The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated in several Th1-mediated inflammatory diseases. Recently, the efficacy of the anti-IP-10 antibody MDX-1100 reported in a phase 2 clinical trial for RA [44
] reinforced the crucial role of the CXCL10-CXCR3 axis in this disease, and the therapeutic potential of small molecule CXCR3 antagonists [45
]. So far, only one of the CXCR3 antagonists, AMG487 (T487), progressed to Phase II clinical trials but has been halted because of lack of efficacy. Since this may have been due to variability in drug exposure, it is clear that this failure is not a misrepresentation of CXCR3 as a drug target. In this regard, SCH 546738 is a small molecule non-competitive CXCR3 antagonist with much higher affinity than AMG487 and therefore may have better chance to achieve the in vivo efficacy.
In the mouse CIA model, SCH 546738 is efficacious in reducing disease development by attenuating leukocyte infiltration into the joint and the structural damage to the bone and cartilage. It is of interest to note that SCH 546738 demonstrated efficacy even though dosing was started after the disease process was initiated and when mice had already started to show signs of paw swelling. It was reported that T487 reduced inflammation and cartilage damage in mouse and rat models of CIA [21
]. In rat adjuvant arthritis, blockade of CXCR3 by anti-CXCR3 mAb significantly inhibits T cell infiltration of arthritic joints and reduces the severity of arthritis [46
]. All these data directly demonstrate an important role of CXCR3 in the development of arthritis and CXCR3 blockade reduces the disease severity in the arthritis. It is likely that small molecule CXCR3 antagonists may achieve the efficacy of the anti-IP-10 antibody MDX-1100 reported in a phase 2 clinical trial for RA.
The available functional data for the role of CXCR3 and its ligands in EAE are contradictory. Different investigators have reported conflicting results when using IP-10-/-
mice, anti-IP-10 antibody, anti-sense RNA and vaccines [47
]. The recent results from CXCR3-/-
mice show that CXCR3 is not required for the recruitment of immune cells to the CNS in MOG-EAE. The work by Liu et al. [49
] showed exacerbation of EAE disease in CXCR3-/-
mice and with neutralizing anti-CXCR3 Abs. It indicates that the exacerbation in the CXCR3-/-
mice correlates with enhanced effector T cell proliferation and reduced peripheral and CNS expression of IFN-γ, but with no impact on leukocyte migration to CNS. A subsequent study by Muller et al. [50
] showed that CXCR3-/-
mice had more severe chronic disease with increased demyelination and axonal damage, although the number of CD4+ and CD8+ T cells infiltrating the CNS were similar in CXCR3-/-
and wild type mice. In contrast to MOG-EAE, CXCR3 appears to promote the lymphocyte accumulation inside the CNS in some virus-induced demyelinating disease models [51
]. This may point to disease-specific functions of CXCR3 and its ligands, which can vary depending on the nature of the pathogenic insult. These varied results probably reflect the complex and perhaps divergent roles for the chemokine system in the pathogenesis of EAE and virus-induced neuroinflammatory diseases. Recently, a nonspecific small molecule antagonist of CCR5, CCR2 and CXCR3 (TAK-779) was reported to reduce incidence and severity of EAE by decreasing migration of inflammatory cells into the CNS [52
]. Our study is the first report that a specific small molecule CXCR3 antagonist SCH 546738 consistently inhibits both mouse and rat EAE clinical disease with no evidence of exacerbation. Furthermore, 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. At least for small molecule antagonists including SCH 546738, the beneficial effect of CXCR3 blockade has been observed in EAE. Maybe studies using CXCR3-/-
mice and neutralizing anti-CXCR3 Abs offer some hints as to other possible function of CXCR3 receptor and its ligands. Beyond leukocyte recruitment, CXCR3 may modulate T cell IFN-γ production, regulation between Th1 vs. Th17 cells, or control T cells at the perivascular space in the CNS. It is not unlikely that a small molecule antagonist, a neutralizing antibody or a genetic deletion can perturb a receptor's activity in different ways, leading to different conclusion about the protein's biological function.
The role of CXCR3 in leukocyte recruitment was first demonstrated in the CXCR3 knockout mouse in year 2000, where the rejection of a cardiac allograft was significantly delayed, and resulted in permanent allograft engraftment with cyclosporine [10
]. In addition, lack of CXCL10 in the graft led to prolonged allograft survival [53
]. However, two recent studies published in 2008 [54
] questioned the importance of CXCR3 in allograft rejection and found moderate to little increase in graft survival using CXCR3-/-
mice or small molecule CXCR3 antagonist MRL-957 and anti-CXCR3 antibody targeting in human CXCR3 knock-in mice. These two studies conclude that CXCR3 is not essential for leukocyte recruitment in the cardiac allograft rejection. In contrast, Uppaluri et al. [56
] demonstrates that a CXCR3 blocking antibody significantly prolonged both cardiac and islet allograft survival, and induced long-term graft survival greater than 100 days when combined with rapamycin. In 2009, one study shows that TAK-779 attenuates cardiac allograft vasculopathy in part by reducing CCR5+
T lymphocyte subset infiltration into the graft [57
]. The other study by Rosenblum et al. [58
] shows that small molecule CXCR3 antagonist AMG1237845 prolongs allograft survival; however, it does not inhibit leukocyte recruitment into the graft. The difference in the contribution of CXCR3 to mouse allograft rejection observed in similar models in different laboratories can not be explained by current data sets and additional experiments are required to clarify these conflicting results.
In the rat cardiac allograft transplant model, a small molecule CXCR3 antagonist TLRK-A was reported to prolong graft survival, but was active only in combination with cyclosporine [59
]. However, another small molecule CXCR3 antagonist NIBR2130 did not prolong graft survival [55
]. In this study, we demonstrate that SCH 546738 delays graft rejection and in combination with cyclosporine, permits permanent engraftment in the rat cardiac allograft transplant model.
In summary, our study demonstrates that administration of SCH 546738 attenuates disease in mouse CIA, rat and mouse EAE, and rat cardiac allograft rejection. Combination of IFN-β therapy and SCH 546738 has an additive effect in the mouse EAE model. Furthermore, in combination with cyclosporine, SCH 546738 permits permanent engraftment in the rat cardiac allograft transplant model.
The findings from our study and others indicate that targeting the CXCR3 receptor by small molecule antagonists and antibodies can be a promising approach to RA. Since the results from CXCR3 inhibition in EAE and allograft rejection remains contradictory, we need to better understand the roles of the chemokine system operating in the pathogenesis of EAE and allograft rejection that truly reflects the molecular mechanism in human diseases and enhance the chance of success in human clinical trials.