碳酸酐酶Ⅸ靶向PET显像探针的合成及初步生物学评价

王乾辉; 仇冰青; 彭幻; 彭莹; 林建国; 邱玲

doi:10.3760/cma.j.cn321828-20241108-00390

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中华核医学与分子影像杂志

• 基础研究 •

ENGLISH ABSTRACT

碳酸酐酶Ⅸ靶向PET显像探针的合成及初步生物学评价

作者及单位信息

出版日期： 2025 -03 -25 ·

DOI： 10.3760/cma.j.cn321828-20241108-00390

Synthesis and preliminary biological evaluation of carbonic anhydrase Ⅸ-targeted PET imaging probe

Authors Info & Affiliations

Wang Qianhui

School of Chemistry and Materials Engineering, Jiangnan University, Wuxi 214122, China

Qiu Bingqing

School of Chemistry and Materials Engineering, Jiangnan University, Wuxi 214122, China

Peng Huan

School of Chemistry and Materials Engineering, Jiangnan University, Wuxi 214122, China

Peng Ying

NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China

Lin Jianguo

NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China

Qiu Ling

School of Chemistry and Materials Engineering, Jiangnan University, Wuxi 214122, China

Published： 2025 -03 -25 ·

DOI： 10.3760/cma.j.cn321828-20241108-00390

APP内阅读

摘要

目的设计合成一种 ¹⁸F标记的靶向碳酸酐酶Ⅸ(CAⅨ)的小分子PET显像探针 ¹⁸F-单-乙酰唑胺(SAZ)，并初步探究其生物学性质。

方法以乙酰唑胺为原料合成探针前体SAZ，通过亲核取代等反应制得目标探针 ¹⁸F-SAZ，测定其标记产率、放化纯、比活度、脂水分配系数和稳定性。利用人肾癌细胞株OS-RC-2(CAⅨ阳性表达)和人结肠癌细胞株HCT116(CAⅨ阴性表达)进行细胞摄取实验，并构建荷瘤鼠模型进行microPET显像，对探针在荷瘤鼠体内的生物分布进行分析。通过重复测量方差分析及Bonferroni法比较组间差异。

结果成功制备探针 ¹⁸F-SAZ，其产率约(5.60±0.51)%，比活度(7.90±0.62)MBq/nmol，放化纯大于99%，脂水分配系数log P＝-0.38±0.01，在PBS或小鼠血清中温育4h后放化纯仍大于99%。细胞摄取实验显示，OS-RC-2细胞对探针的摄取在30min时为(1.47±0.24)百分加入放射性剂量(%AD)，显著高于阻断组和HCT116细胞[(0.60±0.07)%AD、(0.50±0.05)%AD； F＝24.31， P值：0.012、0.013(Bonferroni法校正)]。MicroPET显像示，OS-RC-2荷瘤鼠肿瘤对探针摄取在30min时达到最大[(2.92±0.07)每克组织百分注射剂量率(%ID/g)]，而探针在阻断组和HCT116荷瘤鼠肿瘤摄取最大仅为(1.36±0.02)和(1.12±0.07)%ID/g。 ¹⁸F-SAZ在OS-RC-2荷瘤鼠中主要分布于肿瘤及肾、肠道、肝、胃等器官。

结论成功合成 ¹⁸F-SAZ，其具有较高的放化纯及良好的体外稳定性，可特异性靶向CAⅨ高表达肿瘤细胞，有望成为一种新型靶向CAⅨ的PET显像探针。

关键词：癌，肾细胞;碳酸酐酶Ⅸ;同位素标记;氟放射性同位素;正电子发射断层显像术;肿瘤细胞，培养的;小鼠，裸

ABSTRACT

ObjectiveTo design and synthesize a ¹⁸F-labeled small molecule PET imaging probe targeting carbonic anhydrase Ⅸ (CAⅨ), named as ¹⁸F-single-acetazolamide (SAZ), and to evaluate its biological properties preliminarily.

MethodsAcetazolamide was used as raw material to synthesize the precursor SAZ, and the target probe ¹⁸F-SAZ was obtained through nucleophilic substitution and other reactions. The radiochemical yield, radiochemical purity, specific activity, lipid water partition coefficient log P, and stability of ¹⁸F-SAZ were determined. Cancer cell lines OS-RC-2 (CAⅨ-positive) and HCT116 (CAⅨ-negative) were used for cell uptake experiments, and corresponding tumor-bearing mice were constructed for microPET imaging. Biodistribution of the probe in OS-RC-2 tumor-bearing mice was analyzed. The difference among groups was analyzed by repeated measures analysis of variance and Bonferroni method.

ResultsThe probe ¹⁸F-SAZ was successfully prepared with the labelling yield of (5.60±0.51)%, specific activity of (7.90±0.62)MBq/nmol, radiochemical purity more than 99%, and the lipid water partition coefficient log P of -0.38±0.01. After incubation with PBS or mouse serum for 4 h, the radiochemical purity was still more than 99%. The uptake of ¹⁸F-SAZ in OS-RC-2 cells reached (1.47±0.24) percentage of the added radioactivity dose (%AD) at 30min, which was significantly higher than the uptake in the blocked group and that in HCT116 cells ((0.60±0.07)%AD, (0.50±0.05)%AD; F=24.31, P values: 0.012, 0.013 (Bonferroni correction method)). The results of microPET imaging showed that the uptake of ¹⁸F-SAZ in OS-RC-2 tumors reached the maximum at 30min ((2.92±0.07) percentage activity of injection dose per gram of tissue (%ID/g)), while the maximum uptakes in the blocked group and HCT116 tumors were only (1.36±0.02) and (1.12±0.07)%ID/g, respectively. ¹⁸F-SAZ was mainly distributed in tumors and organs including kidney, intestine, liver, stomach in OS-RC-2 tumor-bearing mice.

ConclusionsThe probe ¹⁸F-SAZ is successfully synthesized. It has high radiochemical purity and good stability in vitro, and can specifically target tumor cells with high expression of CAⅨ. It is expected to be a new CAⅨ-targeting PET imaging probe.

KEYWORDS： Carcinoma, renal cell;Carbonic anhydrase Ⅸ;Isotope labeling;Fluorine radioisotopes;Positron-emission tomography;Tumor cells, cultured;Mice, nude

Corresponding author: Qiu Ling, Email: grodef.mabnisjgniluiq

FUNDING:

Scientific Research Project of Jiangsu Commission of Health（ZD2022036）

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

王乾辉,仇冰青,彭幻,等. 碳酸酐酶Ⅸ靶向PET显像探针的合成及初步生物学评价[J]. 中华核医学与分子影像杂志,2025,45(03)：169-174.

DOI:10.3760/cma.j.cn321828-20241108-00390

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肾细胞癌是一种具有高度侵袭性的恶性肿瘤，占所有肾脏肿瘤的80%～90%，其又以肾透明细胞癌(renal clear cell carcinoma, RCCC)为主 ^{[
1
]} 。多数RCCC患者在发现时已处于中晚期，且30%的患者在初次手术后会发生复发或转移 ^{[
2
,

3
]} 。临床上主要使用CT或MRI对肾癌进行检查，但提供的图像不足以判断肾癌的亚型及分期 ^{[
4
]}，且常用于肾癌检测的螺旋式CT很难鉴别出较小的肾癌病灶 ^{[
5
]} 。PET显像可以快速无创获得高分辨率图像，从分子水平鉴别诊断肿瘤的分型和分期。 ¹⁸F-FDG是目前临床最常用的广谱肿瘤显像剂，但其诊断RCCC原发灶的灵敏度仅为62% ^{[
6
]}，且在鉴别肾癌良恶性方面特异性有限 ^{[
7
,

8
]}。因此，为了提高肾癌的早期鉴别诊断能力，亟需开发特异性的探针。

碳酸酐酶Ⅸ(carbonic anhydrase Ⅸ，CAⅨ)是一种在缺氧肿瘤细胞表面特异性过表达的跨膜蛋白 ^{[
9
,

10
]} 。CAⅨ的表达水平受缺氧诱导因子(hypoxia-inducible factor, HIF)1α的调控，而在RCCC中有高达90%的肿瘤细胞存在肿瘤抑制因子von Hippel-Lindau (VHL)的突变缺失，导致HIF-1α聚集，从而使CAⅨ表达明显上调 ^{[
11
]}。此外，CAⅨ的过表达不仅显著提高了肿瘤转移的可能性，且与化疗耐药性和放疗耐受性有关 ^{[
12
]} 。因此，CAⅨ被视为肾癌诊断、治疗方案选择、疗效预测与预后评估的重要标志物。本研究基于CAⅨ的小分子靶向基团乙酰唑胺，设计合成一种 ¹⁸F标记的靶向CAⅨ的小分子PET显像探针 ¹⁸F-单-乙酰唑胺(single-acetazolamide, SAZ)，并探究其在CAⅨ高表达模型中的特异性摄取情况。

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参考文献

参考文献

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晚期肾透明细胞癌系统性治疗中国专家共识专家组，中国临床肿瘤学会肾癌专家委员会，中国抗癌协会泌尿系统肿瘤专业委员会．晚期肾透明细胞癌系统性治疗中国专家共识(2024版)[J]. 中华肿瘤学杂志， 2024,46(9):844-854. DOI： 10.3760/cma.j.cn112152-20240322-00117 .