CN112142739A - 靶向叶酸受体的放射性叶酸衍生物及其制备方法和应用 - Google Patents
靶向叶酸受体的放射性叶酸衍生物及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及放射性标记技术领域,尤其涉及一种靶向叶酸受体的放射性叶酸衍生物及其制备方法和应用。
背景技术
叶酸(Folic Acid,FA)即蝶酰谷氨酸,在生物体内参与甲硫氨酸循环、甲基化反应、DNA合成等重要过程。叶酸为哺乳动物细胞必需而又不能自身合成,因此必须完全依赖外源性供给。叶酸受体(Folate Receptor,FR)是一种糖基磷脂酰肌醇(Glycosyphosphatidylinositol,GPI)偶联蛋白,除个别组织外,叶酸受体FR在正常组织上表达水平很低,而在许多肿瘤细胞表面过表达。叶酸受体与叶酸及其衍生物,如甲基四氢叶酸等都有高度的亲和性和特异性,基于这种特性,可将显像剂、治疗药物等与叶酸耦连,靶向肿瘤细胞,从而应用于肿瘤的影像诊断如核医学显像、核磁共振显像、荧光显像和肿瘤治疗中。
叶酸及其衍生物与放射性核素偶联形成显影剂,可与叶酸受体FR特异性结合,选择性浓集于FR表达丰富的组织。由于FR在肿瘤和非肿瘤组织分布的显著性差异,FR介导的显影剂可获得肿瘤与正常组织高对比度的图象,以此对肿瘤进行诊断、定位及化疗效果评价,以叶酸为基础的显像剂已经被应用于PET、SPECT、MRI、荧光成像等肿瘤显像中。
目前用于标记FR靶向放射性药物的核素有18F、67/68Ga、99mTc、111In等,其中,67Ga标记叶酸的螯合物应用于人类叶酸受体阳性肿瘤显像的研究较早,但到目前为止还没有相关药物进行临床前研究(Ke,CY,Mathias CJ,Green MA,Adv Drug Deliver Rev,2004.56:1143-1160);111In-DTPA-FA是第一个进入并完成II期临床试验的FR靶向肿瘤显像剂,但由于111In不易制备,价格昂贵,且半衰期较长(t1/2=68h),因而并没有真正进入临床使用[Siegel BA,Dehdashti F,Mutch DG,etc.J Nucl Med.2003.44(5):700-707.];99mTc来源方便,价格便宜,半衰期较短(t1/2=6.02h),可以较大剂量注射以获得高标记率的图像,并且对人体辐射剂量较小,近年来,研究较多的是99mTc标记叶酸螯合物,其中,99mTc(CO)3-DTPA-叶酸的比活度高于111In-DTPA-叶酸和99mTc-DTPA-叶酸(Mathias CJ,Hubers D,LowPS,etc.Bioconjug Chem.200011:253-257.),99mTc-EC20(Müller C,Reddy JA,Leamon CP,etc.Mol Pharm.2010,7:597-604.)是众多以99mTc为基础的显像剂中的研究热点,而随着点击化学的诞生以[99mTc(CO)3(H2O)3]-为基础的99mTc的叶酸标记也已出现(Mindt TL,MüllerC,Melis M,etc.Bioconjug Chem.2008 19:1689-1695);但是上述核素均为单光子核素,成像的标记率比较低。而67/68Ga-DOTA-叶酸(Fani M,Wang X,Nicolas G,etc.Eur J Nucl MedMol Imaging.2011 38:108-119.)由于体内的稳定性较差,临床应用受到极大限制。而同时放射性核素的18F利用直接亲核取代后水解制备新的叶酸分子探针也已经兴起(Ross TL,Honer M,Müller C,etc.J Nucl Med.2010 51:1756-1762.),然而由于放射化学产率仅为4%,大大的限制了临床转化的应用,目前仅限于实验室研究。
因此,本领域的技术人员致力于开发一种制备成本低、标记率高、体内稳定性强的靶向叶酸受体的放射性叶酸衍生物。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是提供一种制备成本低、标记率高、体内稳定性强的靶向叶酸受体的放射性叶酸衍生物。
为实现上述目的,本发明第一方面提供了一种靶向叶酸受体的放射性叶酸衍生物,所述放射性叶酸衍生物具有如式1所示的结构:
其中,放射性核素为68Ga、64Cu、Al18F、89Zr和177Lu中的一种。
本发明第二方面提供了一种上述靶向叶酸受体的放射性叶酸衍生物的制备方法,其特征在于,包括如下步骤:
步骤1:将NH2-PEG6-叶酸溶于二甲基亚砜中,并加入2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸进行反应,反应结束后分离得到NOTA-PEG6-叶酸;
步骤2:将所述NOTA-PEG6-叶酸溶于酸性缓冲液中,加入放射性核素,反应得到式1所示的叶酸衍生物。
进一步地,步骤1中,所述2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸和所述NH2-PEG6-叶酸的摩尔比为(0.8-1.2):1。
进一步地,步骤1中,使用N,N-二异丙基乙胺将pH调节为8.5-9.5。
进一步地,步骤2中,所述NOTA-PEG6-叶酸的质量为0.01-10mg。
进一步地,步骤2中,所述反应的温度为20-120℃,时间为2-20min。
进一步地,步骤2中,所述酸性缓冲液的pH为3.4-4.6。
进一步地,步骤2中,所述酸性缓冲液为盐酸-乙酸钠缓冲溶液。
进一步地,步骤2中,所述放射性核素的添加量为0.37MBq-37GBq。
本发明第三方面提供了上述靶向叶酸受体的放射性叶酸衍生物在靶向叶酸受体的肿瘤的PET诊断或治疗中的应用。
本发明提供的靶向叶酸受体的放射性叶酸衍生物由PEG6-叶酸、双功能螯合剂NOTA和放射性核素构成,其中,放射性核素为68Ga、64Cu、Al18F、89Z以及177Lu中的一种,该放射性化合物可以制备成针剂用于靶向叶酸受体的肿瘤的PET诊断或治疗。PEG6修饰叶酸的目的是调节化合物在体内的代谢途径,在保证与叶酸受体亲和力不变的基础上,增强化合物的水溶性,达到更好的体内显像诊断效果;NOTA作为双功能螯合剂将PEG6-叶酸与放射性核素68Ga、64Cu、Al18F或89Zr结合,从而在体内通过叶酸与叶酸受体的特异性识别,将放射性核素载带到高表达叶酸受体的肿瘤,利用核医学正电子发射计算机断层(PET)技术对病变进行无创的显像诊断,或者将放射性治疗核素177Lu标记到NOTA-PEG6-叶酸上用于高表达叶酸受体的肿瘤的治疗。本发明提供的靶向叶酸受体的放射性化合物的制备成本低,具有标记率高、体内稳定性强等优点。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的提供的NOTA-PEG6-叶酸的HPLC图谱;
图2为本发明的提供的NOTA-PEG6-叶酸的质谱图;
图3是本发明实施例1提供的Al18F-NOTA-PEG6-叶酸的radio-HPLC图和UV-HPLC图;
图4是本发明实施例2提供的68Ga-NOTA-PEG6-叶酸的TLC图;
图5是本发明实施例3提供的177Lu-NOTA-PEG6-叶酸的TLC图;
图6是本发明实施例4提供的64Cu-NOTA-PEG6-叶酸的TLC图;
图7是本发明实施例5提供的89Zr-NOTA-PEG6-叶酸的TLC图;
图8为本发明实施例2提供的68Ga-NOTA-PEG6-叶酸在正常裸鼠体内生物分布图;
图9为本发明实施例2提供的68Ga-NOTA-PEG6-叶酸在SKOV3荷瘤鼠的microPET/CT显像图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
以下实施例所使用的NH2-PEG6-叶酸购自中肽生化有限公司;2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸(S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid(p-SCN-Bn-NOTA))购自美国Macrocyclics公司;N,N-二异丙基乙胺、乙酸钠、N,N-二甲基甲酰胺(DMF)和二甲基亚砜(DMSO)购自美国Sigma-Aldrich公司;放射性核素64Cu、18F、89Zr、68Ga产于上海交通大学医学院附属仁济医院核医学科医用回旋加速器;放射性核素177Lu购自中国同辐股份有限公司。
实施例1
本实施例提供的放射性叶酸衍生物具有如式1所示的结构,其中,放射性核素为Al18F。
本实施例提供的放射性叶酸衍生物的制备方法包括如下步骤:
步骤1、将5mg,10μmol的NH2-PEG6-叶酸溶于100μL的DMSO溶液中,得到浓度为50mg/mL NH2-PEG6-叶酸的DMSO溶液,随后加入5mg,11μmol的p-SCN-Bn-NOTA以及300μL浓度为6.67%的N,N-二异丙基乙胺的DMF溶液,室温反应1h后,用去离子水稀释,用HPLC半制备柱分离纯化得到NOTA-PEG6-叶酸,NOTA-PEG6-叶酸在220nm处有紫外(UV)吸收,其HPLC图谱如图1所示,其质谱检测图如图2所示,分子量为1198.3。
步骤2、在3μL浓度为0.2mol/L的氯化铝的乙酸-乙酸钠的缓冲溶液中加入100μL放射性活度为3.7GBq的回旋加速器上生产的氟-18离子(18F-),混合均匀,在100℃下加热10min;
步骤3、在步骤2得到的反应液中加入含有0.05mgNOTA-PEG6-叶酸的6μL盐酸-乙酸钠缓冲溶液(pH为4),混合均匀,加热到100℃下反应10min,反应结束后用HPLC分离得到Al18F-NOTA-PEG6-叶酸,Al18F-NOTA-PEG6-叶酸的radio-HPLC图和UV-HPLC图如图3所示。
其中,HPLC的条件均为:流动相A:0.1%TFA的水溶液,流动相B为:0.1%TFA的乙腈溶液;流速为每分钟1mL;梯度为0-1min,95%流动相A;1-15min,95%-5%流动相A;15-20min,5%流动相A;20-22min,5%-95%流动相A,PDA检测器紫外为220nm。
实施例2
本实施例提供的放射性叶酸衍生物具有如式1所示的结构,其中,放射性核素为68Ga。
本实施例提供的放射性叶酸衍生物的制备方法包括如下步骤:
步骤1、与实施例1相同;
步骤2、用5ml,0.1M的盐酸水溶液从68Ge/68Ga发生器上淋洗68Ga(1.48GBq)到反应容器中(放射性活度为185MBq),然后加入含有100μg NOTA-PEG6-叶酸的1ml盐酸-乙酸钠溶液(pH为3.9),加热到100℃下反应10min,反应结束后用HPLC分离得到68Ga-NOTA-PEG6-叶酸。
采用Radio-TLC进行放射化学纯度测定,其中展开剂为1M的乙酸铵溶液/甲醇体积比为1:1,纸色谱采用美国安捷伦公司的iTLC-SG硅胶色谱,展开图如图4所示。
实施例3
本实施例提供的放射性叶酸衍生物具有如式1所示的结构,其中,放射性核素为177Lu。
本实施例提供的放射性叶酸衍生物的制备方法包括如下步骤:
步骤1、与实施例1相同;
步骤2、在浓度为0.05M的177LuCl3盐酸溶液(5μL,18.5MBq)中加入含有0.1mgNOTA-PEG6-叶酸的110μL盐酸-乙酸钠缓冲溶液(pH为3.8),混合均匀,加热到95℃下反应20min,反应结束后用HPLC分离得到177Lu-NOTA-PEG6-叶酸。
采用与实施例2相同的方法对其进行检测,其展开图如图5所示。
实施例4
本实施例提供的放射性叶酸衍生物具有如式1所示的结构,其中,放射性核素为64Cu。
本实施例提供的放射性叶酸衍生物的制备方法包括如下步骤:
步骤1、与实施例1相同;
步骤2、在20μL的64CuCl2(3.7MBq)溶液中加入含有50μgNOTA-PEG6-叶酸的1ml盐酸-乙酸钠缓冲溶液(pH为3.9),混合均匀,加热到65℃下反应10min,反应结束后用HPLC分离得到64Cu-NOTA-PEG6-叶酸。
采用与实施例2相同的方法对其进行检测,其展开图如图6所示。
实施例5
本实施例提供的放射性叶酸衍生物具有如式1所示的结构,其中,放射性核素为89Zr。
本实施例提供的放射性叶酸衍生物的制备方法包括如下步骤:
步骤1、与实施例1相同;
步骤2、在20μL的89ZrC2O4(草酸锆,3.7MBq)溶液中加入含有50μg NOTA-PEG6-叶酸的1ml盐酸-乙酸钠缓冲溶液(pH为4.0),混合均匀,加热到100℃下反应10min,反应结束后用HPLC分离得到89Zr-NOTA-PEG6-叶酸。
采用与实施例2相同的方法对其进行检测,其展开图如图7所示。
本发明进一步对实施例2所制备的叶酸衍生物的效果作进一步说明。
(一)取实施例2制备的68Ga-NOTA-PEG6-叶酸100μCi,将其注射到正常小鼠内,分别在30min、1h和90min后取不同的脏器,称量脏器重量和各器官或脏器中的放射性计数,计算脏器的摄取值,各脏器的摄取值的计算结果如图8所示。
(二)68Ga-NOTA-PEG6-叶酸在肿瘤PET/CT成像中的应用:
取实施例2制备的68Ga-NOTA-PEG6-叶酸200μCi,将其注射到荷瘤鼠(卵巢癌SKOV3裸鼠)体内,进行60min的小动物PET/CT成像,图9为68Ga-NOTA-PEG6-叶酸在SKOV3裸鼠荷小鼠卵巢癌BALB/c小鼠模型中的特异性PET显像(箭头指示叶酸受体阳性肿瘤部位)。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (10)
2.一种如权利要求1所述的靶向叶酸受体的放射性叶酸衍生物的制备方法,其特征在于,包括如下步骤:
步骤1:将NH2-PEG6-叶酸溶于二甲基亚砜中,并加入2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸进行反应,反应结束后分离得到NOTA-PEG6-叶酸;
步骤2:将所述NOTA-PEG6-叶酸溶于酸性缓冲液中,加入放射性核素,反应得到式1所示的叶酸衍生物。
3.如权利要求2所述的制备方法,其特征在于,步骤1中,所述2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸和所述NH2-PEG6-叶酸的摩尔比为(0.8-1.2):1。
4.如权利要求2所述的制备方法,其特征在于,步骤1中,使用N,N-二异丙基乙胺将pH调节为8.5-9.5。
5.如权利要求2所述的制备方法,其特征在于,步骤2中,所述NOTA-PEG6-叶酸的质量为0.01-10mg。
6.如权利要求2所述的制备方法,其特征在于,步骤2中,所述反应的温度为20-120℃,时间为2-20min。
7.如权利要求2所述的制备方法,其特征在于,步骤2中,所述酸性缓冲液的pH为3.4-4.6。
8.如权利要求2所述的制备方法,其特征在于,步骤2中,所述酸性缓冲液为盐酸-乙酸钠缓冲溶液。
9.如权利要求2所述的制备方法,其特征在于,步骤2中,所述放射性核素的添加量为0.37MBq-37GBq。
10.权利要求1所述的靶向叶酸受体的放射性叶酸衍生物在靶向叶酸受体的肿瘤的PET诊断或治疗中的应用。
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