CN108743975A - 一种靶向肿瘤vegfr-3分子的近红外荧光显像剂的设计、合成及应用 - Google Patents
一种靶向肿瘤vegfr-3分子的近红外荧光显像剂的设计、合成及应用 Download PDFInfo
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Abstract
本发明涉及一种靶向肿瘤VEGFR‑3分子的近红外荧光显像剂的设计、合成和应用,其结构式为ICG‑OSu‑(PEG)n‑G(CGLARGRGC),其中ICG为近红外荧光显像剂吲哚菁绿,ICG‑OSu为磺酸基吲哚菁绿活化脂,环状多肽G(CGLARGRGC)的核心LARGR为靶向VEGFR‑3分子的多肽TMVP1,为羧基反应性,两者通过聚乙二醇(PEG)桥接,其中n为2到20的整数。本发明提供的近红外分子显像剂,极大地提高了ICG显像宫颈癌、乳腺癌病灶及其淋巴结转移灶的特异性,对以后临床上诊断宫颈癌、乳腺癌以及应用荧光腔镜清除肿瘤转移淋巴结提供了良好指示。
Description
技术领域
本发明涉及医学影像学显像技术领域,特别是在荧光腔镜下恶性肿瘤的精准切除手术。
背景技术
近年来,恶性肿瘤已成为威胁人类健康的首要疾病,2014年《世界癌症报告》中国新诊断癌症病例为307万,占全球总数的21.8%。癌症死亡人数约220万,占全球癌症死亡人数的26.9%。目前实体肿瘤患者治疗方式仍以手术切除为主,辅以化疗、放疗和分子靶向治疗,而影响癌症患者治疗效果和预后最为关键的是早发现、早诊断和早干预。现有的CT/MR/B超等影像学检查已经很好的应用于肿瘤的诊断,但其诊断的准确度取决于医生是否具有丰富的经验和扎实的理论知识,并且其鉴别诊断良恶性肿瘤仍然存在着不足之处。因此,寻找到一种特异的诊断方法或诊断剂依然是每一位科研工作者不懈追求的目标。
肿瘤新生淋巴管的形成是促进肿瘤细胞播散和淋巴结转移关键的环节,特别是转移方式以淋巴结转移为主的宫颈癌、乳腺癌和胃癌等。临床上乳腺癌和宫颈癌等肿瘤患者常规的要进行淋巴结清扫,以提高患者的生存率、决定患者术后的化疗的疗程与方案和评估患者的预后。大量的研究证明在肿瘤细胞转移到淋巴结前,淋巴结微环境已经发生改变促进肿瘤细胞的迁移(种子土壤学说),其中淋巴结内微环境VEGFC大量增加促进大量新生淋巴管的形成。基于新生淋巴管在肿瘤转移及播散的重要性,科学家们希望研发出一种针对肿瘤新生淋巴管的显像剂。VEGFR-3蛋白是一种酪氨酸激酶受体(FLT4),其配体为VEGF-C和VEGF-D,在胚胎的早期发育VEGFR-3在所有的内皮细胞均表达,但是随着胚胎的成熟,其表达仅局限在淋巴管内皮细胞。近年来多项研究证实VEGFR-3在新生淋巴管的生成和维持中起到重要的作用,并与肿瘤的增殖及侵袭转移直接相关,可作为淋巴管转移标志物之一,肿瘤转移的前哨淋巴结和微量肿瘤转移淋巴结高度表达均表明VEGFR-3可作为肿瘤诊断治疗的靶向分子。
公开号为CN103804470A的申请中,利用鞭毛噬菌体肽库筛选出了对VEGFR-3具有亲和性的含有五个氨基酸的肽段LARGR(命名为TMVP1)。通过前期的实验表明,这个五个氨基酸的肽段对淋巴管具有高亲和性,有作为分子靶向诊断剂的潜质,但是对于具体如何应用到肿瘤诊断中并未涉及,该五个氨基酸的肽段LARGR在荧光显像中的应用的效果如何也是未知。
吲哚菁绿(Indocyanine Green,ICG),是一种高灵敏近红外荧光素,其已经被美国FDA批准用于临床。其安全性较高,且已有基于ICG开发的荧光腹腔镜应用于临床。ICG静脉注射至人体后,98%-99%的ICG与血浆高分子蛋白(如白蛋白)结合,ICG-蛋白质复合物的激发波长为750-810nm,在近红外区,此区间生物组织的透射性达到最大。欧美国家主要将其用于眼部血管造影、心输出量测定、肝功能评估,近年来有大量关于ICG在肿瘤的诊断、治疗方面的研究。在荧光腹腔镜下将ICG用于宫颈癌、子宫内膜癌的前哨淋巴结显像,增加了前哨淋巴结活检的阳性率。因此ICG是一个很好的前哨淋巴结指示剂,但是其对淋巴结的显像存在明显的局限性,缺乏特异性,无法区分肿瘤转移的淋巴结与炎性增生的淋巴结。
PEG修饰在蛋白及多肽的药物开发中是一项成熟的技术,以增加水溶性、生物相容性和药物作用时间。PEG(聚乙二醇)是一种pH中性,无毒,水溶性较高的亲水聚合物,其重复单元为氧乙烷基,端基为两个羟基,呈线性或支化链状结构。PEG聚合物是迄今为止已知聚合物中蛋白和细胞吸收水平最低的聚合物,由于PEG无毒及良好的生物相容性,PEG已被FDA批准可作为体内注射药用聚合物。当PEG藕联到药物分子或药物表面时,可以将其优良性质赋予修饰后的药物分子,改变它们在水溶液中的生物分配行为和溶解性,在其修饰的药物周围产生空间屏障,减少药物的酶解,避免在肾脏的代谢中很快消除,从而可以有效地延长药物的半衰期,增强药物的稳定性;PEG修饰后的药物水溶性一般均增加,药物通过改变分子结构从而改进了药物动力学和药效等性质,提高了作用部位的血药浓度。但同时PEG引入也会带来新的问题,比如药物系统清除减慢,药物长期滞留体内可能发生中毒现象,以及药物自血液向目标组织的输送速度也会受到限制。因此,如何设计PEG桥接方式以及PEG链的长短对药物的设计至关重要。
临床上对于分期在IA2期及以上的宫颈癌患者无论是否有淋巴结转移,常规进行淋巴结清扫,造成部分患者术后淋巴回流障碍,下肢水肿,影响患者术后的生活质量。因此迫切需要研发一种技术鉴别有无淋巴结转移,进而决定是否进行淋巴结清扫术。阴道镜最主要是用于评估子宫颈细胞学检查结果异常的女性,其检查的目的是通过识别细胞学检查所发现的异常细胞来源,结合阴道镜下直接活组织检查结果、病理类型以及级别进行诊断,并通过确定子宫颈的病变范围来确定适宜的治疗方法。阴道镜的局限性在于不能看到子宫颈管内的病变,也不易鉴别有无间质浸润,在诊断子宫颈病变方面受到一定限制,尤其是绝经后的妇女。因此,在阴道镜下宫颈活检取样时需要有可靠的指示剂来指导取样。ICG连接TMVP1为解决此类问题提供了一种可能。常规的ICG不能直接连接多肽,我们尝试用ICG的衍生物ICG-OSu通过(PEG)n来桥接TMVP1,ICG-OSu为氨基反应性,而TMVP1为羧基反应性,二者分别连接于(PEG)n的两端,此方式ICG连接多肽属于首创。目前,荧光腹腔镜已在临床上得到应用,ICG-OSu-(PEG)n-TMVP1将是具有良好应用前景的肿瘤分子靶向的荧光显像剂。
发明内容
为了克服传统的ICG显像肿瘤特异性低的缺点,本发明提供了一种新型的靶向肿瘤VEGFR-3分子的近红外荧光显像剂ICG-OSu-(PEG4)n-TMVP1,在原有的靶向多肽TMVP1(LARGR)的基础上进行设计改造,获得了结构式为ICG-OSu-(PEG)n-G(CGLARGRGC)的荧光显像剂,利用该显像剂不仅用于肿瘤诊断,还用于荧光腔镜手术中引导病灶的精准切除,用于荧光阴道镜中指导宫颈活检的取样。
本发明的技术方案如下:
TMVP1能特异性靶向于高表达VEGFR-3的肿瘤细胞,将其设计成环状结构Cyclic(CGLARGRGC)G,以增加其稳定性,通过(PEG)n与ICG-OSu共价连接,构建为本发明所示的新的显像剂ICG-OSu-(PEG)n-TMVP1。一方面此显像剂仍保持对肿瘤的靶向特性,能够特异性结合于恶性肿瘤的原位灶及转移灶。另一方面ICG的荧光特性也保持不变,在带有荧光激发器的成像设备下,能够清晰显示荧光图像。PEG两端的活性羟基可以修饰蛋白和多肽,可以改善生物相容性和药物作用时间,但PEG的引入修饰带来新的技术问题,系统清除越慢,药物长期滞留体内可能发生中毒现象,以及药物自血液向目标组织的输送速度也会受到限制。在此处,我们通过大量的细胞毒性实验、体外的靶向性实验,一方面通过设计PEG桥接结构以及PEG链,其一端连接ICG-OSu的氨基,另一端连接TMVP1的羧基。由此设计而来的显像剂水溶性增强,体内循环时间延长,在体内1-2h肝肾聚集最多,在12h以后主要滞留于靶向的肿瘤部位,既安全有效,又利用此时间差有效的去除了非特异性结合所导致的荧光背景。本发明提供的显像剂为一种稳定的化合物,无明显细胞毒性,副作用少,同时在前期的临床实验研究中,局部给药或静脉给药后在任何配置有荧光检测系统的仪器(荧光腹腔镜、荧光阴道镜等)下能够显像,为临床上微小病灶的检出及精准切除提供了新的思路。
具体地本发明提供一种靶向肿瘤VEGFR-3分子的近红外荧光显像剂,其结构式为ICG-OSu-(PEG)n-G(CGLARGRGC),其中ICG为近红外荧光显像剂吲哚菁绿,ICG-OSu为磺酸基吲哚菁绿活化脂,环状多肽G(CGLARGRGC)的核心序列LARGR为靶向肿瘤VEGFR-3分子的多肽TMVP1,聚乙二醇(PEG)桥接,其中n为2到20的整数。优选n为3-10的整数,更优选n为4、5、6、7,最优选n为4。
其中ICG-OSu是ICG的氨基反应性衍生物,环状多肽TMVP1为羧基反应性。
本发明所述的靶向肿瘤VEGFR-3分子的近红外荧光显像剂ICG-OSu-(PEG)n-TMVP1,具体的合成步骤为:1)利用固相载体根据设计的氨基酸序列合成多肽GCGLARGRGC,共价连接至(PEG)n;2)将多肽从树脂上切除下来,用MEOH/I2进行氧化,将多肽序列通过二硫键搭桥形成环状,即(PEG)n-G(CGLARGRGC),然后进行分离、提纯、冻干;3)将步骤2)所得的产品与ICG-OSu按1∶1混合,溶解在DMF里,然后加2倍体积的DIEA,反应10分钟,LCMS鉴定反应完全后,分离、提纯、冻干,得到本发明所述的显像剂ICG-OSu-(PEG)n-TMVP1。
本发明的肿瘤靶向近红外荧光显像剂还可以进一步用于在制备肿瘤诊断试剂中应用,所述肿瘤选自宫颈癌、乳腺癌、卵巢癌、子宫内膜癌、肺癌、前列腺癌、肾癌,优选宫颈癌、乳腺癌。
本发明的肿瘤靶向近红外荧光显像剂在区分肿瘤转移的淋巴结与炎性增生的淋巴结诊断试剂中的应用。本发明的肿瘤靶向近红外荧光显像剂还可以在制备荧光腔镜手术中引导肿瘤病灶及淋巴结转移灶的切除指示试剂中的应用,所述肿瘤为宫颈癌、乳腺癌。
本发明的肿瘤靶向近红外荧光显像剂可以在制备荧光阴道镜中取样指示试剂中应用,其特征在于,所述肿瘤为宫颈癌。
我们检测了本发明所述显像剂ICG-OSu-(PEG)n-TMVP1的理化性质,根据本显像剂的体外细胞结合效率,最优选n为4,通过细胞实验、动物实验检测了ICG-OSu-PEG4-TMVP1的安全性及肿瘤靶向性。所合成的显像剂纯度高,荧光性质与ICG一致。CCK-8试验证明其对肿瘤细胞系、正常细胞系均没有毒性;小鼠的急性毒性实验证明其对小鼠脏器无毒副作用,安全性高;细胞结合实验证明其能与肿瘤细胞特异性结合,效果明显优于ICG;接种乳腺癌细胞的荷瘤小鼠动物实验证明其能与肿瘤组织特异性结合,效果明显优于ICG;乳腺癌肺转移、淋巴转移模型小鼠动物实验证明其能特异性显像肿瘤转移灶。本显像剂目前正在进行II期临床试验(注册号:ChiCTR-INR-17012474),用于宫颈癌术中指导淋巴结的精准清扫,初步表现出优于ICG的特异性指示效应。
与现有的技术相比,本发明的有益效果是:与目前FDA唯一批准用于人体的近红外荧光显像剂ICG相比,ICG-Osu-PEG4-TMVP1在稳定、安全的前提下,显像肿瘤病灶及淋巴转移灶的特异性更好,应用前景更广泛。
附图说明
图1为ICG-OSu-PEG4-TMVP1的合成路线及质量检测图。
图2为ICG-OSu-PEG4-TMVP1的细胞毒性实验结果。
图3为ICG-OSu-(PEG)n-TMVP1与肿瘤细胞结合效率图。
图4为TMVP1-FITC的肿瘤细胞结合实验及竞争性实验。
图5为ICG-OSu-PEG4-TMVP1的体内分布及药代动力学检测结果。
图6为ICG-OSu-PEG4-TMVP1对原位肿瘤的显像。
图7为ICG-OSu-PEG4-TMVP1对肺转移肿瘤的显像。
图8为ICG-OSu-PEG4-TMVP1对淋巴结转移灶的显像。
图9为ICG-OSu-PEG4-TMVP1临床试验中的一例阳性结果。
具体实施方式
以下结合附图对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例1合成ICG-OSu-PEG4-TMVP1并进行相应的质检
参见图1a,先利用多肽固相载体合成PEG-G(CGLARGRGC),再与ICG-OSu反应,得到本发明所示的显像剂。图1b是通过高效液相色谱仪(HPLC,Agilent 1200,美国)检测本显像剂的纯度(>95%)。图1c和1d通过液相质谱和色谱联用(LC-MS,Agilent 1200HPLC&6410Triple Quad,美国)检测证明了本发明所示显像剂的正确合成。
实施例2细胞毒性实验
1.细胞培养
鼠乳腺癌细胞系4T1(购自美国ATCC细胞库)用含有体积分数10%胎牛血清的RPMI-1640培养基(Gibco,ThermoFish公司,美国)培养。鼠胚胎成纤维细胞系3T3(购自美国ATCC细胞库)用含有体积分数10%胎牛血清的DMEM培养基(Gibco,ThermoFish公司,美国)培养。以上细胞均在37℃,含5%CO2的培养箱培养。
2.CCK-8试验
分别将4T1、3T3细胞按照1*104个/孔的密度种96孔板,在37℃,5%CO2培养箱培养24h后,置换入分别含有0,3.125,6.5,12.5,25,50ug/ml ICG-OSu-PEG4-TMVP1的新鲜培养基,每组设置3个副孔。孵育24h后,吸出培养基,换入CCK-8试剂(Dojindo MolecularTechnologies,Japan),孵育2.5h之后,在酶标仪(Biorad,美国)波长450nm下测OD值。细胞活性度计算公式为:viablerate=(ODtreated-ODblank)/(ODcontrol-ODblank)*100%。
实验结果参加图2,无论是癌细胞还是正常细胞,无论药物浓度高低,均对细胞的增殖无明显影响。
实施例3不同PEG数目下肿瘤细胞结合效率实验
合成不同PEG数目的该显像剂,分别取n=2,4,6,8,10,20。如实施例2中所示方法培养4T1细胞,将4T1细胞按照1*105个/孔的密度种24孔板,隔夜培养后,各孔分别加入500ul 1μM的不同PEG数目的ICG-OSu-(PEG)n-TMVP1或ICG,加入PBS的孔作为空白对照,冰上孵育20分钟后,用冷的PBS洗3遍,置于小动物活体成像仪下采集孔板的荧光图片。再通过IVIS显像软件的ROI工具对各孔的荧光值进行定量分析。
实验结果参见图3,图3a为4T1细胞在不同PEG数目的本显像剂孵育后的荧光图像,图3b为各孔荧光强度的定量分析。说明取n=4时,本显像剂与肿瘤细胞具有最佳的结合效率。该实验表明PEG链及其数目将显著影响显像剂与肿瘤细胞的结合效率。
实施例4TMVP1的体外靶向实验
连接绿色荧光FITC的靶向肽TMVP1-FITC委托上海药明康德新药开发有限公司合成,细胞培养参见实施例2,分别将4T1、3T3细胞按1*105个/孔的密度种24孔板,隔夜培养后,用PBS轻柔洗两遍,然后用200ul的4%多聚甲醛固定10min,PBS洗3遍后,加入50μg/ml的TMVP1-ICG冰上孵育1h。对于阻断实验,在加TMVP1-FITC之前提前30min加入100μg/ml的TMVP1。孵育之后这些细胞用PBS洗3遍,然后用DAPI(1μg/m1)染核,再在倒置荧光显微镜(U-HGLGPS,Olympus,Tokyo Japan)下观察。
结果参见图4,图4a说明TMVP1-FITC结合肿瘤细胞4T1明显多于正常细胞3T3,图4b说明此结合效应能够被靶向肽TMVP1阻断。该实验表明TMVP1能特异性结合于肿瘤细胞。
实施例5体内分布和药代动力学实验
正常的BALB/c小鼠(购自北京华阜康生物科技股份有限公司)尾静脉给100uL、25umol/L的ICG-OSu-PEG4-TMVP1,不同时间点(30min、1h、2h、12h、24h、48h)在小动物成像仪(PerkinElmer,美国)下进行显像(ex/em 745/840nm),在各时间点解剖小鼠,摘取主要脏器(心、肝、脾、肺、肾、结肠)进行显像,肌肉组织为对照组织,再通过IVIS显像软件的ROI工具对各时间点各脏器荧光值进行计量每组3只老鼠。
实验结果参见图5,图5a、5b分别为各个时间点小鼠和脏器的典型显像图,图5c为各脏器荧光量化统计图,表1为对应的统计表。说明ICG-OSu-PEG4-TMVP1通过肝脏、肾脏两条途径代谢,给药后30min在肝脏聚集最多,给药后1h在肾脏聚集最多,之后迅速清除。在给药24h后肝脏清除达91.5%,肾脏清除达93.3%。
表1:ICG-OSu-PEG4-TMVP1的体内分布和药代动力学实验结果
heart | liver | spleen | lung | kidney | |
30min | 4.33E+08 | 6.01E+10 | 5.63E+08 | 2.26E+09 | 8.60E+09 |
1h | 5.41E+08 | 5.52E+10 | 6.06E+08 | 2.81E+09 | 9.27E+09 |
2h | 3.43E+08 | 3.34E+10 | 4.45E+08 | 2.27E+09 | 6.57E+09 |
12h | 5.85E+07 | 9.25E+09 | 9.52E+07 | 4.33E+08 | 1.10E+09 |
24h | 2.57E+07 | 5.09E+09 | 5.26E+07 | 1.78E+08 | 6.17E+08 |
48h | 1.12E+07 | 9.03E+08 | 3.22E+07 | 9.69E+07 | 1.15E+08 |
实施例6体内原位肿瘤靶向实验
将鼠乳腺癌细胞系4T1转染荧光素酶(luciferase)慢病毒,使其能通过检测生物素光来实时监测肿瘤的大小。培养扩增4T1-Luc细胞,1×105个/只接种至4周的BALB/c小鼠(购自北京华阜康生物科技股份有限公司)右侧乳房垫,3周后肿瘤生长至直径约1cm,尾静脉给100uL、25umol/L的ICG-OSu-PEG4-TMVP1或者ICG后,在小动物成像仪下显像(30min、1h、2h、12h、24h),解剖摘取肿瘤组织及各脏器显像,通过IVIS显像软件的ROI工具对肿瘤组织的荧光强度进行定量分析,每只小鼠以肿瘤对侧乳房组织为对照。每组3只老鼠。
实验结果参见图6,图6a为生物素光模式下的显像图,说明了乳腺癌原位模型的成功建立,图6b为ICG荧光模式下各个时间点的典型显像图,图6c是两者在不同时间点的典型脏器荧光显像图。图6d为利用ROI工具测量肿瘤组织及对侧乳房的荧光值,用GraphPadPrism 5.0软件对ICG-OSu-PEG4-TMVP1组和ICG组各时间点T/N ratio作的统计图。说明ICG-OSu-PEG4-TMVP1的肿瘤靶向能力明显优于ICG,能够与肿瘤组织特异性结合。
实施例7体内肺转移肿瘤靶向实验
培养扩增4T1-Luc细胞,1×105个/只尾静脉注射至4周的BALB/c小鼠,2周后在小动物成像仪下确认肿瘤肺转移模型的正确建立。尾静脉给100uL、25umol/L的ICG-OSu-PEG4-TMVP1或者ICG后,在小动物成像仪下显像(30min、1h、2h、12h、24h),解剖摘取肿瘤转移肺组织及各脏器显像,通过IVIS显像软件的ROI工具对肿瘤组织的荧光强度进行定量分析。每组3只老鼠。
实验结果参见图7,图7a为生物素光模式下的显像图,说明了乳腺癌肺转移模型的成功建立,图7b为ICG荧光模式下各个时间点的典型显像图,图7c是两者在不同时间点的典型脏器荧光显像图。图7d为利用ROI工具测量肿瘤转移组织及实施例5中正常肺组织的荧光值,用GraphPad Prism 5.0软件对各组小鼠肺组织的荧光强度值作的统计图。说明ICG-OSu-PEG4-TMVP1对肺转移肿瘤的靶向能力明显优于ICG,能够与转移的肿瘤组织特异性结合。
实施例8体内淋巴结转移灶靶向实验
培养扩增鼠乳腺癌细胞系4T1,以4×105个/ml的密度25ul于双下肢脚垫局部接种至4周的BALB/c小鼠,3周后观察淋巴结明显肿大,脚垫肿瘤局部右侧给药20uL、25umol/L的ICG-OSu-PEG4-TMVP1,左侧给药等量的ICG后,在小动物成像仪下显像(30min、60min、2h),显像结束后解剖淋巴结,石蜡包埋切片后进行HE染色,进一步确定肿瘤淋巴结转移模型的建立。
实验结果参见图8,为ICG荧光模式下各个时间点的典型显像图,小鼠右侧(图片中仰卧位,显示为左侧)为ICG-OSu-PEG4-TMVP1,左侧为ICG,说明ICG-OSu-PEG4-TMVP1的靶向肿瘤转移淋巴结的能力明显优于ICG,能够与肿瘤转移淋巴结特异性结合。
实施例9异常毒性实验
选取4周龄BALB/c小鼠(购自北京华阜康生物有限公司)15只,随机分为3组(尾静脉注射组、脚垫局部注射组、对照组),每组5只,试验前和试验的整个观察期均按正常饲养条件饲养,实验前称老鼠的体重。尾静脉注射组每只小鼠注射100uL、250umol/L的ICG-OSu-PEG4-TMVP1,局部注射组每只小鼠脚垫注射25ul、250umol/L的ICG-OSu-PEG4-TMVP1,空白对照不做处理。7天内观察各实验组和对照组小鼠饮食、呼吸、活动、反射、排便、痛觉、皮肤。7天后称每个小鼠的体重,眼眶取血测肝肾功能,处死、解剖小鼠,观察小鼠各脏器颜色、形态,并固定包埋做HE染色。
实验结果:药物处理组小鼠在整个实验过程中,未出现死亡,未发现饮食、呼吸、活动、反射、排便、痛觉、皮肤异常。参见表2药物处理组小鼠同未处理组小鼠实验前后体重变化无明显差异(P>0.05),器官未见明显病变,肝肾功能未见明显差异。各组小鼠脏器做HE染色亦未见异常。
表2:异常毒性实验结果
实施例10安全性实验
1.细菌内毒素检测:根据《中国药典》(2010版)内容,确定供试品内毒素检测水平λ=0.125EU/ml。先进行灵敏度校验实验,确定鲎试剂灵敏度为0.125EU/m。再进行干扰实验,供试品与检测试剂无干扰反应。
实验结果参见表3,凝胶实验检测样品的内毒素水平,证实样品内毒素水平符合要求。
表3:
2.无菌性检测:取本品1mL,在武汉同济医院检验科微生物临床检验组营养培养基培养3天,未见细菌生长。
实施例11在宫颈癌术中的临床应用
将本试剂用于宫颈癌术中荧光腹腔镜下指导淋巴结的清扫,具体的用法为:术前30min宫颈局部(3°、9°)注射给药,现配现用,用注射用无菌水溶解粉剂,1mg/mL,各点分别注射200ul显象剂。对于宫颈癌分期为IIB期的病人,于阴道中上端左右两侧分别加注200ul显象剂。同样剂量的ICG注射液作为对照组。分别观察术中荧光腹腔镜下淋巴结显像情况,并常规行广泛子宫切除术+盆腔淋巴结清扫术,术后参照病理科报告的淋巴活检结果,对比两者对转移淋巴结显像的敏感性和特异性。
其中一例试验结果参见图9,本发明所述的显像剂能清晰的显像宫颈癌转移阳性的淋巴结和淋巴管。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 武汉凯德维斯生物技术有限公司
<120> 一种靶向肿瘤VEGFR-3分子的近红外荧光显像剂的设计、合成及应用
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 10
<212> PRT
<213> 人工序列()
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<221> DISULFID
<222> (2,10)
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Gly Cys Gly Leu Ala Arg Gly Arg Gly Cys
1 5 10
Claims (8)
1.一种靶向肿瘤VEGFR-3分子的近红外荧光显像剂,其特征在于,结构式为ICG-OSu-(PEG)n-G(CGLARGRGC),其中ICG为近红外荧光显像剂吲哚菁绿,ICG-OSu为磺酸基吲哚菁绿活化脂,是ICG的氨基反应性衍生物,环状多肽G(CGLARGRGC)的核心LARGR为靶向VEGFR-3分子的多肽TMVP1,为羧基反应性,两者通过聚乙二醇(PEG)桥接,其中n为2到20的整数。
2.根据权利要求1所述一种靶向肿瘤VEGFR-3分子的近红外荧光显像剂,其特征在于,优选n为3-10的整数,更优选n为4、5、6、7,最优选n为4。
3.根据权利要求1所述的肿瘤靶向近红外荧光显像剂,其特征在于其制备方法包括以下步骤:
1)利用固相载体合成多肽GCGLARGRGC,连接至(PEG)n;
2)将多肽从树脂上切除下来,用MEOH/I2进行氧化,将多肽序列通过二硫键搭桥形成环状,即(PEG)n-G(CGLARGRGC),然后进行分离、提纯、冻干;
3)将步骤2)所得的产品与ICG-OSu按1∶1混合,溶解在DMF里,然后加DIEA反应,LCMS鉴定反应完全后,分离、提纯、冻干,即得显像剂。
4.根据权利要求1-3任意一项所述的肿瘤靶向近红外荧光显像剂在制备肿瘤诊断试剂中应用。
5.根据权利要求4所述的应用,其特征在于,所述肿瘤选自宫颈癌、乳腺癌、卵巢癌、子宫内膜癌、肺癌、前列腺癌、肾癌。
6.根据权利要求1-3任意一项所述的肿瘤靶向近红外荧光显像剂在区分肿瘤转移的淋巴结与炎性增生的淋巴结诊断试剂中的应用。
7.根据权利要求1-3任意一项所述的肿瘤靶向近红外荧光显像剂在制备荧光腔镜手术中引导肿瘤病灶及淋巴结转移灶的切除指示试剂中的应用,其特征在于,所述肿瘤为宫颈癌、乳腺癌。
8.根据权利要求1-3任意一项所述的肿瘤靶向近红外荧光显像剂在制备荧光阴道镜中取样指示试剂中的应用,其特征在于,所述肿瘤为宫颈癌。
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