title: Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy.
abstract: Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.
authors.affiliation: Laboratory of Molecular Radiopharmacology, Department of Radiology and Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110, USA.
authors.forename: V
authors.initials: V
authors.lastname: Polyakov
authors.forename: V
authors.initials: V
authors.lastname: Sharma
authors.forename: J L
authors.initials: JL
authors.lastname: Dahlheimer
authors.forename: C M
authors.initials: CM
authors.lastname: Pica
authors.forename: G D
authors.initials: GD
authors.lastname: Luker
authors.forename: D
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism*; D015696:Gene Products, tat / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D015609:Organotechnetium Compounds / Q000378:metabolism* / Q000493:pharmacokinetics / Q000627:therapeutic use; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D019275:Radiopharmaceuticals / Q000378:metabolism* / Q000627:therapeutic use; D011878:Radiotherapy; D012211:Rhenium / Q000378:metabolism* / Q000493:pharmacokinetics / Q000627:therapeutic use; D014018:Tissue Distribution; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D002614:Chelating Agents; D015696:Gene Products, tat; D015609:Organotechnetium Compounds; D010446:Peptide Fragments; D019275:Radiopharmaceuticals; D054322:tat Gene Products, Human Immunodeficiency Virus; D012211:Rhenium