细胞凋亡PET探针~（18）F-SFB-C2A-GST的合成及实验研究
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摘要
目的合成满足PET使用的细胞凋亡的分子探针~(18)F-SFB-C2A-GST融合蛋白;并对合成的探针通过动物实验获取正常组织、器官的生理分布数据,为进行细胞凋亡PET显像提供参考数据。
方法利用回旋加速器(PETtrace GE医疗系统)使加速粒子在16.5Mev能量下轰击化学丰度>98%的高纯富氧水H_2O~(18) ,通过~(18)O(p,n)~(18)F核反应生成含~(18)F-;以乙基-4-三甲胺苯甲酸酯-三氟磺酸盐为反应前体,运用TRACERlab FXF-N及TRACERlab FX-FDG两套PET显像药物合成模块的级联,通过亲核氟化取代、氢氧化钠水解、酯化反应“三步法”合成用于标记蛋白、多肽类的中间体N-琥珀酰亚胺-4-[~(18)F]氟苯甲酸酯(~(18)F-SFB)。过程概括为:第一步:利用He气流将反应生成的~(18)F-传输至阴离子交换柱Sep Pak?Light QMA,利用K_2CO_3及氨基聚醚(Kryptofix2.2.2)的混合溶液将QMA柱上的~(18)F-淋洗入化学合成的反应瓶,在95℃温度下把混合液蒸发干燥,保证反应瓶无水环境。第二步:将前体乙基-4-三甲胺苯甲酸酯-三氟磺酸盐(前体溶解于无水二甲基亚砜,DMSO)加入反应瓶,密闭反应管并加热至110℃持续15分钟;冷却反应后混合液至50℃左右,然后向反应瓶中加入氢氧化钠溶液,将混合液温度再次加热到95℃左右以实现水解过程。第三步:将获得的对氟[~(18)F]苯甲酸混合液通过四甲基氢氧化胺处理,再利用O-(N-琥珀酰亚胺)N,N,N,N-四甲基脲四氟硼酸盐在90℃温度下反应5分钟将琥珀酰亚胺基酯化取代四甲胺基进而合成N-琥珀酰亚胺-4-[~(18)F]氟苯甲酸酯(~(18)F-SFB)。利用HPLC对合成产物与标准品~(19)F-SFB进行对比检验,通过比较两者在HPLC分析柱上的保留时间来确定合成的中间产物;利用二氯甲烷低沸点的化学性质去除乙腈等有毒杂质确保融合蛋白C2A-GST的安全标记,同时分别利用HPLC及γ射线薄层色谱扫描仪检测其放射化学纯度。
利用得到的~(18)F-SFB与经2-亚氨基噻吩盐酸盐(2-IT)修饰和0.1mol/l PH值为7.4的硼酸盐缓冲液处理后的C2A-GST融合蛋白在室温下反应,将纯化的~(18)F-SFB的PBS溶液加入已纯化C2A-GST融合蛋白,室温下反应30分钟获得~(18)F-SFB-C2A-GST。最后通过聚丙烯酰胺凝胶柱分离纯化~(18)F-SFB-C2A-GST。
取正常ICR小鼠36只,随机分为6组,自尾静脉注射纯化的0.1-0.2ml的生理盐水注射液3.7 MBq,于注射后5分钟、30分钟、60分钟、120分钟、240分钟分别断头处死解剖,测量心、肝、脾、胃、肾、肺、脑、骨胳、血液、股骨、小肠等组织器官每克组织的百分注射剂量,并绘制时间—生物分布曲线;从而获得~(18)F-SFB-C2A-GST在小鼠的正常生物分布数据;分别于注射~(18)F-SFB-C2A-GST后5分钟、15分钟、60分钟、120分钟四个时相采集部分ICR小鼠的MicroPET图像,分析主要器官的分布及代谢情况。
结果利用本研究方法获得的~(18)F-SFB与标准品~(19)F-SFB对照,在相同的HPLC分析条件下,两者在HPLC柱上的保留时间基本吻合。标记后的重组蛋白~(18)F-SFB-C2A-GST放射化学纯度大于95%,经HPLC分析其放射峰位与紫外色谱峰位一致,且其紫外色谱峰位与C2A-GST紫外色谱峰位一致。合成的放射化学纯度>85%。生物分布显示~(18)F-SFB-C2A-GST在小鼠肾组织、肝组织表现为高摄取;在脾表现为明显摄取,在肺组织早期摄取相对明显但清除较快;在心肌、骨骼、脑等组织呈现低摄取。
结论本方法可以成功合成用于标记蛋白、多肽、抗体等生物活性分子的中间体~(18)F-SFB。~(18)F -SFB可以成功与C2A-GST融合蛋白鳌合,实现C2A-GST融合蛋白的正电子核素标记(分子探针合成),并可获得满意的放射化学纯度和体外稳定性。~(18)F -SFB-C2A-GST在生物体表现为明显的组织摄取差异,主要通过肝肾代谢、清除,不能通过血脑屏障进入脑组织,将其应用于肝、肾、脑等实质组织的凋亡PET显像受到限制;应用于心肌、骨骼、肺等本底摄取低或清除速度较快的组织器官进行凋亡PET显像的前景较好。
Objective synthesize the PET or PET/CT imaging agent-~(18)F-labeled C2A domain of SynaptotagminⅠ(molecular probe )for imaging of apoptosis; Obtain the physiological distribution in normal tissue and organ of rabbit, so as to provide reference data for PET imaging of apoptosis in abnormal tissue and organ of animal or human in which apoptosis cells are existing.
Methods Generate the ~(18)F ions through ~(18)O(p,n)~(18)F nuclear reaction with the positive electronof 16.5Mev high energy incyclotron(PETtrace GE medical system), utilize ethyl4-trimethyl ammonium benzoate trifluoro methanesulfonate as precurosor, TracerlabFX-FN and Tracerlab FX-FDG are deploied by connectio n, they are all used for synthesis of PET imaging agent. The intermedium used for conjugate ~(18)F ions and C2A domain of SynaptotagmInⅠ-N- succinimidyl4 -[~(18)F] ?uoro-benzoate (~(18)F-SFB) is synthesized with three steps: nucleophilic fluorization substitution、hydrolyzed by natrium hydroxydatum esterification reaction .First, an aqueous solution of Kryptofix222, and K2CO3 in MeCN, was used for eluting ~(18)F ions on the Sep Pak?Light QMA Cartridge which are transferred by He gas to a conical screw-cap vial, The reaction vial was heated at 95℃and the liquid evaporated to dry residue. Second, a solution of 10mg ethyl 4-(trimethyl ammonium triflate) benzoate dissolved in 200ul dry DMSO was added into reaction vial, the vial was sealed and heated at 110℃for 15min. Cooling the reaction vial to about 50℃, the hydrolysis step was carried out by the addition of 500 ul 1M NaOH, followed by heating at 95℃for 10 minutes to produce 4-fluorobenzoic acid. Third, 4-fluorobenzoic acid was treated with aqueous solution of Tetramethyl ammonium hydroxide,then a solution of TSTU(15mg;50umol) in 1ml anhydrous acetonitrile was added, and the mixture was heated at 90℃for 5min.
Compared ~(18)F-SFB with standard preparation-19F-SFB by HPLC, according to the remaining time of ~(18)F-SFB and 19F-SFB to judge ~(18)F-SFB is the real production or not.
Removal the acetonitrile in ~(18)F-SFbsolution by dichlormethane to make ~(18)F-SFB safe to label the C2A-GST fusion protein.Detect the radiochemical purity of ~(18)F-SFB by HPLC orγthin-layer chromatography.Before mixing the purified ~(18)F-SFB into the prepared physiologic saline solution of C2A-GST at ordinary temperature, the C2A-GST fusion protein was modified by 2-imin- othiolane and borate buffer solution(0.1mol/l,PH=7.4).keep the reaction time for 30 minutes to make sure of production;of ~(18)F-SFB-C2A- GST.Separating the ~(18)F-SFB-C2A-GST by polyacrylamide gel column.
Adopting thirty-six ICR mouse in next experiment,grouping of all mouse into six groups in randomization. Injection of physiologic saline solution of ~(18)F-SFB-C2A-GST through the vena caudalis of the mouse with a volume of 0.1 to 0.2 milliliters containing intensity of radioactivity about 3.7Mbq.Kill the mouse by the method of decapitation after five minutes、thirty minutes.sixty minutes.one hundred and twenty minutes.two hundreds and forty minutes of injection,obduction of the dead mouse and extirpating the tissue of cardiac muscle.liver.spleen.stomach.kidney.lung.brain.femoral bone.blood and intestina parva.Measuring theγcounts of all the tissues and calculating the percentage of injected dose ratio of per-gram tissues. Then drawing the curve of radioactivity distribution versus time according to the data measured; from the data above we can procure normal biology distribution of ~(18)F-SFB-C2A-GST in ICR mouse. Scaning ICR mouse by MicroPET at the time of 5min.15min.60min and 120min after injection of ~(18)F-SFB-C2A-GST, respectively, analysis the distribution and metabolism of ~(18)F-SFB-C2A-GST in its main organic.
Results The ~(18)F-SFB acquired from our empirical method in this research was compared with standard preparation 19F-SFB, the two compounds have the approximate retention time in the HPLC column at the same analysis condition. ~(18)Flabeled recombination protein ~(18)F-SFB-C2A-GST (molecular probe) has a high radiochemical purity exceeding 95%, analysis of the ~(18)F-SFB-C2A-GST by HPLC,its chromatographic peak is coincidence with its radiation peak, and the chromatographic peak of ~(18)F-SFB-C2A-GST is coincidence with the chromatographic peak of C2A-GST. Measured the radiochemical purity of ~(18)F-SFB-C2A-GST, its radiochemical purity is more than 85%. biology distribution demonstrating: the nephridial tissue,hepatic tissue have a high uptake; lung tissue,spleen tissue showing a evident uptake; the uptake in cardiac muscle,femoral bone,brain and muscle is low .
Conclusions ~(18)F-SFB, an important agent for labeling protein, polypeptide, antibody and etal, can be synthesized by synthesis unit of Tracerlab FXFN and Tracerlab FXFDG with editing and modifying its control sequence and cascading the two units. C2A-GST fusion protein can be labeled by 18-fluorine through the pivotal intermediate—~(18)F-SFB (molecular probe synthesis); The radiochemistry purity and vitro stability of ~(18)F-SFB-C2A-GST are satisfactory. The biology distribution of ~(18)F-SFB-C2A-GST in ICR rat showing a great tissue difference;Liver and kidney are mainly metabolism clearance organ,blood-brain barrier blocks ~(18)F-SFB-C2A-GST entering brain parenchyma, application of ~(18)F-SFB-C2A-GST in Liver ,kidney and brain is limited in PET Visualization to detect apoptosis; it is suitable for PET visualization in cardiac muscle,skeleton and muscle due to these tissue having a low background uptake.
方法利用回旋加速器(PETtrace GE医疗系统)使加速粒子在16.5Mev能量下轰击化学丰度>98%的高纯富氧水H_2O~(18) ,通过~(18)O(p,n)~(18)F核反应生成含~(18)F-;以乙基-4-三甲胺苯甲酸酯-三氟磺酸盐为反应前体,运用TRACERlab FXF-N及TRACERlab FX-FDG两套PET显像药物合成模块的级联,通过亲核氟化取代、氢氧化钠水解、酯化反应“三步法”合成用于标记蛋白、多肽类的中间体N-琥珀酰亚胺-4-[~(18)F]氟苯甲酸酯(~(18)F-SFB)。过程概括为:第一步:利用He气流将反应生成的~(18)F-传输至阴离子交换柱Sep Pak?Light QMA,利用K_2CO_3及氨基聚醚(Kryptofix2.2.2)的混合溶液将QMA柱上的~(18)F-淋洗入化学合成的反应瓶,在95℃温度下把混合液蒸发干燥,保证反应瓶无水环境。第二步:将前体乙基-4-三甲胺苯甲酸酯-三氟磺酸盐(前体溶解于无水二甲基亚砜,DMSO)加入反应瓶,密闭反应管并加热至110℃持续15分钟;冷却反应后混合液至50℃左右,然后向反应瓶中加入氢氧化钠溶液,将混合液温度再次加热到95℃左右以实现水解过程。第三步:将获得的对氟[~(18)F]苯甲酸混合液通过四甲基氢氧化胺处理,再利用O-(N-琥珀酰亚胺)N,N,N,N-四甲基脲四氟硼酸盐在90℃温度下反应5分钟将琥珀酰亚胺基酯化取代四甲胺基进而合成N-琥珀酰亚胺-4-[~(18)F]氟苯甲酸酯(~(18)F-SFB)。利用HPLC对合成产物与标准品~(19)F-SFB进行对比检验,通过比较两者在HPLC分析柱上的保留时间来确定合成的中间产物;利用二氯甲烷低沸点的化学性质去除乙腈等有毒杂质确保融合蛋白C2A-GST的安全标记,同时分别利用HPLC及γ射线薄层色谱扫描仪检测其放射化学纯度。
利用得到的~(18)F-SFB与经2-亚氨基噻吩盐酸盐(2-IT)修饰和0.1mol/l PH值为7.4的硼酸盐缓冲液处理后的C2A-GST融合蛋白在室温下反应,将纯化的~(18)F-SFB的PBS溶液加入已纯化C2A-GST融合蛋白,室温下反应30分钟获得~(18)F-SFB-C2A-GST。最后通过聚丙烯酰胺凝胶柱分离纯化~(18)F-SFB-C2A-GST。
取正常ICR小鼠36只,随机分为6组,自尾静脉注射纯化的0.1-0.2ml的生理盐水注射液3.7 MBq,于注射后5分钟、30分钟、60分钟、120分钟、240分钟分别断头处死解剖,测量心、肝、脾、胃、肾、肺、脑、骨胳、血液、股骨、小肠等组织器官每克组织的百分注射剂量,并绘制时间—生物分布曲线;从而获得~(18)F-SFB-C2A-GST在小鼠的正常生物分布数据;分别于注射~(18)F-SFB-C2A-GST后5分钟、15分钟、60分钟、120分钟四个时相采集部分ICR小鼠的MicroPET图像,分析主要器官的分布及代谢情况。
结果利用本研究方法获得的~(18)F-SFB与标准品~(19)F-SFB对照,在相同的HPLC分析条件下,两者在HPLC柱上的保留时间基本吻合。标记后的重组蛋白~(18)F-SFB-C2A-GST放射化学纯度大于95%,经HPLC分析其放射峰位与紫外色谱峰位一致,且其紫外色谱峰位与C2A-GST紫外色谱峰位一致。合成的放射化学纯度>85%。生物分布显示~(18)F-SFB-C2A-GST在小鼠肾组织、肝组织表现为高摄取;在脾表现为明显摄取,在肺组织早期摄取相对明显但清除较快;在心肌、骨骼、脑等组织呈现低摄取。
结论本方法可以成功合成用于标记蛋白、多肽、抗体等生物活性分子的中间体~(18)F-SFB。~(18)F -SFB可以成功与C2A-GST融合蛋白鳌合,实现C2A-GST融合蛋白的正电子核素标记(分子探针合成),并可获得满意的放射化学纯度和体外稳定性。~(18)F -SFB-C2A-GST在生物体表现为明显的组织摄取差异,主要通过肝肾代谢、清除,不能通过血脑屏障进入脑组织,将其应用于肝、肾、脑等实质组织的凋亡PET显像受到限制;应用于心肌、骨骼、肺等本底摄取低或清除速度较快的组织器官进行凋亡PET显像的前景较好。
Objective synthesize the PET or PET/CT imaging agent-~(18)F-labeled C2A domain of SynaptotagminⅠ(molecular probe )for imaging of apoptosis; Obtain the physiological distribution in normal tissue and organ of rabbit, so as to provide reference data for PET imaging of apoptosis in abnormal tissue and organ of animal or human in which apoptosis cells are existing.
Methods Generate the ~(18)F ions through ~(18)O(p,n)~(18)F nuclear reaction with the positive electronof 16.5Mev high energy incyclotron(PETtrace GE medical system), utilize ethyl4-trimethyl ammonium benzoate trifluoro methanesulfonate as precurosor, TracerlabFX-FN and Tracerlab FX-FDG are deploied by connectio n, they are all used for synthesis of PET imaging agent. The intermedium used for conjugate ~(18)F ions and C2A domain of SynaptotagmInⅠ-N- succinimidyl4 -[~(18)F] ?uoro-benzoate (~(18)F-SFB) is synthesized with three steps: nucleophilic fluorization substitution、hydrolyzed by natrium hydroxydatum esterification reaction .First, an aqueous solution of Kryptofix222, and K2CO3 in MeCN, was used for eluting ~(18)F ions on the Sep Pak?Light QMA Cartridge which are transferred by He gas to a conical screw-cap vial, The reaction vial was heated at 95℃and the liquid evaporated to dry residue. Second, a solution of 10mg ethyl 4-(trimethyl ammonium triflate) benzoate dissolved in 200ul dry DMSO was added into reaction vial, the vial was sealed and heated at 110℃for 15min. Cooling the reaction vial to about 50℃, the hydrolysis step was carried out by the addition of 500 ul 1M NaOH, followed by heating at 95℃for 10 minutes to produce 4-fluorobenzoic acid. Third, 4-fluorobenzoic acid was treated with aqueous solution of Tetramethyl ammonium hydroxide,then a solution of TSTU(15mg;50umol) in 1ml anhydrous acetonitrile was added, and the mixture was heated at 90℃for 5min.
Compared ~(18)F-SFB with standard preparation-19F-SFB by HPLC, according to the remaining time of ~(18)F-SFB and 19F-SFB to judge ~(18)F-SFB is the real production or not.
Removal the acetonitrile in ~(18)F-SFbsolution by dichlormethane to make ~(18)F-SFB safe to label the C2A-GST fusion protein.Detect the radiochemical purity of ~(18)F-SFB by HPLC orγthin-layer chromatography.Before mixing the purified ~(18)F-SFB into the prepared physiologic saline solution of C2A-GST at ordinary temperature, the C2A-GST fusion protein was modified by 2-imin- othiolane and borate buffer solution(0.1mol/l,PH=7.4).keep the reaction time for 30 minutes to make sure of production;of ~(18)F-SFB-C2A- GST.Separating the ~(18)F-SFB-C2A-GST by polyacrylamide gel column.
Adopting thirty-six ICR mouse in next experiment,grouping of all mouse into six groups in randomization. Injection of physiologic saline solution of ~(18)F-SFB-C2A-GST through the vena caudalis of the mouse with a volume of 0.1 to 0.2 milliliters containing intensity of radioactivity about 3.7Mbq.Kill the mouse by the method of decapitation after five minutes、thirty minutes.sixty minutes.one hundred and twenty minutes.two hundreds and forty minutes of injection,obduction of the dead mouse and extirpating the tissue of cardiac muscle.liver.spleen.stomach.kidney.lung.brain.femoral bone.blood and intestina parva.Measuring theγcounts of all the tissues and calculating the percentage of injected dose ratio of per-gram tissues. Then drawing the curve of radioactivity distribution versus time according to the data measured; from the data above we can procure normal biology distribution of ~(18)F-SFB-C2A-GST in ICR mouse. Scaning ICR mouse by MicroPET at the time of 5min.15min.60min and 120min after injection of ~(18)F-SFB-C2A-GST, respectively, analysis the distribution and metabolism of ~(18)F-SFB-C2A-GST in its main organic.
Results The ~(18)F-SFB acquired from our empirical method in this research was compared with standard preparation 19F-SFB, the two compounds have the approximate retention time in the HPLC column at the same analysis condition. ~(18)Flabeled recombination protein ~(18)F-SFB-C2A-GST (molecular probe) has a high radiochemical purity exceeding 95%, analysis of the ~(18)F-SFB-C2A-GST by HPLC,its chromatographic peak is coincidence with its radiation peak, and the chromatographic peak of ~(18)F-SFB-C2A-GST is coincidence with the chromatographic peak of C2A-GST. Measured the radiochemical purity of ~(18)F-SFB-C2A-GST, its radiochemical purity is more than 85%. biology distribution demonstrating: the nephridial tissue,hepatic tissue have a high uptake; lung tissue,spleen tissue showing a evident uptake; the uptake in cardiac muscle,femoral bone,brain and muscle is low .
Conclusions ~(18)F-SFB, an important agent for labeling protein, polypeptide, antibody and etal, can be synthesized by synthesis unit of Tracerlab FXFN and Tracerlab FXFDG with editing and modifying its control sequence and cascading the two units. C2A-GST fusion protein can be labeled by 18-fluorine through the pivotal intermediate—~(18)F-SFB (molecular probe synthesis); The radiochemistry purity and vitro stability of ~(18)F-SFB-C2A-GST are satisfactory. The biology distribution of ~(18)F-SFB-C2A-GST in ICR rat showing a great tissue difference;Liver and kidney are mainly metabolism clearance organ,blood-brain barrier blocks ~(18)F-SFB-C2A-GST entering brain parenchyma, application of ~(18)F-SFB-C2A-GST in Liver ,kidney and brain is limited in PET Visualization to detect apoptosis; it is suitable for PET visualization in cardiac muscle,skeleton and muscle due to these tissue having a low background uptake.
引文
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[13] Enfu Hui, Jihong Bai, Edwin R. Chapman. Ca21-Triggered Simultaneous Membrane Penetration of the TandemC2-Domains of Synaptotagmin I[J]. Biophysical Journal 2006, 91(9):1767–1777.
[14] Shankar Vallabhajosula.18F-Labeled Positron Emission Tomographic Radiopharmaceuticals in Oncology: An Overview of Radiochemistry and Mechanisms of Tumor Localization [J].Seminars Nuclear Med 37:400-419.
[15]张锦明,田嘉禾.国内正电子放射性药物发展现状简介[J].同位素,2006,19(4):240-244.
[16]李林法主编肿瘤靶向分子影像.北京:科学出版社2006,70-126.
[17] Imma Fernandez,Demet Arac,Josep Ubach,et al.Three-Dimensional Structure of the Synaptotagmin 1 C2B-Domain: Synaptotagmin I as aPhos- pholipid Binding Machine . Neuron[J],32:1057–1069.
[18] R. Bryan Sutton,Bazbek A.Davletov,Albert M. Berghuis, etal. Structureof- theFirstC2DomainofSynaptotagminI:ANovelCa2+/Phospholipid-Binding Fold.Cell[J]. 80:929-938.
[19]Fernandez-ChaconR,ShinOH,Konigstorfer,etal.Structure/Function Analysis of Ca2-Binding to the C2A Domain of Synaptotagmin I.The Journ-al of Neuroscience[J].2002, 22(19):8438–8446.
[20] P.M?ding,F.Füchtner,F.Wüst etal. Module-assisted synthesis of the bifunc-tionallabellingagentN-succinimidyl4-[18F]fluorobenzoate ([18F]SFB). Applied Radiation and Isotopes [J].2005,63(3):329-332.
[21] Bazbek A.Davletov,Thomas C.Sudhof.A Single C2 Domain from Synaptotagmin I is Sufficient for High Affinity Ca2+/ Phospholipid Binding [J]. Journal of Biological Chemistry 1993,26(35):26386-26390.
[22] J.萨姆布鲁克D. W.拉塞尔著.黄培堂等译分子克隆实验指南.北京科学出版社2002,1217—1248.
[23] Zhao M, Zhu X,Ji s,et al. 99mTc-labeled C2A domain of synaptotagmin I as a target-specific molecular probe for noninvasive imaging of acute myocar dial infarction. The Journal of Nuclear Medicine [J].2006,47(8):1367 -1375.
[24] Feng Wang, Wei Fang, Ming Zhao, et al. Imaging paclitaxel (chemotherapy) -induced tumor apoptosis with 99mTc-C2A, a domain of synaptotagmin I:a preliminary study[J].Nuclear Medicine and Biology 2008, 35:359-364.