A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

Abstract

Background: The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.

Results: In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.

Conclusions: SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Figure 1

Synthesis of SCH 546738. Synthesis of SCH 546738 was accomplished by the method outlined with details in Methods section.

Figure 2

Effect of concentrations of [¹²⁵I]hCXCL10 and [¹²⁵I]hCXCL11 on the IC₅₀ of SCH 546738 binding to human CXCR3 receptor. The ability of SCH 546738 to compete the binding of [¹²⁵I]hCXCL10 and [¹²⁵I]hCXCL11 to human CXCR3 receptor was determined using various concentrations of [¹²⁵I]hCXCL10 and [¹²⁵I]hCXCL11 as described in Methods. After 3 hr reaction, specific counts relative to input counts (B/B₀) in the presence of increasing concentrations of SCH 546738 are plotted. The IC₅₀ for each concentration of [¹²⁵I]hCXCL10 and [¹²⁵I]hCXCL11 are shown.

Figure 3

Effect of SCH 546738 on human Activated T cell chemotaxis induced by CXCL10, CXCL9, CXCL11 and CCL19. Human peripheral blood lymphocytes were prepared by Ficoll-Hypaque centrifugation, depleted of monocytes, and stimulated by PHA/IL-2. After 7-9 days of stimulation, activated T cell chemotaxis assay was carried out as described in Methods. The medium containing 20% fetal bovine serum was used for all dilutions of chemokines and compounds. Various concentrations of each indicated chemokine were added to the bottom wells in the presence of fixed concentrations of SCH 546738 (1, 10, 100 nM) (added both on the filter with cells and in the bottom wells). Chemotaxis of each sample is expressed relative to spontaneous response (without chemokines) as chemotactic index.

Figure 4

Plasma concentration versus time profiles of SCH 546738 in Lewis rat and C57BL/6 mouse. SCH 546738 in 0.4% methylcellulose was administered orally at 10 mg/kg (mpk) in Lewis rats or 30 mpk in C57BL/6 mice. The plasma concentration of SCH 546738 in the blood was calculated as the mean of 3 animals (n = 3) at indicated time points post-dose. Their AUC from 0 to 24 hr is also calculated and indicated.

Figure 5

SCH 546738 attenuates disease in mouse collagen-induced arthritis. SCH 546738 in 0.4% methylcellulose was administered orally twice daily at 3, 10 and 40 mg/kg (mpk). Dosing was initiated 16 days (day -4) postimmunization and continued through day 9. The disease score was significantly decreased in SCH 546738-treated animals as compared with vehicle-treated animals (* p < 0.05, two-tailed t test) at day 4, 7 and 9.

Figure 6

SCH 546738 in mCIA: Histopathological analysis of paws on day 9. Paws collected at day 9 from the vehicle (0.4% methylcellulose) and 40 mpk SCH 546738 groups of two independent experiments were analyzed by histopathology. (A) compares the histopathological scores of both groups of tissues. Both leukocyte infiltration into the joint and the structural damage to the bone and cartilage was significantly attenuated in SCH 546738-treated animals (* p < 0.05; ** p < 0.005, two-tailed t test). Example images of paw tissue sections collected from both groups of animals are shown in (B) (top, phalanges area; bottom, tarsal area). Massive cellular infiltrates and bone/cartilage erosions were evident in both tarsal and phalanges areas of the vehicle treated mouse paw (left panels). In contrast, cellular infiltrates were mainly observed in the phalanges region (right top), and rarely in the tarsal region (right bottom) of SCH546738-treated animals.

Figure 7

Combination of IFN-β therapy and CXCR3 inhibition has an additive effect on delaying disease onset and attenuating disease severity in the mouse EAE model. IFN-β was administered at 1700 ng by daily intramuscular injection and SCH 546738 was orally twice daily at 30 mpk. The mouse EAE was conducted as described in Methods. Treatment with either IFN-β or SCH 546738 alone or the combination significantly delayed disease onset and attenuated disease severity (p < 0.05, two-tailed t test) at day 16, 17 and 19.

Figure 8

SCH 546738 delays graft rejection and in combination with cyclosporine, permits permanent engraftment in the rat cardiac allograft transplant model. SCH 546738 was administered orally twice daily at 1, 5 and 15 mpk. Cyclosporine was administered daily at 2.5 mpk. In the combination study, 5 mpk SCH 546738 and 2.5 mpk cyclosporine were administered. SCH 546738 significantly increased the mean survival time of the graft at 1 mpk (MST = 11 days; p < 0.05), 5 mpk (MST = 14 days; p < 0.05) and 15 mpk (MST = 14.9 days; p < 0.05) when compared with the vehicle control (MST = 6 days). Graft survival was analyzed using the log-rank test.