CN108250276B - Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法 - Google Patents
Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法 Download PDFInfo
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Abstract
本发明涉及一种Cu‑64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,该方法包括以下步骤:⑴将水溶性修饰的NOTA‑PEG24‑Dimer‑Sansalvamide A多肽溶解于乙腈,配制成浓度为1μg/μL的多肽溶液;⑵在64CuCl2中加入乙酸氨缓冲液进行反应,得到反应液64Cu(OAc)2;⑶将多肽溶液加入到反应液64Cu(OAc)2中加热,并在加热的过程中持续震荡,得到产物64Cu‑NOTA‑PEG24‑Dimer‑San A;⑷产物64Cu‑NOTA‑PEG24‑Dimer‑San A除去溶剂后经磷酸盐缓冲液溶解、滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu‑NOTA‑PEG24‑Dimer‑San A。本发明64Cu‑NOTA‑PEG24‑Dimer‑San A环肽分子探针具有实现胰腺癌特异性诊断与治疗一体化的潜力。
Description
技术领域
本发明涉及一种探针的制备方法,尤其涉及Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法。
背景技术
胰腺癌是恶性程度很高,诊断和治疗都很困难的消化道恶性肿瘤,被称为“癌王”,其中位生存期低于6个月,总的5年生存率低于6%,仅10%的患者适合手术治疗,其预后差与该病恶性度高、早期诊断困难以及缺乏有效的抗癌药物密切相关。随着我国人口的老龄化,胰腺癌在我国的发病率有逐年增加的趋势,其将成为未来几年我国一个主要的健康问题。
目前,胰腺癌的常规影像诊断有超声(包括内镜超声)、CT以及MRI,然而这些结构影像对胰腺癌诊断特异性差,胰腺癌与慢性胰腺炎、尤其是慢性肿块性胰腺炎的鉴别诊断是影像医学与临床医学长期以来亟待解决的难题。
PET/CT(正电子发射型计算机断层显像仪/CT)是21世纪产生的分子影像学新技术,其被誉为探测肿瘤的“雷达”,肿瘤诊断的“福尔摩斯”。巧妙的将PET的功能影像与CT的解剖影像融合,二者优势互补。与单纯结构影像不同的是PET/CT显像需要体内注射放射性药物。18F-FDG(18F-2-氟-2脱氧-D-葡萄糖)是葡萄糖分子的类似物,该显像剂是目前全球使用最广泛的显像剂,18F因其适量的能量(511KeV)及半衰期(109.8min)也成为PET/CT最常用的正电子放射性核素。天然的葡萄糖进入细胞后可以被己糖激酶Ⅱ催化生成6-磷酸-葡萄糖,继而在相应酶的催化下转变为丙酮酸,再参与三羧酸循环最后变成水和二氧化碳。18F-FDG是2-位碳原子上的羟基被18F取代的D-葡萄糖的类似物。18F-FDG由于与天然的葡萄糖的化学结构相似,因此也能通过与葡萄糖相同的转运载体Glut-1转运入细胞,在胞浆内被己糖激酶Ⅱ催化生成6-磷酸-葡萄糖。但是18F-FDG却不能被特异的果糖-1-磷酸酶识别和催化,因而无法生成相应的二磷酸己糖参加有氧和无氧糖代谢。由于6-磷酸-18F-FDG带负电荷,不能反向通过细胞膜离开细胞。所以6-磷酸-18F-FDG最后停留、集聚在肿瘤细胞的胞浆内。通过PET显像仪探测18F湮灭辐射后发射的高能γ光子,再经过计算机的处理,就可以获得反映体内葡萄糖代谢状态和水平的18F-FDG的分布影像。
18F-FDG PET/CT的临床应用,大大提高了肿瘤诊断的准确性。其主要优势在于肿瘤的TNM分期、疗效评估、复发监测以及肿瘤原发灶的探查,但18F-FDG是一种肿瘤非特异性的显像剂,在肿瘤鉴别诊断方面存在不足,对于胰腺癌的鉴别诊断也存在假阳性与假阴性从而导致良恶性诊断的误诊与漏诊。
18F-3'-脱氧-3'-L氟代胸苷(18F-FLT)是一种肿瘤增殖性显像剂。18F-FLT是胸苷的类似物,可被细胞摄取作为合成DNA的原料,故该显像剂可以评估和量化细胞增殖活动。很多临床研究评估了18F-FLT PET/CT在胰腺癌诊断中的潜力,到目前为止,没有研究表明18F-FLT在胰腺癌的诊断中比18F-FDG更有优势。总体而言,18F-FLT在大多数肿瘤中的摄取低于18F-FDG。
为了克服上述显像剂的不足,基于抗体的显像剂应运而生。如124I标记的 CA199抗体片段,64Cu标记的单克隆抗体MAb159,89Zr标记的抗胰岛素样生长因子1受体抗体(Insulin-like growth factor-1 receptor ,IGF-1R)以及64Cu 标记的组织因子单克隆抗体ALT-836等进行胰腺癌的分子影像诊断。但放射免疫显像不论是使用放射性核素标记单克隆抗体或者其片段,均存在分子量较大,血液清除缓慢(4~20h),在较短时间内难以得到较高的肿瘤/非肿瘤(T/NT)比值的问题;再者,CA199是胰腺癌非特异性抗原,在胰腺炎性病变中亦有明显表达。
受体显像所用的标记配体分子量小、血液清除快、组织穿透力强、T/NT比值高、无免疫原性,具有灵敏度高、特异性强和准确性好等优点,是分子影像最活跃的前沿研究领域之一。多肽作为小分子配体标记探针除上述诸多的优势外,还有合成成本相对低廉、容易化学修饰的优点。目前,多肽标记的胰腺癌探针多针对胰腺癌细胞高表达的靶点—整合素 αvβ6以及αvβ3。
热休克蛋白90(heart shock protein 90,Hsp90)是一种高度保守的在生物界普遍存在、有特殊分子伴侣功能的蛋白,分子量约为83~90KDa。Hsp90含有三个高度保守的结构域:即N端三磷酸腺苷结合域、中间结构域、C末端结构域,以同源二聚体形式存在,主要与细胞周期和细胞凋亡调控相关。研究证实,Hsp90在包括胰腺癌在内的多种恶性肿瘤细胞中异常高表达,是正常细胞的2~10倍,并与肿瘤的发生、发展、分级、分期及预后密切相关;Hsp90在肿瘤细胞质中被激活并定位到细胞表面,而在正常细胞中仅驻留在细胞质中。因此,Hsp90作为一潜在的肿瘤治疗研究靶点日益受到关注,也为其成为靶标的分子影像研究奠定了基础。
Sansalvamide A (简称San A) 是1999年由美国Belofsky等从海洋菌属Fusarium中分离出来的一种由两个亮氨酸、一个缬氨酸、一个苯丙氨酸和一个α-羟基异己酸组成的环五肽酯类化合物,具有很高的亲脂性及显著的抗肿瘤能力,其对美国国立癌症研究所的60种恶性肿瘤细胞系具有显著的抗增殖活性,对包括胰腺癌细胞系在内多种肿瘤细胞具有治疗靶向性。将San A分子中的酯键改造成酰胺键,所得化合物为环五肽(称San A环肽)。研究发现,利用氟、氯、甲氧基等基团取代San A环肽分子中苯环对位氢原子,所得San A 环肽衍生物的抗肿瘤生物活性明显优于San A。
San A及其衍生物抗胰腺癌活性的研究,国内尚属空白,而国外研究较多。有学者认为San A具有杀死多种胰腺癌细胞系的重要作用。它的衍生物具有独特的抗癌特性,并与目前抗胰腺癌的药物没有结构同源性。Pan PS等合成了31种San A 环肽衍生物,对两种胰腺癌细胞系(分别是PL45和 BxPC-3)的抗癌活性进行了研究,其中6种衍生物的抗胰腺癌效果是临床常用药物如(如, 5-FU)的140多倍,而正常细胞对其则具有较好的耐受性。
到目前为止,合成的San A环肽衍生物有100余种,其抗肿瘤活性差别明显。大量对San A环肽衍生物构效关系的研究发现,为保证其较高的抗癌活性,该衍生物必须含有两个连续D-氨基酸和/或N-甲氧基。Pan等合成了78种San A环肽衍生物,其中第1号衍生物(简称Diamer San A),显示出最强的抗胰腺癌(PL45)活性,对胰腺癌PL45细胞的半数抑制率(IC50)仅为1-20 nM,是报道的抗胰腺癌活性最强的San A环肽衍生物。
64Cu (T1/2=12.7h)被国际原子能机构(IAEA)称为“新兴PET核素”,具有广阔的应用前景。与18F等PET核素相比,64Cu具有相对较长的半衰期(T1/2 = 12.7 h),可进行较长时间的显像研究,并且便于核素中长程的运输。64Cu同时发射有β+电子(17%)和β-电子(39%),可以同时用于PET成像和放射性治疗,有望集中放射性核素的诊疗一体化研究。
目前抗胰腺癌的一线药物吉西他滨(Gemcitabine),其在改善胰腺癌患者生活质量和延长生存期方面均优于5-Fu,中位生存期为5~ 6个月,1年生存期不到20%。吉西他滨为一种新的胞嘧啶核苷衍生物,进入人体内后由脱氧胞嘧啶激酶活化,由胞嘧啶核苷脱氨酶代谢。其为嘧啶类抗肿瘤药物,主要代谢物在细胞内掺入DNA,主要作用于细胞G1/S期。吉西他滨为水溶性药物,细胞穿透能力差,故不能很好的发挥抗胰腺癌的效果。有研究者对吉西他滨分子进行了修饰,在其结构基础上增加脂溶性多肽,拟提高吉西他滨的抗癌能力。
专利201711070013.6《F-18标记修饰Dimer-San A探针的制备方法》是优化的胰腺癌分子影像探针,但18F本身半衰期短,其多肽标记率低,相比较64Cu克服了上述的不足,且具有治疗的潜力。
发明内容
本发明所要解决的技术问题是提供一种有效提高标记率的Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法。
为解决上述问题,本发明所述的Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,包括以下步骤:
⑴将水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽溶解于乙腈,配制成浓度为1 μg/μL的多肽溶液;所述水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽的序列为:Cyclo[4Aph(NOTA-PEG24)-Leu-Leu-DVal-DLeu-Phe-Leu-Leu-DVal-DLeu],其结构式为:
⑵在37~111 MBq的64CuCl2中加入300 μL 0.1 M 且pH=5.5的乙酸氨(CH3COONH4)缓冲液进行反应,得到反应液64Cu(OAc)2;
⑶将5~10 μg所述多肽溶液加入到所述反应液64Cu(OAc)2中,于45℃加热45 min,并在加热的过程中持续震荡,得到产物64Cu-NOTA-PEG24- -Dimer-Sansalvamide A,简称64Cu-NOTA-PEG24-Dimer-San A,其结构式为:
⑷所述产物64Cu-NOTA-PEG24-Dimer-San A用悬干机于40ºC除去乙腈溶剂,并用含有1%二甲基亚砜(DMSO)的磷酸盐缓冲液(PBS)溶解,最后经0.22 μm滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu-NOTA-PEG24-Dimer-San A。
所述步骤⑷中磷酸盐缓冲液的用量与所述产物64Cu-NOTA-PEG24-Dimer-San A的比例为300~500 μL:5~10 μg。
本发明与现有技术相比具有以下优点:
1、本发明首次以PEG24-Dimer-Sansalvamide A环肽分子作为金属离子64Cu的标记前体,以Hsp90为显像和治疗的靶点,构建64Cu标记的PEG24-Dimer-Sansalvamide A新型分子探针,实现胰腺癌诊断、治疗一体化。
本发明以Sansalvamide A衍生物中活性最强的环肽Dimer-Sansalvamide A (简称Dimer-San A)作为标记前体(IC50为1-20 nM),其结构式如下:
该标记前体在保留该类衍生物重要活性功能基团的前提下,通过在Dimer-San A分子的苯环上引入氨基,增加系列亲水基团聚乙二醇(PEG),并耦联1,4,7-三氮环壬烷-1,4,7-三乙酸(1,4,7-triazacyclononane-1,4,7-triacetic acid,NOTA) 双功能螯合剂,通过MTT实验对其进行生物学活性鉴定后(实验证实修饰后的多肽活性未发生改变),实现长半衰期正电子放射性核素64Cu对其进行标记以构建胰腺癌新型分子探针:64Cu-NOTA-PEG24-Dimer-San A。研究证实,San A及其环肽衍生物是Hsp90的抑制剂,具有显著的抗肿瘤作用,其与靶点结合具有高特异性、高亲和力,且与目前的抗癌药物没有结构同源性。
2、本发明合成的分子探针64Cu-NOTA-PEG24-Dimer-San A以Hsp90作为胰腺癌特异性诊断和治疗的靶点,经体外细胞摄取实验证实,其与靶点有较高的结合率,体外阻断实验进一步证实了该显像剂与靶点结合的特异性(参见图1~2);该靶点存在于肿瘤细胞表面,分子探针不需通过细胞膜脂质双分子层便可以与靶点结合,克服了传统抗胰腺癌药物需要穿过细胞膜才能发挥作用的不足。
【体外细胞摄取与阻断实验】
将PL45细胞收集并种植到24孔板(0.5×105细胞/孔),放至温育箱培养24 h (37ºC-, 5% CO2)。
非阻断实验每孔加入相同浓度的显像剂(5 μCi/100 μL);用于阻断实验的孔先加入100 μL一定浓度的非标记肽,反应60 min后再加入相同浓度的显像剂,进行竞争结合反应,用以证实显像剂与靶点结合有无特异性。加样以后将24孔板温浴不同时间(0、15、30、60、90、120 min),PBS漂洗游离显像剂,胰酶消化、收集细胞后,用γ计数器检测细胞在不同时间显像剂的摄取情况。上述实验重复两次,每孔设置复孔。
图2中摄取高的表示一定浓度的显像剂(64Cu-NOTA-PEG24-Dimer-San A)在不同时间的细胞摄取情况,15 min时显像剂摄取达到峰值,细胞结合率为5.02±0.5%;摄取较低的曲线表示加入一定阻断剂后细胞摄取下降,60min时细胞结合率降为:0.45±0.4%。说明64Cu-NOTA-PEG24-Dimer-San A与靶点Hsp90结合具有特异性。
3、本发明标记方法简单,标记率高,放射高效液相色谱(Radio-HPLC)64Cu-NOTA-PEG24-Dimer-San A的停留时间为16.948min,其标记率为100%(单箭头所示),游离Cu-64基本为0(复合箭头所示)显示标记率达到100% (见图1)。
4、本发明标记的前体为环肽二聚体,与线性单体肽相比较,稳定性高,显像效果好。
5、本发明标记温度较低(仅45 ºC),一般不会导致标记前体的破坏。
6、本发明中64Cu同时发射β+电子和β-电子,可以用于PET显像和放射性核素治疗,而多肽NOTA-PEG24-Dimer-San A本身就显示出高效的抗胰腺癌的效果(IC50为13.61 nM),故 64Cu标记的NOTA-PEG24-Dimer-SanA具有胰腺癌诊疗一体化的潜能。
【MTT实验进行多肽NOTA-PEG24-Dimer-San A的抗癌活性鉴定】
配制MTT浓度为5mg/ml。称取MTT 0.5克,溶于100 ml的磷酸缓冲液(PBS)中,用0.22μm滤膜过滤以除去溶液里的细菌。在配制和保存的过程中,容器用铝箔纸包住。
96孔板铺板(每孔5000个PL45细胞),5% CO2,37℃孵育,24h细胞贴壁后加入浓度梯度的NOTA-PEG24-Dimer-San A多肽,与细胞在温育箱共浴72小时后,每孔加入20μL MTT溶液,继续培养4h;终止培养,小心吸去孔内培养液。每孔加入150μL DMSO,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪OD 570nm处测量各孔的吸光值。同时设置对照孔。实验重复3次,每孔设置复孔。
图3表示该肽的IC50为13.61 nM,表现出很高的抗癌活性。
研究表明修饰后的多肽,假若能保持其生物活性不变,则标记以后发生临床转化的潜力很大,该发明中涉及的原始肽Dimer-San A对胰腺癌PL45细胞的IC50为1-20 nM,而经过修饰后的NOTA- PEG24-Dimer-San A的IC50为13.61 nM,充分说明修饰后的多肽生物活性并没有发生变化。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1为放射高效液相色谱(Radio-HPLC)显示的标记率。
图2为本发明64Cu-NOTA-PEG24-Dimer-San A细胞摄取实验及体外阻断实验结果。
图3为本发明多肽NOTA-PEG24-Dimer-San A 的MTT活性鉴定试验结果。
具体实施方式
实施例1 Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,包括以下步骤:
⑴将水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽溶解于乙腈,配制成浓度为1 μg/μL的多肽溶液;水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽的序列为:Cyclo[4Aph(NOTA-PEG24)-Leu-Leu-DVal-DLeu-Phe-Leu-Leu-DVal-DLeu],其结构式为:
⑵在37 MBq的64CuCl2中加入300 μL 0.1 M 且pH=5.5的乙酸氨(CH3COONH4)缓冲液进行反应,得到反应液64Cu(OAc)2。
⑶将5 μg多肽溶液加入到反应液64Cu(OAc)2中,于45℃加热45 min,并在加热的过程中持续震荡,得到产物64Cu-NOTA-PEG24- -Dimer-Sansalvamide A,简称64Cu-NOTA-PEG24-Dimer-San A,其结构式为:
⑷产物64Cu-NOTA-PEG24-Dimer-San A用悬干机于40ºC除去乙腈溶剂,并用含有1%二甲基亚砜(DMSO)的磷酸盐缓冲液(PBS)溶解,最后经0.22 μm滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu-NOTA-PEG24-Dimer-San A。
其中:磷酸盐缓冲液的用量与产物64Cu-NOTA-PEG24-Dimer-San A的比例为300 μL:5 μg。
实施例2 Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,包括以下步骤:
⑴将水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽溶解于乙腈,配制成浓度为1 μg/μL的多肽溶液;水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽的序列及结构式同实施例1。
⑵在111 MBq的64CuCl2中加入300 μL 0.1 M 且pH=5.5的乙酸氨(CH3COONH4)缓冲液进行反应,得到反应液64Cu(OAc)2。
⑶将10 μg多肽溶液加入到反应液64Cu(OAc)2中,于45℃加热45 min,并在加热的过程中持续震荡,得到产物64Cu-NOTA-PEG24- -Dimer-Sansalvamide A,简称64Cu-NOTA-PEG24-Dimer-San A,其结构式同实施例1。
⑷产物64Cu-NOTA-PEG24-Dimer-San A用悬干机于40ºC除去乙腈溶剂,并用含有1%二甲基亚砜(DMSO)的磷酸盐缓冲液(PBS)溶解,最后经0.22 μm滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu-NOTA-PEG24-Dimer-San A。
其中:磷酸盐缓冲液的用量与产物64Cu-NOTA-PEG24-Dimer-San A的比例为500 μL:10 μg。
实施例3 Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,包括以下步骤:
⑴将水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽溶解于乙腈,配制成浓度为1 μg/μL的多肽溶液;水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽的序列及结构式同实施例1。
⑵在74 MBq的64CuCl2中加入300 μL 0.1 M 且pH=5.5的乙酸氨(CH3COONH4)缓冲液进行反应,得到反应液64Cu(OAc)2。
⑶将7.5 μg多肽溶液加入到反应液64Cu(OAc)2中,于45℃加热45 min,并在加热的过程中持续震荡,得到产物64Cu-NOTA-PEG24- -Dimer-Sansalvamide A,简称64Cu-NOTA-PEG24-Dimer-San A,其结构式同实施例1。
⑷产物64Cu-NOTA-PEG24-Dimer-San A用悬干机于40ºC除去乙腈溶剂,并用含有1%二甲基亚砜(DMSO)的磷酸盐缓冲液(PBS)溶解,最后经0.22 μm滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu-NOTA-PEG24-Dimer-San A。
其中:磷酸盐缓冲液的用量与产物64Cu-NOTA-PEG24-Dimer-San A的比例为400 μL:7.5 μg。
Claims (2)
1.Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,包括以下步骤:
⑴将水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽溶解于乙腈,配制成浓度为1 μg/μL的多肽溶液;所述水溶性修饰的NOTA-PEG24-Dimer-Sansalvamide A多肽的序列为:Cyclo[4Aph(NOTA-PEG24)-Leu-Leu-DVal-DLeu-Phe-Leu-Leu-DVal-DLeu],其结构式为:
⑵在37~111 MBq的64CuCl2中加入300 μL 0.1 M 且pH=5.5的乙酸氨缓冲液进行反应,得到反应液64Cu(OAc)2;
⑶将5~10 μg所述多肽溶液加入到所述反应液64Cu(OAc)2中,于45℃加热45 min,并在加热的过程中持续震荡,得到产物64Cu-NOTA-PEG24- -Dimer-Sansalvamide A,简称64Cu-NOTA-PEG24-Dimer-San A,其结构式为:
⑷所述产物64Cu-NOTA-PEG24-Dimer-San A用悬干机于40ºC除去乙腈溶剂,并用含有1%二甲基亚砜的磷酸盐缓冲液溶解,最后经0.22 μm滤膜过滤除菌,即得64Cu标记的备用胰腺癌分子探针64Cu-NOTA-PEG24-Dimer-San A。
2.如权利要求1所述的Cu-64标记的Hsp90抑制剂胰腺癌诊疗一体化分子探针的制备方法,其特征在于:所述步骤⑷中磷酸盐缓冲液的用量与所述产物64Cu-NOTA-PEG24-Dimer-San A的比例为300~500 μL:5~10 μg。
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