Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580

Abstract

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Fig. 1.

Structure of GW2580

Fig. 2.

Effects of GW2580 on CSF-1-, GMCSF-, and LPS-induced growth of human monocytes. (Upper) Monocytes from each of four donors were split, and effects on CSF-1- and GMCSF-induced growth were compared. (Lower) Monocytes from each of four donors were split, and effects on CSF-1- and LPS-induced growth were compared.

Fig. 3.

Plasma concentrations of GW2580 after oral dosing in mice. Mice were dosed orally, and three were killed at different times for measurements of GW2580 concentrations in plasma.

Fig. 4.

Effect of GW2580 on CSF-1-priming of LPS-induced TNF and IL-6 production in vivo in mice. GW2580 was dosed 45 min before an i.p. injection of vehicle (Upper) or CSF-1 (Lower)at1.7 μg per mouse. Three and a half hours later, all mice received an i.p. injection of LPS, followed by measurement of TNF (○) and IL-6 (▪) in the plasma 1.5 h after LPS. Each point represents data from six to seven mice. Statistical changes relative to oral-dosing vehicle are shown. *, P <0.01; †, P <0.001.

Fig. 5.

Effect of GW2580 on the growth of M-NFS-60 cells in vivo in mice. Mice were treated b.i.d. with vehicle or GW2580 starting 1 h before i.p. injection with 1 × 10⁷ M-NFS-60 cells. The number of i.p. cells was counted on day 4. Statistical changes relative to the tumor-only group are shown (*, P <0.05; †, P <0.001). In a second study, GW2580 also caused statistically significant inhibition of tumor load, with no-tumor, tumor-only, tumor-plus-20 mg/kg-GW2580, and tumor-plus-80 mg/kg-GW2580 groups yielding 5.6 ± 0.6 (†), 33.8 ± 2.8, 19.3 ± 4.5 (*), and 10.9 ± 1.7 (†) million i.p. cells per mouse, respectively.