Novel Bifunctional Chelators And Their Applications In PET Imaging

ObjectiveIn the past decades, radiopharmaceuticals, especially emission tomography (PET) and radioimmunotherapy (RIT), have been growing very fast. Bifunctional chelators (BFCs), which connect metallic radionuclide and functional agents, play an important role in the development of radiopharmaceuticals. Various BFCs have been investigated during past decades. However, harsh labeling conditions such as high temperature and long labeling time are usually required and they surfferred from the low specific chelation with corresponding radiometal and the low specific activity. Functional agents are another important factor to radiopharmaceuticals. Given the inherent properties of mono-specific or mono-modality probes, they usually failed to provide a satisfactory diagnosis due to the complexity and promiscuity of tumors. Recently, multivalent and multimodal imaging have rapidly emerged as very promising imaging approaches due to their intrinsic advantages, such as the avidity effects and complementary imaging abilities, respectively. However, the synthesis of such probes usually involves several complicated synthetic procedures, such as the extensive use of protections-deprotections and multi-chromatography purifications and low synthesis yields, which heavily hinders the wide application of such promising probes in preclinical and/or clinical studies.Therefore, there remains a need for development of BFCs with mild labeling conditions, while the labeled chelates having high stability and high specific acitivity, which is also one of the aims of this thesis. We also want to develope a universal small-molecule based scaffold for facile construction of multivalent/multimodal imaging probes.MethodsThree new NE3TA-based BFCs were designed and synthesized. All three BFCs were conjugated to peptidomimetic ligand LLP2A, and the resulting conjugates were labeled with Cu-64 and Ga-68. The specific activity and serum stability of radiolabeled bioconjugates were evaluated. A LC-MS method was applied to determine the Cu(II) chelating selectivity of p-NO2-PhPr-NE3TA over Fe(Ⅲ). B16F10 cell line was used to evaluate the cell uptake and cell efflux of two Cu and Ga labeled conjugates. Both 64Cu and 68Ga labeled conjugates were used for the PET/CT imaging of B16F10 tumor bearing mice.BFC-based scaffold NOTA2tBu-N3 bearing two different bioconjugatable groups, were designed and synthesized. Using this scaffold, two different peptides AE105 (targeting uPAR receptor) and c(RGDyK) (targeting integrinαvβ3) were successfully linked, yielding several peptidic heterodimers with various length of PEG linker. All heterodimers were labeled with 64Cu and in vitro screenings were applied. The heterodimer possessing best in vitro properties was selected and used for the PET/CT imaging of U87MG tumor bearing mice. For comparison purpose, PET/CT imaging of two monomers (64Cu-AE105 and 64Cu-RGD) were also performed. In addition, peptide homodimer, PET/Optical dual-modal probes were also prepared via scaffold NOTA2tBu-N3 through metal free click chemistry or solid phase synthesis.Resultsp-NO2-PhPr-NE3TA exhibited high Cu(II) chelation selectivity even at the present of 10 folds of Fe(Ⅲ). Its conjugate NE3TA-LLP2A could be labeled at room temperature with high specific activity (3-4 mCi/nmol), which was much higher than that of NOTA-LLP2A (～1 mCi/nmol) at the same labeling conditions, while demonstrated good serum stability. The other two BFCs synthesized were not as good as p-SCN-PhPr-NE3TA when they were used for Copper-64 labeling. But p-SCN-PhPr-NE2P1A exhibited good Gallium-68 labeling properties. Its bioconjugate NE2P1A-LLP2A could be quantitatively labeled with Ga-68 even at room temperature in neutral pH within 15 mins. While under the same labeling conditions, NOTA-LLP2A could only be labeled with Ga-68 at acidic conditions.The results of ex vivo biodistribution of 64Cu-NE3TA-LLP2A and 68Ga-NE2P1A-LLP2A were similar and both showed high uptake in B16F10 tumor and high tumor-to-muscle ratios. The tumor-to-muscle ratios at 2 h,4 h and 24 h post injection of 64Cu-NE3TA-LLP2A were 5.81±0.99、10.50±2.02 and 25.19± 7.78%ID/g, respectively. The tumor-to-muscle ratios at 1 h,2 h and 4 h post injection of 68Ga-NE2P1A-LLP2A were 6.66±1.87、10.62±2.98 and 10.25±1.45, respectively. All tumors were clearly visible on PET/CT images at both 2 h and 4 h post injection of 64Cu-NE3TA-LLP2A or at 1 h and 2 h post injection of 68Ga-NE2P1A-LLP2A. Good blocking were observed after coinjection of 64Cu-NE3TA-LLP2A/68Ga-NE2P1A-LLP2A with unlabeled LLP2A, demonstrating the high specificity of two tracers to B16F10 tumor.By using the scaffold NOTA2tBu-N3, four peptidic heterodimers AE105-PEGn-NOTA-PEG4-RGD (n=0,4,8,12, denoted T1, T2, T3, T4, respectively), one homodimers (AE105-NOTA-PEG4-AE105) and two PET/Optical dual-modal probes (AE105-NOTA-noclick-Cy3 and AE105-NOTA-click-Cy5) were conveniently prepared with high yields. All tracers could be labeled with Cu-64 at high specific activity (～1 mCi/nmol), and the resulting radiolabeled conjugates exhibited good stability in human serum. The in vitro cell studies of four heterodimers were performed and the results showed that the heterodimer bearing a longer PEG linker could be uptake more by U87MG cells at the order of T4>T3>T2≥T1; whereas, the T3 heterodimer showed the highest cell retention in U87MG cell at the order of T3>T2≥T1>T4. The results of ex vivo biodistribution showed that high uptake in U87MG tumor and high tumor-to-muscle ratios were achieved after injection of 64Cu-T3. The tumor-to-muscle ratios at 1 h,4 h and 24 h post injection (p.i.) were 4.37±1.46,12.69±3.08, and 15.47±3.32%ID/g, respectively. All tumors were clearly visible on PET/CT images at 1 h and 4 h post injection of 64Cu-T3, 64Cu-RGD and 64Cu-AE105, while the tumor uptakes of 64Cu-T3 were significantly higher than those obtained by injection of 64Cu-RGD or 64Cu-AE105.ConclusionThree novel BFCs have been successfully developed. p-SCN-PhPr-NE3TA exhibited high Cu2+coordinating selectivity, mild labeling conditions and high specific activity. p-SCN-PhPr-NE2P1A was a good alternate BFC for Ga-68, especially for labeling temperature-and pH-sensitive biomolecules. Both 64Cu-NE3TA-LLP2A and 68Ga-NE2P1A-LLP2A exhibited high accumulation in B16F10 tumors, making them very promising tracers for imaging VLA-4 overexpressed diseases.A universal BFC-based molecular scaffold was also successfully developed for the facile construction of multivalent and multimodal imaging probes. Compared to other reported small-molecule scaffolds, this new BFC scaffold demonstrated several advantages, including:simple chemistry (no extensive protection-deprotection and no optimization on reaction conditions required), easy purifications (fewer or only one chromatographic purification needed) and better synthetic yields. More importantly, this molecular scaffold can be used to easily prepare a variety of multivalent and/or multimodal imaging probes with any interested ligands/moieties using various PET radioisotopes. One of the PET tracers (e.g., 64Cu-T3) built upon this scaffold demonstrated promising in vitro and substantially improved in vivo performance. Therefore, the reported molecular scaffold provides a universal and reliable platform to construct multivalent and multimodal imaging probes concisely and will be extremely useful as a simple and modular platform for structural optimization or routine preclinical/clinical use of multivalent/multimodal imaging probes.