ABR-215757 (5757) is a quinoline-3-carboxamide (Q-compound) currently in clinical development for systemic sclerosis. Q-compounds have shown efficacy in several different models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis. However, the mechanism of action is still poorly understood. In this thesis we have tried to find the mechanism of action of 5757 using different inflammatory models. We studied the effect of 5757 on generation of cells from bone marrow in cultures supplemented with different colony stimulating factors (paper I). CD11c+ cells increased in presence of 5757 while GR1+ cells decreased and also proliferated less in presence of 5757. These results were seen in GM-CSF and M-CSF stimulated cultures but not in cultures stimulated with G-CSF. 5757 did not affect the function of the generated cells. We also studied the effect of 5757 in amelioration of inflammatory disease. We used EAE, the animal model for the human autoimmune disease multiple sclerosis for this purpose (paper II). Treatment of mice with 5757 effectively reduces induction of EAE. This mechanism is an early effect since treatment with 5757 during the first five days after induction also reduced EAE development. Influx of T cells and myeloid cells to CNS was significantly reduced in these mice. EAE is a T cell dependent disease and the activation of antigen-specific disease-causing T cells occurs early in the peripheral lymphoid organs. In vivo proliferation of antigen-specific T cells and production of the effector cytokines IFN-γ and IL-17 were reduced in mice treated with 5757. To study the effect of 5757 on cell migration we used a sterile peritonitis model were necrotic cells or alum were used to induce inflammation (paper III). 5757 reduced migration of inflammatory monocytes and eosinophils to inflamed tissue. We also showed that the effect of 5757 on cell recruitment to inflamed tissue is dependent of Receptor of Advanced Glycation End products (RAGE) and that expression of this receptor by hematopoietic cells is important for the effect of 5757. Taken together this thesis shows that i) 5757 affects generation of immune cells in vitro, ii) 5757 treatment affects T cell activation and thereby reducing EAE and ii) that 5757 reduce migration of inflammatory cells to inflamed tissue in a RAGE-dependent way. (Less)

Quinoline-3-carboxamides (Q-compounds) are currently in clinical development for both autoimmune disease and cancer. We have previously shown that the Q-compound paquinimod (ABR-215757) significantly ameliorates disease symptoms in several mouse models of human inflammatory disease. Considering that recruitment of inflammatory cells into tissue is a common denominator of these models, we have in this report investigated whether paquinimod would interfere with cell accumulation during sterile peritoneal inflammation. To mimic the cell recruitment elicited by tissue injury, we used necrotic cells to induce the acute inflammatory response. We show that per oral treatment with paquinimod significantly reduced the accumulation of Ly6C(hi) inflammatory monocytes and eosinophils, but not neutrophils, in this model, and that this correlated with reduced number of such cells also in the omentum. Treatment also reduced the accumulation of these cell populations at a subcutaneous site of inflammation. In alum-induced inflammation, however, neutrophils were the dominant cell population and paquinimod failed to reduce the accumulation of inflammatory cells. Taken together, our results indicate that paquinimod selectively inhibits cell recruitment during acute sterile inflammation, but that this effect is context-dependent. These data have important implications for the understanding of the mechanism of action of Q-compounds in both pre-clinical and clinical settings.

Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6C(hi) inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4(+) T cells and reduced frequency of IFN-γ- and IL-17-producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6C(hi) CD115(+) inflammatory monocytes and CD11b(+)CD11c(+) dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte-derived DCs and CD4(+) T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4(+) T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b(+) myeloid cells.

Quinoline-3-carboxamides are currently in clinical development for treatment of both autoimmune disease and cancer. Carboxamides such as ABR-215757 (5757) have shown efficacy in several in vivo mouse models of human inflammatory autoimmune disease. Some microbial infections in mice cause GM-CSF dependent accumulation of dendritic cells expressing TNFα and inducible nitric oxide synthase (iNOS; Tip-DCs) in lymphoid organs. Functionally similar DCs develop in GM-CSF stimulated bone marrow (BM) cell cultures and offered an in vitro model that allowed us to study the impact of 5757 on cellular development of relevance for in vivo inflammatory conditions. We show in here that addition of 5757 to such cultures, in a dose-dependent way increased the frequency of DCs, while it reduced the frequency of Gr-1(+) cells by inhibiting their proliferation. This effect was specific as the compound neither influenced DC development from myeloid progenitors, nor the development of granulocytes in G-CSF stimulated BM cell cultures. Importantly, we also show that 5757 treatment reduced the accumulation of Gr-1(+) cells during inflammation in vivo. We therefore propose that this compound may ameliorate autoimmune disease by blocking proliferation of Gr-1(+) cells during inflammation-induced mobilization of myeloid cells.