title: Preclinical evaluation of a new, stabilized neurotensin(8--13) pseudopeptide radiolabeled with (99m)tc.
abstract: The rapid degradation of neurotensin (NT) limits its clinical use in cancer imaging and therapy. Thus, a new NT(8--13) pseudopeptide, NT-VIII, was synthesized. Some changes were introduced in the sequence of NT(8--13) to stabilize the molecule against enzymatic degradation: Arg(8) was N-methylated, and Lys and Tle replaced Arg(9) and Ile(12), respectively. Finally, (NalphaHis)Ac was coupled to the N-terminus for (99m)Tc(CO)(3) labeling. This peptide was characterized both in vitro and in vivo.
  
  
  METHODS
  The new analog was labeled with (99m)Tc(CO)(3). Its metabolic stability was analyzed both in human plasma and in HT-29 cells. Binding properties, receptor downregulation, and internalization were tested with HT-29 cells. Biodistribution was evaluated in nude mice with HT-29 xenografts.
  
  
  RESULTS
  (99m)Tc(CO)(3)NT-VIII showed a high stability in plasma, where most of the peptide remained intact after 24 h of incubation at 37 degreesC. However, the degradation in HT-29 cells was more rapid (46% of intact (99m)Tc(CO)(3)NT-VIII after 24 h at 37 degreesC). Binding to NT1 receptors (NTR1) was saturable and specific. Scatchard analysis showed a high affinity for (99m)Tc(CO)(3)NT-VIII, with a dissociation constant similar to (125)I-NT (1.8 vs. 1.6 nmol/L). After interacting with NTR1, (99m)Tc(CO)(3)NT-VIII was rapidly internalized, with more than 90% internalized after 30 min. It also distributed and cleared rapidly in nude mice bearing HT-29 xenografts. The highest rates of accumulation were found in kidney and tumor at all time points tested. Tumor uptake was highly specific because it could be blocked by coinjection with a high dose of (NalphaHis)Ac-NT(8--13). Tumors were clearly visualized in scintigraphy images.
  
  
  CONCLUSIONS
  The changes that were introduced stabilized the molecule against enzymatic degradation without affecting binding properties. Moreover, the increase in stability enhanced tumor uptake, making this derivative a promising candidate for clinical use.
authors.affiliation: Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Elisa
authors.initials: E
authors.lastname: García-Garayoa
authors.forename: Peter
authors.initials: P
authors.lastname: Bläuenstein
authors.forename: Matthias
authors.initials: M
authors.lastname: Bruehlmeier
authors.forename: Alain
authors.initials: A
authors.lastname: Blanc
authors.forename: Koen
authors.initials: K
authors.lastname: Iterbeke
authors.forename: Peter
authors.initials: P
authors.lastname: Conrath
authors.forename: Dirk
authors.initials: D
authors.lastname: Tourwé
authors.forename: P August
authors.initials: PA
authors.lastname: Schubiger
mesh_terms: D000230:Adenocarcinoma / Q000000981:diagnostic imaging; D000818:Animals; D005260:Female; D019073:HT29 Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009496:Neurotensin / Q000138:chemical synthesis / Q000493:pharmacokinetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000493:pharmacokinetics; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis; D018028:Receptors, Neurotensin / Q000378:metabolism; D013329:Structure-Activity Relationship; D013667:Technetium / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: D010446:Peptide Fragments; D019275:Radiopharmaceuticals; D018028:Receptors, Neurotensin; D009496:Neurotensin; C054381:neurotensin (8-13); D013667:Technetium