Neurotensin-derived branched peptides and uses thereof

0001] The instant invention refers to in vivo stable branched peptides derived from the sequence of Neurotensin (NT). The peptides may be conjugated to functional units for specific targeting of cancer cells. Thus they can be used for diagnosis and/or therapy of tumors. BACKGROUND ART [0002] One of the major problems in classic chemotherapy is the non-specific toxicity of most anticancer agents even for normal cells. Then, specific targeting of tumors is the main challenge in the research on cancer therapy and diagnosis. [0003] Presently, innovative tumor-specific therapies follow the strategy of targeting tumor associated proteins, specifically expressed or over expressed on tumor cells. [0004] The observation that receptors for different endogenous regulatory peptides are expressed in a number of primary human cancers, opened new perspectives on the use of synthetic peptides for tumor-selective targeting. [0005] Neurotensin (NT) is a 13 amino acid peptide that has the dual function of neurotransmitter or neuromodulator in the central nervous system and local hormone in the periphery. NT receptors are overexpressed in severe malignancies such as small cell lung cancer, colon, pancreatic and prostate carcinomas. NT stabilized analogues have been proposed for tumor therapy several years agoand NT is still considered the best possible candidate for a peptide-based therapy of exocrine pancreatic carcinomasin consideration of the high incidence and density of NT receptors in these tumors. Over 75% of all ductal pancreatic carcinomas over-express NT receptors, whereas normal pancreas tissue, pancreatitis and endocrine pancreas do not. [0006] Tumor receptor-targeting is fundamental in approaching the problem of non-specific toxicity of cancer chemotherapies and it is a precious tool for tumor localization by radioisotopes. Nonetheless, the in vivo use of peptides has largely been limited by their short half-life. [0007] The inventors of the present invention previously demonstrated that synthesis of peptides in branched dendrimeric form results in molecules that can retain peptide biological activity and are very resistant to proteolytic activity of biological fluids, thus having a markedly higher half-life with respect to monomeric peptides. [0008] The instant invention refers to in vivo stable dendrimeric peptides derived from NT. Such peptides are also conjugated to functional units to be used to target cancer cells. In particular, conjugation of NT4(8-13) with either the photosensitizer Che6 or the chemotherapic molecule MTX has shown to be specific for tumor cells and non-toxic to healthy ones, then overcoming the secondary effects of the classic chemotherapeutics when given systemically to mice. [0009] In the present invention, in addition to the specificity of the molecules, an increased activity of the conjugated molecules compared to free uncoupled drug was observed. The molecules of the present invention were selected among a pool of numerous analogues that the inventors synthesized. The 'carrier' peptide (neurotensin) is the best carrier among a number of others. For example, luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH) (QHWSYGLRPG, SEQ ID No. 3), presented in vitro binding profiles lower than NT. In addition, the linkers used in the present invention were selected for each new molecule because of their large influence on the activity of the molecule. For example NT(8-13)4-beta-Ala-Biotin was shown to loose binding activity to NT receptor compared to NT(8-13)4; whereas NT4-peg-Biotin maintained IC50 values comparable to that of NT4. NT(1-13)4-Fluorescein and NT(8-13)4-Fluorescein are able to stain cancer cells and tissue more efficiently than NT(8-13)4-beta-Ala-K(PEG-Fluorescein) and the fluorophore is also more stable. Finally, the chemotherapeutic moiety was chosen on the basis of its functional groups such as to be selectively and univocally used for the coupling. The strength of the bond was modulated depending on the site and mode of action of the drug itself, the best conjugation was selected. As an example 6-mercaptopurine conjugated to neurotensin through an 'uncleavable' linker was completely non cytotoxic, the same drug conjugated through a 'cleavable' linker was active, but the activity resulted insufficient to be considered for further development. Similarly, monastrol derivative is not the first choice of compound in the present invention. Taken together, these specific features render the objects of the present invention unique and preferable over any possible analogue