18F-FP-peptide用于化疗后肿瘤细胞凋亡显像

目的 研发含天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)核心的正电子核素标记的caspase-3多肽活性显像剂,以在体检测肿瘤细胞凋亡.方法 用国产合成模块通过点击化学合成( 18S,21S,24S,27S,30S)-27-(2-羧乙基)-21-(羧甲基)-30-( (2S,3R,4R,5R,6S)-6-((2-(4-(3-18F]氟戊基)-1H-1,2,3三唑-1-yl)乙酰氨基)甲基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酰氨)-24-异丙基-18-甲基-17,20,23,26,29-五羰基-4,7,10,13-四氧-16,19,22,25,28-五氨杂三十烷-1,32-二酸(18 F-FP-peptide).在体外18F-FP-peptide与经卡铂化疗的A549肺癌细胞混合60 min后,检测细胞放射性摄取率,摄取率=(放射性活度平均值×10×100％)/(细胞计数×细胞体积×放射性对照值).将18F-FP-peptide注射到经卡铂化疗的荷A549肿瘤小鼠体内,60 min后测其生物学分布,并行micro PET/CT显像.应用流式细胞仪检测细胞凋亡率,评价18 F-FP-peptide放射性摄取率与细胞凋亡率的相关性.数据处理使用SPSS 13.0统计软件,计量资料采用x-±s表示,组间比较采用单因素方差分析,相关性研究采用线性相关与回归分析.结果 18 F-FP-peptide的合成效率为(21.0±4.5)％(n=6,不校正),放化纯为99％,比活度为870 GBq/μmol.体外细胞实验研究表明,各组细胞凋亡率随卡铂化疗时间延长逐渐升高,经卡铂处理后0、1、2和24h后细胞凋亡率分别为(1.02±0.31)％、(4.85±1.26)％、(6.37±2.21)％和(10.23±2.43)％,对应的细胞摄取率分别为(0.06±0.01)％、(0.31±0.04)％、(0.44±0.02)％和(0.86±0.04)％,各组细胞凋亡率与放射性摄取率之间呈明显正相关(y=1.1244+11.0949x,r=0.9850,P＜0.01)；荷A549肿瘤卡铂化疗后24h,肿瘤组织放射性摄取率为(0.33±0.02) ％ID/g,明显高于未化疗肿瘤组织的(0.08±0.01) ％ID/g(F=31.25,P＜0.01)；而两者的细胞凋亡率分别为(15.50±1.47)％和(1.92±0.31)％,差异有统计学意义(F=33.54,P＜0.01)；荷A549肿瘤化疗后细胞凋亡率和肿瘤对18 F-FP-peptide的放射性摄取率呈明显正相关(y=-1.9055+54.4341x,r=0.9907,P＜0.01)；Micro PET显像示,未经化疗的肿瘤基本无放射性摄取,SUVmax与对侧比值为1.32±0.01,而经卡铂化疗的肿瘤明显摄取显像剂,SUVmax与对侧比值为3.46±0.02,两者比较差异有统计学意义(F=11.05,P＜0.01).结论 18F-FP-peptide是有临床潜在价值的caspase-3 活性显像剂.

Imaging of apoptosis with 18 F.FP-peptide focused on the evaluation of tumor response to chemother-apy

Objective To investigate the feasibility of a PET radionuclide, caspase-3-activatable probe containing Asp-Glu-Val-Asp （DEVD）, for imaging evaluation of tumor apoptosis in vivo. Methods（ 18 S, 21 S, 24 S, 27 S, 30 S ） -27- （ 2 -carboxyethyl ） -21 - （ carboxymethyl ） -30 - （ （ 2 S, 3 R, 4 R, 5 R, 6 S ） -6 - （ （ 2 - （ 4 - （ 3- 18 F ] fluoropropyl ） - 1 H- 1,2,3-triazol- 1 -yl ） acetamido ） methyl ） -3,4,5 -trihydroxytetrahydro-2H-py- ran-2 - carboxamido ） -24 -isopropyl- 18 -methyl- 17,20,23,26,29 -pentaoxo-4,7,1 O, 13 -tetraoxa- 16,19,22,25, 28-pentaazadotriacontane-1,32-dioic acid （lSF-FP-peptide） was automatically synthesized using a domestie synthesis module by ＂click chemistry＂. In vitro cellular uptake of ISF-FP-peptide was evaluated by mixing the peptide with lung adenocarcinoma A549 cells after chemotherapy by earboplatin. Apopto~is was monitored by flow cytometry. The correlation between cellular uptake and the apoptotic ratio was analyzed. In vivo bio- distribution and micro PET imaging were performed on A549 tumor bearing mice with or without chemothera- py. SPSS 13.0 was used for statistical analysis. Comparison among groups was conducted by one-way analy- sis of variance, and correlation was analyzed by linear correlation and regression. Results The yield of ~s F-FP-peptide was （21.0 ＋ 4.5 ） % （ n = 6, end of synthesis （EOS） ）. The radioehemistry purity was over 99% and the specific activity was 870 GBq/p~nol. After the in vitro treatment of A549 cells, the apoptotie ratio was （1.02 ＋0.31）%, （4.85 ~ 1.26）%, （6.37 ＋2.21）% and （10.23 ＋2.43）% at 0, 1, 2 and 24 h, respectively. The corresponding cellular uptake of lSF-FP-peptide was （0. 06 -＋0. 01 ）%, （0. 31 ＋ 0. 04）%, （0.44 ＋0. 02）% and （0.86 ~0.04）%. Significant positive correlation was found between the apoptosis ratio and cellular uptake of is F-FP-peptide （y = 1. 1244 ＋ 11. 0949x, r = 0. 9850, P 〈 0. 01 ）. The tumor uptake of A549 bearing mice at 24 h after chemotherapy was （0.33 ＋0.02） %ID/g, which was signifi- cantly higher than that of the control （ （0. 08 ~0.01 ） % ID/g, F = 31.25, P 〈 0.01 ）. The cell apoptotic rati- os were （ 15.50 ~ 1.47） % for tumors undergoing chemotherapy and （ 1.92 ＋0.31 ） % for the control group （ F = 33.54, P 〈 0.01 ）. Significant positive correlation was also found between the apoptotic ratio and the tumor uptake of 18 F-FP-peptide in vivo （y = - 1. 9055 ＋ 54. 4341x, r = 0. 9907, P 〈 0.01 ）. Micro PET ima- ges displayed that there was almost no uptake in the untreated tumor （ SUV ratio = 1.32 ：e0. 01 ） but signifi- cantly increased uptake of 18 F-FP-peptide in the treated tumor （ SUV ratio = 3.46 ~ 0. 02, F = 11.05, P 〈 0. O1 ）. Conclusion lSF-FP-oeptide can be a potential radiotracer for caspase-3 imaging in tissue apoptosis.