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报告基因HSV1-tk的PET探针的合成及其生物学评价
Alternative TitleSynthesis and biological evaluation of PET probe for imaging HSV1-tk reporter gene expression
蔡汉成
Subtype博士
Thesis Advisor尹端沚
2007-05-18
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral上海应用物理研究所
Keyword报告基因 单纯疱疹病毒胸腺嘧啶核苷激酶基因 正电子发射断层显像 合成 分子探针 氟-18标记
Abstract报告基因HSV1-tk的PET显像可以定量、反复、无损伤地反映活体体内治疗基因的转染效率、转染组织特异性和体内表达持续时间,以及对基因治疗中载体的分布和转导基因的有效性等进行评价,因而可指导基因治疗方案的优化和评价基因治疗的效果。报告基因与其分子探针是实现该报告基因PET分子显像必备的两个因素,研制报告基因HSV1-tk特异性PET分子探针是其PET显像成功关键所在。为了发展制备方法更简单、灵敏度更高、选择性更好的PET报告基因HSV1-tk分子探针,本课题发展了一种新颖的、高效的一步法F-18亲核取代标记方法,运用此标记方法制备了报告基因HSV1-tk新型PET分子探针核苷类似物2-氨基-6-[18F]氟-9-(4-羟基-3-羟甲基丁基)嘌呤(6-[18F]FPCV),并对其进行了质量控制和体外体内生物学评价以及micro-PET显像研究。同时制备报告基因HSV1-tk的PET经典分子探针[18F]FHBG。 本文首先通过对原料药2-氨基-6-氯-9-(4-乙酰氧基-3-乙酰氧甲基丁基)嘌呤水解脱去乙酰基、与三甲胺乙醇溶液反应得到相应的氯化铵盐、再与KF反应,制得6-[18F]FPCV的参比化合物2-氨基-6-氟-9-(4-羟基-3-羟甲基丁基)嘌呤(6-FPCV)。以核苷氯化三甲基铵盐作为标记前体,一步法F-18亲核取代三甲基铵盐,放射性合成6-[18F]FPCV。其优化的实验条件是:m(K2.2.2): m(K2CO3)=10: 1、1 mL DMF 作溶剂、反应温度为60℃、反应时间为15 min。经Silica plus Sep-Pak cartridge分离纯化,得到纯6-[18F]FPCV,总合成时间约为35~42 min。放射性化学产率约为45–55%(n= 6,未经衰变校正),放射化学纯度大于98%,比活度大于5.62GBq/ μmol。 其次研究6-[18F]FPCV体外生物学评价实验,包括分配系数的测定、体外稳定性实验、药代动力学实验以及体外细胞摄取实验。6-[18F]FPCV体外生物学实验表明:6-[18F]FPCV在PBS、小牛血清中稳定性较好,在孵育360 min后,其放射化学纯度仍保持在90%以上;6-[18F]FPCV的分配系数 Log P = -0.517;6-[18F]FPCV在小鼠体内的药代动力学符合简单的二室模型,计算得到6-[18F]FPCV的分布相半衰期T1/2(α)=1.2 min,清除相半衰T1/2(β)= 73.7min;探针6-[18F]FPCV在C6-tk细胞中摄取率比对照组C6细胞高5.5~18.8倍,其细胞摄取率的增加是由于细胞中HSV1-tk表达量的增加,说明探针可以应用活体内HSV1-tk的表达的显像。 然后研究6-[18F]FPCV的体内生物学行为及micro-PET显像,6-[18F]FPCV体内生物学评价实验表明:探针6-[18F]FPCV在正常小鼠体内能迅速地分布到全身,并能较快地清除,经肝、肾代谢,但探针6-[18F]FPCV在体内60 min后代谢易脱氟;探针6-[18F]FPCV在肿瘤模型裸鼠体内15 min前的稳定性较好,未见放射性代谢物,在C6-tk肿瘤和C6肿瘤中吸收值也比较高,其比值最高为1.69,经放射自显像证实6-[18F]FPCV在C6-tk肿瘤中吸收明显高于在C6肿瘤中的吸收,说明探针6-[18F]FPCV在转染报告基因HSV1-tk的C6-tk肿瘤中有特异性聚集。micro-PET显像实验表明:与[18F]FDG的micro-PET显像相比,注射6-[18F]FPCV 15 min后,其在C6-tk肿瘤和对照组C6肿瘤中摄取差异明显(其比值为1.5左右),可用于HSV1-tk的早期micro-PET显像,但由于6-[18F]FPCV在体内60 min后脱氟行为明显等原因,尚需作进一步研究后,才能用探针6-[18F]FPCV对体内报告基因HSV1-tk进行较长时间的micro-PET连续显像。 最后从抗疱疹病毒药物喷昔洛韦出发,与4-甲氧基氯化三苯甲烷反应保护氨基及一个羟基、另一个羟基磺酯化,再经氟化、水解四步反应合成了9-(4-[18F]氟-3-羟基甲基丁基)鸟嘌呤([18F]-FHBG)的标记前体Tosyl-FHBG及参比化合物9-(4-氟-3-羟基甲基丁基)鸟嘌呤(FHBG)。标记前体Tosyl-FHBG经两步放射性反应合成报告基因HSV1-tk的PET经典分子探针[18F]FHBG,并建立了Sep-Pak Cartridge分离方法取代常用的HPLC分离纯化方法。[18F]FHBG整个反应时间90~100 min,经衰变校正总产率为9 ± 3%(n= 6), 放化纯度>97%。
Other AbstractRepeated, quantitative, noninvasive imaging of reporter gene expression in vivo imaging with positron emission tomography (PET) could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy, such as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time, a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors. PET-based non-invasive imaging of gene expression involves reporter gene and PET probe. The herpes simplex virus thymidine kinase (HSV1-tk) gene has frequently been applied as a reporter gene for monitoring transgene expression in animal models. Consequently, sensitive and selective PET probe for imaging are required. In this study, the novel probe 2-amino-6-[18F]fluoro-9- (4-hydroxy-3-hydroxymethylbutyl) purine (6-[18F]FPCV) were prepared by one-step nucleophilic radiofluorination, and evaluated as a novel probe for imaging the expression of HSV1-tk reporter gene. 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine([18F]FHBG), a classical probe of HSV1-tk PET imaging, were prepared by an improved way, too. First, 2-amino-6-chloro-9-(4-acetoxy-3-acetoxymethylbutyl) purine was hydrolyzed to 2-amino-6-chloro-9-(4-hydroxy-3-hydroxymethylbutyl) purine, then was converted to the corresponding trimethylammonium chloride by treatment with trimethylamine (TMA) in ethanolic solution in a mild condition, then was reacted with anhydrous KF to produce 6-[18F]FPCV referencer compound 2-amino-6-fluoro- 9-(4-hydroxy-3-hydroxymethylbutyl)purine(6-FPCV). 6-[18F]FPCV was prepared by an one-step nucleophilic substitution with [18F]KF from label precursor nucleoside trimethylammonium chloride and isolated by a silica Sep-Pak cartridge. The radiochemical yield of 6-[18F]FPCV was 45–55% decay corrected (d.c) in six runs with radiochemical purity >98% with an average specific activity 5.62 GBq/μmol, and the radiosynthesis time was 35–42 minutes from end of bombardment. Second, the quality control and basic properties were further investigated according to requirements of PET radiopharmaceuticals. the log P of 6-[18F]FPCV was -0.517 and the RCP of 6-[18F]FPCV in PBS or BSA was more than 90% after 6 hours. The time-activity curve of 6-[18F]FPCV was fitted as pharmacokinetics two compartments model and the plasma half-life (t1/2) of the tracer in normal mice blood was the T1/2(α) of 1.2 min and T1/2(β) of 73.7 min. C6 rat glioma cells transfected with HSV1-tk (C6-tk) and control C6 cells were incubated with 6-[18F]FPCV for 30 min to 180 min. The in vitro tracer uptake in HSV1-tk containing C6-tk cells was 5.56 to 18.88 times higher than that in control cells and reached up to 8.6 ± 0.38%dose/106 cells after three hours incubation, whereas very low uptake was found in control C6 cells (0.45 ± 0.03 %dose/106 cells). At the same time, in vitro cellular 6-[18F]FPCV accumulation linearly increased with increasing portions of C6-tk cells expressing HSV1-tk. Subsequently, 6-[18F]FPCV was evaluated as a potential new probe for monitoring the expression of HSV1-tk reporter gene in vivo by small animal PET (micro-PET) imaging. For in vivo studies, in nude rats carrying both a C6 and a C6-tk tumor, the average ratio of tracer accumulation between the tumors was 1.5 at 15 min post injection. The tracer is rapidly cleared from nontarget tissue into the urine because only the HSV1-tk-expressing tumor, kidneys, and bladder remained visible on the late micro-PET images. In conclusion, we have demonstrated that 6-[18F]FPCV is a promising tracer for monitoring HSV1-tk enzyme activity early in vivo with micro-PET. Finally, Penciclovir was converted to Ditrityl-Penciclovir by treatment with methoxytrityl chloride, then followed by tosylation with high yield. Ditrityl-4-Tosyl-Penciclovir was reacted with tetrabutylammonium fluoride to afford Ditrityl-4-fluoro-Penciclovir. Removal of the methoxytrityl groups by acidic hydrolysis produced 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG), which is reference compound of PET classical probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine ([18F]FHBG) for HSV1-tk imaging. [18F]FHBG were prepared by nucleophilic substitution of the tosylated precursor Ditrityl-4-Tosyl-Penciclovir with [18F]KF/K2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Sep-Pak Cartridge method in 9 ± 3%(n= 6) radiochemical yield with radiochemical purity >97%, and the total radiosynthesis time was 90~100 min.
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7190
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
蔡汉成. 报告基因HSV1-tk的PET探针的合成及其生物学评价[D]. 上海应用物理研究所. 中国科学院上海应用物理研究所,2007.
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