CN109705193B - 一种放射性标记tEB-TMTP1化合物及其制备方法和应用 - Google Patents
一种放射性标记tEB-TMTP1化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种放射性标记tEB‑TMTP1化合物，通过DOTA和NOTA双功能螯合剂修饰，合成DOTA‑tEB‑TMTP1和NOTA‑tEB‑TMTP1，可分别标记放射性核素68Ga、64Cu和177Lu等放射性金属核素，合成特异性靶向高转移肿瘤的核医学诊疗探针。本发明设计合成的探针具有两大优势，首先标记步骤简单，又较高的标记产率，为临床应用打下基础，其次获得的DOTA‑tEB‑TMTP1和NOTA‑tEB‑TMTP1探针具有优异的药动学性质，显著延长其在体内的半衰期，可以在体内长时间循环，增加肿瘤部位摄取，实现对高转移的肿瘤进行靶向治疗。
Description
技术领域
本发明涉及一种放射性标记tEB-TMTP1化合物及其制备方法和应用，属于放射性标记化合物领域。
背景技术
据统计，我国肿瘤患者五年生存率仅有20％，远低于发达国家的70％。当前，以手术、放化疗为主的治疗，只能切除已有癌灶和杀死部分癌细胞，无法杀灭体内所有癌细胞，且半年内复发转移率高达69％。因此，对治疗肿瘤来说，提高疗效、控制复发和转移及降低死亡率显得非常重要。
单光子发射计算机断层成像术(SPECT)和正电子发射计算机断层扫描(PET)由于其具有灵敏、准确及定位精确等特点，在诊断和指导治疗肿瘤等疾病方面已显示其优越性。但是，由于SPECT和PET技术依然缺乏高靶向性特异性的诊断治疗探针，不能完全满足临床的要求。
TMTP1(NVVRQ)是通过细胞表面展示系统(bacterial peptide display system)来筛选得到的一种肿瘤靶向肽，它对高转移性细胞，包括前列腺癌、乳腺癌、肺癌、胃癌、卵巢癌高转移细胞株均有靶向性，而对低转移细胞和正常细胞无明显靶向性(Yang,W.,etal.,TMTP1,anovel tumor-homing peptide specifically targeting metastasis.ClinCancer Res,2008.14(17):p.5494-502)。有文献报道99mTc标记的TMTP1作为SPECT显像剂，可以靶向高转移的卵巢癌(Li,F.,et al.,Evaluation of(99m)Tc-HYNIC-TMTP1as atumor-homing imaging agent targeting metastasis with SPECT.Nucl Med Biol,2015.42(3):p.256-62)，但是其肝脏摄取本底高，且SPECT分辨率远低与PET。我们前期的工作利用18F标记TMTP1，合成PET探针[18F]AlF-NOTA-G-TMTP1进行体内评价，结果证明其可以特异性靶向高转移肝癌，具有良好的应用前景(李业森,et al.,18F-AlF-NOTA-G-TMTP1的合成及对高转移潜能肝癌细胞荷瘤鼠的显像研究.中华核医学与分子影像杂志,2015.35(5):p.351-354；Li,Y.,et al.,Syntheses and preliminary evaluation of[18F]AlF-NOTA-G-TMTP1for PET imaging of high aggressive hepatocellular carcinoma.ContrastMedia Mol Imaging,2016.11(4):p.262-71)。
伊凡蓝(Evans blue,EB)可以结合体内的白蛋白，可以用于评价细胞活性(Saunders,N.R.,et al.,Markers for blood-brain barrier integrity:howappropriate is Evans blue in the twenty-first century and what are thealternatives？Front Neurosci,2015.9:p.385.)，文献报道末端截短的EB(truncatedEvans Blue，tEB)用于MRI显像评价内皮血管受损情况(Yamamoto,T.,et al.,Firstfunctionalized MRI contrast agent recognizing vascular lesions.Anal Sci,2004.20(1):p.5-7)，陈小元课题组首先应用一种NOTA共轭的末端截短的EB，即[18F]AlF-NOTA-NEB作为血池和前哨淋巴结显像剂，并成功应用于人体试验(Zhang,J.,et al.,Clinical Translation of an Albumin-Binding PET Radiotracer 68Ga-NEB.J NuclMed,2015.56(10):p.1609-14)。
但是，目前并没有将TMTP1和tEB结合起来作为PET或SPECT诊断治疗探针的报道。
发明内容
本发明的目的在于克服现有技术的不足之处，提供了一种放射性标记tEB-TMTP1化合物及其制备方法，所述放射性标记tEB-TMTP1化合物可以作为PET或SPECT诊断治疗探针。
本发明解决其技术问题所采用的技术方案之一是：
一种放射性标记tEB-TMTP1化合物，包括连接在一起的tEB-TMTP1和NOTA或者连接在一起的tEB-TMTP1和DOTA，其结构式如下：
或
本发明解决其技术问题所采用的技术方案之二是：
一种上述的放射性标记tEB-TMTP1化合物的制备方法，包括下列步骤：
(1)将Fmoc-H-Gln(Trt)-2-Wang Resin树脂放入反应管中加入二氯甲烷进行溶胀，然后加入哌啶DMF溶液脱保护，加入的HBTU活化羧基，再加入Fmoc-Arg(Pbf)-OH进行偶联完成一个循环的缩合；
(2)重复步骤(1)的操作，依次加入Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH，最后加入DOTA-tris(tBu)easter或NOTA-bis(tBu)easter进行缩合；
(3)利用Pd(OAc)2、PPh3、N-甲基吗啉和PhSiH3对侧链OAll进行脱保护，然后加入EB染料、HBTU和DIPEA的条件下进行缩合，停止多肽反应后，将所述Fmoc-H-Gln(Trt)-2-WangResin树脂取出，放入砂芯漏斗，加入二氯甲烷洗涤，吹干，加入切割液，室温裂解；
(4)停止反应后，砂芯漏斗过滤，再用所述切割液洗涤一次，加入乙醚沉淀，然后静置25-35min，5000r/min离心4-6min，重复三次，得到多肽在进行HPLC纯化，得到DOTA-tEB-TMTP1或NOTA-tEB-TMTP1；
(5)对步骤(4)中所得的DOTA-tEB-TMTP1或NOTA-tEB-TMTP1进行放射性核素[177Lu]、[64Cu]或[68Ga]标记。
优选地，所述步骤(1)为：将Fmoc-H-Gln(Trt)-2-Wang Resin树脂放入反应管中加入二氯甲烷进行溶胀，然后加入20％哌啶DMF溶液脱保护，加入的0.4mmol的HBTU活化羧基，再加入Fmoc-Arg(Pbf)-OH进行偶联完成一个循环的缩合。
优选地，所述步骤(2)为：重复步骤(1)的操作，依次加入0.3-0.5mmol的Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH，最后加入0.3-0.5mmol的DOTA-tris(tBu)easter或NOTA-bis(tBu)easter进行缩合。
优选地，所述步骤(3)为：利用0.02-0.04mmol的Pd(OAc)2，0.01-0.02mmol的PPh3，0.8-1.2mmol的N-甲基吗啉和0.8-1.2mmol的PhSiH3对侧链OAll进行脱保护，然后加入0.35-0.45mmol的EB染料、0.35-0.45mmol的HBTU和0.35-0.45mmol的DIPEA条件下缩合，停止多肽反应后，将所述Fmoc-H-Gln(Trt)-2-Wang Resin树脂取出，放入砂芯漏斗，加入二氯甲烷洗涤，吹干，加入切割液，室温裂解110-130min。
优选地，所述切割液为TFA、茴香硫醚、H2O、苯酚、1，2-乙二硫醇的混合液，重量比为(80.0-83.0)∶(4.5-5.5)∶(4.5-5.5)∶(4.5-5.5)∶(2.0-3.0)。
优选地，所述步骤(5)为：所述DOTA-tEB-TMTP1或NOTA-tEB-TMTP1进行[177Lu]、[64Cu]或[68Ga]标记的方法为：向反应容器中加入0.1～1mL 0.1M pH值为5～8的醋酸钠缓冲液，取1～20mCi[177Lu]、[64Cu]或[68Ga]加入上述缓冲液中，然后加入20～80μg所述步骤(4)制得的DOTA-tEB-TMTP1或者NOTA-tEB-TMTP1，混合物摇匀后在20～100℃反应10～60min，冷却至常温；将以上反应液缓慢注入事先活化过的Sep-Pak C18柱，再用10～20mL蒸馏水淋洗除去水溶性杂质，吹干后用200～600μL的乙醇淋洗，淋洗液用生理盐水稀释至乙醇含量小于10％。
本发明解决其技术问题所采用的技术方案之三是：
一种上述的放射性标记tEB-TMTP1化合物作为肿瘤显像和治疗的应用。
本技术方案与背景技术相比，它具有如下优点：
1、本发明的放射性标记tEB-TMTP1化合物是一种64Cu、177Lu或68Ga标记的TMTP1，可以利用其特异性靶向高转移肿瘤的生物学特性，对高转移肿瘤进行早期诊断，特异性高，灵敏度高。
2、本发明对TMTP1进行了结构改造，利用EB进行修饰，使其在血液中的滞留时间增长，延长体内的半衰期，增加肿瘤的摄取量，可以达到对对肿瘤进行放射性治疗的效果。
3、本发明利用DOTA或者NOTA两种双功能螯合剂分别对TMTP1进行修饰，使其可以实现对64Cu、177Lu、68Ga放射性金属核素的标记，其标记步骤简单，反应条件温和，标记产率高，稳定性好，易于实现自动合成，另外纯化步骤简单，有利于放射性标记化合物的商业应用与临床推广。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为本发明实施例中[64Cu]DOTA-tEB-TMTP1利用HPLC测定其放化纯度的结果图。
图2为本发明实施例中[64Cu]DOTA-tEB-TMTP1在荷瘤小鼠(B143)中microPET显像冠状面断层图。
图3为本发明实施例中[64Cu]DOTA-tEB-TMTP1在荷瘤小鼠(PC3)中microPET显像冠状面断层图。
具体实施方式
下面通过实施例具体说明本发明的内容：
(1)制备DOTA-tEB-TMTP1或者NOTA-tEB-TMTP1
将0.1mmol的Fmoc-H-Gln(Trt)-2-Wang Resin树脂放入反应管中加入二氯甲烷进行溶胀、然后加入20％哌啶DMF溶液脱保护、加入HBTU活化羧基，再加入Fmoc-Arg(Pbf)-OH进行偶联完成一个循环的缩合，重复上述操作，依次加入0.4mmol的Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH、DOTA-tris(tBu)easter或NOTA-bis(tBu)easter进行缩合。利用0.03mmol的Pd(OAc)2，0.015mmol的PPh3，1mmol的N-甲基吗啉和1mmol的PhSiH3对侧链OAll进行脱保护，然后加入0.4mmol的EB染料、0.4mmol的HBTU和4mmol的DIPEA条件下缩合，停止多肽反应后，将树脂取出，放入砂芯漏斗，加入二氯甲烷洗涤，吹干，加入2mL切割液TFA–茴香硫醚–H2O–苯酚–1，2-乙二硫醇(重量比：82.5∶5∶5∶5∶2.5)，室温裂解2h。停止反应，砂芯漏斗过滤，用切割液洗涤一次，加入乙醚沉淀，然后静置30min，再5000r/min离心5min，重复三次，得到多肽经HPLC分离纯化，收集目标产物的馏分，合并后冻干，得目标产物DOTA-tEB-TMTP1或NOTA-tEB-TMTP1，经过质谱分析确认。
上述HPLC分析，流动相A为0.1％三氟乙酸水溶液，B为0.1％三氟乙酸乙腈，梯度洗脱条件，0～10min，95％A；10～25min，95％A～15％A；25～40min，15％A流速为1mL/min，检测波长为220nm。
上述质谱检测，m/z:1712.43([M+H]+)。
(2)制备[64Cu]DOTA-tEB-TMTP1：向反应容器中加入0.2mL 0.1M pH值5.5的醋酸钠缓冲液，取2mCi 64Cu加入上述缓冲液中，然后加入60μg DOTA-tEB-TMTP1或者NOTA-tEB-TMTP1，混合物摇匀后在90℃反应30min，冷却至常温，用HPLC测定其标记率。
(3)纯化[64Cu]DOTA-tEB-TMTP1：将步骤2中反应液缓慢注入事先活化过的Sep-PakC18柱，再用10～20mL蒸馏水淋洗除去水溶性杂质，吹干后用200～600μL的乙醇淋洗，淋洗液用生理盐水稀释至乙醇含量小于10％，用HPLC测定其保留时间和放化纯度，观察其外观性状是否为无色澄清透明液体。未经衰变校正的放化产率为83.7％，放化纯度＞95％(如图1所示)。
(4)荷肿瘤裸鼠(PC3)MicroPET显像：PC3荷瘤裸鼠麻醉状态下经尾静脉分别注射0.1ml[64Cu]DOTA-tEB-TMTP1(3.7MBq)并于注射后1.5h、4h、24h、38、48h后进行静态microPET/CT断层扫描(Siemens Inveon)，图像采集后通过二维有序子集最大期望值法(ordered subset expectation maximization，OSEM)进行空间重构。在衰变校正的冠状面显像图中，用ASI Pro 5.2.4.0软件圈出肿瘤、正常组织和器官的感兴趣区域(region ofinterest，ROI)，从图中可以看出，[64Cu]DOTA-tEB-TMTP1在血液中的半衰期显著增加，随着时间的延迟，肿瘤部位的摄取显著性增加，在14h后，肿瘤/肌肉比值可达2.7，肿瘤部位对[64Cu]DOTA-tEB-TMTP1的摄取一直保持很高的量，在48h时，肿瘤/肌肉比值依然可达3.7(如图2所示)。
(6)荷肿瘤裸鼠(B143)MicroPET显像：B143荷瘤裸鼠麻醉状态下经尾静脉分别注射0.1ml[64Cu]DOTA-tEB-TMTP1(3.7MBq)并于注射后8h、20h、33h、44h进行静态microPET/CT断层扫描(Siemens Inveon)，图像采集后通过二维有序子集最大期望值法(orderedsubset expectation maximization，OSEM)进行空间重构。在衰变校正的冠状面显像图中，用ASI Pro 5.2.4.0软件圈出肿瘤、正常组织和器官的感兴趣区域(region of interest，ROI)，计算肿瘤模型中肿瘤/非肿瘤(T/NT)的放射性比值，在8h时，肿瘤/肌肉比值即可达到3.9，并且一直持续至44h，依然可以保持肿瘤/肌肉为3.8(如图3所示)。
以上所述，仅为本发明较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
Claims (8)
1.一种放射性标记tEB-TMTP1化合物，其特征在于：其结构式如下：
2.一种权利要求1所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于，包括下列步骤：
(1)将Fmoc-H-Gln(Trt)-2-Wang Resin树脂放入反应管中加入二氯甲烷进行溶胀，然后加入哌啶DMF溶液脱保护，加入HBTU活化羧基，再加入Fmoc-Arg(Pbf)-OH进行偶联完成一个循环的缩合；
(2)重复步骤(1)的操作，依次加入Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH，最后加入DOTA-tris(tBu)easter或NOTA-bis(tBu)easter进行缩合；
(3)利用Pd(OAc)2、PPh3、N-甲基吗啉和PhSiH3对侧链OAll进行脱保护，然后加入EB染料、HBTU和DIPEA进行缩合，停止多肽反应后，将所述Fmoc-H-Gln(Trt)-2-Wang Resin树脂取出，放入砂芯漏斗，加入二氯甲烷洗涤，吹干，加入切割液，室温裂解；
(4)停止反应后，砂芯漏斗过滤，再用所述切割液洗涤一次，加入乙醚沉淀，然后静置25-35min，5000r/min离心4-6min，重复三次，得到多肽再进行HPLC纯化，得到DOTA-tEB-TMTP1或NOTA-tEB-TMTP1；
(5)对步骤(4)中所得的DOTA-tEB-TMTP1或NOTA-tEB-TMTP1进行放射性核素[177Lu]、[64Cu]或[68Ga]标记。
3.如权利要求2所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于：所述步骤(1)为：将Fmoc-H-Gln(Trt)-2-Wang Resin树脂放入反应管中加入二氯甲烷进行溶胀，然后加入20％哌啶DMF溶液脱保护，加入的0.4mmol的HBTU活化羧基，再加入Fmoc-Arg(Pbf)-OH进行偶联完成一个循环的缩合。
4.如权利要求2所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于：所述步骤(2)为：重复步骤(1)的操作，依次加入0.3-0.5mmol的Fmoc-Val-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH、最后加入0.3-0.5mmol的DOTA-tris(tBu)easter或NOTA-bis(tBu)easter进行缩合。
5.如权利要求2所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于：所述步骤(3)为：利用0.02-0.04mmol的Pd(OAc)2，0.01-0.02mmol的PPh3，0.8-1.2mmol的N-甲基吗啉和0.8-1.2mmol的PhSiH3对侧链OAll进行脱保护，然后加入0.35-0.45mmol的EB染料、0.35-0.45mmol的HBTU和0.35-0.45mmol的DIPEA条件下缩合，停止多肽反应后，将所述Fmoc-H-Gln(Trt)-2-Wang Resin树脂取出，放入砂芯漏斗，加入二氯甲烷洗涤，吹干，加入切割液，室温裂解110-130min。
6.如权利要求2所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于：所述切割液为TFA、茴香硫醚、H2O、苯酚、1，2-乙二硫醇的混合液，重量比为(80.0-83.0)∶(4.5-5.5)∶(4.5-5.5)∶(4.5-5.5)∶(2.0-3.0)。
7.如权利要求2所述的放射性标记tEB-TMTP1化合物的制备方法，其特征在于：所述步骤(5)为：所述DOTA-tEB-TMTP1或NOTA-tEB-TMTP1进行[177Lu]、[64Cu]或[68Ga]标记的方法为：向反应容器中加入0.1～1mL 0.1M pH值为5～8的醋酸钠缓冲液，取1～20mCi[177Lu]、[64Cu]或[68Ga]加入上述缓冲液中，然后加入20～80μg所述步骤(4)制得的DOTA-tEB-TMTP1或者NOTA-tEB-TMTP1，混合物摇匀后在20～100℃反应10～60min，冷却至常温；将以上反应液缓慢注入事先活化过的Sep-Pak C18柱，再用10～20mL蒸馏水淋洗除去水溶性杂质，吹干后用200～600μL的乙醇淋洗，淋洗液用生理盐水稀释至乙醇含量小于10％。
8.一种如权利要求1所述的放射性标记tEB-TMTP1化合物在制备肿瘤显像和治疗剂中的应用。
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