Angiogenesis, Jul 2013; 16(3): 503-24 DOI: 10.1007/s10456-013-9347-8 Tumour vasculature targeting agents in hybrid/conjugate drugs E.M. Prokopiou, S.A. Ryder and J.J. Walsh School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland E-mail: jjwalsh@tcd.ie Note: This is an author-created version of an article accepted for publication in Angiogenesis following peer review. The final publication is available at http://link.springer.com. Direct links to the definitive publisher-authenticated version [Prokopiou EM, Ryder SA, Walsh JJ. Tumour vasculature targeting agents in hybrid/conjugate drugs. Angiogenesis. 2013 Jul; 16(3): 503-24]: http://link.springer.com/article/10.1007%2Fs10456-013-9347-8 http://dx.doi.org/10.1007/s10456-013-9347-8 Abstract Tumour vasculature targeting has been a very active area of cancer drug discovery over the last decade. Growth of solid tumours beyond a certain point requires a sufficient blood supply in order for them to develop and metastasise. While novel anti-angiogenic and vascular disrupting agents represent an important contribution to the armoury of anti-cancer agents, they nevertheless usually require combination with standard cytotoxic therapy in order to demonstrate positive clinical outcomes. In line with this consensus, a new concept has arisen, namely the design of functional hybrids where at least one component of the design targets a tumour angiogenic/vasculature pathway. This review will outline examples of such hybrid/conjugate-based approaches. Emphasis will be placed on their preclinical evaluation with particular focus on the arginine-glycine-aspartic acid/asparagine-glycine-arginine (RGD/NGR) conjugates, heparin-related hybrids and antibody- drug conjugates. In conclusion, the benefits and shortcomings of hybrids under development will be discussed in the context of future directions and applications. Keywords Angiogenesis • Combination therapy • Conjugates • Hybrids • Tumour vasculature • Vascular disrupting agents Angiogenesis Angiogenesis is defined as the process in which new blood vessels form from the existing vasculature [1]. During this process, endothelial cell proliferation is induced, which results in alignment of endothelial cells into capillary tubes (vasculogenesis). Physiologically, this process takes place during embryogenesis and the female reproductive cycle, as well as in wound healing. During tumour angiogenesis, the “angiogenic switch” is turned on causing the normally quiescent vasculature to constantly sprout new vessels, thus facilitating tumour growth [1, 2]. As evident from earlier in vivo work in rabbits with implanted tumours and non-tumour tissue in non-vascularised cornea, tumour growth was shown to be angiogenesis-dependent. Tumour tissue was able to grow once newly formed vasculature was developed, whereas non- tumour tissue did not attract new blood vessels [3]. Angiogenesis is a complex process which involves a number of steps including the production and release of angiogenic factors (upon activation by hypoxia or genetic mutations) [4–6], and the binding of these factors to vascular endothelial cell receptors, causing activation and cell proliferation. Extracellular metalloproteinases mediate many of the changes in the microenvironment by degrading the extracellular matrix (ECM) in front of the proliferating endothelial cells [7]. Endothelial cells then subsequently migrate towards the tumour tissue where they align to form new blood vessels and connect to create a loop that allows the blood to circulate. Specialised muscle cells (e.g. smooth muscle cells and pericytes) stabilise the vessel tubes providing structural support [8]. Angiogenic regulators Many endogenous molecules are involved in the control of angiogenesis and several of these have been studied for potential therapeutic applications. Pro-angiogenic regulators include vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs) and their associated tyrosine kinase receptors, VEGFRs, FGFRs, PDGFRs and EGFRs and matrix metalloproteinases (MMPs). Endogenous angiogenesis inhibitors include thrombospondin-1 (TSP-1), angiostatin, endostatin, tumstatin and canstatin. TSP-1 counteracts pro-angiogenic stimuli by evoking suppressive signals through activation of endothelial cell receptors [9]. Angiostatin and endostatin are produced in the tumour stroma through the action of proteinases which are induced as part of the angiogenic cascade [10]. Angiostatin (an internal fragment of plasminogen containing at least three of the kringles of plasminogen) was shown to be inversely correlated with VEGF and inhibited endothelial cell migration, tube formation and aortic ring sprouts [11]. In an in vivo assay, angiostatin was found to maintain metastases in a dormant state when administered exogenously [12] and was associated with longer patient survival [13]. A Phase II trial using recombinant