Studies on radiometal chelator-bombesin peptide-based radiopharmaceuticals for tumor GRP-receptor subtype mediated radioimaging
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Bombesin-like peptides bind to gastrin-releasing peptide receptor subtype 2 (BB2) with significantly high binding affinity and specificity. In an attempt to develop non-mitogenic bombesin analogs with antagonist targeting-vectors at the C-terminus, seven bombesin analogs were synthesized using HMBA resin. The antagonist targeting-vectors include: methyl ester, ethyl ester, propyl ester, ethyl amide, propyl amide, butyl amide and hydrazide. The synthetic procedures were optimized for convenient preparation of bombesin analogs possessing different moieties at the C-terminus. The prepared analogs were characterized using LC-MS. Three C-terminally modified bombesin analogs were synthesized and conjugated to a 6-bromohexanoic acid linker and subsequently to the cyclic tetraazabifunctional chelate, cyclen, using optimized solid-phase strategy. The C-terminal modification include: ethyl ester, propyl amide and des-Met14 amide. The prepared conjugates were analyzed, purified and characterized using HPLC and ES-MS. Net peptide contents of the developed analogs were assessed using UV absorption. The binding affinity profiles of the developed C-terminus modified bombesin-cyclen conjugates to the tumors overexpressing GRP-receptors were assessed using in-vitro competitive displacement of ₁₂₅I-BBN from human PC-3 prostate cancer cell line. Radiolabeling of bombesin conjugates with ₉₉mTc were optimized. The propyl amide C-terminally modified bombesin analog was synthesized and conjugated to the Fmoc-6-aminohexanoic acid linker and subsequently to DOTA, using solid-phase strategy. The prepared conjugate was analyzed, purified and characterized using HPLC and ES-MS. The net peptide content was assessed by UV absorption. The binding affinity profile of the developed propyl amide C-terminus modified bombesin-DOTA conjugate to the tumors overexpressing GRP-receptors was assessed using in-vitro competitive displacement of ₁₂₅I-BBN from the human PC-3 prostate cancer cell line. Labeling with natIn and ₁₁₁In were successfully performed. The degree of in vitro internalization and efflux of ₁₁₁In-DOTA-Conjugate in PC-3 cell line were assessed. The biodistribution and uptake studies of ₁₁₁In-DOTA-Conjugate were performed in healthy CF-1 mice. Pharmacokinetic studies were performed in PC-3 tumor-bearing SCID mice. Micro SPECT/CT imaging of the prostate cancer PC-3 xenografts in SCID mouse was performed at 1-hr post-injection of ₁₁₁In-DOTA-Conjugate. The results obtained from the presented study demonstrate the potential of using bombesin analogs possessing antagonist C-terminal vectors as a candidate of choice for specific in vivo targeting of the tumors overexpressing GRP-receptors. The positive outcome from this study may be the driving force for switching the focus to the development of the receptor targeting research using antagonist peptide vectors. Further investigations of the role that chain lengths of the C-terminal vector play on the biological activity of the bombesin analogs in terms of being agonist, antagonist, or partial agonist are needed.
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