Clinical & Experimental Metastasis 21: 129–138, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 129 Anti-metastatic properties of RGD-peptidomimetic agents S137 and S247 Kristen E. Shannon, Jeffery L. Keene, Steven L. Settle, Tiffany D. Duffin, Maureen A. Nickols, Marisa Westlin, Sally Schroeter, Peter G. Ruminski & David W. Griggs Pfizer Global Research and Development, Chesterfield, Missouri, USA Received 31 October 2003; accepted in revised form 27 January 2004 Key words: anti-metastatic, arg-gly-asp, breast cancer, integrin antagonists, peptidomimetic, RGD Abstract Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against α 5 β 1 and α IIb β 3 . Both endothelial and tumor cell migration mediated by α v β 3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists. Abbreviations: GFP – green fluorescent protein; LAP – latency associated peptide; RGD – arg-gly-asp Introduction Disseminated tumor cells are frequently present in patients at the time of first disease diagnosis [1–4]. For this reason, agents that only target early metastatic steps such as the es- cape of cells from the primary tumor or the attachment of tumor cells to the endothelium in distant organs may fail to gain widespread clinical utility. A more desirable approach may be to combine antagonism of early events with a di- minished capability of already-migrated cells to survive and proliferate in their non-native environment. In addition to inhibiting the potential outgrowth of undetected tumor foci, such therapy could in principle reduce ‘metastasis of meta- stases’, a process with often fatal consequences that may recapitulate the early steps of primary metastasis. Integrins are principal mediators of cell attachment, mi- gration, differentiation and survival, and therefore have the potential to impact multiple steps in the metastatic cascade. Many integrins including α IIb β 3 , α 5 β 1 and the entire family of alpha v subunit-containing integrin bind to extracellular Correspondence to: David W. Griggs, PhD, Pfizer Global Research and Development, Mail Code BB3K, 700 Chesterfield Parkway W, Chesterfield, MO 63017, USA. Tel: +1-636-247-6413; Fax: +1-636-247-6313; E-mail: david.w.griggs@pfizer.com matrix proteins in a manner that is dependent on the lig- and amino acid sequence arg-gly-asp (RGD). In particular, α v β 3 has been extensively studied due in part to its reported overexpression on both endothelial cells in neovasculature and on a subset of highly aggressive human carcinomas [5, 6]. Expression of constitutively activated α v β 3 directly pro- moted metastasis in preclinical models using human breast cancer cells [7]. Specific inhibitors of α v β 3 , as well as in- hibitors of both α v β 5 and α 5 β 1 which are also expressed on endothelial and tumor cells, affect angiogenesis, tumor growth, and metastasis [6, 8–10]. Studies with integrins α v β 6 and α v β 8 suggest functions in the activation of TGFβ , which in turn has pleiotropic effects on many inflammatory and tumor cell types [11–13]. Finally, the integrin α IIb β 3 is expressed on some solid tumors and may contribute to invasion and metastasis [14]. However, this integrin also has a critical role in mediating platelet aggregation and blood clotting, and thus most designers of RGD-based inhibitors have sought to spare this molecule for applications in cancer [6, 15]. We have previously described integrin antagonists that inhibit angiogenesis, primary tumor growth and the devel- opment of tumor-induced hypercalcemia, and colon cancer