Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis

Abstract

Introduction: CP-690550 is a small molecule inhibitor of Janus kinase 3 (JAK3), a critical enzyme in the signaling pathway of multiple cytokines (interleukin (IL)-2, -7, -15 and -21) that are important in various T cell functions including development, activation and homeostasis. The purpose of this study was to evaluate CP-690550 in murine collagen-induced (CIA) and rat adjuvant-induced (AA) models of rheumatoid arthritis (RA).

Methods: CIA and AA were induced using standard protocols and animals received the JAK3 inhibitor via osmotic mini-pump infusion at doses ranging from 1.5-15 mg/kg/day following disease induction. Arthritis was assessed by clinical scores in the CIA models and paw swelling monitored using a plethysmometer in the AA model until study conclusion, at which time animals were killed and evaluated histologically.

Results: CP-690550 dose-dependently decreased endpoints of disease in both RA models with greater than 90% reduction observed at the highest administered dose. An approximate ED50 of approximately 1.5 mg/kg/day was determined for the compound based upon disease endpoints in both RA models examined and corresponds to CP-690550 serum levels of 5.8 ng/ml in mice (day 28) and 24 ng/ml in rats (day 24). The compound also reduced inflammatory cell influx and joint damage as measured histologically. Animals receiving a CP-690550 dose of 15 mg/k/d showed no histological evidence of disease.

Conclusion: The efficacy observed with CP-690550 in CIA and AA suggests JAK3 inhibition may represent a novel therapeutic target for the treatment of RA.

Figure 1

Clinical scores from murine collagen-induced arthritis study 1. Animals were given initial injection of type II collagen on day -21 and disease was induced with a second injection on day 0. On day 3 (arrow), pumps were implanted and clinical signs measured twice a week from day 10 to day 28. By day 10, a statistically significant, dose-dependent decrease in the clinical score was observed with all doses of CP-690550 and these remained significant throughout the remainder of the study.

Figure 2

Clinical scores from murine collagen-induced arthritis study 2. Animals were given initial injection of type II collagen on day -21 and disease was induced with a second injection on day 0. On day 3 (arrow), pumps were implanted and clinical signs measured twice a week from day 6 to day 31. By day 9, a statistically significant, dose-dependent decrease in the clinical score was observed with all doses of CP-690550 and these remained significant to day 28. On day 31, only the high and mid dose of CP-690550 contained a statistically significant decrease in clinical score. At no time point during the course of the study did the anti-tumor necrosis factor (TNF) antibody treatment result in a statistically significant decrease in clinical score.

Figure 3

Histological evaluation of damage to murine knees. Histological sections of knee samples from murine collagen-induced arthritis study 1 were graded as described in Table 1. CP-690550 produced a dose-dependent inhibition of knee damage that reached statistical significance (p < 0.0001) at the 15 mg/kg/day dose relative to vehicle.

Figure 4

Representative histological sections from murine collagen-induced arthritis study 1. In all figures, the arrowheads point to the femoral condyle (top) and tibial plateau (bottom) articular cartilages and the asterisks highlight the inflammatory cells in the soft tissue surrounding the joint. Panel (a) is from a naïve control containing no damage to the articular cartilage and few cells in the soft tissue surrounding the joint. Panel (b) is from a vehicle treated animal, demonstrating significant influx of inflammatory cells into the synovial tissue and cavity as well as significant proteoglycan loss and erosion of the articular cartilage. Panels (c) and (d) are from animals that have been dosed with CP-690550 at 15 and 1.5 mg/kg/day, respectively. At both dose levels, CP-690550 decreased cell influx, synovial hypertrophy, articular cartilage damage and proteoglycan loss. The knees from animals dosed with CP-690550 15 mg/kg/day were very similar to the knees from the naïve animals. Bar = 500 μm.

Figure 5

Serum interleukin (IL)-6 levels from murine collagen-induced arthritis study 2. Blood was drawn from mice 15 days following the second type II collagen injection and serum IL-6 measured by enzyme-linked immunosorbent assay (ELISA). Data are mean ± standard error of the mean of values from 6–8 animals/treatment group, except the naïve group (n = 3).

Figure 6

Measurement of rat foot pad swelling in rat adjuvant-induced arthritis (AA) model. Animals were injected at the base of the tail with Mycobacterium butyricum in mineral oil on day 0 to induce disease. On day 10 (arrow), pumps were implanted and foot swelling measured twice a week from day 14 to day 24. CP-690550 decreased hind paw swelling in a dose dependent manner. In the rat AA model, data were expressed as percent control with the diseased vehicle treatment group on day 24 being set to 100%. As early as day 14, both the 5 and 15 mg/kg dose of CP-690550 resulted in a statistically significant decrease in foot volume. By day 17, all doses of CP-690550 resulted in a statistically significant decrease in foot volume that remained significant for the rest of the study. Data are mean ± standard error of the mean of 10 animals per group, except for the naïve group (n = 5).

Figure 7

Histological evaluation of damage to foot pad in rat adjuvant-induced arthritis (AA) model. Representative sections from vehicle (a) and 15 mg/kg/day CP-690550 (b) treated rats on day 24. In the vehicle treated animal there was significant inflammation (asterisk) and destruction of the metatarsal bone (arrow) and joint spaces (arrowhead). In contrast, there was no inflammation and the metatarsal joint spaces appeared normal in the CP-690550 treated animal.