[CANCER RESEARCH 60, 2178 –2189, April 15, 2000] PTK787/ZK 222584, a Novel and Potent Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases, Impairs Vascular Endothelial Growth Factor- induced Responses and Tumor Growth after Oral Administration Jeanette M. Wood, 1 Guido Bold, Elizabeth Buchdunger, Robert Cozens, Stefano Ferrari, Jo ¨rg Frei, Francesco Hofmann, Ju ¨ rgen Mestan, Helmut Mett, Terence O’Reilly, Elke Persohn, Johannes Ro ¨sel, Christian Schnell, David Stover, Andreas Theuer, Harry Towbin, Fritz Wenger, Kathie Woods-Cook, Andreas Menrad, Gerhard Siemeister, Michael Schirner, Karl-Heinz Thierauch, Martin R. Schneider, Joachim Drevs, Georg Martiny-Baron, Frank Totzke, and Dieter Marme ´ Oncology Research, Novartis Pharma AG, CH-4002 Basel, Switzerland [J. M. W., G. B., E. B., R. C., S. F., J. F., F. H., J. M., H. M., T. O., E. P., J. R., C. S., D. S., A. T., H. T., F. W., K. W-C.]; Research Laboratories of Schering AG, D-13342 Berlin, Germany [A. M., G. S., M. S., K-H. T., M. R. S.]; and Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany [J. D., G. M-B., F. T., D. M.] ABSTRACT PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthala- zine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor  tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor fami- lies, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-. PTK787/ZK 222584 inhibits VEGF-induced au- tophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 M, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 M for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25–100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cyto- toxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role. INTRODUCTION Angiogenesis, the formation of new vessels from an existing vas- cular network, is an essential event in a variety of physiological and pathological processes. Under physiological conditions, angiogenesis is restricted to processes such as embryogenesis, ovulation, and wound healing. Angiogenesis also occurs in pathological processes such as inflammation (1– 4), rheumatoid arthritis (5– 8), ocular neo- vascularization (9 –13), psoriasis (14 –16), tumor growth, and the formation of metastases (17, 18). Of the numerous growth factors and cytokines that have been shown to have angiogenic effects, VEGF 2 appears to be a key factor in pathological situations that involve neovascularization as well as enhanced vascular permeability (19, 20). The VEGF receptors, Flt-1 (VEGF-R1; Ref. 21) and KDR (VEGF-R2; Ref. 22), are almost exclusively located on endothelial cells (23). Expression of these receptors is low in normal tissues and only up-regulated during the development of these pathological states when neovascularization occurs (24, 25). Both receptors have seven immunoglobulin-like domains in their extracellular region, a single transmembrane-span- ning domain, and an intracellular split tyrosine kinase domain and belong to the same family of receptors as PDGFR, c-Kit ( a receptor for stem cell factor), c-Fms (a receptor for colony-stimulating factor), Flt-3, and Flt-4. Flt-1 binds VEGF-A and VEGF-B (26, 27) and the related placenta growth factor (28, 29), whereas KDR binds VEGF-C and VEGF-D (30, 31) in addition to VEGF-A. VEGF-C and VEGF-D are both ligands and activators of Flt-4 (VEGF-R3), which is ex- pressed on the endothelial cells of lymphatic vessels (32–34). Al- though activation of Flt-1 was shown to mediate biological responses, such as endothelial and monocyte cell migration and tissue factor induction (27, 28, 35–38), in cells expressing only Flt-1 or in Flt-1- deficient cells transfected with Flt-1 cDNA, stimulation with VEGF induces only weak receptor phosphorylation and no significant mito- genic response (37, 38). In contrast to Flt-1, KDR is strongly auto- phosphorylated upon VEGF stimulation and mediates a mitogenic response (39, 40). Gene knock-out experiments for VEGF (41, 42) as well as its receptors (43– 46) have highlighted the pivotal role of the VEGF/ VEGF receptor system in the development of the embryonic vascular system. Various different approaches have been used to interfere with the VEGF/VEGF receptor system in adult animals and thereby deter- mine the role of VEGF receptors in the various pathological states. These approaches include VEGF neutralizing antibodies (47– 49), antibodies against the VEGF receptors (50, 51), recombinant soluble VEGF receptor proteins (52, 53), a tetracycline-regulated VEGF ex- pression system (54, 55), and dominant-negative mutants of the VEGF receptors (56). Results from these approaches suggest the VEGF/VEGF receptor system is a novel and attractive therapeutic target for suppression of pathological neovascularization and, in par- ticular, for inhibiting tumor growth (57, 58). Our aim was to design a low molecular weight synthetic molecule Received 5/14/99; accepted 2/18/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Oncology Research, K-125 2.10, Novartis AG, CH 4002 Basel, Switzerland. 2 The abbreviations used are: VEGF, vascular endothelial growth factor; Flt, fms-like tyrosine kinase; KDR, kinase insert domain-containing receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PTK787/ZK 222584, 1-[4-chloroanilino]-4-[4- pyridylmethyl] phthalazine succinate; HUVEC, human umbilical cord endothelial cell; CHO, Chinese hamster ovarian cells; GST, glutathione S-transferase; PKC, protein kinase C; BrdUrd, 5-bromo-2-deoxyuridine; bFGF, basic fibroblast growth factor; Cdc2, cell cycle-dependent kinase 2; Flk, fetal liver kinase; Fms, feline myelosarcoma; mAb, monoclonal antibody; Tie, tyrosine kinase with immunoglobulin. 2178