CN113150032B - 一种锝-99m标记含异腈的叶酸衍生物及其制备方法和应用 - Google Patents
一种锝-99m标记含异腈的叶酸衍生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[99mTc‑(CNMBFA)6]+配合物及制备方法和应用。通过配体CNMBFA的合成以及[99mTc‑(CNMBFA)6]+配合物的制备两个步骤，得到[99mTc‑(CNMBFA)6]+配合物。该配合物制备简单、放射化学纯度高、稳定性好、其在荷KB肿瘤裸鼠的肿瘤部位有特异性摄取，并且肾脏摄取较低，减少了对肾脏的损伤作用，是性能优良的靶向叶酸受体的新型肿瘤显像剂。
Description
技术领域
本发明属于放射性药物技术领域，特别涉及一种锝-99m标记含异腈的叶酸衍生物及其制备方法和应用。
背景技术
肿瘤是一类由于机体细胞在各种因素的影响下失去控制地持续增殖导致的病变。从发展程度上讲，肿瘤可分为良性肿瘤和恶性肿瘤，恶性肿瘤又称为癌症。目前，癌症已经成为仅次于心脑血管疾病的威胁人类健康的第二大杀手。近几年来，核医学显像具有简便、灵敏、特异、安全、无损伤、易于重复等优点，可以检测体内的分子生物学行为以及病理生理的变化，已经成为肿瘤诊断的一大类检测手段。随着单光子发射计算机断层成像术(Single-Photon Emission Computed Tomography，SPECT)和正电子发射断层成像术(Positron Emission Tomography，PET)的广泛使用，放射性核素肿瘤显像已经成为核医学诊断的优势之一。
叶酸受体(FR)是一种通过聚糖磷酯酰肌醇连接在细胞膜上的糖蛋白，平均分子量为38kDa，主要包括α，β和γ三种亚型。叶酸受体，特别是叶酸α受体在健康细胞中的表达高度保守，但是在许多源于上皮组织的恶性肿瘤，如卵巢癌、乳腺癌、子宫内膜癌、肺癌和鼻咽癌等肿瘤细胞中却高度表达，因此叶酸α受体成为国内外放射性靶向药物的研究热点，用于实现对其高度表达的肿瘤的诊断和治疗。根据研究发现，目前报道的靶向叶酸受体的放射性药物，虽然肿瘤部位中有较高摄取，能够清晰识别肿瘤的位置，但是这些药物在肾脏中的摄取很高，导致肾脏接受的辐照吸收剂量过高，使肾脏受到损伤，并且多数药物制备均需要高效液相色谱纯化，上述因素限制了这些药物在临床上的广泛应用。
放射性核素99mTc具有良好的核素性质，半衰期为6.02h，发射140keV的γ射线，且具有配位化学多样性的优点，廉价易得，是目前使用最广泛的SPECT显像核素。因此，临床上常使用99mTc的配合物进行某些疾病的诊断，占核医学SPECT显像的90％以上。异氰基(-NC)属于强配位基团，可以与99mTc(Ⅰ)形成六配位的稳定99mTc配合物。由于配合物中含有6个异氰基配体分子，可增强标记物的靶向性。
综上所述，为研发靶向性好，肾脏摄取低且制备不需要纯化的99mTc标记叶酸受体显像剂，本发明通过合成含异腈的叶酸衍生物，对其进行99mTc标记来探求新型性能优良的靶向FR显像剂，具有重要的科学意义和广阔的临床应用前景，也是本领域面临的重要任务。
发明内容
本发明的目的是提供一种制备简便、高放射化学纯度且特异性靶向FR的一种锝-99m标记含异腈的叶酸衍生物，同时提供其制备方法。
为了实现上述目的，本发明采用以下技术方案：一种锝-99m标记含异腈的叶酸衍生物，简写为：[99mTc-(CNMBFA)6]+配合物，其结构如下式所示：
该结构式中：以99mTc+核为中心核，CNMBFA配体分子中异腈的碳原子与99mTc配位形成六配位的[99mTc-(CNMBFA)6]+配合物。n为大于或等于0的整数，m为大于或等于0的整数。
本发明还提供[99mTc-(CNMBFA)6]+配合物的制备方法，其制备步骤如下：
a:配体CNMBFA的合成:
称取适量化合物1于25mL圆底烧瓶中，加入适量二甲基亚砜(DMSO)溶解，然后加入适量氢氧化钠，氮气保护下加入适量化合物2，黑暗条件下室温反应6h。反应结束后用冷乙醚反复洗涤，真空干燥后得到配体CNMBFA。
具体合成路线为：
b:[99mTc-(CNMBFA)6]+配合物的制备：
将适量的柠檬酸钠、L-半胱氨酸溶于生理盐水中，加入SnCl2·2H2O，调节溶液pH8.0左右，向其中依次加入10ug CNMBFA和新鲜淋洗的Na99mTcO4溶液，100℃下反应20min即得到所述的[99mTc-(CNMBFA)6]+配合物。
通过上述方法制备的[99mTc-(CNMBFA)6]+配合物的放射化学纯度大于90％，为亲水性物质，体外稳定性良好。抑制实验结果显示该配合物在肿瘤部位摄取被明显抑制，表明[99mTc-(CNMBFA)6]+与叶酸受体是特异性结合。其在荷瘤小鼠肿瘤部位有较高的摄取与良好的滞留，肿瘤/肌肉和肿瘤/血比值较好。尤其是其在肾脏摄取较低，注射2h后肾脏摄取值为16.02±0.72ID％/g。而目前进入临床研究的靶向FR的99mTc-EC20(EC20 is a folate-containing peptide consisting in sequence ofpteroic acid(Pte),D-Glu,β-Ldiaminopropionic acid(βDpr),Asp,and Cys.)在注射4h后肾脏摄取值为138±12.4ID％/g(Christopher P.Leamon,Matthew A.Parker,Iontcho R.Vlahov,Le-Cun Xu,JosephA.Reddy,Marilynn Vetzel,andNikki Douglas.Bioconjugate Chemistry,2002,13(6):1200-1210)，因此，[99mTc-(CNMBFA)6]+配合物在保持靶向FR的特性外还显著降低了肾脏摄取，减少了对肾脏的损伤作用，是一种性能优良且便于推广的靶向FR的新型SPECT分子探针，达到了发明目的。
实验表明，[99mTc-(CNMBFA)6]+配合物的性能如下：
1.[99mTc-(CNMBFA)6]+的鉴定
(1)[99mTc-(CNMBFA)6]+配合物薄层色谱(TLC)鉴定
薄层层析色谱(TLC)鉴定：使用双层析体系，体系一用聚酰胺薄膜作为支持体，用乙腈作为展开剂，体系二用快速硅胶层析试纸作为支持体，用丙酮：生盐：1.1(v/v)作为展开剂，测定的层析结果见表1。
表1各组分的层析结果(Rf值)
由上述层析鉴定所测得的标记物的放射化学纯度大于90％。
(2)[99mTc-(CNMBFA)6]+配合物高效液相色谱(HPLC)鉴定
高效液相色谱(HPLC)鉴定：用C18反向柱，SCL-10AVP型高压液相色谱仪，A相为纯水(含0.1％TFA)，B相为乙腈(含0.1％TFA)，梯度为0-5min B相相由10％变为90％，5-20minB相为90％，20-25min B相由90％变为10％，25min B相为10％。进样量为10μL，流速为1mL/min。测定的各组分的保留时间(Rt)分别为：99mTcO4 -：2.95min；[99mTc-(CNMBFA)6]+：9.73min，所得的色谱结果表明有放射性主峰，[99mTc-(CNMBFA)6]+配合物的放射化学纯度大于90％。
2.[99mTc-(CNMBFA)6]+配合物的脂水分配系数的测定
取1.9mLpH 7.4的磷酸盐缓冲液(0.025mol/L)于5mL离心试管中，在离心试管中加入2.0mL正辛醇和0.1mL[99mTc-(CNMBFA)6]+溶液，盖上塞子，涡旋5min，离心5min(5000r/min)。然后分别从有机相和水相中取出3×0.1mL，测定二相的放射性计数，并计算其分配系数P(P＝有机相的放射性活度/水相的放射性活度)，重复三次，测得logP＝-1.72±0.13，表明[99mTc-(CNMBFA)6]+是一种水溶性物质。
3.[99mTc-(CNMBFA)6]+配合物的稳定性测定
将[99mTc-(CNMBFA)6]+配合物分别在室温下放置和37℃小鼠血清中放置4小时后测定其放射化学纯度，结果表明[99mTc-(CNMBFA)6]+配合物在室温下和37℃小鼠血清中放置4小时后放射化学纯度大于90％，说明其体外稳定性良好。
4.[99mTc-(CNMBFA)6]+配合物的体外细胞竞争结合实验
(1)将培养后贴壁生长的KB细胞用胰酶消化后，铺于48孔板中，每孔细胞数为5×104个。置于37℃恒温培养箱中生长24h。
(2)将标记液用相应的培养基稀释为5μCi/mL。将叶酸配制成不同浓度的原液。将48孔板分为8组，每组孔板中分别加入不同浓度的100μL的叶酸原液，100μL稀释后的标记液(5μCi/1mL)和200μL的培养基。置于37℃恒温培养箱中孵育2h。
(3)将细胞板从培养箱取出，吸掉培养基并用含有0.5％BSA的PBS(0.1M，pH 7.4)缓冲盐清洗细胞(2×0.5mL)。将清洗过的细胞用1mL的1M NaOH裂解细胞5min。收集NaOH裂解液于软胶管中，用γ-计数仪测定各个管的计数，并取100μL稀释后标记液作为Total放射性计数。数据用GraphPad Prism 5.0处理，符合非线性拟合Log IC50值。体外细胞竞争结合实验表明，[99mTc-(CNMBFA)6]+配合物与叶酸受体高表达的KB细胞的竞争结合IC50值为1.145nM，表明该配合物与叶酸受体是特异性结合。
5.[99mTc-(CNMBFA)6]+配合物在荷瘤小鼠中的生物分布实验
从荷KB肿瘤裸鼠的尾静脉注射0.10mL[99mTc-(CNMBFA)6]+配合物溶液(约7.4×105Bq)，注射后30和120min时断头处死。取其心、肝、肺、肾、脾、骨、肠、胃、肌肉、血、肿瘤等有关组织和器官，擦净后称重，并在γ-Counter上测其放射性计数，计算各组织的每克百分注射剂量(％ID/g)。每个时相的小鼠数为3只。结果见表2。
表2[99mTc-(CNMBFA)6]+配合物在S180小鼠中的生物分布
从表2可以看出，[99mTc-(CNMBFA)6]+配合物在实验组中肿瘤和肾脏有较好的摄取，而在抑制组中肿瘤和肾脏摄取均降低了30％以上，表明[99mTc-(CNMBFA)6]+配合物与FR是特异性结合。
6.[99mTc-(CNMBFA)6]+在荷KB肿瘤小裸鼠SPECT/CT扫描显像结果
显像实验分为正常组和抑制组。在荷KB肿瘤裸鼠的尾静脉注射[99mTc-(CNMBFA)6]+配合物溶液(约18.5MBq)，其中抑制组提前30min注射0.1ml叶酸溶液(1mg/ml)进行抑制，2小时后，使用异氟烷麻醉。将小鼠俯卧固定，使用SPECT/CT进行显像。SPECT显像结果表明[99mTc-(CNMBFA)6]+在正常组老鼠肿瘤中有明显浓集，除了肾脏中有较高浓集外，其他非靶器官中摄取较低，而在抑制组老鼠肿瘤与肾脏中摄取明显降低，表明其可作为特异性靶向FR的分子探针。
具体实施方式
下面通过实施例详述本发明：一种[99mTc-(CNMBFA)6]+配合物：
a.配体CNMBFA的合成：
称取0.145g(0.3mmol)化合物1于25mL圆底烧瓶中，加入适量二甲基亚砜(DMSO)溶解，然后加入0.130g(0.33mmol)氢氧化钠，氮气保护下加入0.116g(0.36mmol)化合物2(m＝1,n＝1)，黑暗条件下室温反应6h。反应结束后用冷乙醚反复洗涤，真空干燥后得到配体CNMBFA。1H-NMR(400MHz,DMSO)δ(ppm):δ8.59(m,1H),7.70–7.48(m,2H),7.23(m,5H),6.66–6.52(m,1H),4.76(s,2H),4.49–4.36(m,1H),3.29(s,4H),3.06(s,4H),1.92–1.52(m,2H),1.04(d,J＝6.9Hz,2H)；IR(KBr)/cm-1:2150.72(-NC)；HR-MS(ESI)for C31H33N10O6:found641.2583,calcd 641.2579.
b.[99mTc-(CNMBFA)6]+配合物的制备
将2.6mg柠檬酸钠、1mg L-半胱氨酸溶于适量生理盐水中，向其中加入0.10mgSnCl2·2H2O，调节溶液pH 8.0左右，向其中依次加入10ug CNMBFA和新鲜淋洗的Na99mTcO4溶液，100℃下反应20min即得到所述的[99mTc-(CNMBFA)6]+配合物。
Claims (3)
1.一种99mTc标记含异腈的叶酸衍生物，结构通式为[99mTc-(CNMBFA)6]+，
其结构式为：
该结构式中：以99mTc+核为中心核，CNMBFA配体分子中异腈的碳原子与99mTc配位形成六配位的[99mTc-(CNMBFA)6]+配合物；n为大于或等于0的整数，m为大于或等于0的整数。
2.如权利要求1所述一种99mTc标记含异腈的叶酸衍生物的制备方法，其工艺步骤如下：
a:配体CNMBFA的合成:
称取适量化合物1于25mL圆底烧瓶中，加入适量二甲基亚砜溶解，然后加入适量氢氧化钠，氮气保护下加入适量化合物2，黑暗条件下室温反应6h；反应结束后用冷乙醚反复洗涤，真空干燥后得到配体CNMBFA；
具体合成路线为：
b:[99mTc-(CNMBFA)6]+配合物的制备：
将适量的柠檬酸钠、L-半胱氨酸溶于生理盐水，加入适量的SnCl2·2H2O，调节溶液pH8.0左右，向其中依次加入适量的配体CNMBFA和新鲜淋洗的Na99mTcO4溶液，沸水浴下反应20min即得到所述的[99mTc-(CNMBFA)6]+配合物。
3.如权利要求1所述一种99mTc标记含异腈的叶酸衍生物在制备靶向叶酸受体肿瘤显像药物中的应用。
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