Effects of the cFMS kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in normal and arthritic rats.

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.

Phosphorylation of p70s6 kinase is implicated in androgen-induced levator ani muscle anabolism in castrated rats.

Androgens are known to increase muscle mass, strength and muscle protein synthesis. However, the molecular mechanisms by which androgens regulate skeletal muscle development remain poorly understood. The ribosomal protein kinase p70(s6k) is a regulator of ribosome biogenesis and plays an important role in the regulation of growth-related protein synthesis. The phosphorylation of p70(s6k) has been implicated in load-induced skeletal muscle hypertrophy. In the current study, we determined the effect of DHT on the phosphorylation of p70(s6k) in the androgen-sensitive levator ani muscle of castrated rats. DHT induced a rapid increase in the phosphorylation of p70(s6k), which was detectable within 6 h after a single injection. Interestingly, DHT-induced phosphorylation of p70(s6k) occurred only in androgen-sensitive muscles, but not prostate and seminal vesicle. Co-administration of flutamide, an AR antagonist, inhibited DHT-induced p70(s6k) phosphorylation. While serum IGF-I levels were not changed by DHT treatment, IGF-I gene expression levels increased and the mRNA levels of IGFBP3 and IGFBP5 were suppressed in the LA muscle after DHT replacement in castrated rats. These results suggest that the phosphorylation of p70(s6k), likely via the IGF-I pathway, may play an important role in androgen-induced skeletal muscle hypertrophy.

Regulation of TNF-alpha secretion by a specific melanocortin-1 receptor peptide agonist.

The lack of specific pharmacological tools has impeded the evaluation of the role of each melanocortin receptor (MCR) subtype in the myriad physiological effects of melanocortins. 154N-5 is an octapeptide (MFRdWFKPV-NH(2)) that was first identified as an MC1R antagonist in Xenopus melanophores [J. Biol. Chem. 269 (1994) 29846]. In this manuscript, we show that 154N-5 is a specific agonist for human and murine MC1R. The peptide has negligible activity at MC3R and MC4R and is 25-fold less potent and a weak agonist at MC5R. 154N-5 was tested in both a cellular and an animal model of tumor necrosis factor-alpha (TNF-alpha) secretion. The inhibitory efficacy of 154N-5 on TNF-alpha secretion in both models was similar to the nonselective agonist NDP-alpha-melanocyte stimulating hormone (NDP-alphaMSH), thus, we conclude that inhibition of TNF-alpha secretion by melanocortin peptides is mediated by MC1R. 154N-5 is a valuable new tool for the evaluation of specific contribution of MC1R agonism to physiological and pathological processes.

Ileal bile acid transporter inhibition, CYP7A1 induction, and antilipemic action of 264W94.

264W94 was designed to inhibit the ileal bile acid transporter (IBAT). Evaluated in vitro, 264W94 dose-dependently inhibited sodium-dependent uptake of 10 micro M [(3)H]taurocholic acid (TC) by rat and monkey brush border membrane vesicles with IC(50)s of 0.24 micro M and 0.41 micro M, and had a competitive profile with K(i) of 0.2 micro M against TC in Chinese hamster ovary cells expressing human IBAT. In distal ileum in situ, 1-10 micro M of 264W94 rapidly decreased uptake of 3mM TC by 24-39%, with corresponding decreases in biliary recovery. In rats and mice in vivo, oral 264W94 decreased absorption of TC analog, 23,25-(75)Se-homocholic acid taurine ((75)SeHCAT; quantitated in feces), with ED(30) of 0.02 mg/kg bid. (75)SeHCAT traced through the GI-tract revealed that peak (97%) inhibition of (75)SeHCAT absorption by the distal quarter of small intestine occurred at 4 h after single dose of 264W94 (0.1 mg/kg). Inhibition of IBAT by 264W94 in rats was associated with compensatory, same-day, 4-fold induction of hepatic cholesterol 7alpha-hydroxylase (CYP7A1) activity, exhibiting normal diurnal fluctuation for 3 days of dosing. In diet induced hypercholesterolemic rats, 264W94 (0.03-1.0 mg/kg bid) dose-dependently reduced serum LDL+VLDL cholesterol up to 61%. In conclusion, 264W94 is a potent new cholesterol lowering agent that acts through inhibition of IBAT and exhibits activity in a human model.