Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis

Abstract

Background: The Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment.

Methods: Potency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of in vitro assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed in vitro using colony forming assays. In vivo the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model.

Results: CP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in in vitro enzyme assays. Dose-dependent inhibition of paw edema was observed in vivo with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the in vitro whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2.

Conclusion: Results from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.

Figure 1

Peripheral blood neutrophil count and the inflammatory cytokines IL-6 and IL-17 increase with disease in the rat AIA model. Normal and AIA rats (n = 12 rats per group per timepoint) were characterized temporally post-adjuvant immunization. Hind paw volume was measured by volume displacement as an indication of joint arthritis, days 13 to 26 (A). Peripheral blood neutrophils were quantitated using a Cell-Dyne 3700 analyzer, days -1 to 21 (B). IL-17 (C) and IL-6 (D) were quantitated using immunoassays, days -1 to 21. (*) indicates statistical significance p ≤ 0.04 compared to normal controls. Data represented as the mean ± SEM.

Figure 2

CP-690,550 dose-dependently inhibits hind paw edema, PBNC, and serum inflammatory cytokine levels in the rat AIA model. AIA rats were treated orally with CP-690,550 (n = 12 rats per treatment group), twice daily, starting on day 14 and continuing through day 21 post-adjuvant immunization. Arthritis was monitored by measuring hind paw volume using volume displacement (A). (*) indicates statistical significance p < 0.014 compared to vehicle control treated disease animals. Drug exposure-paw volume response relationship was determined (B). CP-690,550 ED₅₀ plasma exposures were compared to in vitro rat whole blood IC₅₀ potencies for JAK1/2, JAK2, and JAK1/3 (C). Whole blood was analyzed for PBNC using a Cell-Dyne analyzer on day 21 post immunization (D). (*) indicates statistical significance p < 0.002 compared to vehicle control treated disease animals. IL-17 and IL-6 were quantitated using immunoassays (E). IL-17 (#) indicates statistical significance p ≤ 0.059 and IL-6 (*) indicates statistical significance p ≤ 0.013 compared to disease vehicle control. Day 21 dose-related correlation between reductions in hind paw edema and PBNC in AIA rats (F). Data represented as the mean ± SEM (A-D) or the mean percent control ± SEM (E and F).

Figure 3

CP-690,550 inhibits the increase in bone marrow myeloid precursors in the rat AIA model. Bone marrow from normal and AIA rats (n = 12 rats per group) was analyzed for maturing myeloid precursors by flow cytometry at day 7 and day 21 post-adjuvant immunization (A). (*) indicates statistical significance p ≤ 0.0008 compared to normal animals. AIA rats were treated with CP-690,550 or vehicle control orally, once daily, days 14 to 21, and bone marrow was collected and analyzed on day 21 for maturing myeloid cells by flow cytometry (B). (*) indicates statistical significance p ≤ 0.05 compared to the disease vehicle control. Data represented as the mean ± SEM.