CN113813405A - 基于赖诺普利的分子影像探针及其应用 - Google Patents
基于赖诺普利的分子影像探针及其应用 Download PDFInfo
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Abstract
本发明公开了基于赖诺普利的分子影像探针及其应用。通过赖诺普利构建的探针可用于恶性肿瘤的近红外荧光成像和核医学显像。赖诺普利偶联近红外荧光染料可作为肿瘤特异性靶向荧光分子探针,用于肿瘤边界精准定位,可为术前和术中影像导航带来实时性,具有提高手术精确度的优点。赖诺普利偶联具有诊断/治疗功能的放射性核素,可实现在体实时检测及定点靶向恶性肿瘤,以达到疾病诊断和治疗的目的。
Description
技术领域
本发明属于荧光造影剂、放射性药物及核医学技术领域,具体涉及荧光/核素标记的赖诺普利,并进一步公开其制备方法和应用。
背景技术
肿瘤己经成为威胁人类健康和生命的罪魁祸首。因此,肿瘤的诊断以及针对肿瘤有效的治疗显得尤为重要并且迫切。周所周知,肿瘤靶向的分子影像探针是肿瘤诊断、分期及术中导航的有利的工具。其中,肿瘤特异性靶向的配体是肿瘤靶向分子探针的关键所在。目前设计筛选靶向配体的方法主要包括计算机辅助药物设计、先导化合物修饰改造、从代谢产物中发现、药物合成中间体中发现、组合化学和高通量筛选、天然化合物中分离提取、噬菌体展示库筛选以及“老药新用”。不可否认,“老药新用”是一个重要的药物发现途径,1988诺贝尔生理学或医学奖获得者詹姆斯·布莱克提出,新药发现的最佳之路起始于老药。“老药”已经有确定的药代动力学信息和毒理学信息,安全性高是其最明显的特点,只需验证其对新适应症的有效性。追溯“老药新用”的发展历程,临床应用的偶然发现促成了许多成功的经典案例。詹姆斯·布莱克开发的非选择性β受体拮抗剂普萘洛尔和第一个组胺H2受体拮抗剂西咪替丁是“老药新用”的典型例子。普萘洛尔是治疗冠心病和高血压的经典药物,现被用于骨质疏松症和黑色素瘤的治疗;西咪替丁是治疗消化性胃溃疡的革命性药物,经证明适用于治疗慢性阻塞性肺疾病、HIV病毒感染等;《Nature》指出二甲双胍与另一种“老药新用”血红素联用,可用于治疗三阴乳腺癌;又如俗称“砒霜”的三氧化二砷是一种剧毒,最新研究发现其可用于治疗急性早幼粒细胞白血病;前列腺特异性抗原(PSMA)抑制剂早期应用于神经学领域,近年来核素及荧光标记的PSMA小分子抑制剂广泛用于原位及转移性前列腺癌的精准诊疗中。由此可见,“老药新用”策略在药物开发中具有重要指导意义,因此运用“老药新用”策略筛选肿瘤的靶向药物是一种快速有效的方法。
赖诺普利,为血管紧张素转换酶抑制剂。用于治疗原发性高血压及肾血管性高血压,可单独使用或与其他类的抗高血压药如利尿药合并使用。《国家基本药物目录(2018年版)》将其列入心血管系统用药的基本用药目录。
基于“老药新用”策略,我们对大量处于临床和上市药物进行了近红外荧光染料/放射性同位素标记,构建了相应的分子影像探针,并考察了这些探针对肿瘤的靶向性,以期找到具有肿瘤靶向性的药物,能够通过偶联示踪物质或放射性物质制备肿瘤显像试剂或治疗药物。
发明内容
本发明的首要目的在于提供基于赖诺普利在肿瘤诊疗试剂中的应用。本发明的另一目的在于提供基于赖诺普利构建的荧光及放射性探针。本发明的目的可通过以下技术方案实现:
赖诺普利或基于赖诺普利构建的肿瘤靶向分子影像探针在制备癌症诊断试剂或治疗药物中的应用。
作为本发明的一种优选,基于赖诺普利构建的肿瘤靶向分子影像探针在制备肿瘤诊断/治疗显像剂中的应用;
作为本发明的进一步优选,赖诺普利或基于赖诺普利构建的肿瘤靶向分子影像探针在制备肿瘤边界精准定位或术中影像导航光学显像试剂,或制备具有诊断/治疗功能的放射性药物中的应用。
作为本发明的进一步优选,所述的基于赖诺普利构建的肿瘤靶向分子影像探针在制备肿瘤分子成像诊断显像剂中的应用;所述的分子成像包括在各种细胞、组织或其他活体生物成像。
肿瘤诊断/治疗试剂,其特征在于具备以下通式:
M-L-G,
其中,M表示光标记、金属螯合剂与金属放射性核素络合物、非金属放射性核素18F及11C中的任意一种;
L为连接基团;
G为赖诺普利单体或二聚体。
作为本发明的一种优选,所述的L选自6-氨基己酸、PEG4、PEG6或G6中的任意一种或多种:
作为本发明的一种优选,所述的光标记选自有机发色团、有机荧光团、光吸收化合物、光反射化合物、光散射化合物或生物发光分子。
作为本发明的进一步优选,所述的光标记选自近红外一区荧光染料MPA、IRDye800、Cy7.5、ICG、Cy5.5或其类似物或近红外二区荧光染料。
作为本发明的进一步优选,所述的金属螯合剂选自联肼尼克酰胺、1,4,7-三氮杂环壬烷-1,4,7-三乙酸、7-[(4-羟基丙基)亚甲基]-1,4,7-三氮杂化壬烷-1,4-二乙酸、1,4,7,10-四氮杂环四氮杂环十二烷-1,4,7,10-四乙酸、巯基乙酰三甘氨酸、二乙基三胺五乙酸或它们的组合修饰。
作为本发明的进一步优选,所述的肿瘤诊断/治疗试剂为荧光标记的单体化合物或二聚体化合物,优选自以下任意一种结构物质:
其中,近红外荧光染料优选MPA、IRDye800、Cy7.5。
作为本发明的进一步优选,所述的肿瘤诊断/治疗试剂为放射性核素标记的赖诺普利单体配合物或赖诺普利二聚体配合物,优选自以下任意一种结构物质:
或者将式(II)中的双功能螯合剂联肼尼克酰胺替换为1,4,7-三氮杂环壬烷-1,4,7-三乙酸、7-[(4-羟基丙基)亚甲基]-1,4,7-三氮杂化壬烷-1,4-二乙酸、1,4,7,10-四氮杂环四氮杂环十二烷-1,4,7,10-四乙酸、巯基乙酰三甘氨酸或二乙基三胺五乙酸中的任一种,放射性核素99mTc替换为68Ga、64Cu、67Ga、90Y、111In、89Zr或177Lu。
本发明还提供了制备所述近红外荧光探针的方法,包括:
1)近红外荧光染料的前体MPA-Cl的合成
将冰醋酸、对肼基苯磺酸、甲基异丙基酮和醋酸钠混合反应,纯化后得产物2,2,3-三甲基[3H]-吲哚-5-磺酸;再将邻二氯苯加入到2,2,3-三甲基[3H]-吲哚-5-磺酸与1,3-丙磺酸内脂的混合物中,制得2,2,3-三甲基-5-磺酸-1-(3-磺酸-丙基)-[3H]-吲哚。再将该产物与N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]-anilinemonohydrochlorid反应并分离纯化得到绿色MPA-Cl染料。
2)NH2-L-Lisinopril的合成
将Boc保护羧基活化的Boc-NH-L-NHS与赖诺普利在DMSO溶液于室温中反应2小时,反应完成后经制备液相纯化并冻干得到中间体Boc-NH-L-Lisinopril。中间体Boc-NH-L-Lisinopril经体积相等的三氟乙酸和二氯甲烷混合物脱Boc,最后将溶剂旋蒸得到中间体NH2-L-Lisinopril。
3)3-(三苯甲硫基)丙酸-L-Lisinopril的合成
将中间体NH2-L-Lisinopril与3-(三苯甲硫基)丙酸溶于DMSO中,加入缩合剂EDCI和NHS,室温反应,反应完成后通过制备液相分离纯化并冻干,冻干完成后通过加入三氟乙酸和三乙基硅烷脱掉巯基保护基三苯基,最后分离纯化得到中间体3-硫基丙酸-L-Lisinopril。
4)MPA-L-Lisinopril的合成
将分离纯化得到的近红外染料前体MPA-Cl与关键中间体3-硫基丙酸-L-Lisinopril溶于二甲基亚砜中,加入适量的N,N-二异丙基乙胺(DIPEA),室温反应过夜,反应完成后经制备液相纯化分离得到目标荧光化合物。
单体形式的靶向配合物较二聚体形式制备较简单,其二聚体结构中含有用于靶向肿瘤的赖诺普利和用于放射性标记的双功能螯合剂联肼尼克酰胺(HYNIC),以及起到增加赖诺普利与放射性核素配体N-三(羟甲基)甲基甘氨酸(Tricine)和三苯基膦三间磺酸钠盐(TPPTS)之间距离并调节体内药代动力学特性的连接剂L,L选自6-氨基己酸、PEG4、PEG6或G6中的任意一种或多种。
通过对双功能螯合剂的改变,比如将联肼尼克酰胺替换成双功能螯合剂1,4,7-三氮杂环壬烷-1,4,7-三乙酸、7-[(4-羟基丙基)亚甲基]-1,4,7-三氮杂化壬烷-1,4-二乙酸、1,4,7,10-四氮杂环四氮杂环十二烷-1,4,7,10-四乙酸、巯基乙酰三甘氨酸或二乙基三胺五乙酸中的任一种,放射性核素99mTc替换为68Ga、64Cu、67Ga、90Y、111In、89Zr或177Lu。
本发明还提供了制备所述单体放射性核素探针制备的方法,包括:
1)双功能螯合剂HYNIC-L-NHS的合成
将6-氯烟酸和80%水合肼加入到乙醇中,加热回流反应,反应完成后减压旋蒸溶剂,得到的粘稠物加入到蒸馏水中,调PH=5.5左右,析出固体,抽滤烘干得到黄色固体,产品经ESI-MS质谱和核磁氢谱确定为6-联肼烟酸。得到的6-联肼烟酸和对氨基苯甲醛加入到二甲基亚砜(DMSO)中,加热反应5-6小时,反应完成后加入到水中析出,抽滤得固体,此固体烘干后与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)以及N-羟基琥珀酰亚胺(NHS)一起加入到DMSO中室温反应,反应完成后加入水中析出固体,此固体通过硅胶柱纯化后经ESI-MS质谱和核磁氢谱确定为中间体HYNIC-NHS,然后此中间体和连接剂L在碱性条件下反应,最后用活化剂EDCI和NHS活化,纯化后得到HYNIC-L-NHS固体待用。
2)HYNIC-L-Lisinopril的合成
将纯化的中间体HYNIC-L-NHS溶于DMSO中,加入1-1.5摩尔量的赖诺普利,然后再加入2-3摩尔量的DIPEA,室温反应1-2小时,反应完成后通过制备液相进行分离纯化并通过质谱确证。
3)99mTc-HYNIC-L-Lisinopril的合成
分别配制浓度为100.0-120mg/mL的TPPTS(三苯基磷三间磺酸钠)溶液,浓度为130.0-150mg/mL的Tricine(三甲基甘氨酸),浓度为102.4-110mg/mL丁二酸-丁二酸钠缓冲液(其中丁二酸77.0-88.8mg,丁二酸钠25.4-29.3mg),分别取10.0uL TPPTS溶液,10.0uLTricine溶液,10.0uL丁二酸-丁二酸钠缓冲液分别和10.0uL(1.0mg/mL)所述HYNIC-L-Lisinopril混合于西林瓶中,然后加入10mCi Na 99mTcO4于100℃金属浴加热20-30分钟,待反应结束后冷却至室温,产品经Agilent ZORBAX SB-Aq分析柱分析鉴定。
另外,本发明还提供了制备所述二聚体放射性核素探针制备的方法,包括:
1)双功能螯合剂HYNIC-L-NHS的合成
方法同单体放射性核素探针制备步骤1)。
2)支架(2L-谷氨酸)的合成
取适量的Boc-谷氨酸溶于DMSO中,加入2-3倍摩尔量的EDCI和NHS 60度加热反应0.5-1小时后HPLC分析谷氨酸双活化酯已生成,然后往溶液中加入2-3倍摩尔量的连接剂L和2-3倍摩尔量的DIPEA 60℃加热反应0.5-1小时后HPLC分析2分子连接剂L已连接上谷氨酸,然后加入等体积的TFA室温反应过夜脱除Boc保护,最后粗品经制备液相分离后冷冻干燥待用。
3)中间体2L-E-HYNIC-NHS的合成
将制备得到的支架2L-谷氨酸溶于DMSO中,然后加入相同摩尔量的HYNIC-L-NHS,再加入2-3倍摩尔量的DIPEA,室温反应1-2小时,反应完成后通过制备液相分离纯化并通过质谱对目标化合物进行确证,纯化后的产物用EDCI和NHS活化,纯化后得到2L-E-HYNIC-NHS待用。
4)(Lisinopril)2-2L-E-HYNIC的合成
将纯化的中间体2L-E-HYNIC-NHS溶于DMSO中,加入1-1.5摩尔量的赖诺普利,然后再加入2-3摩尔量的DIPEA,室温反应1-2小时,反应完成后通过制备液相进行分离纯化并通过质谱确证。
5)放射性探针99mTc-HYNIC-2L-E-(Lisinopril)2的合成
分别配制浓度为100.0-120mg/mL的TPPTS(三苯基磷三间磺酸钠)溶液,浓度为130.0-150mg/mL的Tricine(三甲基甘氨酸),浓度为102.4-110mg/mL丁二酸-丁二酸钠缓冲液(其中丁二酸77.0-88.8mg,丁二酸钠25.4-29.3mg),分别取10.0uL TPPTS溶液,10.0uLTricine溶液,10.0uL丁二酸-丁二酸钠缓冲液分别和10.0uL(1.0mg/mL)所述HYNIC-2L-E-(Lisinopril)2混合于西林瓶中,然后加入10mCi Na 99mTcO4于100℃金属浴加热20-30分钟,待反应结束后冷却至室温,产品经Agilent ZORBAX SB-Aq分析柱分析鉴定。
本发明所述的基于赖诺普利构建的分子探针可特异性靶向至肿瘤部位,并且在肿瘤部位有良好的摄取和滞留能力,具有高的靶/非靶比值,适合用于制备肿瘤术中影像导航试剂以及制备放射性药物,用于肿瘤核医学诊断及精准放疗。
本发明所述的荧光及放射性核素探针与现有技术相比的有益效果为:
1、上市药物赖诺普利在临床使用已有近百年的历史,已经有确定的药代动力学信息和毒理学信息,具有高的体内安全性。因此,基于赖诺普利构建体内分子探针在安全性方面具有独特的优势,可显著降低药物的研发成本及风险。
2、本发明前期研究发现赖诺普利能特异性靶向肿瘤,通过小动物活体成像筛选结果显示,基于上市药物赖诺普利设计的分子影像探针能特异性靶向识别多种肿瘤,并且在正常肝组织中无明显摄取。借助赖诺普利能特异性靶向肿瘤的性质,通过偶联荧光染料构建荧光探针,可在术中协助医生精确定位肿瘤边界,达到对肿瘤精准切除的目的。另外,赖诺普利还可偶联具有诊断/治疗功能的放射性核素构建相应的放射性药物,可达到对肿瘤诊断和精准放射治疗的目的。
3、基于赖诺普利构建的分子探针经体内光学和放射性核素显像结果证实对多种肿瘤具有优异的靶向效果,包括肝癌、结直肠癌、乳腺癌、肺癌、胰腺癌、胃癌等。所述探针可特异性靶向肿瘤部位的特性将有可能实现对恶性肿瘤的核医学诊断、治疗以及光学手术导航。
4、本发明中使用了稳定性以及水溶性更理想的近红外荧光染料MPA作为光学显像基团,改善了药物在体内的药代动力学。
5、本发明中引入了多个水溶性的PEG4或PEG6分子,以进一步改善药代动力学特性,特别是从非肿瘤组织的清除动力学。
6、本发明中使用HYNIC作为双功能螯合剂,同时使用Tricine和TPPTS作为协同配体从而使“99mTc-HYNIC”核具有更好的体内外稳定性。
附图说明
图1、MPA-PEG4-Lisinopril的质谱图。
图2、制备的单体荧光化合物MPA-PEG4-Lisinopril在荷瘤鼠体内的2h荧光成像。A为在肝癌MHCC97-H荷瘤鼠体内的荧光成像;B为在结直肠癌HT29荷瘤鼠体内的荧光成像;C为在乳腺癌MDA-MB-231荷瘤鼠体内的荧光成像;D为在乳腺癌MCF-7荷瘤鼠体内的荧光成像;E为在结直肠癌HCT116荷瘤鼠体内的荧光成像;F为在胃癌MG3-803荷瘤鼠体内的荧光成像;G为在肝癌HepG2荷瘤鼠体内的荧光成像;H为在胰腺癌MiaPaCa-2荷瘤鼠体内的荧光成像;I为在肺癌A549荷瘤鼠体内的荧光成像。
图3、制备的单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在荷瘤鼠体内1h的SPECT-CT成像。A为在肝癌MHCC97-H荷瘤鼠体内的SPECT-CT成像;B为在宫颈癌HeLa荷瘤鼠体内的SPECT-CT成像;C为在胰腺癌CFPAC-1荷瘤鼠体内的SPECT-CT成像;D为在肝癌Bel-7404荷瘤鼠体内的SPECT-CT成像;E为在胃癌MGC-803荷瘤鼠体内的SPECT-CT成像。
图4、二聚体99mTc-HYNIC-2PEG4-(Lisinopril)2的放射分析液相图。
图5、制备的二聚体放射性化合物99mTc-HYNIC-2Aca-(Lisinopril)2在肝癌MHCC97-H荷瘤鼠体内的SPECT-CT成像。
具体实施方式
以下通过具体的实施例和应用例来进一步说明本发明:其中合成步骤中所使用的化学物质均为现有物质或市售商品。
实施例1 MPA-PEG4-Lisinopril的合成
称取市售的赖诺普利水合物50mg以及45mg Boc-NH-PEG4-COOH加入到800μL DMSO中,然后加入24mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)偶联剂以及14.2mgN-羟基丁二酰亚胺(NHS),混匀后再加入32mg N,N-二异丙基乙胺(DIPEA),室温反应2小时,反应完成后用制备液相进行分离纯化,冷冻干燥得到中间体Boc-NH-PEG4-Lisinopril。此中间体经200μL等体积的三氟乙酸和二氯甲烷混合液室温脱Boc,可得到中间体NH2-PEG4-Lisinopril。取此中间体30mg以及16mg 3-(三苯甲硫基)丙酸加入到加入到500μLDMSO中,然后加入9mg缩合剂EDCI以及5.5mg NHS,混匀后再加入21mg DIPEA,室温反应2小时,反应完成后用制备液相进行分离纯化,冷冻干燥得到中间体3-(三苯甲硫基)丙酸-PEG4-Lisinopril。取20mg中间体3-(三苯甲硫基)丙酸-PEG4-Lisinopril加入到200μL等体积的三氟乙酸和三乙基硅烷中室温搅拌1小时脱掉巯基保护基三苯基,后经制备液相分离纯化,冷冻干燥得到关键中间体3-硫基丙酸-PEG4-Lisinopril。取10mg关键中间体3-硫基丙酸-PEG4-Lisinopril与12mg近红外染料前体MPA-Cl溶于二甲基亚砜中,加入适量的DIPEA,氮气保护下室温反应过夜,反应完成后经制备液相纯化分离得到荧光化合物,制备的荧光化合物经ESI-MS质谱分析确认为目标化合物MPA-PEG4-Lisinopril,ESI-MS:[M-2H]3-=780.28以及[M-3H]3-=520.25(图1)。
制备液相条件如下所示:使用了Agilent 1220Infinity II系列HPLC系统配备Agilent ZORBAX SB-C18半制备柱(9.4×250mm,5um),梯度淋洗60分钟,流速2mL/min,其中流动相A为超纯水(0.01%TFA),B为乙腈(0.01%TFA)。淋洗梯度设定为:0-5分钟时95%A和5%B,15分钟时80%A和20%B,45分钟时50%A和50%B,60分钟时5%A和95%B。
实施例2制备的单体荧光化合物MPA-PEG4-Lisinopril在肝癌MHCC97-H荷瘤鼠体内的荧光成像。
取制备好的荧光化合物MPA-PEG4-Lisinopril并配制成生理盐水溶液(100nmol/mL),取0.1mL(约10nmol)分别注射于3只肝癌MHCC97-H荷瘤裸鼠(体重约22克)尾静脉,并于给药后1h、2h、4h、6h、8h、10h和12h进行光学信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2A,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肝癌(MHCC97-H)部位。
实施例3制备的单体荧光化合物MPA-PEG4-Lisinopril在结直肠癌HT29荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只结肠癌HT29荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2B,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向结直肠癌(HT29)部位。
实施例4制备的单体荧光化合物MPA-PEG4-Lisinopril在乳腺癌MDA-MB-231荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只乳腺癌MDA-MB-231荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2C,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向乳腺癌(MDA-MB-231)部位。
实施例5制备的单体荧光化合物MPA-PEG4-Lisinopril在乳腺癌MCF-7荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只乳腺癌MCF-7荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2D,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向乳腺癌(MCF-7)部位。
实施例6制备的单体荧光化合物MPA-PEG4-Lisinopril在结直肠癌HCT116荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只结直肠癌HCT116荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2E,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向结直肠癌(HCT116)部位。
实施例7制备的单体荧光化合物MPA-PEG4-Lisinopril在胃癌MGC-803荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只胃癌MGC-803荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2F,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向胃癌(MGC-803)部位。
实施例8制备的单体荧光化合物MPA-PEG4-Lisinopril在肝癌HepG2荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只肝癌HepG2荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2G,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肝癌(HepG2)部位。
实施例9制备的单体荧光化合物MPA-PEG4-Lisinopril在胰腺癌MiaPaCa-2荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只胰腺癌MiaPaCa-2荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2H,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向胰腺癌(MiaPaCa-2)部位。
实施例10制备的单体荧光化合物MPA-PEG4-Lisinopril在肺癌A549荷瘤鼠体内的荧光成像
按实施例2相同方法将MPA-PEG4-Lisinopril分别注射于3只肺癌A549荷瘤裸鼠,并于给药后1h、2h、4h、6h、8h、10h和12h进行荧光信号采集。观察荧光药物在模型鼠体内的分布以及在肿瘤区域的富集情况。结果见图2I,可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肺癌(A549)部位。
实施例11单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在肝癌MHCC97-H荷瘤鼠体内的SPECT-CT成像
1)双功能螯合剂HYNIC-PEG4-NHS的合成
将1g 6-氯烟酸和2.0mL 80%水合肼加入到10mL乙醇中,加热回流反应4小时,反应完成后减压旋蒸溶剂,得到的粘稠物加入到蒸馏水中,调pH=5.5左右,析出固体,抽滤烘干得到黄色固体0.86g,产品经ESI-MS质谱和核磁氢谱确定为6-联肼烟酸。得到的0.86g 6-联肼烟酸和0.61g对氨基苯甲醛加入到3.0mL二甲基亚砜(DMSO)中,加热反应5-6小时,反应完成后加入到水中析出,抽滤,烘干得固体1.2g。此烘干的1.2g固体后与2.5g EDCI及1.5gNHS一起加入到DMSO中室温反应,反应完成后加入水中析出固体,此固体通过硅胶柱纯化后干燥,称重1.3g,经ESI-MS质谱和核磁氢谱确定为HYNIC-NHS,ESI-MS:[M+H]=382.1508。此产物纯化后加入到含有DIPEA的PEG4中,室温反应2小时,反应完成后往溶液中加入2倍摩尔量的EDCI和NHS,反应完成后通过制备液相分离纯化,冷冻干燥后经质谱验证为目标产物HYNIC-PEG4-NHS,ESI-MS:[M+H]=630.3及[M+Na]+=652.3。
2)将纯化的5mg中间体HYNIC-PEG4-NHS溶于0.3mL DMSO中,往混合液中加入5mg赖诺普利以及5.6mg DIPEA,室温反应2小时。反应完成后通过制备液相进行分离纯化产物,最后得到黄色固体2.8mg,通过质谱确证为目标产物HYNIC-PEG4-Lisinopril,ESI-MS:[M+H]+=919.38及[M-H]-=917.53。
3)放射性化合物99mTc-HYNIC-PEG4-Lisinopril的合成
分别配制浓度为100.0mg/mL的TPPTS(三苯基磷三间磺酸钠)溶液,浓度为130.0mg/mL的Tricine(三甲基甘氨酸),浓度为102.4mg/mL丁二酸-丁二酸钠缓冲液(其中丁二酸77.0mg,丁二酸钠25.4mg),分别取10.0uL TPPTS溶液,10.0uL Tricine溶液,10.0uL丁二酸-丁二酸钠缓冲液分别和10.0uL(1.0mg/mL)以及HYNIC-PEG4-Lisinopril混合于西林瓶中,然后加入10mCi Na 99mTcO4于100℃金属浴加热20分钟,待反应结束后冷却至室温,制得放射性药物99mTc-HYNIC-PEG4-Lisinopril,产品经Agilent ZORBAX SB-Aq分析柱分析鉴定。
使用的HPLC法为配备了放射性在线检测器(Flow-RAM)和Agilent ZORBAX SB-Aq分析柱(4.6×250mm,5um)的Agilent 1220Infinity II系列HPLC系统。梯度淋洗45分钟,流速1mL/min,其中流动相A为超纯水(0.01%TFA),B为乙腈(0.01%TFA)。淋洗梯度设定为:0-5分钟时95%A和5%B,15分钟时70%A和30%B,20分钟时65%A和35%B,25分钟时45%A和55%B,45分钟时5%A和95%B。
放射性化合物99mTc-HYNIC-PEG4-Lisinopril配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只肝癌MHCC97-H荷瘤裸鼠尾静脉,并于给药后0.5h、1h、2h、3h、4h进行SPECT-CT信号采集。观察放射性核素探针在小鼠体内的分布以及在肿瘤区域的富集情况。结果见图3A,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肝癌(MHCC97-H)部位。
实施例12单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在宫颈癌HeLa荷瘤鼠体内的SPECT-CT成像
按实施例11相同方法将放射性化合物99mTc-HYNIC-PEG4-Lisinopril配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只宫颈癌HeLa荷瘤裸鼠,并于给药后0.5h、1h、2h及4h进行SPECT-CT信号采集。结果见图3B,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向宫颈癌(HeLa)部位。
实施例13单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在胰腺癌CFPAC-1荷瘤鼠体内的SPECT-CT成像
按实施例11相同方法将放射性化合物99mTc-HYNIC-PEG4-Lisinopril配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只胰腺癌CFPAC-1荷瘤裸鼠,并于给药后0.5h、1h、2h及4h进行SPECT-CT信号采集。结果见图3C,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向胰腺癌(CFPAC-1)部位。
实施例14单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在肝癌Bel-7404荷瘤鼠体内的SPECT-CT成像
按实施例11相同方法将放射性化合物99mTc-HYNIC-PEG4-Lisinopril配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只在肝癌Bel-7404荷瘤裸鼠,并于给药后0.5h、1h、2h及4h进行SPECT-CT信号采集。结果见图3D,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肝癌(Bel-7404)部位。
实施例15单体放射性化合物99mTc-HYNIC-PEG4-Lisinopril在胃癌MGC-803荷瘤鼠体内的SPECT-CT成像
按实施例11相同方法将放射性化合物99mTc-HYNIC-PEG4-Lisinopril配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只在胃癌MGC-803荷瘤裸鼠,并于给药后0.5h、1h、2h及4h进行SPECT-CT信号采集。结果见图3E,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向胃癌(MGC-803)部位。
实施例16二聚体99mTc-HYNIC-2PEG4-(Lisinopril)2的放射合成
1)双功能螯合剂HYNIC-NHS的合成
将1g 6-氯烟酸和2.0mL 80%水合肼加入到10mL乙醇中,加热回流反应4小时,反应完成后减压旋蒸溶剂,得到的粘稠物加入到蒸馏水中,调pH=5.5左右,析出固体,抽滤烘干得到黄色固体0.86g,产品经ESI-MS质谱和核磁氢谱确定为6-联肼烟酸。得到的0.86g 6-联肼烟酸和0.61g对氨基苯甲醛加入到3.0mL二甲基亚砜(DMSO)中,加热反应5-6小时,反应完成后加入到水中析出,抽滤,烘干得固体1.2g。此烘干的1.2g固体后与2.5g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)以及1.5g N-羟基琥珀酰亚胺(NHS)一起加入到DMSO中室温反应,反应完成后加入水中析出固体,此固体通过硅胶柱纯化后干燥,称重1.3g,经ESI-MS质谱和核磁氢谱确定为目标产物HYNIC-NHS,ESI-MS:[M+H]=382.15。
2)支架(Aca)2-E的合成
将5.0g叔丁氧羰基(t-Butyloxy carbony)保护的谷氨酸(E),8.3g二环己基碳二亚胺(DCC)以及4.6g NHS加入到100mL有机溶剂四氢呋喃(THF)中,室温搅拌过夜进行双羧基活化,反应完成后抽滤,滤液用THF进行洗涤,洗涤完成后不进一步纯化,直接加入到50mL的二甲基亚砜(DMSO)中溶解,然后加入10g氨基己酸(Aca),最后加入14.6g DIPEA室温反应2小时,检测反应完成后,往反应中加入3.0mL三氟乙酸(TEA)进行Boc保护基的脱除,反应完成后通过制备液相进行分离纯化,最后烘干得到稠状固体7.8g,通过质谱验证为预期目标物(Aca)2-E。
3)中间体HYNIC-E-(Aca)2-2NHS的合成
将制备得到的0.5g支架(Aca)2-E溶于DMSO中,然后加入0.28g HYNIC-NHS,再加入0.32g DIPEA,室温反应2小时,然后加入EDCI和NHS活化,反应完成后通过制备液相分离纯化并冷冻干燥,得到黄的固体0.34g,通过质谱验证为预期目标化合物HYNIC-E-(Aca)2-2NHS。
4)HYNIC-2Aca-E-(Lisinopril)2的合成
将纯化的5mg中间体HYNIC-E-(Aca)2-2NHS溶于0.3mL DMSO中,反应完成后加入5.8mg赖诺普利,然后再加5.6mg DIPEA,室温反应2小时,反应完成后通过制备液相进行分离纯化,最后得到黄色固体3.5mg,通过质谱确证为目标产物,ESI-MS:[M+2H]2+=708.1以及[M+3H]3+=472.4。
5)99mTc-HYNIC-2Aca-E-(Lisinopril)2的制备
分别配制浓度为100.0mg/mL的TPPTS(三苯基磷三间磺酸钠)溶液,浓度为130.0mg/mL的Tricine(三甲基甘氨酸),浓度为102.4mg/mL丁二酸-丁二酸钠缓冲液(其中丁二酸77.0mg,丁二酸钠25.4mg),分别取10.0uL TPPTS溶液,10.0uL Tricine溶液,10.0uL丁二酸-丁二酸钠缓冲液分别和10.0uL(1.0mg/mL)以及HYNIC-2Aca-E-(Lisinopril)2混合于西林瓶中,然后加入10mCi Na99mTcO4于100℃金属浴加热20分钟,待反应结束后冷却至室温,制得放射性药物99mTc-HYNIC-2Aca-E-(Lisinopril)2,产品经Agilent ZORBAX SB-Aq分析柱分析鉴定。
使用的HPLC法为配备了放射性在线检测器(Flow-RAM)和Agilent ZORBAX SB-Aq分析柱(4.6×250mm,5um)的Agilent 1220Infinity II系列HPLC系统。梯度淋洗45分钟,流速1mL/min,其中流动相A为超纯水(0.01%TFA),B为乙腈(0.01%TFA)。淋洗梯度设定为:0-5分钟时95%A和5%B,15分钟时70%A和30%B,20分钟时65%A和35%B,25分钟时45%A和55%B,45分钟时5%A和95%B。二聚体99mTc-HYNIC-2PEG4-(Lisinopril)2的放射分析液相图见图4.
实施例17二聚体放射性化合物99mTc-HYNIC-2Aca-E-(Lisinopril)2在肝癌MHCC97-H荷瘤鼠体内的SPECT-CT成像
按实施例11相同方法将放射性化合物99mTc-HYNIC-2Aca-E-(Lisinopril)2配制成生理盐水溶液(3mCi/mL),取0.1mL(约300μCi)分别注射于3只在肝癌MHCC97-H荷瘤裸鼠,并于给药后0.5h、1h、2h及4h进行SPECT-CT信号采集。结果见图5,由图可见荧光探针MPA-PEG4-Lisinopril能特异性靶向肝癌(MHCC97-H)部位。
Claims (10)
1.赖诺普利或基于赖诺普利构建的肿瘤靶向分子影像探针在制备癌症诊断试剂或治疗药物中的应用。
2.根据权利要求1所述的应用,其特征在于基于赖诺普利构建的肿瘤靶向分子影像探针在制备肿瘤诊断/治疗显像剂中的应用;优选在制备肿瘤边界精准定位或术中影像导航光学显像试剂,或制备具有诊断/治疗功能的放射性药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于:权利要求1中所述的基于赖诺普利构建的肿瘤靶向分子影像探针在制备肿瘤分子成像诊断显像剂中的应用;所述的分子成像包括在各种细胞、组织或其他活体生物成像。
4.肿瘤诊断/治疗试剂,其特征在于具备以下通式:
M-L-G,
其中,M表示光标记、金属螯合剂与金属放射性核素络合物、非金属放射性核素18F及11C中的任意一种;
L为连接基团;
G为赖诺普利单体或二聚体。
6.根据权利要求5所述的肿瘤诊断/治疗试剂,其特征在于所述的光标记选自有机发色团的化合物、有机荧光团的化合物、光吸收化合物、光反射化合物、光散射化合物或生物发光分子。
7.根据权利要求6所述的肿瘤诊断/治疗试剂,其特征在于所述的光标记选自近红外一区荧光染料MPA、IRDye800、Cy7.5、ICG、Cy5.5或其类似物或近红外二区荧光染料。
8.根据权利要求5所述的肿瘤诊断试剂,其特征在于所述的金属螯合剂选自联肼尼克酰胺、1,4,7-三氮杂环壬烷-1,4,7-三乙酸、7-[(4-羟基丙基)亚甲基]-1,4,7-三氮杂化壬烷-1,4-二乙酸、1,4,7,10-四氮杂环四氮杂环十二烷-1,4,7,10-四乙酸、巯基乙酰三甘氨酸、二乙基三胺五乙酸或它们的组合修饰。
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Non-Patent Citations (4)
Title |
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FATMA BEYAZIT等: "Assessment of serum angiotensin-converting enzyme in patients with epithelial ovarian cancer", 《ARCH GYNECOL OBSTET》 * |
FRANK J. FEMIA等: "Synthesis and Evaluation of a Series of 99mTc(CO)3+Lisinopril Complexes for In Vivo Imaging of Angiotensin-Converting Enzyme Expression", 《THE JOURNAL OF NUCLEAR MEDICINE》 * |
SAJJAD ABBASI POUR等: "Design and characterization of lisinopril-loaded superparamagnetic nanoparticles as a new contrast agent for in vitro, in vivo MRI imaging, diagnose the tumors and drug delivery system", 《J MATER SCI: MATER MED》 * |
YUANBIAO TU等: "AT1R-Specific Ligand Angiotensin II as a Novel SPECT Agent for Hepatocellular Carcinoma Diagnosis", 《ACS SENS》 * |
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