T-614, a novel immunomodulator, attenuates joint inflammation and articular damage in collagen-induced arthritis

Abstract

Introduction: T-614 is a novel oral antirheumatic agent for the treatment of rheumatoid arthritis. Whether it has immunomodulatory or disease-modifying properties and its mechanism of action are largely undetermined.

Methods: Rats with collagen-induced arthritis (CIA) were treated with T-614 (5 and 20 mg/kg) daily. Animals receiving methotrexate (1 mg/kg every 3 days) and the nonsteroidal anti-inflammatory agent nimesulide (10 mg/kg per day) were used as controls. A combination therapy group was treated with both T-614(10 mg/kg per day) and methotrexate (1 mg/kg every 3 days). Hind paw swelling was evaluated and radiographic scores calculated. Serum cytokine levels were assessed by Bio-plex analysis. Quantitative PCR was used to evaluate expression of mRNA for interferon-gamma, IL-4 and IL-17. Serum IL-17 and anti-type II collagen antibodies (total IgG, IgG1, IgG2a, IgG2b and IgM) were measured using ELISA.

Results: Oral T-614 inhibited paw swelling and offered significant protection against arthritis-induced cartilage and bone erosion, comparable to the effects of methotrexate. CIA rats treated with T-614 exhibited decreases in both mRNA expression of IL-17 in peripheral blood mononuclear cells and lymph node cells, and circulating IL-17 in a dose-dependent manner. T-614 also reduced serum levels of tumor necrosis factor-alpha, IL-1beta and IL-6. A synergistic effect was observed for the combination of methotrexate and T-614. In addition, T-614 (20 mg/kg per day) depressed production of anti-type II collagen antibodies and differentially affected levels of IgG2a subclasses in vivo, whereas IgM level was decreased without any change in the IgG1 level. Together, the findings presented here indicate that the novel agent T-614 has disease-modifying effects against experimental arthritis, as opposed to nimesulide.

Conclusions: Our data suggested that T-614 is an effective disease-modifying agent that can prevent bone/cartilage destruction and inflammation in in CIA rats. Combination with methotrexate markedly enhances the therapeutic effect of T-614.

Figure 1

Effects of therapeutic treatment with T-614 on disease progression in rats with established CIA. Rats were orally treated daily with T-614 at 5 mg/kg per day or 20 mg/kg per day; MTX at 1 mg/kg every 3 days; nimesulide at 10 mg/kg per day; T-614 at 10 mg/kg per day and MTX at 1 mg/kg every 3 days; or vehicle. Treatment began on day 12 after immunization with type II collagen until day 36. Data are expressed as mean ± standard error of the mean (n = 5 to 7). *P < 0.05, **P < 0.01, versus day 12 or the vehicle-treated rats. CIA, collagen-induced arthritis; MTX, methotrexate.

Figure 2

Effects of therapeutic treatment with T-614 on inflammation in the CIA rats. (a) STIR magnetic resonance images of hind paws from CIA rats. The presence of soft tissue swelling (yellow arrow) and localization of bone marrow edema (yellow triangle) are highlighted. Neither paw swelling nor bone marrow edema was seen in normal rats (subpanel a). Severe soft tissue swelling and bone erosion were seen in CIA rats treated with vehicle (subpanel b). Similar damage was observed in rats treated with nimesulide (subpanel d), but much less damage was seen in rats treated with MTX (subpanel c), T-614 (subpanels e and f), and combination treatment with T-614 and MTX (subpanel g). (b) Magnetic resonance imaging score of soft tissue swelling in treated CIA rats. Data are expressed as mean ± standard error of the mean (n = 3 to 5). *P < 0.05, **P < 0.01, versus vehicle-treated arthritic rats. CIA, collagen-induced arthritis; MTX, methotrexate.

Figure 3

Effects of treatment with T-614 on structural integrity in CIA rats. (a) Macroradiographs of rat hind paws. Neither paw swelling nor joint damage was observed in normal rats (subpanel a). Severe bone matrix resorption and erosion were seen in CIA rats treated with vehicle (subpanel b). Similar damage was observed in rats treated with nimesulide (subpanel d), but the damage was much less in rats treated with MTX (subpanel c), T-614 (subpanels e and f), and both of them (subpanel g). (b) Radiologic score of bone erosion in treated CIA rats. Data are expressed as mean ± standard error of the mean (n = 3 to 5). *P < 0.05, **P < 0.01 versus the vehicle-treated rats. (c) All images were obtained using a RS-9 in Vivo Micro-CT. Neither joint damage nor bone loss was seen in normal rats (subpanel a). Severe bone matrix resorption, erosion joint, and bone loss were seen in CIA rats treated with vehicle (subpanel b). Similar bone loss was seen in rats treated with nimesulide (subpanel d) but this was much less in rats treated with MTX (subpanel c), T-614 (subpanels e and f), and the combination of T-614 and MTX (subpanel g). CIA, collagen-induced arthritis; MTX, methotrexate.

Figure 4

Effects of T-614 on cytokine levels in CIA rats. Rats were orally treated with different doses of T-614, nimesulide, MTX and vehicle, beginning on day 12 after the immunization with CIA until day 36. (a) Effects of T-614 on mRNA levels of IFN-γ, IL-4 and IL-17 in lymph node and PBMCs from CIA rats. (b) Effects of T-614 on serum levels of TNF-α, IL-1β, IL-6 and IL-17 in the CIA rats. CIA, collagen-induced arthritis; IFN, interferon; IL, interleukin; LN, lymph node; MTX, methotrexate; PBMC, peripheral blood mononuclear cell.

Figure 5

Effects of T-614 on serum IgG levels in CIA rats. Serum was collected on day 36. Anti-CII (total IgG, IgM, IgG₁, IgG_2a, and IgG_2b) levels increased as disease progressed. Combination therapy reduced the total anti-CII antibody level significantly, as well as levels of IgM, IgG₁, IgG_2a, and IgG_2b. High dose T-614(20 mg/kg per day) also decreased levels of total IgG, IgM and IgG_2a, whereas low-dose of T-614 (5 mg/kg per day) or MTX (1 mg/kg every 3 days) had an effect only on IgG_2a level. Data are expressed as mean ± standard error of the mean (n = 5 to 7). *P < 0.05, versus the vehicle-treated rats.