A methionine aminopeptidase-2 inhibitor, PPI-2458, for the treatment of rheumatoid arthritis

Abstract

The hallmark of rheumatoid arthritis (RA) is the progressive destruction of articular joints, characterized by invasive synovial hyperplasia and pathological neovascularization. Here we report that PPI-2458, a member of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibits the proliferation of human fibroblast-like synoviocytes (HFLS-RA), derived from RA patients, with a growth inhibitory concentration 50 (GI(50)) of 0.04 nM and a maximum inhibition of >95% at 1 nM. Human umbilical vein endothelial cells (HUVEC) are similarly inhibited in proliferation by PPI-2458 (GI(50), 0.2 nM). We developed a method to measure the level of MetAP-2 enzyme inhibition after exposure to PPI-2458 and demonstrate that growth inhibition of PPI-2458-sensitive HFLS-RA and HUVEC is linked to MetAP-2 enzyme inhibition, in a dose-dependent fashion. The secretion of several inflammatory mediators such as IL-6 and vascular endothelial growth factor from activated HFLS-RA was not inhibited by PPI-2458. The CNS toxicity profile of PPI-2458, determined by the incidence of seizures, is significantly improved over that of the parental compound TNP-470. In the rat model of peptidoglycan-polysaccharide-induced arthritis, PPI-2458 significantly attenuated paw swelling when therapeutically administered after the onset of chronic disease. We suggest that the mechanism of PPI-2458 action, highly selective and potent anti-proliferative activity on HFLS-RA and HUVEC in vitro, a significantly improved CNS toxicity profile, and marked attenuation of chronic disease in the rat peptidoglycan-polysaccharide arthritis model in vivo, positions this compound as a drug for the treatment of RA.

Fig. 1.

Chemical structures of fumagillin, TNP-470, and PPI-2458.

Fig. 2.

PPI-2458-induced growth inhibition in HFLS-RA (•) and HUVEC (○), but not NHDF-Ad (▪) (A), is linked to MetAP-2 enzyme inhibition (B). (A) HFLS-RA and NHDF-Ad (8 × 10³ cells) and HUVEC (8 × 10³ cells) were incubated with increasing concentrations (10^-12 to 10^-6 M) of PPI-2458 for 7 and 4 days, respectively. For the final 24 h, 1 μCi/well of [³H]thymidine was added, and proliferation was determined by [³H]thymidine incorporation. The results are representative of at least five independent experiments. (B) The MetAP-2 assay was performed with cell lysates from HFLS-RA, NHDF-Ad, and HUVEC treated exactly as described in A, except that no [³H]thymidine was added. The amount of free MetAP-2 in each cell lysate was measured as described in Materials and Methods. Total amounts of MetAP-2 in PPI-2458-free cell lysates (=100%) were 47 pg/mg of cellular protein in HFLS-RA, 53 pg/mg in NHDF-Ad, and 7 pg/mg in HUVEC.

Fig. 3.

Incidence of seizures after PPI-2458 and TNP-470 administration. Sprague–Dawley rats were given PPI-2458 or TNP-470 at doses of 6 or 60 mg/kg i.v. for 14 consecutive days. The animals were observed daily after dosing for clinical signs of toxicity, and the incidence of seizures was recorded for each treatment group (n = 8; four per sex).

Fig. 4.

PPI-2458 decreases paw volumes (swelling) in PG-PS-induced arthritis in rats. Arthritis was induced in female Lewis rats by a single injection of PG-PS (25 mg/kg, i.p.) (▪). Treatment was started at day 15, after the onset of chronic disease. Rats were dosed po/qod with vehicle (□), dex (1 mg/kg, po) (▾) or PPI-2458 at 0.25 mg/kg, po (Δ) 1 mg/kg, po (224), 5 mg/kg, po (o), 50 mg/kg, po (•). At day 31, the rats were killed, and paw volumes were measured as described in Materials and Methods. Each point on the graph represents mean ± SEM (n = 8, except vehicle: n = 4). **, P < 0.01, *, P < 0.05 compared with PG-PS-treated rats.