Introduction of a fatty acid chain modification to prolong circulatory half-life of a radioligand towards glucose-dependent insulinotropic polypeptide receptor

IF 3.6 4区 医学 Q1 RADIOLOGY, NUCLEAR MEDICINE & MEDICAL IMAGING
Amina Khalil , Sona Hakhverdyan , Pierre Cheung , Martin Bossart , Michael Wagner , Olof Eriksson , Irina Velikyan
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引用次数: 0

Abstract

Background

The beneficial role of glucose-dependent insulinotropic polypeptide receptor (GIPR) in weight control and maintaining glucose levels has led to the development of several multi-agonistic peptide drug candidates, targeting GIPR and glucagon like peptide 1 receptor (GLP1R) and/or the glucagon receptor (GCGR). The in vivo quantification of target occupancy by these drugs would accelerate the development of new drug candidates. The aim of this study was to evaluate a novel peptide (GIP1234), based on previously reported ligand DOTA-GIP-C803, modified with a fatty acid moiety to prolong its blood circulation. It would allow higher target tissue exposure and consequently improved peptide uptake as well as in vivo PET imaging and quantification of GIPR occupancy by novel drugs of interest.

Method

A 40 amino acid residue peptide (GIP1234) was synthesized based on DOTA-GIP-C803, in turn based on the sequences of endogenous GIP and Exendin-4 with specific amino acid modifications to obtain GIPR selectivity. A palmitoyl fatty acid chain was furthermore added at Lys14 via a glutamic acid linker to prolong its blood circulation time by the interaction with albumin. GIP1234 was conjugated with a DOTA chelator at the C-terminal cysteine residue to achieve 68Ga radiolabeling. The resulting PET probe, [68Ga]Ga-DOTA-GIP1234 was evaluated for receptor binding specificity and selectivity using HEK293 cells transfected with human GIPR, GLP1R, or GCGR. Blocking experiments with tirzepatide (2 μM) were conducted using huGIPR HEK293 cells to investigate binding specificity. Ex vivo and in vivo organ distribution of [68Ga]Ga-DOTA-GIP1234 was studied in rats and a pig in comparison to [68Ga]Ga-DOTA-C803-GIP. Binding of [68Ga]Ga-DOTA-GIP1234 to albumin was assessed in situ using polyacrylamide gel electrophoresis (PAGE). The stability was tested in formulation buffer and rat blood plasma.

Results

[68Ga]Ga-DOTA-GIP1234 was synthesized with non-decay corrected radiochemical yield of 88 ± 3.7 % and radiochemical purity of 97.8 ± 0.8 %. The molar activity for the radiotracer was 8.1 ± 1.1 MBq/nmol. [68Ga]Ga-DOTA-GIP1234 was stable and maintained affinity to huGIPR HEK293 cells (dissociation constant (Kd) = 40 ± 12.5 nM). The binding of [68Ga]Ga-DOTA-GIP1234 to huGCGR and huGLP1R cells was insignificant. Pre-incubation of huGIPR HEK293 cell sections with tirzepatide resulted in the decrease of [68Ga]Ga-DOTA-GIP1234 binding by close to 90 %. [68Ga]Ga-DOTA-GIP1234 displayed slow blood clearance in pigs with SUV = 3.5 after 60 min. Blood retention of the tracer in rat was 2-fold higher than that of [68Ga]Ga-DOTA-C803-GIP. [68Ga]Ga-DOTA-GIP1234 also demonstrated strong liver uptake in both pig and rat combined with decreased renal excretion. The concentration dependent binding of [68Ga]Ga-DOTA-GIP1234 to albumin was confirmed in situ by PAGE.

Conclusion

[68Ga]Ga-DOTA-GIP1234 demonstrated nanomolar affinity and selectivity for huGIPR in vitro. Addition of a fatty acid moiety prolonged blood circulation time and tissue exposure in both rat and pig in vivo. However, the liver uptake was also increased which may make PET imaging of abdominal tissues such as pancreas challenging. The investigation of the influence of fatty acid moiety on the biological performance of the peptide ligand paved the way for further rational design of GIPR ligand analogues with improved characteristics.

Abstract Image

Abstract Image

引入脂肪酸链修饰,延长葡萄糖依赖性促胰岛素多肽受体放射性配体的循环半衰期
背景由于葡萄糖依赖性胰岛素多肽受体(GIPR)在控制体重和维持血糖水平方面的有益作用,开发出了几种以 GIPR 和胰高血糖素样肽 1 受体(GLP1R)和/或胰高血糖素受体(GCGR)为靶点的多拮抗剂候选药物。在体内量化这些药物的靶点占据率将加速候选新药的开发。本研究的目的是评估一种新型多肽(GIP1234),它以之前报道的配体 DOTA-GIP-C803 为基础,并用脂肪酸修饰以延长其血液循环。方法以 DOTA-GIP-C803 为基础合成了一种 40 个氨基酸残基的多肽(GIP1234),该多肽又以内源性 GIP 和 Exendin-4 的序列为基础,进行了特定的氨基酸修饰,以获得 GIPR 选择性。此外,还通过谷氨酸连接体在 Lys14 处添加了棕榈酰脂肪酸链,以通过与白蛋白的相互作用延长其血液循环时间。GIP1234 的 C 端半胱氨酸残基与 DOTA 螯合剂共轭,以实现 68Ga 放射性标记。利用转染了人类 GIPR、GLP1R 或 GCGR 的 HEK293 细胞,对所得到的 PET 探针 [68Ga]Ga-DOTA-GIP1234 的受体结合特异性和选择性进行了评估。使用 huGIPR HEK293 细胞进行了替氮帕肽(2 μM)阻断实验,以研究结合特异性。在大鼠和猪体内研究了[68Ga]Ga-DOTA-GIP1234与[68Ga]Ga-DOTA-C803-GIP的体内外器官分布。使用聚丙烯酰胺凝胶电泳(PAGE)原位评估了[68Ga]Ga-DOTA-GIP1234 与白蛋白的结合情况。结果合成了[68Ga]Ga-DOTA-GIP1234,非衰变校正放射化学收率为 88 ± 3.7 %,放射化学纯度为 97.8 ± 0.8 %。放射性示踪剂的摩尔活度为 8.1 ± 1.1 MBq/nmol。[68Ga]Ga-DOTA-GIP1234与huGIPR HEK293细胞的亲和力稳定(解离常数(Kd)= 40 ± 12.5 nM)。68Ga]Ga-DOTA-GIP1234 与 huGCGR 和 huGLP1R 细胞的结合不明显。用替氮帕肽预孵育 huGIPR HEK293 细胞切片会导致[68Ga]Ga-DOTA-GIP1234 的结合率下降近 90%。[68Ga]Ga-DOTA-GIP1234在猪体内的血液清除缓慢,60分钟后SUV = 3.5。该示踪剂在大鼠体内的血液滞留率是[68Ga]Ga-DOTA-C803-GIP的2倍。[68Ga]Ga-DOTA-GIP1234在猪和大鼠体内也表现出很强的肝脏摄取能力,同时肾脏排泄减少。结论[68Ga]Ga-DOTA-GIP1234 在体外对 huGIPR 具有纳摩尔亲和力和选择性。在大鼠和猪体内,添加脂肪酸分子可延长血液循环时间和组织暴露时间。不过,肝脏摄取量也增加了,这可能会给胰腺等腹部组织的 PET 成像带来挑战。研究脂肪酸分子对多肽配体生物学性能的影响,为进一步合理设计具有更好特性的 GIPR 配体类似物铺平了道路。
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来源期刊
Nuclear medicine and biology
Nuclear medicine and biology 医学-核医学
CiteScore
6.00
自引率
9.70%
发文量
479
审稿时长
51 days
期刊介绍: Nuclear Medicine and Biology publishes original research addressing all aspects of radiopharmaceutical science: synthesis, in vitro and ex vivo studies, in vivo biodistribution by dissection or imaging, radiopharmacology, radiopharmacy, and translational clinical studies of new targeted radiotracers. The importance of the target to an unmet clinical need should be the first consideration. If the synthesis of a new radiopharmaceutical is submitted without in vitro or in vivo data, then the uniqueness of the chemistry must be emphasized. These multidisciplinary studies should validate the mechanism of localization whether the probe is based on binding to a receptor, enzyme, tumor antigen, or another well-defined target. The studies should be aimed at evaluating how the chemical and radiopharmaceutical properties affect pharmacokinetics, pharmacodynamics, or therapeutic efficacy. Ideally, the study would address the sensitivity of the probe to changes in disease or treatment, although studies validating mechanism alone are acceptable. Radiopharmacy practice, addressing the issues of preparation, automation, quality control, dispensing, and regulations applicable to qualification and administration of radiopharmaceuticals to humans, is an important aspect of the developmental process, but only if the study has a significant impact on the field. Contributions on the subject of therapeutic radiopharmaceuticals also are appropriate provided that the specificity of labeled compound localization and therapeutic effect have been addressed.
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