CN104667306A - 99mTc标记RGD多肽三聚体肿瘤显像药剂的化学结构及制备方法 - Google Patents
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Abstract
本发明是关于一种肿瘤显像药剂的化学结构及制备方法,特别是关于一项SPECT锝(99mTc)标记RGD多肽三聚体药物:99mTc-4P-RGD3,此肿瘤显像药物以配体方式与整合素αvβ3受体结合,而达到肿瘤部位显像的用途。实施步骤包括:将HYNIC-0su与4P-RGD3进行连接步骤以获得配体HYNIC-4P-RGD3、利用配体中的HYNIC和99mTc螯合作用步骤制成99mTc-4P-RGD3肿瘤显像药物、通过动物体内分布研究及肿瘤显像步骤证实较高的肿瘤摄取及更低的全身正常脏器本底摄取,可提高全身肿瘤图像的清晰度、通过药物代谢实验步骤显示99mTc-4P-RGD3经过动物体内代谢后仍可由原型方式排出体外。经本发明所采用的技术手段所制备的锝标记整合素αvβ3肿瘤显像药剂(99mTc-4P-RGD3),解决了其他锝标记RGD药物在动物体内所产生的高本底摄取问题,使锝标记RGD药物更适用于全身肿瘤的评估。
Description
【技术领域】
本发明是关于单光子发射型计算机断层显像(single photon emission computedtomography,SPECT)中的一种肿瘤显像药剂的化学结构及其制备方法,特别是关于一项锝(99mTc)标记RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)多肽三聚体药物:99mTc-4P-RGD3,此药物通过静脉注射后可经由肿瘤内的整合素(integrin)αvβ3表达,以配体方式与整合素αvβ3受体互相结合,而达到肿瘤部位显像的目的。
【背景技术】
根据世界卫生组织WHO所发布的[世界癌症报告]中统计,近年来中国癌症发病人数为306.5万,约占全球发病的1/5,而癌症死亡人数为220.5万,约占全球癌症死亡人数的1/4,使得中国的癌症发病率及死亡率已经攀升为世界首位,因此中国在癌症的诊断及预防方面急需成本较低廉和制备较简便的肿瘤诊断药物。
目前临床上常规使用氟-18(F-18)标记的去氧葡萄糖(fludeoxyglucose,18F-FDG)显像剂于评价肿瘤的糖代谢情况,可对肿瘤的恶性程度进行分层,对于肿瘤的诊断和疗效评估具有重要的临床价值,然而该显像剂为氟-18标记的药物,其制备过程较复杂且成本较高,同时需要使用昂贵的正电子发射型计算机断层显像(positron emission tomography,PET)或正电子发射型计算机断层显像/X线计算机断层成像(PET/Computed Tomography,PET/CT)仪器才可进行显像,因此造成诊断费用较昂贵,临床应用上难以普及,特别是在基层医院中无法得到广泛推广使用。
锝标记RGD多肽体和18F-FDG相比,同样可用于肿瘤的评估,其制备过程较简便,且可以通过较低廉的SPECT或单光子发射型计算机断层显像/X线计算机断层成像(SPECT/CT)仪器进行显像,因此具有成本较低廉和技术简便的特点,在未来的临床应用拓展中,更具有优势。另外锝标记RGD多肽体显像可对于肿瘤内的整合素αvβ3的高低程度进行评估,有别于18F-FDG的模式,在肿瘤生物学行为表达的多样性层面中,对肿瘤恶性程度可提供另一种重要的诊断价值。
整合素αvβ3是目前研究最多的整合素分子,在骨肉瘤、成神经细胞瘤、肺癌、乳腺癌、***癌、膀胱癌、胶质母细胞瘤及浸润性黑色素瘤等多种实体肿瘤细胞表面和肿瘤新生血管均有丰富的表达,在成熟血管内皮细胞和绝大多数正常组织表达缺乏或几乎不表达。新生血管和***的形成在肿瘤发生、发展和转移中发挥着重要的作用,肿瘤新生血管形成促进肿瘤生长和转移,淋巴道形成与肿瘤转移直接相关。αvβ3受体介导肿瘤细胞粘附和移行,在肿瘤新生血管生成和淋巴道转移发挥重要的作用。αvβ3的表达水平与恶性肿瘤的浸润转移能力等恶性表型有关,也可以作为评价肿瘤恶性预后的指标。
整合素是一组跨膜糖蛋白,有一个α亚单位和β亚单位通过非共价键组成的异二聚体,也是细胞外基质蛋白的受体,与细胞外基质蛋白(如纤维结合蛋白、玻璃结合蛋白、层粘蛋白和胶原等)的受体识别序列RGD特异结合。整合素调控新生血管和淋巴道的形成,诸多研究表明,整合素调控金属基质蛋白酶等蛋白水解酶,直接参与细胞外基质的降解,促使肿瘤转移;整合素的丰富表达是促成肿瘤细胞和血管内皮细胞迁移和侵袭的重要分子,可直接介导肿瘤细胞与基质蛋白的粘附结合;并参与调控细胞内的细胞骨架构成及细胞增殖。
现有的研究结果认为锝标记RGD多肽二聚体(99mTc-3P-RGD2)及锝标记RGD半乳糖二聚体(99mTc-Galacto-RGD2)显像剂与恶性肿瘤的integrinαvβ3表达具有高亲和力,以SPECT或SPECT/CT成像,可成为肿瘤诊断的经济型药剂,但此两项药剂在动物体内的非靶器官摄取值较高,特别是腹部的器官形成较高的放射性本底,可干扰了腹部器官的肿瘤部位成像效果。
【发明内容】
本发明所使用的技术方法提供一种针对整合素αvβ3表达而显像的锝标记放射性RGD多肽三聚体肿瘤显像药物(99mTc-4P-RGD3),通过新型的多肽三聚体配体结构,在动物实验中发现药物在肿瘤靶点的摄取比现有其他锝标记RGD药物较高,且在全身脏器的放射性本底比现有其他锝标记RGD药物更低,因此在动物体内可产生更清晰的全身肿瘤标靶图像,供肿瘤评估或诊断使用。
本发明的主要技术方案如下:将HYNIC-Osu与4P-RGD3的多肽三聚体进行相连接,通过制备程序而获得配体HYNIC-4P-RGD3,并利用配体中的HYNIC和99mTc的螯合作用,而制成RGD多肽三聚体的整合素αvβ3肿瘤显像药物:99mTc-4P-RGD3。
【附图说明】
图1显示本发明实施中标记前体化合物HYNIC-4P-RGD3的合成路线示意图及化学结构。
图2显示本发明实施中99mTc标记的肿瘤显像药物99mTc-4P-RGD3合成路线示意图及化学结构。
图3显示本发明实施中99mTc-4P-RGD3的放射性HPLC图谱。
图4显示本发明实施中99mTc-4P-RGD3(n=7)、99mTc-3P-RGD2(n=6)及99mTc-半乳糖RGD2(n=8)在全身器官中,於60分钟后的生物分布数据比较结果。
图5显示本发明实施中负U87MG胶质瘤裸鼠于注射99mTc-4P-RGD3(约37MBq)后的3D及横切面SPECT/CT显像结果。
图6显示99mTc-4P-RGD3注射前在生理盐水的放射性HPLC图谱,注射后30分钟尿液样本的放射性HPLC图谱,以及注射120分钟后尿液样本及粪便样本的放射性HPLC图谱。
【具体实施方式】
RGD多肽三聚体单光子放射药物制备方法:
一.材料和仪器:使用已经纯化的化学试剂均向美国的Sigma/Aldrich公司购买(St.Louis,MO)。RGD多肽三聚体(4P-RGD3)是向美国的Peptides International,Inc公司专门定制(Louisville,KY)。HYNIC-Osu是根据文献所记载的方法进行配制。MALDI(matrix-assisted laser desorption ionization)质谱数据通过Applied BiosystemsVoyager DE PRO质谱仪(Framingham,MA)进行采集。99mTcO4 -通过贩卖药商取得。
二.HPLC(高效液相色谱)分析方法:
1.方法1:半制备性高效液相色谱(HPLC)分析方法所使用的配备为含紫外光检测器(波长=254nm)的LabAlliance高效液相色谱分析***和Zorbax C18半制备色谱柱(9.4nm×250mm,孔径大小,Agilent Technologies,Santa Clara,CA)。梯度洗脱条件如下:流速为2.5mL/分钟,起始流动相为90%的溶液A(0.1%TFA的水溶液)和10%的溶液B(0.1%TFA的乙腈溶液)从0-5分钟时变成80%的溶液A和20%的溶液B,在20分钟时变成50%的溶液A和50%的溶液B。
2.方法2:放射性高效液相色谱(radio-HPLC)分析方法使用LabAlliance HPLC***,此***配备β-Ram IN/US探测器(Tampa,FL)和Zorbax C18圆柱(4.6mm×250mm,孔径大小;Agilent Technologies,Santa Clara,CA)。梯度洗脱条件如下:流速为1mL/分钟,从0至5分钟起始流动相为90%的溶液A(25mM的醋酸铵溶液)和10%溶液B(乙腈溶液),在5至20分钟接着用梯度流动相将溶液B的溶度从10%提高到60%。
三.HYNIC-4P-RGD2标记物前体化合物的制备方法:
将13.5mg HYNIC-Osu(30.0μmol)和9.0mg 4P-RGD2(3.0μmol)溶解在2.0mL的二甲基甲酰胺(DMF)中。滴加过量的二异丙基乙胺(DIEA)(5滴)后,在室温下将混合物持续搅拌直至反应完成(约24小时)。向该混合物中加入2.0mL水并使用纯TFA将pH值调整到3-4范围。该产物经由HPLC分离纯化(HPLC方法1),收集保留时间约18分钟的组分。收集液体经冷冻干燥后得到目标产物为6.5mg(约50%)的HYNIC-4P-RGD2白色粉末,化学结构如图1,(MALDI-MS):m/z[C148H227N35O48S]的分子量=3295.8240。
四.99mTc-4P-RGD2的制备方法:
将含有20-25μg的HYNIC-4P-RGD3冻干小瓶注入5mg TPPTS、6.5mg甘氨酸、40mg甘露糖醇、38.5mg琥珀酸二钠六水合物、12.7mg的琥珀酸溶液及1.0-1.5ml的Na99mTcO4溶液(具1,110-1,850MBq活度的生理盐水)。将上述反应液的小瓶在沸水浴中加热10-20分钟,然后将静置在室温下约5分钟,可得99mTc-4P-RGD2溶液样品(化学结构如图2),以radio-HPLC方法(HPLC方法2)分析其放射化学纯度。从99mTc-4P-RGD2的放射性HPLC图谱(图3)可见99mTc-4P-RGD2具备高放射化学纯度的特性(纯度>90%),因此可用于生物分布和肿瘤显像等相关研究。
五.生物分布及肿瘤显像研究的99mTc-4P-RGD2剂量制备:
生物分布研究的99mTc-4P-RGD2剂量制备是将99mTc-4P-RGD3溶液加入生理盐水调整至10-30Ci/mL的活性浓度。对于肿瘤显像的剂量制备是将99mTc-4P-RGD2溶液加入生理盐水至约10mCi/mL的活性浓度。实验时每只动物的注射剂量约0.1mL的溶液。
六.动物模型:
生物分布和肿瘤显像研究是根据符合美国国家卫生研究院动物实验的指导方针进行(出自于1985年修订,NIH出版号86-23,实验动物护理)。U87MG细胞购置于美国ATCC公司(Manassas,VA),细胞培养条件如下:以含有10%胎牛血清和1%青霉素和链霉素的DMEM培养基(非必需氨基酸丙酮酸钠),在含有5%CO2的条件下37℃培养,当细胞生长至90%融合状态时开始持续倍数增长,细胞生长为单层、多层或分割层。使用4-5周的雌性无胸腺nu/nu裸鼠,在无菌条件下将5×106肿瘤细胞加入0.1mL的生理盐水种植在其肩背部皮下。细胞种植约4周后,肿瘤体积生长至0.1-0.5g时用于进行生物分布和显像的研究。
七.生物分布研究:
将7只随机选择的负U87MG胶质瘤无胸腺裸鼠(20-25g),每一只裸鼠以尾静脉注射约3μCi的99mTc-4P-RGD3,在60钟后分别以大量的戊巴比妥钠(约200mg/kg)将动物处死,并且从小鼠心脏中取得血液样本,收集肿瘤和正常器官(脑,眼睛,心脏,脾,肺,肝,肾,肌肉和肠)及肿瘤的组织,再用盐水洗涤,用吸水薄纸吸干、用Perkin Elmer Wizard 1480γ计数器(Shelton,CT)计算重量,器官摄取计算为每克组织注射剂量百分数(%ID/g)。下列表格直接比较99mTc-4P-RGD2及现有己被开发药物99mTc-Galacto-RGD2与99mTc-3P-RGD2在负U87MG胶质瘤无胸腺裸鼠体内注射后60分钟的生物分布结果。
八.图4绘出99mTc-4P-RGD3(n=7)、99mTc-3P-RGD2(n=6)和99mTc-半乳糖-RGD2(n=8)在全身器官注射后60分钟的生物分布数据比较结果。注射后60分钟,99mTc-4P-RGD3(7.34±1.66%ID/g)的肿瘤摄取与99mTc-3P-RGD2(7.24±0.95%ID/g)和99mTc-半乳糖-RGD2(6.86±1.33%ID/g)非常相似,然而99mTc-4P-RGD3在全身正常器官:如肠、肝、肺、肌肉、脾脏、肾脏中的攝取本底值顯著降低,因此99mTc-4P-RGD3可产生比99mTc-3P-RGD2和99mTc-半乳糖-RGD2更高的全身肿瘤图像质量,对于全身肿瘤的诊断应用可提供更高的优势。
九.图5显示负U87MG胶质瘤裸鼠于注射99mTc-4P-RGD3(约37MBq)后的3D及横切面SPECT/CT显像结果。于99mTc-4P-RGD3注射后发现动物腹部的放射性累积为最小(特别是在肠道内),其肿瘤摄取在SPECT量化的基础上测量约11.5%ID/cm3,SPECT/CT图像显示肿瘤和背景区具有极佳的对比度,因此肿瘤部位清晰可见。通过SPECT/CT数据可清楚地表明99mTc-4P-RGD3是一种极优的放射性全身肿瘤显像药剂。
十.选用正常小鼠(n=2)来显示99mTc-4P-RGD3在体内的稳定性进行代谢研究,每只动物注射99mTc-4P-RGD3约10MBq,分别在30分钟和120分钟手动挤压膀胱,收集尿液样本,与等体积的50%乙腈水溶液混合,将混合物经8,000rpm离心,收集上层清液并用0.20μm的Millex-LG过滤器进行过滤,滤液以HPLC进行分析。采集注射后120分钟的粪便样本,使用20%乙腈水溶液进行均一化处理,将所得到混合物涡旋约5分钟,同样经8,000rpm离心,收集上层清液并用0.20μm的Millex-LG过滤器进行过滤,滤液以HPLC进行分析。尿样本和粪便样品的放射性回收的百分率>95%(按γ计数计算)。
十一.图6显示了典型的99mTc-4P-RGD3代谢结果,分别为99mTc-4P-RGD3注射前在生理盐水中(A),以及注射后30分钟收集的尿液样本(B)和注射后120分钟收集的尿液样本(C)和粪便样本(D)的HPLC图谱,结果发现超过2小时的研究期间内,没有在尿液样本和粪便样本中检测到99mTc-4P-RGD3的代谢产物,结果显示99mTc-4P-RGD3经过动物体内代谢后仍可由原型方式排出体外。
Claims (2)
1.一种99mTc标记的RGD多肽三聚体放射性药物(99mTc-4P-RGD3),该药物在通过功能螯合剂HYNIC进行放射性核素99mTc标记,其主要特征是采用RGD的多肽三聚体结构和99mTc结合,从而提高了药物在肿瘤靶点的摄取及明显降低了药物在动物全身非靶器官中的放射性本底,从而提高了全身肿瘤显像的图像质量。
2.权利要求1中所述99mTc标记的RGD多肽三聚体放射性药物的制备方法步骤如下:
a.HYNIC-4P-RGD3标记物前体化合物的制备方法
将HYNIC-Osu(13.5mg,30.0μmol)和4P-RGD3(9.0mg,3.0μmol)溶解在2.0mL的二甲基甲酰胺(DMF)中;滴加过量的二异丙基乙胺(DIEA)(5滴);在室温下将混合物搅拌直至反应完成(约24小时);向该混合物中加入2.0mL水,用纯三氟乙酸(TFA)将pH值调整到3-4范围;产物经由HPLC分离纯化,收集保留时间约18分钟的组分;液体经冷冻干燥后得到目标产物为6.5mg(约50%)HYNIC-4P-RGD3白色粉末,(MALDI-MS):m/z[C148H227N35O48S]的分子量=3295.8240;
b.99mTc-4P-RGD3的制备方法
将含有20-25μg的HYNIC-4P-RGD3冻干小瓶注入5mg TPPTS、6.5mg甘氨酸、40mg甘露糖醇、38.5mg琥珀酸二钠六水合物、12.7mg的琥珀酸溶液及1.0-1.5ml的Na99mTcO4溶液(具1,110-1,850MBq活度的生理盐水);将上述反应液在沸水浴中反应10-20分钟;在室温下静置约5分钟,可得99mTc-4P-RGD3溶液样品。
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