Introduction: The membrane-bound metalloproteinase aminopeptidase N (APN) is involved in several different cellular processes associated with the malignant phenotype. Furthermore APN has also been reported to have a functional role in tumor angiogenesis and there appear to be a strong correlation between the expression of APN and the invasive capacity of a numerous tumor cell types. The melphalan-derived prodrug melphalan-flufenamide (previously designated J1) can be activated by APN suggesting possible anti-angiogenic properties.

Experimental: A series of experiments were performed to evaluate the effects of melphalan-flufenamide on angiogenesis; cytotoxicity on bovine endothelial cells, the TCS Cellworks AngioKit model with human endothelial cells co-cultured with fibroblasts, the chicken embryo chorioallantoic membrane assay, and finally in vivo in mice using the Cultrex DIVAA angio-reactor assay.

Results: Melphalan-flufenamide displayed high cytotoxic activity against endothelial cells, about hundred times more potent than the parental substance melphalan. This difference was also demonstrated in the more complex assays. Melphalan-flufenamide-treated AngioKit co-cultures showed significant superiority compared to melphalan regarding total tubule number, total tubule length, mean tubule length, and number of junctions. Finally mice were implanted subcutaneously with DIVAA angio-reactors, i.e. sterile tubes with a VEGF gel matrix, and treated with drugs. Melphalan-flufenamide was the most effective compound inhibiting in-growth of host micro-vessels in the tube, significantly better than melphalan and slightly better than the positive control bevacizumab.

Conclusion: APN is associated with several characteristics of the malignant phenotype, including angiogenesis. Melphalan-flufenamide is an APN-activated prodrug of melphalan, currently being evaluated in clinical trials. This study suggests that melphalan-flufenamide besides being an alkylating agent also demonstrates anti-angiogenic effects in different preclinical models in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B6.