Organometallic 99mTc(III) ‘4+1’ Bombesin(7-14) Conjugates: Synthesis, Radiolabeling and In Vitro/In Vivo Studies


Organometallic 99mTc(III) ‘4+1’ Bombesin(7-14) Conjugates: Synthesis, Radiolabeling and In Vitro/In Vivo Studies

Künstler, J.-U.; Veerendra, B.; Figueroa, S. D.; Sieckmann, G. L.; Rold, T. L.; Hoffman, T. J.; Smith, C. J.; Pietzsch, H.-J.

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin releasing peptide receptor (GRPr). The GRPr is over-expressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc ‘4+1’ mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is co-ordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2’,2’’-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, ßAla and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2) resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanides ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc-complexes were obtained with a radiochemical yield of 30 to 80% depending on the amount of the isocyanide (20 to 100 nmol) used. These new conjugates were purified by reversed-phased high performance liquid chromatography (RP-HPLC) to give a radiochemical purity of ≥ 95 %. The 99mTc-conjugates exhibited high in vitro stability (> 90 %, 24 h). Analogous nonradioactive Re-conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the new Re-conjugates indicated that they exhibited identical retention times to the corresponding 99mTc-conjugates under identical HPLC conditions, demonstrating structural similarity between the two metallated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that ~ 65% of [99mTc(NS3)(L2-βAla-BBN(7-14))] conjugate was either surface bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc-conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-βAla-BBN(7-14))], L = L1, L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic ‘4+1’ complex unit.

  • Bioconjugate Chemistry 18(2007), 1651-1661

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Publ.-Id: 9382