Suppression of immunoglobulin production by a novel dihydroorotate dehydrogenase inhibitor, S-2678.

We discovered a novel dihydroorotate dehydrogenase (DHO-DH) inhibitor, S-2678 ([2-fluoro-2',5'-dimethyl-4'-[6-(3-methyl-2-butenyloxy) pyridin-3-yl] biphenyl-4-yl]-(3-methyl-2-butenyl) amine). Its inhibitory activity against DHO-DH was more potent than that of A77 1726, an active metabolite of the anti-rheumatic drug leflunomide. S-2678 suppressed immunoglobulin production in mouse B cells and human peripheral blood mononuclear cells in vitro, with little or no inhibition of cell proliferation, probably through inhibition of class switch recombination in the immunoglobulin heavy chain loci in B cells. In vivo antibody production induced by systemic immunization with ovalbumin was dramatically suppressed by oral administration of S-2678, without any toxicological signs. However, S-2678 did not affect T-cell activation in vitro, and cytokine production induced by intravenous anti-CD3 antibody in mice. S-2678 did not affect host defense in a mouse model of Candida infection, whereas leflunomide severely impaired it. In conclusion, S-2678 selectively acts on B cells, resulting in antibody production, which suggests that it is useful for the treatment of humoral immunity-related diseases.

Discovery of novel non-peptide thrombopoietin mimetic compounds that induce megakaryocytopoiesis.

We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.

Synergistic effect of PGD2 via prostanoid DP receptor on TNF-alpha-induced production of MCP-1 and IL-8 in human monocytic THP-1 cells.

Prostaglandin (PG) D2, a major cyclooxygenase metabolite generated predominantly from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases via the two PGD2 receptors, prostanoid DP receptor and chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2). Monocytes are known to express the prostanoid DP receptor, however, the role of it in inflammatory responses is still unclear. In the present study, to clarify the functional roles of prostanoid DP receptor on monocytes, we examined the effect of PGD2 on the production of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 from a human monocytic cell line, THP-1. Single activation of prostanoid DP receptor hardly produced any cytokines or chemokines. However, activation with PGD2 in the presence of tumor necrosis factor (TNF)-alpha mediated significant production of MCP-1 and IL-8, but not the other cytokines and chemokines, in comparison to single stimulation with TNF-alpha. In addition, the selective prostanoid DP receptor antagonist, pinagladin ((Z)-7-[(1R,2R,3S,5S)-2-(benzothiophen-3-ylcarbonylamide)-10-norpinan-3-yl]hept-5-enoic acid) inhibited the production of MCP-1 and IL-8 upon combined stimulation with PGD2 and TNF-alpha. The synergistic production of MCP-1 and IL-8 by PGD2 was mimicked by dibutyryl cAMP (db-cAMP) and was inhibited by a protein kinase A (PKA) inhibitor. Our findings suggest that activation of the prostanoid DP receptor on THP-1 cells enhances TNF-alpha-induced MCP-1 and IL-8 production via the cAMP/PKA signaling pathway.

Preclinical combination chemotherapy of nedaplatin with gemcitabine against gemcitabine-refractory human lung cancer.

The antitumor efficacy of the combination of nedaplatin (NDP) with gemcitabine (GEM) was evaluated against Ma44/GEM, a GEM-refractory subline of Ma44 human lung cancer, which was established by serial in vitro passage of Ma44 cells in the presence of GEM.Ma44/GEM showed less sensitivity to GEM and cytosine arabinoside with resistance factors of 7.7 and 8.3, respectively, but not to Taxol, Irinotecan, Mitomycin C and NDP. Flow cytometry analysis demonstrated that membrane transporter molecules such as multidrug-resistant, multidrug-resistant related protein or lung resistant protein were not induced in Ma44/GEM cells. In vivo experiments confirmed the less sensitivity of Ma44/GEM to GEM. The resistant factor of Ma44/GEM to GEM in vivo was estimated to be 6.7 in terms of ED(50).MA44/GEM-implanted athymic mice were treated with GEM i.v. once followed by i.v. injection of NDP at an interval of approximately 30 min. The mice were treated again with GEM after 3 or 4 days. The combined dosing of NDP with GEM resulted in synergistically enhanced inhibition of tumor growth against Ma44/GEM. The antitumor efficacy of the combination of NDP and GEM was superior to the best effect of either monotherapy. These results demonstrate the effectiveness of the combination of NDP with GEM against the GEM-refractory human lung cancer model.