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Volume 39 Issue 6
Nov.  2021
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ZUO Feng, HUO Hua, WANG Zhiguo, ZHANG Guoxu, SHI Qingxue, ZHANG Zongpeng. Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(6): 525-528. doi: 10.12206/j.issn.1006-0111.202009023
Citation: ZUO Feng, HUO Hua, WANG Zhiguo, ZHANG Guoxu, SHI Qingxue, ZHANG Zongpeng. Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(6): 525-528. doi: 10.12206/j.issn.1006-0111.202009023

Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer

doi: 10.12206/j.issn.1006-0111.202009023
  • Received Date: 2020-09-08
  • Rev Recd Date: 2021-09-09
  • Available Online: 2021-12-27
  • Publish Date: 2021-11-25
  •   Objective  To optimize the synthesis method of 18F-T807 and study preliminary biodistribution.   Methods  18F-T807 was synthesized using an optimized method in TRACERlab FXFN synthesizer with a t-BOC(t-Butyloxy carbonyl)-protected 18F-T807 precursor NPPI-9 as starting material, improving experimental conditions for synthesis, then QC and biodistribution study in Wistar rats conducted.   Results  The improved synthesis conditions increased the synthesis yield from 20.5%±6.1% to 25.7%±5.8%. QC met the standard. Wistar rats had higher intake in kidney, liver, blood and lowest intake in brain, heart, lung.   Conclusion  The optimized synthesis method to synthesize 18F-T807 is simple and easy, and high yield, which can meet the needs of scientific research and clinical practice.
  • [1] CHO H, CHOI J Y, HWANG M S, et al. Tau PET in Alzheimer disease and mild cognitive impairment[J]. Neurology,2016,87(4):375-383. doi:  10.1212/WNL.0000000000002892
    [2] CHIEN D T, SZARDENINGS A K, BAHRI S, et al. Early clinical PET imaging results with the novel PHF-tau radioligand [F18]-T808[J]. J Alzheimers Dis,2014,38(1):171-184.
    [3] WEINER M W, VEITCH D P, AISEN P S, et al. The Alzheimer's Disease Neuroimaging Initiative: a review of papers published since its inception[J]. Alzheimers Dement,2012,8(1suppl):S1-68.
    [4] MACCIONI R B, FARÍAS G, MORALES I, et al. The revitalized tau hypothesis on Alzheimer's disease[J]. Arch Med Res,2010,41(3):226-231. doi:  10.1016/j.arcmed.2010.03.007
    [5] SABRI O, SEIBYL, ROWE C, et al. Use of florbetapir-PET for imaging beta-amyloid pathology[J]. JAMA,2011,305(3):275-283. doi:  10.1001/jama.2010.2008
    [6] OKAMURA N, FURUMOTO S, FODERO-TAVOLETTI M T, et al. Non-invasive assessment of Alzheimer's disease neurofibrillary pathology using 18F-THK5105 PET[J]. Brain,2014,137(Pt6):1762-1771.
    [7] FODERO-TAVOLETTI M T, OKAMURA N, FURUMOTO S, et al. 18F-THK523: a novel in vivo tau imaging ligand for Alzheimer's disease[J]. Brain,2011,134(Pt4):1089-1100.
    [8] OKAMURA N, FURUMOTO S, HARADA R, et al. Characterization of 18F-THK-5351, a novel PET tracer for imaging tau pathology in Alzheimer's disease[EB/OL]. 2014
    [9] HONER M, GOBBI L, KNUST H, et al. Preclinical evaluation of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 as novel PET radiotracers for imaging tau aggregates in alzheimer disease[J]. J Nucl Med,2018,59(4):675-681. doi:  10.2967/jnumed.117.196741
    [10] WONG D F, COMLEY R A, KUWABARA H, et al. Characterization of 3 novel tau radiopharmaceuticals, 11C-RO-963, 11C-RO-643, and 18F-RO-948, in healthy controls and in alzheimer subjects[J]. J Nucl Med,2018,59(12):1869-1876. doi:  10.2967/jnumed.118.209916
    [11] KUWABARA H, COMLEY R A, BORRONI E, et al. Evaluation of 18F-RO-948 PET for quantitative assessment of tau accumulation in the human brain[J]. J Nucl Med,2018,59(12):1877-1884. doi:  10.2967/jnumed.118.214437
    [12] GAO M Z, WANG M, ZHENG Q H. Fully automated synthesis of [18F]T807, a PET tau tracer for Alzheimer's disease[J]. Bioorg Med Chem Lett,2015,25(15):2953-2957. doi:  10.1016/j.bmcl.2015.05.035
    [13] MARUYAMA M, SHIMADA H, SUHARA T, et al. Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls[J]. Neuron,2013,79(6):1094-1108. doi:  10.1016/j.neuron.2013.07.037
    [14] 王治国, 左峰, 张国旭, 等. 新型阿尔茨海默症Tau蛋白PET显像剂18F-T807的合成[J]. 中国医学装备, 2019, 16(2):125-127. doi:  10.3969/J.ISSN.1672-8270.2019.02.034
    [15] HUANG Y Y, CHIU M J, YEN R F, et al. An one-pot two-step automated synthesis of [18F]T807 injection, its biodistribution in mice and monkeys, and a preliminary study in humans[J]. PLoS One,2019,14(7):e0217384. doi:  10.1371/journal.pone.0217384
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Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer

doi: 10.12206/j.issn.1006-0111.202009023

Abstract:   Objective  To optimize the synthesis method of 18F-T807 and study preliminary biodistribution.   Methods  18F-T807 was synthesized using an optimized method in TRACERlab FXFN synthesizer with a t-BOC(t-Butyloxy carbonyl)-protected 18F-T807 precursor NPPI-9 as starting material, improving experimental conditions for synthesis, then QC and biodistribution study in Wistar rats conducted.   Results  The improved synthesis conditions increased the synthesis yield from 20.5%±6.1% to 25.7%±5.8%. QC met the standard. Wistar rats had higher intake in kidney, liver, blood and lowest intake in brain, heart, lung.   Conclusion  The optimized synthesis method to synthesize 18F-T807 is simple and easy, and high yield, which can meet the needs of scientific research and clinical practice.

ZUO Feng, HUO Hua, WANG Zhiguo, ZHANG Guoxu, SHI Qingxue, ZHANG Zongpeng. Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(6): 525-528. doi: 10.12206/j.issn.1006-0111.202009023
Citation: ZUO Feng, HUO Hua, WANG Zhiguo, ZHANG Guoxu, SHI Qingxue, ZHANG Zongpeng. Synthesis method optimization and biodistribution study of 18F-T807 on TRACERlab FXFN synthesizer[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(6): 525-528. doi: 10.12206/j.issn.1006-0111.202009023
  • 阿尔茨海默症(Alzheimer's disease,AD)是常见的神经系统变性疾病之一,是一种持续性神经功能障碍,也是痴呆最常见的病因,其发生可导致进行性记忆减退、认知障碍、人格改变等症状。65岁以上患病率约5%,85岁以上患病率高于20%,是老年人死亡的主要原因之一[1-3]。AD的病理特征主要是老年斑(senile plaques,SP)、神经纤维缠结(neurofibrillary tangles,NFTs)和广泛神经元缺失。tau蛋白是一种微管相关蛋白,过度磷酸化tau蛋白是造成神经纤维缠结的主要原因,且AD患者病情严重程度与tau蛋白具有明显相关性。因此,tau蛋白显像剂的研究逐渐受到关注[4-5]

    近年来,研究者们研发了多种tau蛋白的PET显像剂,如“THK系列”(包括18F-THK5105、18F-THK523、18F-THK5117、18F-THK5351)[6-8],“RO系列”(包括18F-RO6958548、11C-RO6931643、11C-RO6924963)[9-11],“T系列”(包括18F-T807、18F-T808)[12]以及11C-PBB3[13]等。其中“T系列”18F-T807和18F-T808是由Simens公司开发的tau蛋白的分子探针。

    课题组在参考相关文献的基础上[14-15],使用GE公司的TRACERlab FXFN氟多功能合成模块(图1),以18F-T807前体(BOC保护)NPPI-95(1)为原料,使用改进一锅法自动合成了18F-T807(2),提高了产品产率,并开展初步的正常大鼠生物分布实验,探索其分布特征。

    • 18F-T807前体(BOC保护)NPPI-95(江苏华益公司);强阴离子交换固相萃取柱(QMA柱)、K222、碳酸钾水溶液、乙腈、(德国ABX公司);乙醇(国药化学试剂);盐酸(国药分析质检中心);DMSO(北京百灵威);Ultimate C18柱(美国Waters: 4.6 mm×250 mm,5 μm);富18O水(日本大阳日酸株式会社);0.22 μm MILLEX-GS液体滤膜、0.2 μm Millex-25空气滤膜(德国默克);0.7 mm×40 mm针头(西班牙BD Microlance);Wistar大鼠(北部战区总医院实验动物科)。溶剂乙醇为色谱纯,其余均为分析纯。

      TRACERlab FXFN(美国GE)配备半制备VP 250×16高效液相色谱(德国MN)和紫外检测器及放射性检测器;正电子示踪剂质量控制薄层扫描仪(美国Bioscan),配塑料闪烁体晶体探测器;分析用HPLC(北京优联);GC-7900气相色谱(北京天美);CRC 25R活度计(美国Capintec),合成条件满足药品生产质量管理规范(GMP)的要求。

    • 合成开始前,合成器各溶剂瓶预装溶剂如表1图2所示。

      溶剂瓶溶剂
      1号瓶(V1)1.5mg K2CO3溶于0.5 ml水
      2号瓶(V2)1.5 mg K222溶于1ml乙腈
      3号瓶(V3)1 mg前体溶于1.2 ml DMSO溶剂
      5号瓶(V5)1.5 ml HPLC流动相
      6号瓶(V6)1.5 ml HPLC流动相
      圆底烧瓶2 ml 84%NaHCO3水溶液和30 ml水
      7号瓶(V7)9 ml 0.9%生理盐水
      8号瓶(V8)1 ml 乙醇
      9号瓶(V9)10 ml 水

      18F-T807自动化合成主要有以下几步:①18F-离子的柱分离纯化及蒸馏干燥。②T807前体的18F-离子亲核取代反应。③18F-T807的HPLC分离纯化。④18F-T807 C18柱溶剂转换与再纯化。

      自动合成的具体步骤如下:

      (1)共2.5 ml含18F-离子的18O水由MINItrace加速器经由18O(p, n)18F反应制备,轰击束流45μA,轰击时间40 min,18F-离子混合液由氦气作为载气经过TARGET管线传输到TRACERlab FXFN合成模块的锥形瓶内。

      (2)V10、V11号阀门开启,18F-离子及18O水混合液中的18F-离子在真空泵抽取下被QMA柱(由1 ml乙醇,2 ml水活化)捕获滞留,18O水回收进入18O水回收瓶。

      (3)V1、V13、V24号阀门开启,V1号瓶内的K2CO3溶液流经V1、V10、QMA柱、V11、V13,将18F-离子交换抽入反应瓶。

      (4)关V1、V13号阀门,开启V2号阀门将V2号瓶内穴醚K222乙腈溶剂抽入反应管,18F-离子进入穴醚形成复合物。

      (5)关V2号阀门,开启V20号阀门混合液在氦气吹拂下于85 ℃共沸蒸馏8 min,然后加热到110 ℃,在氦气吹拂下共沸蒸馏4 min除水。

      (6)开启V3、V19号阀门,在氦气推动下V3号瓶内的前体流入反应管,V3、V19、V24号阀门关闭,反应管加热到140 ℃,反应10 min。

      (7)反应瓶降温到50 ℃,开V24、V25号阀门恢复大气压。

      (8)反应后混合液经由V5、V6号瓶内的共3 ml HPLC流动相(25%乙醇水溶液,调整pH至2.0)冲洗到V26号阀门下的中转瓶内,然后打开V26、V12号阀门,在氦气压力下经由Fluid进入HPLC进样环,在Fluid控制下进样环旋转,产物进入HPLC半制备柱,Eluent1号瓶内流动相以5 ml/min的流速通过柱子分离。流动相以紫外(UV,λ=254 nm)和放射计数器监测。图318F-T807的HPLC及UV图。

      (9)18F-T807溶液通过V18号阀门进入圆底瓶,圆底瓶内装有2 ml 84% NaHCO3水溶液和30 ml无菌注射用水。然后经V21、V15、V17号阀门,产物溶液通过V15、V17号阀门间的C18柱(以5 ml乙醇和10 ml水活化),产物会被捕获滞留在柱子上,然后打开V9阀门,用V9号瓶内10 ml水冲洗柱子到废液瓶(WASTE)内,然后C18柱经由V8号瓶内的1 ml乙醇冲洗进入V15阀门下的产品瓶,再经由V7号瓶内装有9 ml生理盐水再次冲洗。

      (10)手动打开V22和V16号阀门,18F-T807在氦气压力下经过0.22μm液体滤膜过滤进入分装热室的收集瓶。

    • 对3批连续生产的产物进行了质量控制。质控项目包括澄明度、pH、核素半衰期、核素纯度、放化纯度、K222和残留溶剂、细菌内毒素、无菌测试,测试结果均符合标准要求。

    • 选择健康雄性Wistar大鼠30只,分为6组,每组5只,实验前6 h禁食禁水,每只通过尾静脉注入0.2 ml(约7.4 MBq)的18F-T807后,分别在5、15、30、60、90、120 min断头处死,取出脑、心、肝、肺、肾、肌肉、骨和血,去污、称重、计数,数据经衰减校正后计算放射性摄取率(每克组织的放射性摄取剂量占注射剂量的百分比)。

    • 18F-T807有多种合成方法,本文在参考相关文献报道基础上,优化反应条件,改变前体用量为1 mg,同时使用HPLC分离条件为25%乙醇水溶液, pH调整至2.0,在线脱BOC保护。C18柱溶剂转换与再纯化,应用经改进的合成方法使合成产率由(20.5±6.1)%提高到(25.7±5.8)%,总反应时间为70 min。

      连续3批产品,其质量控制结果如下:肉眼观察溶液无色透明,6 h后pH值为7,半衰期满足要求,不包含长半衰期核素(t1/2>5天),核素纯度大于99.5%,HPLC和TLC分析结果,即化学纯度和放化纯度合格,流动相是50%甲醇/水(HCl调节pH至2,),流速1.3 ml/min,紫外检测波长为254 nm,TLC条件为NH3H2O-甲醇-CH2Cl2 (1∶5∶94),气相色谱结果显示残留的丙酮、乙腈、DMSO等溶剂均在检测线下,细菌内毒素实验(鲎试剂法)合格,无菌检查合格。各项结果表明产品符合人体使用标准。

      正常大鼠18F-T807在体内的生物分布如表2所示,可见大部分器官在给药5 min后摄取率最高,其中肾、肝、血的摄取率较高,超过5.56%ID/g(%ID/g为放射性摄取率,即各器官的每克放射性摄取值),在肌肉、骨骼摄取率相对较低,因此推断18F-T807主要是经过肝肾排出体外。18F-T807的脑、心、肺摄取率最低,120 min已降低至本底水平(1.08% ID/g),各器官的放射性摄取率随时间的推移逐渐降低,但清除较慢,在120 min 时大部分器官仍有较高的摄取率。

      器官放射性摄取率(% ID/g)
      5 min15 min30 min60 min90 min120 min
      2.25±0.182.03±0.861.81±0.541.59±0.621.20±0.571.11±0.38
      2.05±0.581.99±0.661.78±0.311.55±0.251.19±0.741.08±0.36
      5.79±2.585.95±1.175.48±0.665.29±0.714.83±0.844.27±0.86
      2.12±0.912.01±0.561.91±0.191.57±0.731.21±0.521.09±0.23
      7.36±4.015.11±1.213.89±1.993.63±1.823.17±1.682.99±0.98
      肌肉2.34±0.862.57±1.182.44±0.952.19±1.362.04±1.031.51±0.89
      2.58±0.912.67±0.752.02±0.681.99±0.821.52±0.461.27±0.55
      5.56±0.355.41±0.564.73±0.744.57±1.314.22±0.374.01±0.45
    • 在TRACERlab FXFN合成器上使用优化条件的一锅法自动合成了18F-T807,提高了产品产率。合成后进行的各种质量控制检测均显示产品符合质控标准。初步的正常大鼠生物分布实验,显示了其不同时间放射性摄取率的分布情况,为应用该产品开展人体显像提供了重要基础。

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