Martin W. Rowbottom

CEP-32496: A Novel Orally Active BRAF V600E Inhibitor with Selective Cellular and In Vivo Antitumor Activity

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non–small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAFV600E (Kd BRAFV600E = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal–regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC50 = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAFV600E versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10–55 mg/kg) of both pMEK and pERK in BRAFV600E colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30–100 mg/kg twice daily) against BRAFV600E melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development. Mol Cancer Ther; 11(4); 930–41. ©2012 AACR.

4-Quinazolinyloxy-diaryl ureas as novel BRAFV600E inhibitors.

Aryl phenyl ureas with a 4-quinazolinoxy substituent at the meta-position of the phenyl ring are potent inhibitors of mutant and wild type BRAF kinase. Compound 7 (1-(5-tert-butylisoxazol-3-yl)-3-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)urea hydrochloride) exhibits good pharmacokinetic properties in rat and mouse and is efficacious in a mouse tumor xenograft model following oral dosing.

Abstract 903: AC708 is a potent and selective inhibitor of CSF1R and reduces tumor associated macrophage infiltration in a breast tumor model .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor-associated macrophages (TAMs) are thought to be regulators of solid tumor development based on their capacity to enhance metastatic, invasive, and angiogenic programming of neoplastic tissue. Colony stimulating factor-1 (CSF-1) is a key cytokine involved in recruitment and activation of tissue macrophages, exerting these effects through binding to a high-affinity receptor tyrosine kinase, the CSF-1 receptor. We have developed a small molecule CSF1R inhibitor AC708 in an attempt to impact the TAM-related progression of human tumors. Here we demonstrate that AC708 possesses significant specificity for CSF1R relative to the rest of the kinome, and to the closely related PDGFR family receptors PDGFRα and β, FLT3, and KIT. In cell based assays, AC708 potently inhibited CSF1R phosphorylation mediated by CSF-1 (IC50 = 26 nM) and by IL-34 (IC50 = 33 nM). It also inhibited the viability of growth-factor dependent cells cultured in CSF-1 (IC50 = 38 nM) or IL-34 (IC50 = 40 nM), and inhibited the CSF-1-mediated differentiation and survival of primary human osteoclast with an IC50 of 15 nM. In cytokine release experiments where enriched human monocytes were stimulated with either CSF-1 or IL-34, AC708 inhibited MCP-1 release with nearly identical IC50 regardless of which cytokine was used (CSF-1 (93 nM), IL-34 (88 nM)), and with a lower IC50 than that obtained with the benchmark compound GW-2580 (CSF-1 (148 nM), IL-34 (140 nM)). In vivo, AC708 was assessed for its ability to inhibit the intraperitoneal growth of M-NFS-60 cells in mice. In these experiments, AC708 inhibited M-NFS-60 growth in a dose-dependent manner, with a greater than 80% reduction in cell number at 100 mg/kg, similar to that achieved with the benchmark compound Ki-20227. Two assays were employed to assess the ability of AC708 to modulate endogenous CSF1R. In the first model, AC708 inhibited CSF-1-mediated MCP-1 release in vivo by 60% when dosed at 100 mg/kg. In the second model, plasma levels of TRAP5b were determined following injection of recombinant Parathyroid hormone-related protein (PTHrP) with or without co-administration of AC708. PTHrP-induced increases of plasma TRAP5b were reduced by AC708 in a dose-dependent manner, with levels falling to below baseline in the 100 mg/kg dose group. Lastly to assess the ability of AC708 to modulate TAMs we utilized the 4T-1 breast cancer line implanted orthotopically. Although primary tumor growth was relatively unchanged by AC708 treatment in this model, administration of drug for two weeks resulted in a dose-dependent reduction of tumor resident macrophages, with a 70% reduction at the 100 mg/kg dose relative to vehicle control. AC708 impact on tumor angiogenesis and metastatic potential is currently under investigation and will be reported. These studies further validate CSF1R as a potential target in cancer, and support the development of AC708 as a therapeutic in oncology. Citation Format: Robert C. Armstrong, Barbara Belli, Martin W. Rowbottom, Ron R. Nepomuceno, Alan Q. Dao, Allison M. Rooks, Dan Brigham, Craig W. McMannus, Michael D. Hocker, Mark W. Holladay, Gang Liu. AC708 is a potent and selective inhibitor of CSF1R and reduces tumor associated macrophage infiltration in a breast tumor model . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2013-903