新型双功能金属螯合剂及在PET成像中的应用

发布时间：2018-07-31 08:57

【摘要】：目的近年来,放射性药物的发展十分迅速,尤其是正电子发射成像(PET)诊断药物和放射免疫治疗药物。双功能金属螯合剂作为连接放射性金属核素与功能性配体的纽带,在放射药物的研究中发挥着重要的作用。64Cu和68Ga是科研和临床中两种常用的金属核素,目前已有多种双功能金属螯合剂可用于对二者的标记。然而这些螯合剂通常需要较为苛刻的标记条件,且对放射性金属的选择性低,放射比活度低等,严重限制了新型放射性药物的研究。功能性配体是研究放射药物的另一个重要的因素。受制于传统单价的/单靶向和单模态的分子探针的固有属性,很难应对肿瘤等疾病的复杂性和多样性。近年来,多价的和多模态的新型分子探针由于它们较高的受体亲和力和相互补充的成像能力等优点而备受关注。然而它们的制备过程常常涉及到繁杂的保护和脱保护、多次HPLC纯化和较低的产率等问题,严重的制约了这些分子探针的应用和发展。针对这些问题,有必要设计并合成出一些具备标记条件温和,所得标记物稳定性高、放射比活度高等性质的双功能金属螯合剂,并以之为平台应用于放射性药物的研究中,这也是本研究的主要目的之一。本研究另一个主要的期望是设计并合成出一个基于双功能金属螯合剂结构的通用平台来简化多价的和多模态的分子探针的合成过程,从而促进这一领域的发展。方法设计并合成三种基于NE3TA结构的双功能螯合剂,并将其与多肽LLP2A偶联并分别进行64Cu和6SGa标记,测试其放射比活度、血浆稳定性等性质。利用LC-MS研究p-NO2-PhPr-NE3TA对Cu2+螯合的选择性。测试被64Cu和68Ga标记的分子探针在B16F10细胞中的摄取及流出情况。建立荷B16F10肿瘤小鼠模型,并将被64Cu和6SGa标记的分子探针应用于该模型的肿瘤靶向PET/CT显像。接着设计并合成具有两个不同偶联功能基团的双功能金属螯合剂NOTA2tBu-N3,然后利用该平台将靶向uPAR的多肽AE105及靶向整合素αvβ3受体的c(RGDyk)偶联,从而制备具有不同长度PEG连接基团的多肽异源二聚体分子探针。将各异源二聚体分子探针进行64Cu标记后,通过细胞实验,筛选出一个性质较好的多肽异源二聚体分子探针,并应用于荷U87MG肿瘤小鼠的PET/CT成像。其结果与它相应的单聚体分子探针64Cu-AE105及64Cu-RGD的成像结果进行比较。另外,利用NOTA2tBu-N3为平台,通过点击化学或者固相合成的方法制备其他类型的分子探针,比如多肽同源二聚体和PET/光学多模态分子探针等。结果即使在十倍量Fe(Ⅲ)存在的情况下,p-NO2-PhPr-NE3TA依然能优先与Cu(Ⅱ)进行螯合,显示出对Cu(Ⅱ)较高的选择性。其多肽偶联物NE3TA-LLP2A可在室温下被64Cu标记,并具备良好的血浆稳定性,同时其放射性比活度值达3～4mCi/nmol,远高于NOTA-LLP2A的～1 mCi/nmol。虽然所合成的另外两个双功能金属螯合剂与64Cu的标记结果不理想,但p-SCN-PhPr-NE2P1A表现出优秀的68Ga标记属性。其偶联物NE2P1A-LLP2A可在室温、中性的pH下在15 min内完成6SGa标记,且具备较好的血浆稳定性；而在相同的条件下NOTA-LLP2A的68Ga标记率低。64Cu-NE3TA-LLP2A和68Ga-NE2P1A-LLP2A在荷B16F10小鼠的生物分布结果类似,均在肿瘤部位有较高的摄取,且肿瘤/肌肉比值高。64Cu-NE3TA-LLP2A的肿瘤/肌肉比值在2h,4h和24 h分别达到5.81±0.99、10.50±2.02和25.19±7.78。68Ga-NE2P1A-LLP2A的肿瘤/肌肉的比值在1 h,2 h和4h分别达到6.66±1.87、10.62±2.98和10.25±1.45。将二者用于同一肿瘤模型的PET/CT成像中时,在注射64Cu-NE3TA-LLP2A后的2h,4h和注射68Ga-NE2P1A-LLP2A后的1 h,2 h均能明显分辨出肿瘤。同时注射了阻断剂量的LLP2A的阻断组小鼠的生物分布及PET/CT成像均充分体现了B16F10肿瘤对LLP2A摄取的特异性。利用NOTA2tBu-N3平台,方便地合成出四个多肽异源二聚体分子探针AE105-PEGn-NOTA-PEG4-RGD(n=0,4,8,12,分别简写为T1, T2, T3, T4),一个多肽同源二聚体分子探针(AE105-NOTA-PEG4-AE105)及两个PET/光学双模态分子探针(AE 105-NOTA-nonclick-Cy3和AE105-NOTA-click-Cy5)。所有分子探针均能与64Cu在放射比活度～1 mCi/nmol下进行标记,且标记后在人血浆中稳定性较好。细胞实验表明,随着PEG链的增长,各分子探针在U87MG细胞中的摄取率有逐渐增加的趋势,顺序为T4T3T2≥T1,而与U87MG细胞结合的稳定程度顺序为T3T2≥T1T4。在荷U87MG裸鼠模型的生物分布试验中,64Cu-T3在肿瘤组织中摄取比较高,其肿瘤/肌肉比值在1 h,4 h,24 h分别为4.37±1.46,12.69±3.08,和15.47±3.32。PET/CT成像实验结果显示,64Cu-T3在肿瘤中的摄取率显著高于64Cu-RGD和64Cu-AE105。64Cu-T3的肿瘤/肌肉比值显著高于64Cu-AE105,与64Cu-RGD接近。结论及创新点设计并合成了多个新颖的双功能螯合剂,其中成功p-SCN-PhPr-NE3TA表现出对Cu(Ⅱ)较高的选择性,且与64Cu标记的条件温和,放射比活度高。所合成的p-SCN-PhPr-NE2P1A是一个很好的68Ga螯合剂,可以应用于温度和酸敏感的生物分子的68Ga标记。64Cu-NE3TA-LLP2A和68Ga-NE2P1A-LLP2A均对B16F10肿瘤具有出色的靶向性,在黑色素瘤的诊断中具有很好应用前景。我们也成功的合成了一个基于双功能螯合剂结构的合成平台,可极大的简化多肽异源二聚体等分子探针制备过程。与其它平台比较,我们所合成的这个平台具有多重优势,包括简单的合成(无需大量的保护和脱保护及反应条件优化过程)、简单的纯化过程(需要更少的或只需一次色谱分离),更高的产率。更重要的是,这个平台还可以用于制备多肽同源二聚体、PET/光学双模态等分子探针,用途广泛。多肽异源二聚体分子探针64Cu-T3展示了比其单聚体分子探针更高的肿瘤摄取率以及更好的体内肿瘤成像效果。总而言之,这个平台提供了一条通用和可靠的方法用于制备多模式的分子探针,并且可以作为一个模块化的平台用于多模式分子探针的结构优化或者日常的临床前/临床研究。
[Abstract]:Objective in recent years, the development of radiopharmaceuticals is very rapid, especially positron emission imaging (PET) for the diagnosis of drugs and radioimmunotherapies. Double functional metal chelating agents as a link between radionuclides and functional ligands, the important role of.64Cu and 68Ga in the study of radiopharmaceuticals is scientific and clinical. There are two common metallic nuclides. There are a variety of double functional metal chelating agents that can be used to mark the two. However, these chelating agents usually require more stringent labeling conditions, low selectivity to radioactive metals and low radiological activity, which seriously restrict the study of new radiative drugs. Functional ligands are the study of radiation drugs. Another important factor in objects is the inherent properties of the traditional monovalent / single target and single modal molecular probes, which are difficult to cope with the complexity and diversity of cancer and other diseases. In recent years, multivalent and multimodal molecular probes have been closely related to their higher receptor affinity and complementary imaging capabilities. However, their preparation processes often involve complex protection and deprotection, multiple HPLC purification and low yield, which seriously restrict the application and development of these molecular probes. It is necessary to design and synthesize some mild labeling conditions, high stability and high radioactivity. One of the main expectations of this study is to design and synthesize a common platform based on the structure of a bifunctional metal chelating agent to simplify the synthesis of multivalent and multimodal molecular probes. In order to promote the development of this field, three kinds of double functional chelating agents based on NE3TA structure were designed and synthesized, and they were coupled to the peptide LLP2A and were labeled with 64Cu and 6SGa respectively. The properties of their radioactivity and plasma stability were tested. LC-MS was used to study the selectivity of p-NO2-PhPr-NE3TA for Cu2+ chelation. The test was tested by 64Cu and 68Ga standard. The uptake and outflow of molecular probes in B16F10 cells were recorded. A B16F10 tumor bearing mouse model was established and the molecular probes labeled by 64Cu and 6SGa were applied to the tumor targeting PET/CT imaging of the model. Then, a dual functional metal chelating agent, NOTA2tBu-N3, with two different coupling functional groups, was designed and synthesized, and then the platform was used to make use of the platform. The polypeptide AE105 of the target uPAR and the C (RGDyk) of the target integrin alpha v beta 3 receptor were coupled to prepare a polypeptide heterogenous two polymer molecular probe with different length PEG connecting groups. After 64Cu labeling of the heterogenous two polymer molecular probes, a polypeptide heterologous two polymer molecular probe with good properties was screened and should be screened. PET/CT imaging for U87MG tumor bearing mice. The results were compared with the imaging results of its homopolymer molecular probe 64Cu-AE105 and 64Cu-RGD. In addition, using NOTA2tBu-N3 as a platform, other types of molecular probes were prepared by clicking on chemical or solid phase synthesis, such as polypeptide homopolymer two and PET/ optical multimodality Molecular probe, and so on. Even in the presence of ten times Fe (III), p-NO2-PhPr-NE3TA still preferred to chelate with Cu (II) and showed a high selectivity to Cu (II). Its polypeptide coupling NE3TA-LLP2A could be labeled at room temperature by 64Cu, and had good plasma stability, and its radioactivity value was 3 to 4mCi/nmol, Far higher than NOTA-LLP2A to 1 mCi/nmol., although the other two other bifunctional metal chelating agents synthesized were not ideal with 64Cu, p-SCN-PhPr-NE2P1A showed excellent 68Ga marker properties. The coupling agent NE2P1A-LLP2A could complete 6SGa marking at room temperature, neutral pH in 15 min, and had better plasma stability; Under the same conditions, the 68Ga marker rate of NOTA-LLP2A was low,.64Cu-NE3TA-LLP2A and 68Ga-NE2P1A-LLP2A were similar in the biological distribution of B16F10 mice, all of which were higher in the tumor site, and the tumor / muscle ratio of high.64Cu-NE3TA-LLP2A / muscle ratio in 2H, 4H and 24 h was 5.81 + 0.99,10.50 + 2.02 and 25.19 + 7.78.68Ga. The tumor / muscle ratio of -NE2P1A-LLP2A was 1 h, 2 h and 4H reached 6.66 + 1.87,10.62 + 2.98 and 10.25 + 1.45. for the same PET/CT imaging in the same tumor model. The tumor was clearly identified by the 2H, 4h, and 1 h after the injection of 68Ga-NE2P1A-LLP2A 64Cu-NE3TA-LLP2A, and 2. The biological distribution and PET/CT imaging of the block mice fully reflect the specificity of the B16F10 tumor to LLP2A uptake. Using the NOTA2tBu-N3 platform, four polypeptide heterogenous two polymer probes AE105-PEGn-NOTA-PEG4-RGD (n=0,4,8,12, respectively, T1, T2, T3, T4), a polypeptide homologous two polymer molecular probe (AE105-NOT) A-PEG4-AE105) and two PET/ optical dual mode molecular probes (AE 105-NOTA-nonclick-Cy3 and AE105-NOTA-click-Cy5). All molecular probes can be labeled with 64Cu at the radiological specific activity to 1 mCi/nmol, and the stability is better in human plasma. Cell experiments show that with the increase of PEG chain, the molecular probes are in U87MG cells. The uptake rate has a gradual increase in the order of T4T3T2 > T1, and the stability of the binding with U87MG cells is T3T2 > T1T4. in the biodistribution test of the nude mice bearing U87MG. The uptake of 64Cu-T3 in the tumor tissues is higher, and the ratio of the tumor / muscle is 1 h, 4 h, and 24 h is 4.37 + 1.46,12.69 + 3.08, and 15.47 + 3.32.PET/CT. The results of the experiment showed that the uptake rate of 64Cu-T3 in tumor was significantly higher than that of 64Cu-RGD and 64Cu-AE105.64Cu-T3. The ratio of tumor / muscle was significantly higher than that of 64Cu-AE105 and was close to 64Cu-RGD. Conclusion and innovation point design and synthesis of several novel double functional chelating agents, of which the successful p-SCN-PhPr-NE3TA showed high selectivity to Cu (II), and The conditions of the 64Cu markers are mild and the radioactivity is high. The synthesized p-SCN-PhPr-NE2P1A is a good 68Ga chelating agent, which can be applied to the 68Ga markers of temperature and acid sensitive biomolecules.64Cu-NE3TA-LLP2A and 68Ga-NE2P1A-LLP2A, both of which have excellent targeting of the B16F10 tumor, and have a good prospect in the diagnosis of melanoma. We have also successfully synthesized a biosynthesis platform based on the structure of a dual functional chelating agent, which can greatly simplify the preparation of molecular probes such as polypeptides, heterogenous two polymers. Compared with other platforms, the platform has multiple advantages, including simple synthesis (without a large amount of protection and deprotection and optimization of reaction conditions. A simple purification process (requiring less or only one chromatographic separation), higher yield, and more importantly, the platform can also be used to prepare polypeptide homooligomers, PET/ optical dual mode and other molecular probes, widely used. The polypeptide heteropolymer molecular probe 64Cu-T3 exhibits a higher tumor uptake than its monopolymer molecular probe. In summary, this platform provides a general and reliable method for preparing multimodal molecular probes, and can be used as a modular platform for structural optimization of multi mode molecular probes or daily pre bed / clinical studies.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R981

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