Atsushi Konishi

A synergistic increase in transplantable peripheral blood stem cells in mice by co-administration of recombinant human interleukin 6 and recombinant human granulocyte colony-stimulating factor.

We examined the effects of co-administration of re-combinant human (rh) IL-6 (10 μg/day) and rh granulocyte colony-stimulating factor (G-CSF) (0.35 μg/day) on the number of peripheral blood cells and peripheral progenitor cells in mice. Among blood cells counts, only white blood cells were synergistically enhanced by co-administration of rhIL-6 and rhG-CSF. Moreover, it was found that coadministration of rhIL-6 and rhG-CSF also caused a marked synergistic increase in the number of peripheral blood progenitor cells. Namely, in combination with rhG-CSF, which alone induced a 170-fold increase in peripheral granulocyte-macrophage colonyforming units (CFU-GM) on day 14, rhIL-6 synergistically increased the number of CFU-GM to more than 1600-fold higher than the number in control mice. Administration of rhIL-6 alone induced a 46-fold increase in CFU-GM. Similar synergistic increases of other hematopoietic progenitors, such as colony-forming units in spleen and megakaryocyte colony-forming units in blood, were also observed in mice co-administered rhIL-6 and rhG-CSF. The survival rate of lethally irradiated recipient mice transplanted with mononuclear cells from 100 μl of blood from mice administered rhIL-6 and/or rhG-CSF was examined. When mononuclear cells from mice co-administered rhIL-6 and rhG-CSF were injected, survival rate at day 100 was 92%. In contrast, recipient mice transplanted with mononuclear cells from mice administered either rhIL-6 or rhG-CSF alone showed a survival rate of 31% or 46%, respectively, although transplantation of mononuclear cells from control mice failed to rescue any lethally irradiated recipient mice. These results suggest that co-administration of rhIL-6 and rhG-CSF may be useful for peripheral blood stem cell transplantation.

APC0576, a novel inhibitor of NF-κB-dependent gene activation, prevents pro-inflammatory cytokine-induced chemokine production in human endothelial cells ☆

Endothelial cells participate in the inflammatory and immune reactions. Endothelial cell activation is a recurrent phenomenon linked to the pathogenesis of diverse human diseases, such as acute and chronic inflammation and cardiovascular disorders. Pro-inflammatory cytokines (e.g., IL-1, TNF) are well-known activators of endothelial cells, since they strongly induce production of chemokines (e.g., IL-8, MCP-1) and cell adhesion molecules, resulting in an activation of inflammatory transcription factors such as NF-kappaB. We have established a cell-based reporter assay for the NF-kappaB-dependent gene activation in HUVEC. Using this assay system, we have identified a novel synthetic small molecule, APC0576, 5-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)-2-(4-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)phenoxy)pyridine, as an inhibitor of IL-1-induced NF-kappaB-dependent gene activation without any adverse effects on the cell viability. APC0576 represses the IL-1-induced release of chemokines (e.g., IL-8, MCP-1) in HUVEC. This inhibitory effect occurred at the level of mRNA expression. Despite having a strong inhibitory effect on the NF-kappaB-dependent transcriptional activation, APC0576 does not inhibit the IL-1-induced DNA binding of NF-kappaB, degradation of I-kappaB-alpha, or phosphorylation of RelA (p65). Although its molecular mechanism of action is not yet clear, APC0576 is a promising therapeutic candidate for diverse diseases involved in the pathogenic endothelial activation.

APC0576: A novel small molecule, immunosuppressive agent effective in primate models

Background. APC0576, 5-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)-2-(4-(((S)-2,2-dimethylcyclopropanecarbonyl)amino)phenoxy)pyridine is a novel synthetic compound with an inhibitory activity on NF-&kgr;B–dependent gene activation and chemokine synthesis in human endothelial cells. This article describes the effect of APC0576 on T-cell–dependent immune functions in vitro and in vivo in primate models, because NF-&kgr;B is known to be one of the potent mediators in T-cell activation. Methods. The effects of APC0576 on interleukin-2 production and proliferative responses in human peripheral blood mononuclear cells were studied under various stimuli with in vitro culture assay. Next, female rhesus monkeys were immunized with tetanus toxoid (TTx), and APC0576 was orally administered for 4 weeks. Serum-specific antibody for TTx was monitored weekly using an enzyme-linked immunosorbent assay, and delayed-type hypersensitivity reaction was examined after 4 weeks of APC0576 treatment. To evaluate the immunosuppressive activity, APC0576 was orally administered for 32 days to rhesus monkeys that received transplants of allogeneic kidney. Results. APC0576 effectively suppressed interleukin-2 production and proliferation in activated human peripheral blood mononuclear cells. Both delayed-type hypersensitivity reaction and specific antibody formation evoked by TTx was significantly and dose-dependently attenuated by 4 weeks treatment of APC0576 without any serious toxicologic signs. Allogeneic kidneys grafted in rhesus monkeys were not rejected and fully functioned during the 32 days of APC0576 treatment, although they were rapidly rejected after the withdrawal of the drug. Conclusions. A novel, orally available immunosuppressive agent, APC0576, effectively inhibited T-cell–based immune responses both in vitro and in vivo. APC0576 may have potential for a therapeutic agent in clinical organ transplantation and various cytokine-mediated diseases.