CN106039327A - 一种grpr靶向性分子探针及其制备方法 - Google Patents
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Abstract
本发明属于诊断显像剂领域，涉及一种GRPR靶向性分子探针及其制备方法。具有通式M‑X‑JMV594，其中M是信号组分，X是连接体，JMV594是GRPR的靶向亲和组分；所述JMV594是多肽序列D‑Phe‑Gln‑Trp‑Ala‑Val‑Gly‑His‑Sat‑Leu‑NH2，所述的信号组分M，为放射性同位素与金属螯合剂、荧光染料、量子点、顺磁性材料、超顺磁性材料、磁性纳米粒子、超声微泡、光声纳米颗粒的一种或者几种材料的结合。本发明的GRPR靶向性分子探针可以作为影像显像剂或放射治疗药物，具有良好的药代动力学特性和生物学分布特征，检测结果更加准确，临床应用更有前景。
Description
技术领域
本发明属于诊断显像剂领域，特别涉及一种GRPR靶向性分子探针及其制备方法。
背景技术
常用的分子影像技术包括核磁共振成像(Magnetic resonance imaging,MRI)，计算机X射线断层成像(CT)，正电子发射断层扫描成像(positron emission tomography,PET)，单光子发射型计算机断层成像(single photon emission computed tomography,SPECT)，光学成像(包括多光子成像、近红外一区成像、近红外二区成像和共聚焦成像等)和超声成像。每种显像技术在灵敏度、原理、分辨率和采集时间等方面各有优缺点。
表1各种成像模式比较
分子影像的核心是实时获取高质量、特异靶点分子的图像，因此它需要设计各种靶向性好、肿瘤摄取高、显像信噪比高的分子探针，以满足临床研究的需求。而相对于纳米分子探针，目前多肽小分子探针仍是临床应用的最佳选择。多肽小分子探针通常通过肝脏和肾脏系统代谢，清除时间较短，从而避免由于长期保留在肝脏和肾脏产生的相关毒性。因此高性能的多肽小分子探针的设计和制备成为近年来分子影像学科中最活跃的研究领域。
肿瘤形成是在各种致瘤因素的作用下，细胞生长调控发生严重紊乱的结果。近几年来，研究表明胃泌素释放肽(GRP)作为一种生长因子在多种肿瘤组织中异常表达，并以自主分泌、异常分泌、神经内分泌等方式作用于其特异性受体(GRPR)，从而影响多种信号传导途径，直接刺激肿瘤的生长。GRPR表达情况与肿瘤的性质、类型、分化程度、级别、侵袭力等息息相关。GRPR在正常组织和肿瘤组织中的激活作用有重要的生长效应，且在恶性肿瘤中的表达比在正常的人体组织更为普遍。在多种人类癌症细胞中异常或过度表达，包括在小细胞肺癌、非小细胞肺癌、前列腺癌、乳腺癌、神经胶质母细胞瘤和肺癌等。例如研究证明在人胃、小肠和直肠肿瘤组织中，84％的肿瘤表达GRPR。因此GRPR成为热门的肿瘤标志物应用于癌症的早期诊断和治疗。
蛙皮素(Bombesin，BBN)是一种由14个氨基酸残基组成的生物活性多肽，具有广泛的生物学功能，大量研究证明，BBN与GRPR具有很高的纳摩尔亲和力。但是天然的BBN在体内存在代谢不稳定性，有鉴于此，基于BBN结构研究人员开发各种BBN多肽类似物构建的小分子探针尤其是放射性核素标记(111ln、68Ga、177Lu、64Cu和99Tc等)的PET探针已在临床前实验和临床实验中得到广泛的应用。其中氨基酸序列(Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2)的BBN类似物：JMV594构建的小分子探针一直被认为是最有前途的肿瘤受体GRPR靶向显像和治疗造影剂。例如64Cu标记NODAGA偶联的JMV594的PET小分子探针在体外和体内具有良好的稳定性，并且活体显像结果表明在4小时后肿瘤对该探针摄取值为3.5±1.0％ID/g。但是，对于临床应用而言，该类分子探针普遍存在肿瘤摄取率不高，肿瘤/本底对比度低，主要通过肾脏代谢，药代动力学不佳等缺点。因此如何开发肿瘤摄取高、肿瘤/本底对比度高和药代动力学性质突出的GRPR靶向的小分子探针仍然是一项具有挑战性的工作。
发明内容
本发明所要解决的技术问题是提供一种GRPR靶向性分子探针及其制备方法。该类探针包含靶向GRPR受体肽JMV594和信号组分，两者之间通过不同的连接基团连接从而改善该类化合物在肿瘤部位摄取，肿瘤/本底信号比值以及调节该类化合物体内药代动力学特性，从而达到更好的显像诊断效果。
本发明的分子探针可以具有以下通式：
M-X-JMV594，其中M是信号组分，X是连接体，JMV594是GRPR的靶向亲和组分；
所述JMV594是多肽序列D-Phe-Gln-Trp-Ala-Val-Gly-His-Sat-Leu-NH2，其结构如下：
所述的信号组分M，为用于PET/CT显像的放射性同位素(例如64Cu、68Ga、177Lu、99Tc等)与金属螯合剂(DOTA、DOTAGA、DTPA、PCTA、NOTA、NODAGA、TETA、CB-TE2A等)、荧光染料(包括传统的近红外I区和近红外II区荧光染料)、量子点、顺磁性材料、超顺磁性材料、磁性纳米粒子、超声微泡、光声纳米颗粒的一种或者几种材料的结合。
所述的连接体X，为以下任一种或者两种的结合：
n为0-18的整数。
本发明还提供所述GRPR靶向性小分子探针的制备方法，所述制备方法包括以下步骤：
1)JMV594多肽合成
采用固相Fmoc合成方法，从MBHA树脂开始逐步合成JMV594，多肽合成完毕后，TFA/Tips/H2O(95:2.5:2.5)条件下从树脂上切下(D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)，粗产品用制备HPLC分离纯化后经过质谱分析确认为预期产物JMV594。
2)X-JMV594合成
a)当X为单一连接体：A1,A2,A3时：
将上述步骤获得的JMV594溶于DMF中，依次加入过量的A1,A2,A3和HBTU，室温搅拌过夜，获得的粗产品通过制备HPLC分离纯化后经过质谱分析确认为预期产物SCH。
b)当X为两种连接体组合：B1+C1,B2+C1,B3+C1,B4+C1,B1+C2,B2+C2,B3+C2,B4+C2,
将上述步骤获得的JMV594多肽溶于DMF中，依次加入过量的B和HBTU,室温搅拌过夜，获得的粗产品通过制备HPLC分离纯化后经过质谱分析确认为预期产物ZH。
将上述步骤获得的ZH溶于DMF中，依次加入过量的C，室温搅拌2-4h后，通过制备HPLC分离纯化后经过质谱分析确认为预期产物ZHS。
3)M-X-JMV594的制备
a)用于PET显像/放射治疗的分子探针M-X-JMV594制备(其中M＝放射核素的螯合剂，例如NOTA,NODAGA,DOTA,DOTAGA等)
将上述步骤获得SCH或者ZHS溶于DMF中，依次加入过量的放射核素螯合剂活性酯和DIPEA，室温搅拌2-12h，通过制备HPLC分离纯化后经过质谱分析确认为预期产物。
b)用于其他模态显像的分子探针M-X-JMV594制备(其中M＝其他模态显像的信号组分)
将上述步骤获得SCH或者ZHS溶于DMF中，依次加入过量的信号组分的活性酯分子和DIPEA，室温搅拌2-12h，通过制备HPLC分离纯化后经过质谱分析确认为预期产物。
本发明的GRPR靶向性化合物，可用于对对象待测位置实施光学成像(包括近红外I区，近红外II区)、正电子发射断层成像、单光子发射断层成像、磁共振成像、光学成像、超声成像等。
本发明开发的一类GRPR靶向性影像/放射治疗分子探针，具有良好的药代动力学特性和生物学分布特征，检测结果更加准确，临床应用更有前景。
本发明靶向特异性分子探针具有如下优点：
1)具有GRPR靶向特异性，确保GRPR分子成像靶向检测的准确性。
2)GRPR的活体可视化，确保GRPR分子成像检测的安全，无创和直观性。
3)分子探针与靶向GRPR的高亲和力，从而确保在几个血液循环周期后，探针在靶点达到理想的聚集状态。
4)分子探针检测GRPR的高灵敏度，从而确保其产生的生物学影响或药理学作用尽量低。
5)GRPR活体分子成像的高对比度，从而确保高靶/背景比值和信号/噪声比值。
6)分子探针的高活体稳定性。
7)GRPR活体分子成像的高肿瘤摄取值，从而确保拿到高质量显像。
8)分子探针的生产过程简便易行，成本低，确保分子探针的临床转化。
附图说明
图1为68Ga标记NODAGA-SCH1和NODAGA-JMV594分子探针的结构示意图。
图2为HPLC分析68Ga-NODAGA-SCH1和68Ga-NODAGA-JMV594分子探针在体外小鼠血清稳定性结果。
图3为NODAGA-SCH1和NODAGA-JMV594分子探针在前列腺癌PC-3细胞中IC50值以及68Ga-NODAGA-SCH1和68Ga-NODAGA-JMV594分子探针在不同时间点(0.5h，1h，2h)前列腺癌PC-3细胞摄取值。
图4为68Ga-NODAGA-SCH1和68Ga-NODAGA-JMV594分子探针在前列腺癌模型的PET/CT显像。
图5为68Ga-NODAGA-SCH1和68Ga-NODAGA-JMV594分子探针在体内主要器官的生物学分布。
图6为近红外II区GRPR靶向分子探针SCH1100的结构示意图。
图7为SCH1100分子探针在前列腺癌模型的近红外II区显像
具体实施方式
实施例1：合成化合物68Ga-NODAGA-JMV594
在2mL试管中称取多肽JMV594(1.13mg,0.01mmol)和NODAGA-NHS酯(0.95mg,0.02mmol,2.0当量)溶于DMF溶剂中，加入2当量的DIPEA。室温反应4小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末NODAGA-JMV594，0.80mg，收率55％。MS计算值：C70H103N17O18 +([M+H]+):1470.8,实测值：ESI-MS:m/z 1470.8。
2nmol的NODAGA-JMV594化合物溶解于1mL NaOAc缓冲液(pH＝4.5)在37℃下加入2mCi核素68Ga标记反应15min。标记后的化合物68Ga-NODAGA-JMV594用HPLC纯化后，氮气吹干有机溶剂乙腈，PBS溶液稀释后，直接用于细胞摄取实验和活体成像实验。
实施例2：合成68Ga-NODAGA-SCH1
在2mL试管中称取多肽SCH1(1.25mg,0.01mmol)和NODAGA-NHS酯(0.95mg,0.02mmol,2.0当量)溶于DMF溶剂中，加入2当量的DIPEA。室温反应2小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末NODAGA-SCH1，1.30mg，收率80％。MS计算值：C78H119N18O19 +([M+H]+):1611.9,实测值：MALDI-TOF-MS:1611.9。
2nmol的NODAGA-SCH1化合物溶解于1mL NaOAc缓冲液(pH＝4.5)在37℃下加入2mCi核素68Ga标记反应15min。标记后的化合物68Ga-NODAGA-SCH1用HPLC纯化后，氮气吹干有机溶剂乙腈，PBS溶液稀释后，直接用于细胞摄取实验和活体成像实验。
实施例3：合成化合物68Ga-NODAGA-ZHS1
在2mL试管中称取多肽ZH1(1.28mg,0.01mmol)和化合物C1a(0.56mg,0.02mmol,2.0当量)溶于DMF溶剂中。室温反应2小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末ZHS1，1.13mg，收率75％。MS计算值：C74H111N18O16 +([M+H]+):1506.8,实测值：ESI-MS:1506.9。
在2mL试管中称取多肽ZHS1(1.56mg,0.01mmol)和NODAGA-NHS酯(0.95mg,0.02mmol,2.0当量)溶于DMF溶剂中，加入2当量的DIPEA。室温反应4小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末NODAGA-ZHS1，1.50mg，收率80％。MS计算值：C89H134N21O23 +([M+H]+):1865.0,实测值：ESI-MS:1865.3。
2nmol的NODAGA-ZHS1化合物溶解于1mL NaOAc缓冲液(pH＝4.5)在37℃下加入2mCi核素68Ga标记反应15min。标记后的化合物68Ga-NODAGA-ZHS1用HPLC纯化后，氮气吹干有机溶剂乙腈，PBS溶液稀释后，直接用于细胞摄取实验和活体成像实验。
实施例4：合成化合物68Ga-NODAGA-ZHS2
在2mL试管中称取多肽ZH1(1.28mg,0.01mmol)和化合物C3-1(0.15mg,0.015mmol,1.5当量)溶于DMF溶剂中。室温反应1小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末ZHS2，1.06mg，收率78％。MS计算值：C68H100N15O13S+([M+H]+):1365.7,实测值：ESI-MS:1365.4。
在2mL试管中称取多肽ZHS2(1.36mg,0.01mmol)和NODAGA-NHS酯(0.95mg,0.02mmol,2.0当量)溶于DMF溶剂中，加入2当量的DIPEA。室温反应2小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末NODAGA-ZHS2，1.37mg，收率80％。MS计算值：C83H123N18O20S+([M+H]+):1722.8,实测值：ESI-MS:1722.3。
2nmol的NODAGA-ZHS2化合物溶解于1mL NaOAc缓冲液(pH＝4.5)在37℃下加入2mCi核素68Ga标记反应15min。标记后的化合物68Ga-NODAGA-ZHS2用HPLC纯化后，氮气吹干有机溶剂乙腈，PBS溶液稀释后，直接用于细胞摄取实验和活体成像实验。
实施例5：合成化合物68Ga-NODAGA-ZHS3
在2mL试管中称取多肽ZH1(1.28mg,0.01mmol)和化合物C2(0.28mg,0.015mmol,1.5当量)溶于DMF溶剂中。室温反应1小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末ZHS3，1.17mg，收率80％。MS计算值：C75H102N17O13 +([M+H]+):1447.7,实测值：ESI-MS:1447.4。
在2mL试管中称取多肽ZHS3(1.47mg,0.01mmol)和NODAGA-NHS酯(0.95mg,0.02mmol,2.0当量)溶于DMF溶剂中，加入2当量的DIPEA。室温反应2小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物为白色固体粉末NODAGA-ZHS3，1.44mg，收率80％。MS计算值：C90H125N20O20 +([M+H]+):1805.9,实测值：ESI-MS:1806.1。
2nmol的NODAGA-ZHS3化合物溶解于1mL NaOAc缓冲液(pH＝4.5)在37℃下加入2mCi核素68Ga标记反应15min。标记后的化合物68Ga-NODAGA-ZHS3用HPLC纯化后，氮气吹干有机溶剂乙腈，PBS溶液稀释后，直接用于细胞摄取实验和活体成像实验。
实施例6：合成化合物SCH1100
在2mL试管中称取多肽ZH1(1.28mg,0.01mmol)和化合物C1b(0.66mg,0.015mmol,1.5当量)溶于DMF溶剂中，加入室温反应4小时后。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物ZHS4，1.38mg，收率80％。MS计算值：C84H132N18O21 +([M+H]+):1728.9,实测值：ESI-MS:1728.4。
化合物Q4-1(1.13mg,0.01mmol)溶于DMF溶剂中，加入HBTU(0.38mg，0.01mmol,1.0当量)在2mL试管中室温反应4小时后，加入化合物ZHS4(1.73mg,0.01mmol)室温反应过夜。反应液直接用制备HPLC纯化后真空冷冻干燥得到的产物SCH1100，0.38mg，收率30％。MS计算值：C146H176N24O28S4 +([M+H]+):2843.3,实测值：MALDI-TOF-MS:2844.1。
Claims (4)
1.一种GRPR靶向性分子探针，具有通式M-X-JMV594，其中M是信号组分，X是连接体，JMV594是GRPR的靶向亲和组分；
所述JMV594是多肽序列D-Phe-Gln-Trp-Ala-Val-Gly-His-Sat-Leu-NH2，
所述的信号组分M，为放射性同位素与金属螯合剂、荧光染料、量子点、顺磁性材料、超顺磁性材料、磁性纳米粒子、超声微泡、光声纳米颗粒的一种或者几种材料的结合；
所述的连接体X，为以下任一种或者两种的结合：
n为0-18的整数。
2.权利要求1所述的GRPR靶向性分子探针作为分子影像的显像剂的用途。
3.根据权利要求2所述的用途，其特征在于，所述的分子影像包括正电子发射断层成像、近红外I区光学成像、近红外II区光学成像、单光子发射断层成像、磁共振成像、超声成像或光声成像。
4.权利要求1所述的GRPR靶向性分子探针在制备治疗肿瘤药物上的用途。
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