The finding that receptors for different endogenous regulatory peptides, like the neuropeptide neurotensin (NT), are over-expressed in different human cancers, has opened new perspectives on the use of synthetic peptides for tumor-selective targeting (1). Peptide ligands carrying cytotoxic moieties or radiotracers might act as drugs or diagnostics. Nonetheless, the use of peptides as drugs has largely been limited by their short half-life. Peptides are physiologically hydrolysed by proteases and peptidases and the chemical modifications necessary to stabilize endogenous sequence can deeply modify peptide affinity and specificity.

We demonstrated that NT, like several other endogenous peptides, retains its biological activity and becomes resistant to proteolysis when synthesized in a branched form (2).

NT receptors are over-expressed in different human tumors with high incidence and mortality rate, like small cell lung cancer, colon, prostate and pancreas carcinomas. For this reason, high affinity and stable NT peptides can have important applications as specific tumor-targeting agents.

We synthesized the short functional fragment NT(8-13) in a tetra-branched form (NT4), which was coupled during the synthesis to different units designed for tumor tracing or therapy.

Cell internalization of branched NT4 molecules together with NT receptor regulation and trafficking, were followed by confocal microscopy. NT4 conjugated to fluorescent probes was also used for NT receptor tracing in human specimens from colon cancer surgical resections. Comparison with normal tissues from the same patients showed a clear over-expression of NT receptor in a high percentage of tumors even at early differentiation stages.

Cytotoxicity of NT4 conjugated to methotrexate or to the photosensitizer chlorine e6, was tested on human colon adenocarcinoma cell lines. In both cases, conjugation to branched NT dramatically decreased non-specific toxicity of the drugs by inducing receptor-specific uptake of the toxic moiety by target cells.

NT4 conjugated to methotrexate was also tested in in vivo experiments using HT29 human colon adenocarcinoma xenografts in nude mice. Animals treated with NT4 conjugated to methotrexate showed a clear decrease in tumor growth with respect to untreated mice or mice treated with equimolar amount of the unconjugated drug.

Branched NT molecules are good candidates for the development of peptide tumor-targeting agents that might have the dual use of specific NT receptor tracing followed by targeted therapy of NT receptor-expressing tumors.

  1. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003; 24(4): 389-427.

  2. Bracci L. et al. Synthetic peptides in the form of dendrimers become resistant to protease activity. J Biol Chem. 2003; 278(47): 46590-5.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA