CN108314678A - 以磷脂酰丝氨酸为靶点的分子探针及其用途 - Google Patents
以磷脂酰丝氨酸为靶点的分子探针及其用途 Download PDFInfo
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Abstract
本发明公开了一种具有DPA‑NOTA结构的分子(I),以及同位素标记的核医学分子探针/显像剂(II)和(III):分子(I)制备简单,可用于多种核素标记(包括18F、64Cu、68Ga等成像类核素以及177Lu等治疗类核素等);分子探针(II)和(III)标记条件温和,步骤简便,便于实现自动化合成,便于推广,具有较高体内稳定性和出色的显像效果,有良好的应用前景。
Description
(一)技术领域
本发明涉及一种以磷脂酰丝氨酸为靶点的分子探针、合成方法及其作为PET显像分子探针的应用。
(二)背景技术
分子探针是指在细胞和分子水平上与靶点相互作用的功能性分子,借助高亲和性、高特异性和高灵敏度的分子探针,能够在细胞和分子水平对生物学过程进行定性和定量研究,分子探针在疾病诊断与分期(分层)、疗效检测、个体化治疗以及新药研发等领域中具有重要的应用前景。PET/SPECT核医学分子影像是最成熟的分子影像技术,具有灵敏度高、可定量,以及易于临床转化的优势,核医学分子探针是分子影像研究的一个关键内容。
磷脂酰丝氨酸(phosphatidylserine,PS)是一类磷脂化合物,通常位于细胞膜内层。PS带有负电荷的头部为亲水性,由脂肪酸组成的尾部为亲脂性,正常情况下,PS带负电荷的头部朝向细胞内。在某些情况下,PS带负电荷的头部翻转朝向细胞外。比如,当发生细胞死亡(包括细胞凋亡和细胞坏死)时,大量PS分子翻转到细胞膜外层,因此PS是细胞死亡过程中的一个特异性分子标志物;某些病原微生物的胞外披膜也表达PS,所以PS也是某些病原微生物的一个表面标志物;在某些肿瘤细胞表面,也出现PS大量翻转的情况。
综上所述,PS带负电荷的头部翻转到细胞外是与重要生物学事件相关联的,研究者开发了一些以PS为靶点的分子探针和成像方法。这些分子探针的靶向基团可分为纳米微粒、抗体、蛋白分子、多肽、小分子等多种类型。其中,膜联蛋白AnnexinV是应用最多的一个靶向基团,将Annexin V进行荧光标记后制备的荧光分子探针被广泛应用于细胞凋亡的细胞成像、体外检测以及活体成像,将Annexin V进行99mTc、18F、64Cu等核素标记后的核医学分子探针也已经被用于肿瘤显像、心肌梗死显像、肿瘤治疗后疗效评估等研究,一些分子探针进入了临床实验阶段。2002年,Hamachi等报道了一类含有二甲基吡啶胺(Dipicolylamine,DPA)结构并与锌离子(Zn2+)配位结合的小分子荧光探针PSVue-380,这一类探针的靶向基团ZnDPA能以很高的亲和力与PS特异性结合。近年来,研究者将ZnDPA分别标记了不同波长的荧光团,并用于细胞凋亡、细菌显像和肿瘤显像等研究。研究者还对ZnDPA进行了同位素标记,制备了99mTc、18F、111In等标记的核医学分子探针,这些以ZnDPA为靶向基团的核医学分子探针存在很多问题,比如:合成时间长,一些18F标记方法需要先通过有机相反应合成18F标记的中间体,然后再与靶向基团偶联,整个合成过程比较复杂,反应条件苛刻,产率不高;除此之外,显像剂体内稳定性差,显像效果不佳,分子结构需要优化。
本发明针对以上的技术难题,提供了一种分子探针,其制备简单;具有较高体内稳定性;适合多种核素标记(包括18F、64Cu、68Ga等成像类核素、177Lu等治疗类核素等);还提供了以PS为靶点的核医学分子影像探针,可以在肿瘤成像或死亡细胞成像中进行应用。
上述涉及的参考文献如下(1-7):
1.Wyffels L,Gray BD,Barber C,Woolfenden JM,Pak KY,Liu ZL.Synthesisand preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine)coordination complexes as cell death imaging agents.Bioorganic&MedicinalChemistry.2011;19:3425-33.
2.Sun T,Tang GH,Tian H,et al.Positron emission tomography imaging ofcardiomyocyte apoptosis with a novel molecule probe F-18 FP-DPAZn2.Oncotarget.2015;6:30579-91.
3.Smith BA,Gammon ST,Xiao SZ,et al.In Vivo Optical Imaging of AcuteCell Death Using a Near-Infrared Fluorescent Zinc-DipicolylamineProbe.Molecular Pharmaceutics.2011;8:583-90.
4.Palmowski K,Rix A,Lederle W,et al.A low molecular weight zinc(2+)-dipicolylamine-based probe detects apoptosis during tumour treatment betterthan an annexin V-based probe.European Radiology.2014;24:363-70.
5.Li JL,Gerlach RL,Jonsson CB,Gray BD,Pak KY,Ng CK.Characterizationof F-18-dipicolylamine(DPA)derivatives in cells infected with influenzavirus.Nuclear Medicine and Biology.2015;42:283-91.
6.Lakshmi C,Hanshaw RG,Smith BD.Fluorophore-linked zinc(II)dipicolylamine coordination complexes as sensors for phosphatidylserine-containing membranes.Tetrahedron.2004;60:11307-15.
7.Kwong JMK,Hoang C,Dukes RT,et al.Bis(Zinc-Dipicolylamine),Zn-DPA,aNew Marker for Apoptosis.Investigative Ophthalmology&Visual Science.2014;55:4913-21.
(三)发明内容
本发明要解决的技术问题是提供一种以PS为靶点的分子探针,要求其制备过程简单,稳定性高,并具有良好的显像效果。
本发明采用的技术方案为:
一种具有DPA-NOTA结构的分子(I);以及同位素标记的核医学分子探针(II)和(III)
本发明式(I)所示的DPA-NOTA化合物的合成方法包括如下步骤:带活性酯的1,4,7-三氮杂环壬烷-1,4,7-三乙酸(NOTA)化合物IV与带氨基的DPA化合物V偶联,经过HPLC分离、纯化得到相应的式(I)所示的分子
合成路线如下所示:
本发明式(II)所示的分子探针合成方法包括如下步骤:
1)将64Cu离子(2mCi,74MBq)与分子(I)在乙酸钠缓冲溶液(0.1M,pH 5.0),37℃反应30分钟;
2)步骤1所得反应液用去离子水稀释后,经半制备HPLC纯化,收集相应组分并旋干,即得到中间产物(I-1);
3)步骤2所得的中间产物I-1室温下与2倍摩尔量的Zn2+反应10分钟,得到分子探针(II)。
合成路线如下所示:
本发明式(III)所示的分子探针合成方法包括如下步骤:
1)18F离子用QMA柱吸附,再用生理食盐水溶液洗脱,用冰醋酸/醋酸钠缓冲液调节pH到4;
2)在步骤1处理过的洗脱液中依次加入AlCl3和分子(I),于100℃反应10分钟;
3)步骤2所得反应液用去离子水稀释后,经半制备HPLC纯化,收集相应组分并旋干,即得到中间产物(II-1);
4)步骤3所得的中间产物(II-1)室温下与2倍摩尔量的Zn2+反应10分钟,得到分子探针(III)。
合成路线如下所示:
本发明的优点在于:本发明所述的分子(I)制备简单,适合多种核素标记(包括18F、64Cu、68Ga等成像类核素、177Lu等治疗类核素等);分子探针(II)和(III)制备条件温和,步骤简便,便于实现自动化合成,便于推广,具有较高体内稳定性和出色的显像效果,有良好的应用前景。
(四)附图说明
图1:分子探针(I)的核磁共振谱图,氢谱。
图2:图中横坐标为相对信号强度,纵坐标为时间(0-30分钟)。分子探针(II)在血清中37C孵育2h后的HPLC谱图,表明该显像剂稳定性好。
图3:图中横坐标为相对信号强度,纵坐标为分组,A,B,C三组细胞接受了不同的处理。分子探针(II)在肿瘤细胞(A组),经抗菌药物处理后(B组)以及被Zn-DPA竞争PS位点(C组)后的细胞摄取情况,证明探针是以PS为结合靶点的。
图4:荷瘤裸鼠,在注射探针(II)1h,2h后的PET图像,表明该显像剂具有比较好的显像效果。
(五)具体实施方式
下面以具体实施例对本发明的技术方案做进一步说明,但本发明的保护范围不限于此:具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例1:制备探针(I):
1)将7.3mg化合物IV和5.9mg化合物V溶于50μL DMF中,混合均匀,加入2μL DIPEA,在室温下反应30分钟;
2)将步骤1的反应溶液用1ml(0.1%TFA:5%乙腈:94.9%水)混合液中,使用HPLC分离、旋干、干燥后得到探针分子(I)。其中HPLC分离条件是:Vydac C18半制备柱(218TP510;C18,5μm,250×10mm),流动相A(去离子水,含0.1%TFA),流动相B(乙腈,0.1%TFA),流速4ml/min,流动相梯度设定0-5min:95%A,5%B,5-35min:35%A,65%B。HPLC系统带UV检测器,记录214nm紫外吸收,产物HPLC保留时间是14.8min-15.8min。分子探针(I)的核磁共振氢谱图见附图1.质谱数据:MS(ESI):m/z计算值945.1,测定值945.1.
实施例2:制备探针(II)
使用到的试剂还有:0.1M醋酸钠/醋酸缓冲液(pH 5.0);0.1M磷酸盐缓冲液PBS(pH7.4);1M NaOH溶液和1M HCl溶液;使用到的仪器有:可控温振荡孵育器,涡旋振荡器,微量移液器,旋转蒸发仪,高效液相系统(配紫外检测器和伽玛检测器),伽玛检测器。64Cu的标记过程如下:首先将64CuCl2调节pH到5.0~6.0,然后将10μg化合物I溶解于50μL 0.1M醋酸钠/醋酸缓冲液(pH 5.0)中,加入2mCi(74MBq)64Cu溶液,置于振荡孵育器上37℃,300rpm反应30min。反应结束后,进行HPLC纯化。其中HPLC分离条件是:流动相A(去离子水,含0.1%TFA),流动相B(乙腈,0.1%TFA),流速1ml/min,流动相梯度设定0-5min:95%A,5%B,5-35min:35%A,65%B。产物HPLC保留时间是15min左右,产品通过旋转蒸发仪除去有机溶剂后。然后再与溶于200μL去离子水的Zn(NO3)2(物质的量为20nmol)反应10分钟,反应产物用0.22μm滤膜过滤得到探针(II)。取10μCi探针II,加入到1ml小鼠血清中,置于振荡孵育器上37℃,300rpm孵育2h。孵育完成后加入100μL乙腈,混匀离心过滤后进行HPLC,根据HPLC的出峰情况评价探针的血清稳定性。HPLC结果见附图2(HPLC条件同上)。
实施例3:制备探针(III)
使用到的试剂有:0.1M醋酸钠/醋酸缓冲液(pH 4.0);0.1M磷酸盐缓冲液PBS(pH7.4);0.9%NaCl溶液,5mM的AlCl3溶液。使用到的仪器有:可控温振荡孵育器,涡旋振荡器,微量移液器,旋转蒸发仪,高效液相系统(配紫外检测器和伽玛检测器),伽玛检测器.18F的标记过程如下:将20μg化合物I溶于10μL去离子水中,加入50μL 0.1M醋酸钠/醋酸缓冲液(pH 4.0),加入2μL 5mM的AlCl3溶液,加入10mCi(370MBq)18F离子(体积50μL),100℃反应10分钟,反应产物经过HPLC分离,旋转蒸发仪除去溶剂后重新溶于PBS中。然后再与40nmol Zn(NO3)2反应10分钟,反应产物用0.22μm滤膜过滤得到探针(III)。
实施例4:探针(II)的细胞实验
肿瘤细胞采用PC3细胞,培养在RPMI 1640培养基中,培养基中添加10%小牛血清和1%抗生素,于37℃,5%CO2培养箱中培养。细胞测定时,细胞转入96孔板贴壁培养。A组细胞不做处理,B组细胞经过抗肿瘤药物Staurosporine(10μM*50μL)处理,C组细胞加入过量的Zn-DPA(1μmol)与探针(II)竞争PS位点。
实施例5:探针(II)用于肿瘤显像
按照动物实验规范,接种PC3细胞(1×106,50μL)于雄性裸鼠皮下,5~6周后肿瘤长至0.6~1cm,可以进行肿瘤PET显像。显像前小鼠尾静脉注射1.85MBq(50μCi)标记的探针(II)。在1h、和2h时间点进行PET成像。PET成像结果见附图4。
Claims (5)
1.一种具有分子式(I)结构的化合物,其特征在于分子结构中具有可以与同位素金属离子配位的NOTA基团,以及与锌离子配位的DPA基团。
2.如权利要求1所述的化合物的用途,其特征在于以该化合物作为同位素金属离子标记的前体。
3.一种64Cu同位素标记的核医学分子探针,其特征在于其具有如分子式(II)的结构。
4.一种18F同位素标记的核医学分子探针,其特征在于其具有如分子式(III)的结构
5.如权利要求3和权利要求4所述的分子探针的用途,其特征是以其作为PET显像剂用于细胞凋亡或细胞死亡显像,或者肿瘤显像。
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