its conjugation to the NODAGA chelator, and of the presence of 67Ga-P04087 uptake on autoradiography, the absence of detectable foci in vivo in the valve area may be attributed to both the low intensity of the signal and the presence of background activity originating from blood pool and surrounding tissues in the living animals. Further modifications are necessary to design a radiolabeled peptide with higher binding potencies to PS while possessing enhanced metabolic stability in vivo.

中文翻译：

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P04087（一种功能化的磷脂酰丝氨酸结合肽）的合成、镓标记和表征。

放射性标记的磷脂酰丝氨酸 (PS) 结合肽代表了一种用于细胞凋亡和血栓分子成像的创新策略。六肽 PGDLSR 被描述为 PS 的选择性和高亲和力配体。在这项工作中，我们合成并评估了一种镓标记的 PGDLSR 肽作为核成像的潜在和选择性放射性药物。使用基于 Fmoc 的合成制备 PGDLSR-β-丙氨酸-NODAGA (P04087)，然后与冷镓、68Ga 和 67Ga 螯合。Ga-P04087 对 PS 的亲和力通过使用生物素化的 AnnexinV 的竞争性结合试验进行评估。放射性示踪剂的体外稳定性在室温下和在人血清中孵育后在 37 °C 下使用和不使用金属蛋白酶抑制剂进行检查。在感染性心内膜炎的大鼠模型中评估了 67Ga-P04087 与磷脂酰丝氨酸的体内结合。PGD​​LSR成功制备，收率为31%。P04087 的产率为 28%，化学纯度高（>95%）。67Ga-P04087和68Ga-P04087的放射化学纯度均超过98%。P04087 和 Ga-P04087 的 IC50 处于同一数量级 (10-7M)。放射性标记产物在室温下可稳定 24 小时，但在没有具有稳定作用的蛋白酶抑制剂的情况下，在人血清中会非常迅速地降解。在 SPECT 图像上，在心脏区域中无法视觉检测到局灶性摄取。然而，在放射自显影上，在瓣膜区域存在 67Ga-P04087 的局部摄取，组织切片显示肽结合在植被外周层的定位。尽管在与 NODAGA 螯合剂缀合后保留了肽对 PS 的亲和力，并且在放射自显影中存在 67Ga-P04087 摄取，但在瓣膜区域体内没有可检测到的病灶可能归因于低信号强度和来自活体动物血池和周围组织的背景活动的存在。需要进一步的修饰来设计一种放射性标记的肽，该肽对 PS 具有更高的结合能力，同时具有增强的体内代谢稳定性。并且在放射自显影中存在 67Ga-P04087 摄取，瓣膜区域体内没有可检测到的病灶可能是由于信号强度低以及存在源自血池和活体周围组织的背景活动动物。需要进一步的修饰来设计一种放射性标记的肽，该肽对 PS 具有更高的结合能力，同时具有增强的体内代谢稳定性。并且在放射自显影中存在 67Ga-P04087 摄取，瓣膜区域体内没有可检测到的病灶可能是由于信号强度低以及存在源自血池和活体周围组织的背景活动动物。需要进一步的修饰来设计一种放射性标记的肽，该肽对 PS 具有更高的结合能力，同时具有增强的体内代谢稳定性。