The utility of pathway selective estrogen receptor ligands that inhibit nuclear factor-kappa B transcriptional activity in models of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease that produces synovial proliferation and joint erosions. The pathologic lesions of RA are driven through the production of inflammatory mediators in the synovium mediated, in part, by the transcription factor NF-kappaB. We have identified a non-steroidal estrogen receptor ligand, WAY-169916, that selectively inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. The activity of WAY-169916 was monitored in two models of arthritis, the HLA-B27 transgenic rat and the Lewis rat adjuvant-induced model, after daily oral administration. In both models, a near complete reversal in hindpaw scores was observed as well as marked improvements in the histological scores. In the Lewis rat adjuvant model, WAY-169916 markedly suppresses the adjuvant induction of three serum acute phase proteins: haptoglobin, alpha1-acid glycoprotein (alpha1-AGP), and C-reactive protein (CRP). Gene expression experiments also demonstrate a global suppression of adjuvant-induced gene expression in the spleen, liver, and popliteal lymph nodes. Finally, WAY-169916 was effective in suppressing tumor necrosis factor-alpha-mediated inflammatory gene expression in fibroblast-like synoviocytes isolated from patients with RA. Together, these data suggest the utility of WAY-169916, and other compounds in its class, in treating RA through global suppression of inflammation via selective blockade of NF-kappaB transcriptional activity.

Figure 1

WAY-169916 improves joint scores in HLA-B27 transgenic rat model of arthritis. HLA-B27 transgenic rats, 26 to 28 weeks old, presenting signs of arthritis were treated orally daily with vehicle, prednisolone (0.6 mg/kg), or WAY-169916 (10 mg/kg) for 29 days. Joint scores were assessed by evaluating hindpaws for erythema and swelling (0 to 3 each; maximal score of 12).

Figure 2

WAY-169916 improves joint scores in a dose-dependent fashion in rat adjuvant-induced arthritis model. (a) Male Lewis rats were injected with complete Freund's adjuvant on day 1 and maximal inflammation was allowed to develop. Beginning on day 8 and continuing until day 22, rats were treated daily with oral vehicle or WAY-169916 (0.3 and 0.1 mg/kg). Joint scores were assessed by evaluating hindpaws for erythema and swelling (0 to 3 each; maximal score of 12). (b) WAY-169916 improves joint scores in rat adjuvant-induced arthritis model in both males and females. Experiments were performed as in (a) except that WAY-169916 was dosed daily at 5 mg/kg orally in both intact male and intact female rats.

Figure 3

WAY-169916 inhibits the adjuvant-induced expression of serum acute-phase protein. Serum from control male rats or adjuvant-induced rats treated with either vehicle or WAY-169916 (5 mg/kg) was analyzed for the expression of (a) haptoglobin, (b) α1-acid glycoprotein (α1-AGP) or (c) C-reactive protein (CRP) by radial immunodiffusion assay. Results are expressed as means ± SEM from six rats per group. *P < 0.05 compared with vehicle control. CFA, complete Freund's adjuvant.

Figure 4

The effect of WAY-169916 in spleen cells. (a) The regulation of LBS binding protein (LBP), haptoglobin and S100A9 gene expression by WAY-169916 from spleens from the rat adjuvant model were confirmed by real-time RT–PCR (grey bars) compared with the regulation observed in the gene-profiling experiments (black bars). Results are expressed as means ± SEM from six rats per group. *P < 0.05 compared with vehicle control. (b) Treatment with WAY-169916 does not interfere with the binding of NF-κB to DNA. Nuclear extracts from primary mouse spleen cell cultures were co-treated for 18 hours with vehicle or concanavalin A (ConA; 5 μg/ml) and either WAY-169916 (1 μM) or pyrrolidine dithiocarbamate (PDTC) (100 μM) as indicated. CFA, complete Freund's adjuvant.

Figure 5

Inhibitory effect of treatment with WAY-169916 on inflammatory gene expression in FLS induced by TNF-α. Fibroblast-like synoviocytes (FLS) were pretreated for 1 hour with WAY-169916 (1 μM) or pyrrolidine dithiocarbamate (PDTC) (100 μM) before treatment with tumor necrosis factor-α (TNF-α) for 1 hour. The mRNA levels for TNF-α, IL-6 and intercellular cell-adhesion molecule-1 (ICAM-1) were determined by real-time RT–PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase. Results are reported as means ± SEM for each group, with the mean level of the stimulated cells treated with vehicle defined as 1. *P < 0.05 compared with vehicle control.