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

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• 作者：Jens-Uwe Kunstler ; Bhadrasetty Veerendra ; Said D. Figueroa ; Gary L. Sieckman ; Tammy L. Rold ; Timothy J. Hoffman ; Charles J. Smith ; Hans-Jurgen Pietzsch
• 刊名：Bioconjugate Chemistry
• 出版年：2007
• 出版时间：September 2007
• 年：2007
• 卷：18
• 期：5
• 页码：1651 - 1661
• 全文大小：209K
• 年卷期：v.18,no.5(September 2007)
• ISSN：1520-4812

文摘

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr).The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle forradionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using thenovel Tc '4 + 1' mixed-ligand system, [Tc(NS₃)(CN-R)], in which Tc(III) is coordinated by a monodentateisocyanide 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, Ala, and Ser-Ser-Ser spacer groups (X) andfunctionalized 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 isocyanide ligand frameworks were introducedusing novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentateNS₃ 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 reactionswith the isocyanides and NS₃ or NS₃COOH ligand frameworks. The ^99mTc complexes were obtained with aradiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These newconjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give aradiochemical purity of 95%. The ^99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogousnonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry(ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to thecorresponding ^99mTc conjugates under identical HPLC conditions, demonstrating structural similarity betweenthe two metalated species. The [Re(NS₃)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolarrange as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitrointernalization/externalization assays indicated that ~65% of [^99mTc(NS₃)(L2-Ala-BBN(7-14))] conjugatewas either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other ^99mTc conjugates wasbelow 20%. Biodistribution studies of [^99mTc(NS₃)(L-Ala-BBN(7-14))], L = L1 or L2, in normal, CF-1mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodentmodels) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS₃ ligand frameworkhad only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominatedby the relatively lipophilic '4 + 1' complex unit.