Technetium and rhenium labeled cyclic melanotropin analogues as imaging and therepeutic agents for melanoma.

摘要

Radiolabeled bioactive peptides which bind specifically to surface receptors overexpressed in tumor cells are considered as alternatives for tumor detection and treatment. In developing imaging and radiotherapeutic agents for melanoma skin cancer, two cyclic analogs of alpha-melanocyte stimulating hormone, NAc-Cys-Glu-His-DPhe-Arg-Trp-Cys-Lys-Pro-Val-NH2 (MSH), and NAc-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Lys-Pro-Val-NH2 (CCMSH), the so called first generation and second generation analogs, have been labeled directly with technetium and rhenium. The nonradioactive rhenium complexes have been characterized by NMR, IR and mass spectrometry to determine the metal binding site. The complexes were found cyclized through metal-thiolate bonds. The stability of Tc-99m and Re-188 complexes were tested by challenging the complexes with 10 mM cysteine and 100 mM glutathione and found to be reasonably stable. The cell binding assays conducted with B16-F1 murine melanoma cells indicated a significant improvement in receptor binding affinity from the first generation to the second generation, with the corresponding Ki values of 66 nM and 2.9 nM, respectively. These observations demonstrated that addition of cysteine on the N-terminus of the cyclic analog increased the in vitro stability and receptor binding affinity of the metal complex by moving the metal binding site away from peptide receptor-recognition sequence. Both Tc-99m and Re-188 labeled CCMSH shown rapid accumulation in melanoma tumor in a murine tumor model system. The tumor was effectively imaged with Tc-99m-CCMSH at 30 minutes post injection of radiotracer.;The second part of the thesis reported the preliminary results of direct Tc-99m radiolabeling of DNA recombinant antibody fragments with genetically engineered metal chelation site. Three different mutants with different chelation cysteinyl peptide sequences on their heavy chain C-terminal were tested and compared for their labeling properties. These mutants can be labeled with Tc-99m with 30 to 40% yields. To achieve consistent and better labeling, pretreatment with stannous ion was necessary. The radiolabeled antibody fragments were found with significant amount of nonspecific labeling. All three mutants demonstrated their DNA binding immunoreactivity after radiolabeling by ELISA.