NGR-peptide−drug conjugates with dual targeting properties

Peptides containing the asparagine-glycine-arginine (NGR) motif are recognized by CD13/aminopeptidase N (APN) receptor isoforms that are selectively overexpressed in tumor neovasculature. Spontaneous decomposition of NGR peptides can result in isoAsp derivatives, which are recognized by RGD-binding integrins that are essential for tumor metastasis. Peptides binding to CD13 and RGD-binding integrins provide tumor-homing, which can be exploited for dual targeted delivery of anticancer drugs. We synthesized small cyclic NGR peptide–daunomycin conjugates using NGR peptides of varying stability (c[KNGRE]-NH(2), Ac-c[CNGRC]-NH(2) and the thioether bond containing c[CH(2)-CO-NGRC]-NH(2), c[CH(2)-CO-KNGRC]-NH(2)). The cytotoxic effect of the novel cyclic NGR peptide-Dau conjugates were examined in vitro on CD13 positive HT-1080 (human fibrosarcoma) and CD13 negative HT-29 (human colon adenocarcinoma) cell lines. Our results confirm the influence of structure on the antitumor activity and dual acting properties of the conjugates. Attachment of the drug through an enzyme-labile spacer to the C-terminus of cyclic NGR peptide resulted in higher antitumor activity on both CD13 positive and negative cells as compared to the branching versions.