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A Novel Orally Active Platelet-Derived Growth Factor Receptor/Raf Pathway Inhibitor Attenuates Post-Intervention Intimal Hyperplasia

Joshua I. Greenberg, Eric Murphy, Samuel Barillas, Niren Angle, David Cheresh.
University of California, San Diego, San Diego, Calif.

OBJECTIVES: Currently, no effective small molecule tyrosine kinase inhibitors are available to prevent post-injury arterial remodeling. At the molecular level, endovascular interventions produce pathological activity of PDGF receptors and Raf/MAP kinase substrates. Our goal was to identify a potent inhibitor of these two pathways in vascular smooth muscle cells (VSMC).

METHODS: Physiological cell-based screening identified a Raf kinase inhibitor, KG5, which was predicted by molecular modeling to bind both B-RAF and PDGF Receptor-β in the inactive state. The effect of KG5 on cell viability of VSMCs was examined using an XTT assay. Phosphorylation of receptor tyrosine kinase and Raf pathway targets including phosphorylation of MEK and ERK was assessed by immunoblotting. The effect of KG-5 on PDGF, VEGF, and FGF-mediated chemotaxis was assessed using transwell assays. Finally, C57/Bl6 mice were subjected to endovascular wire injury and treated for 28 days with either vehicle or KG5 (50 mg/kg, ip, bid) followed by histo-morphometric arterial analysis.

RESULTS: KG-5 inhibited VSMC cell viability (EC50=566 nM) at much lower concentrations then the broad spectrum kinase inhibitor Sorafenib (EC50=27.34 µM). KG5 inhibited the PDGF or bFGF induced phosphorylation of C-Raf (S338) and the phosporylation of the PDGF receptor β at 500 nM and 750 nM, respectively. Importantly phosphorylation of VEGFR2 was not observed with KG5 at 10 µM. Chemotaxis to PDGF and FGF was potently suppressed at similar concentrations. Finally, wire-injured femoral arteries in mice treated with KG-5 (n=4/group) demonstrated markedly preserved intimal/medial (I/M) ratios and luminal areas (p<0.05 for both vs. vehicle). Mice tolerated the drug at high concentrations with no complications.

CONCLUSIONS: This report is the first to demonstrate the efficacy of a combined PDGFR/Raf pathway inhibitor against post-intervention intimal hyperplasia. KG5 represents a novel, orally bioavailable small molecule that inhibits VSMC migration and proliferation via dual inhibition of PDGF-Rβ and Raf pathway signaling without affecting VEGFR activation. We propose that this novel compound offers a promising alternative to drugs currently available.

AUTHOR DISCLOSURES: J.I. Greenberg, None; E. Murphy, None; S. Barillas, None; N. Angle, None; D. Cheresh, TargeGen, Inc.; NIH.