Organometallic 99mTc(III) '4 + 1' bombesin(7-14) conjugates: synthesis, radiolabeling, and in vitro/in vivo studies.
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 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 coordinated 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, betaAla, 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 isocyanide 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 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=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 Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated 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 approximately 65% of [99mTc(NS3)(L2-betaAla-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-betaAla-BBN(7-14))], L = L1 or 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.
Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, 01314 Dresden, Germany.
Jens-Uwe
JU
Kunstler
Bhadrasetty
B
Veerendra
Said D
SD
Figueroa
Gary L
GL
Sieckman
Tammy L
TL
Rold
Timothy J
TJ
Hoffman
Charles J
CJ
Smith
Hans-Jurgen
HJ
Pietzsch
D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D002264:Carboxylic Acids / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003486:Cyanides / Q000737:chemistry; D007553:Isotope Labeling; D008024:Ligands; D008297:Male; D051379:Mice; D015609:Organotechnetium Compounds / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D010455:Peptides / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000000981:diagnostic imaging / Q000378:metabolism / Q000473:pathology*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000493:pharmacokinetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013667:Technetium / Q000737:chemistry*; D013997:Time Factors; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D002264:Carboxylic Acids; D002614:Chelating Agents; D003486:Cyanides; D008024:Ligands; D015609:Organotechnetium Compounds; D010455:Peptides; D019275:Radiopharmaceuticals; D013667:Technetium; D001839:Bombesin