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Chimie

1-（2-[18F]氟乙基）-L-色氨酸的放射性合成，采用一锅两步实验方案

Published: September 21, 2021
doi:
10.3791/63025
, , , , , ,
1Diagnostic & Research PET/MRI Center,Nemours/Alfred I. duPont Hospital for Children, 2Department of Radiology,Nemours/Alfred I. duPont Hospital for Children, 3Department of Neurology,Nemours/Alfred I. duPont Hospital for Children, 4Department of Biomedical Research,Nemours/Alfred I. duPont Hospital for Children

Summary

在这里，我们描述了1-（2-[18F]氟乙基）-L-色氨酸的放射合成，这是一种用于研究色氨酸代谢的正电子发射断层扫描成像剂，在放射化学合成系统中使用一锅两步策略，具有良好的放射化学产率，高对映体过量和高可靠性。

Abstract

犬尿氨酸途径 （KP） 是色氨酸代谢的主要途径。有证据表明，KP的代谢物由于其免疫调节作用，神经调节和神经毒性作用，在肿瘤增殖，癫痫，神经退行性疾病和精神疾病中起着至关重要的作用。用于绘制色氨酸代谢的最广泛使用的正电子发射断层扫描（PET）剂 ，α-[11C]甲基-L-色氨酸（[11C]AMT），在费力的放射合成程序中具有20分钟的短半衰期。需要现场回旋加速器对[11C]AMT进行无线电合成。只有有限数量的中心生产[11C]AMT用于临床前研究和临床研究。因此，迫切需要开发一种具有更长半衰期，有利的 体内 动力学且易于自动化的替代成像剂。1-（2-[18F]氟乙基）-L-色氨酸（一种氟-18 标记色氨酸类似物）的效用和价值已在细胞系来源的异种移植物、患者来源的异种移植物和转基因肿瘤模型的临床前应用中得到报道。

本文提出了一种采用一锅两步策略的1-（2-[18F]氟乙基）-L-色氨酸的放射合成方案。使用该协议，放射性示踪剂可以以20±5%（合成结束时衰变校正，n>20）放射性化学产率生产，放射化学纯度和对映体过量均超过95%。该协议具有小前体量，每步反应溶剂不超过0.5mL，潜在毒性4，7，13，16，21，24-六氧沙-1，10-二氮杂双环[8.8.8]六十二烷（K222）的低负载量，以及用于纯化的环境良性和可注射流动相。该协议可以很容易地配置为在市售模块中生产1-（2-[18F]氟乙基）-L-色氨酸用于临床研究。

Introduction

在人类中， 色氨酸是日常饮食的重要组成部分.色氨酸主要通过犬尿氨酸途径（KP）代谢。KP由两种限速酶催化，吲哚胺2，3-双加氧酶（IDO）和色氨酸2，3-双加氧酶（TDO）。超过95%的色氨酸转化为犬尿氨酸及其下游代谢物，最终产生烟酰胺腺嘌呤二核苷酸，这对细胞能量转导至关重要。KP是免疫系统的关键调节剂，也是神经可塑性和神经毒性作用的重要调节剂1，2。色氨酸代谢异常与各种神经系统、 肿瘤、 精神和代谢紊乱有关;因此， 放射性标记色氨酸类似物已被广泛用于临床研究.临床上最常见的两种色氨酸放射性示踪剂是11C-α-甲基-L-色氨酸（[11C]AMT）和11C-5-羟基色氨酸（11C-5-HTP）3。

在20世纪90年代，11C-5-HTP用于可视化分泌5-羟色胺的神经内分泌肿瘤4，并诊断和监测转移性激素难治性前列腺癌的治疗5。 后来，它被用作内分泌胰腺中5-羟色胺能系统定量的成像工具6。12C-5-HTP也是门内胰岛移植和2型糖尿病中可行胰岛的无创检测的有希望的示踪剂7，8。在过去的二十年中，许多放射性标记的氨基酸已进入临床研究9，10。特别是，碳-11标记的色氨酸类似物[11C]AMT在绘制脑5-羟色胺合成图谱11，12，13，14和定位癫痫病灶，致痫性肿瘤，结节性硬化症复合体，胶质瘤和乳腺癌方面受到广泛关注15，16，17，18，19，20，21，22，23，24，25，26.[11C]AMT在儿童各种低级和高级别肿瘤中也具有高摄取率27。此外，人类受试者中[11C]AMT的动力学示踪剂分析已被用于区分和分级各种肿瘤，并将胶质瘤与辐射诱导的组织损伤区分开来15。[11C]AMT 引导的影像学检查显示，在脑部疾病方面有显著的临床益处3，25。然而，由于碳-11的半衰期短（20分钟）和费力的放射合成程序，[11C]AMT的使用仅限于少数具有现场回旋加速器和放射化学设施的PET中心。

氟-18的半衰期为109.8分钟，而碳-11的半衰期为20分钟。人们越来越关注开发用于色氨酸代谢的氟-18标记放射性示踪剂3，28。在放射性标记，转运机制，体外和体内稳定性，生物分布和异种移植物中的肿瘤摄取方面，共报道了15种独特的氟-18放射性标记色氨酸放射性示踪剂。然而，观察到几种示踪剂的快速体内脱氟，包括4-，5-和6-[18F]氟三磷酸吡喃，排除了进一步的临床转化29。5-[18F]氟-α-甲基色氨酸（5-[18F]FAMT）和1-（2-[18F]氟乙基）-L-色氨酸（L-[18F]FETrp，也称为（S）-2-氨基-3-（1-（2-[18F]氟乙基）-1H-吲哚-3-基）丙酸，分子量为249.28克/摩尔），是两种最有前途的放射性示踪剂，在动物模型中具有良好的体内动力学，并且具有超越[111]的巨大潜力C]AMT用于评估色氨酸代谢失调的临床状况28.5-[18F]FAMT在免疫功能低下小鼠的IDO1阳性肿瘤异种移植物中显示出高摄取率，并且比[11C]AMT28，30对KP成像更具特异性。然而，5-[18F]FAMT的体内稳定性仍然是一个潜在的问题，因为在注射示踪剂30后30分钟以上没有报告体内脱氟数据。

基因工程髓母细胞瘤小鼠模型的临床前研究表明，与18F-氟脱氧葡萄糖（18F-FDG）相比，L-[18F]FETrp在脑肿瘤中的积累率高，体内脱氟可忽略不计，背景摄取低，显示出优越的靶向与非靶向比31，32。小鼠辐射剂量测定研究表明，L-[18F]FETrp的有利剂量测定暴露比临床18F-FDG PET示踪剂低约20%。与其他研究人员的发现一致，临床前研究数据提供了大量证据，以支持L-[18F]FETrp的临床翻译，用于研究患有癫痫，神经肿瘤学，自闭症和结节性硬化症等脑部疾病的人类色氨酸代谢异常28，31，32，33，34，35，36.表1显示了三种最广泛研究的色氨酸代谢示踪剂11C-5-HTP，[11C]AMT和L-[18F]FETrp之间的总体比较。 11C-5-HTP和[11C]AMT都有较短的半衰期和费力的放射性标记程序。这里描述了使用一锅两步法对L-[18F]FETrp进行无线电合成的协议。该协议的特点是使用少量放射性标记前体，少量反应溶剂，低毒性K222的负载以及环境友好和可注射的流动相用于纯化和易于配制。

Protocol

注意：该协议涉及放射性物质。任何额外剂量的放射性物质都可能导致癌症等不良健康影响的机会成比例增加。研究人员必须遵循“尽可能低的合理可实现”（ALARA）剂量实践，以指导无线电合成方案，并在热电池或引线罩中提供足够的保护。在无线电合成过程中，最大限度地减少直接接触时间，使用铅屏蔽，并在任何辐射暴露步骤中保持最大距离至关重要。在整个实验过程中佩戴辐射剂量学徽?…

Representative Results

反应方案如图 1所示。放射性标记包括以下两个步骤：1）甲苯磺酸盐放射性标记前体与[18F]氟化物的反应提供 18F标记的中间体，以及2）中间体中 叔丁氧基羰基和 叔丁基保护基团的脱保护，得到最终产物L-[18F]FETrp。两个反应步骤在100°C下继续10分钟。 在从商业供应商处收到[18F]氟化物之前，请组装试剂瓶，?…

Discussion

色氨酸是人体必需的氨基酸.它在情绪，认知功能和行为的调节中起着重要作用。放射性标记的色氨酸衍生物，特别是碳-11标记的[11C]AMT，由于其在绘制血清素合成图谱38，39，检测和分级肿瘤40，指导癫痫手术41，42以及评估糖尿病治疗反应方面的独特作用，已被广泛研究…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了诊断与研究PET / MRI中心以及Nemours / Alfred I. DuPont儿童医院生物医学研究和放射学部门的支持。

Materials

[18F]Fluoride in [18O]H2O PETNET Solutions Inc. N/A
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane ACROS 291950010 Kryptofix 222 or K222, 98%
Acetic acid ACROS 222142500 99.8%
Acetonitrile Sigma-Aldrich 271004 anhydrous, 99.8%
Agilent 1260 HPLC system Agilent Technologies Agilent 1260 Agilent 1260 series
Analytcial chiral HPLC column Sigma-Aldrich 12024AST Astec CHIROBIOTIC T, 25 cm × 4.6 mm
Carbon dioxide, 60 LBS Airgas REFR744R200S 99.99%
D-FETrp standard reference Affinity Research Chemicals Inc N/A Custom synthesis
Empty sterile vial Jubilant HollisterStier 7515 20 mm closure, 10 mL
Ethanol Decon Labs 2716 200 proof, USP grade. ≥99.9%
Fisherbrand 13 mm Syringe Filter, 0.22 µm, PVDF, sterile Fisher Scientific 09-720-3
Hydrochloric acid Sigma-Aldrich 30721 ≥37%
Isopropanol Decon Labs 8316 70%, sterile
L-[18F]FETrp radiolabeling precursor Affinity Research Chemicals Inc N/A Custom synthesis
L-FETrp standard reference Affinity Research Chemicals Inc N/A Custom synthesis
Light C8 cartridge Waters WAT036770 Sep-Pak  C8 plus light cartridge
Needle, 20 G x 1 Becton-Dickinson & Co. 305175
Needle, 20 G x 1 ½ Becton-Dickinson & Co. 305176
Needle, 21 G x 2 Becton-Dickinson & Co. 305129
Neutral aluminum oxide Waters WAT023561 Sep-Pak alumina N plus light
Nylon membrane (0.20 µm ) MilliPore GNWP04700 47 mm
Pall Acrodisc 25 mm syringe sterile filter Pall Corporation 4907
PETCHEM radiochemistry synthesis system PETCHEM Solutions Inc. Pinckney, MI N/A Radiosynthesizer
pH strips 2.0 – 9.0 EMD Millipore 1.09584.0001
Potassium carbonate Sigma-Aldrich 367877 99.995%
Quaternary methylammonium light cartridge Waters 186004051 Sep-Pak QMA light
Semi-preparative C18 HPLC column Phenomenex 00D-4253-N0 100 × 10 mm
Semi-preparative chiral HPLC column Sigma-Aldrich 12034AST Astec CHIROBIOTIC T, 25 cm × 10 mm
Sodium chloride injection 23.4% APP Pharmaceutical, LLC 18730 USP grade
Sodium chloridei injection 0.9% Hospira NDC 0409-4888-10 USP grade
Sodium hydroxide Honeywell 306576 99.99%
Spinal needle, 20 G x 3 ½ Becton-Dickinson & Co. 405182
Sterile alcohol prep pads BioMed Resource Inc. PC661
Sterile empty vials, 2 mL Hollister Stier 7505ZA 13 mm closure
Sterile empty vials, 30 mL Jubilant HollisterStier 7520ZA 20 mm closure
Syringe PP/PE, 3 mL, Luer Lock Air-Tite 4020-X00V0
Syringe PP/PE, 5 mL, Luer Lock Becton-Dickinson & Co. 309646
Syringe,  PP/PE, 10 mL, NORM-JECT Air-Tite 4100-000V0
Syringe, 1 mL, Luer Slip Becton-Dickinson & Co. 309659
Syringe, 3 mL, Luer-Lock Becton-Dickinson & Co. 309657
Ultra high purity argon Airgas AR UHP300 99.999%
Ultrapure water MilliporeSigma ZRQSVP300 Direct-Q 3 tap to pure and ultrapure water purification system
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Tags

Keywords: Radiosynthesis1-(2-[18F]Fluoroethyl)-L-TryptophanOne-potTwo-step ProtocolF18 Radiolabeled Tryptophan TracerTryptophan MetabolismRadioisotopeQMA Light CartridgeK222Potassium CarbonateReactorArgonEvaporationCompressed Carbon Dioxide
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Yue, X., Nikam, R. M., Kecskemethy, H. H., Kandula, V. V. R., Falchek, S. J., Averill, L. W., Langhans, S. A. Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol. J. Vis. Exp. (175), e63025, doi:10.3791/63025 (2021).
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