Design, synthesis, and optimization of a series of 2-azaspiro[3.3]heptane derivatives as orally bioavailable fetal hemoglobin inducers.

Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (HbF) is a promising approach for the management of ß-thalassemia and sickle cell disease (SCD). We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules that could induce HbF, which resulted in identification of the hit compound 1. Exploration of structure-activity relationships and optimization of ADME properties led to 2-azaspiro[3.3]heptane derivative 18, which is more rigid and has a unique structure. In vivo using cynomolgus monkeys, compound 18 induced a significant dose-dependent increase in globin switching, with developable properties. Moreover, compound 18 showed no genotoxic effects and was much safer than hydroxyurea. These findings could facilitate the development of effective new therapies for the treatment of ß-hemoglobinopathies, including SCD.

Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

Phenotypic-screening generates active novel fetal globin-inducers that downregulate Bcl11a in a monkey model.

Heritable disorders associated with hemoglobin production are the most common monogenic disorders. These are mainly represented by disorders such as ß-thalassemia and sickle cell disease. Induction of fetal hemoglobin (HbF) has been known to ameliorate the clinical severity of these ß hemoglobinopathies. A high throughput phenotypic screening was used in this study to isolate novel compounds that may enhance the expression of γ-globin, the component of HbF, in human erythroid cell lines and primary erythroid progenitors derived from human CD34+ cells. The effect of lead compounds on epigenetic enzymes and key transcriptional factors was evaluated to identify their mode of action. One hit compound was further evaluated in vivo using monkey models. Among the ~18,000 compounds screened, 18 compounds were selected and tested to determine their ability to induce HbF in human erythroid cell lines and primary erythroid cells. One of these compounds, a 3-phenyl-isoxazole derivative, could potentially induce HbF in monkey bone marrow cells when administered orally. The compound downregulated negative transcriptional regulators of HbF, Bcl11a and LRF without inhibiting the known epigenetic enzymes. These studies demonstrated the advantages associated with phenotype-screening and identified novel fetal globin inducers that may be useful for treating hemoglobinopathies.

G protein-coupled receptor 39 plays an anti-inflammatory role by enhancing IL-10 production from macrophages under inflammatory conditions.

The possible role of G protein-coupled receptor 39 (GPR39) in inflammation was examined in macrophages. Gpr39 expression increased in thioglycollate-induced peritoneal macrophages. TC-G 1008, a G protein-coupled receptor 39 agonist, enhanced interleukin (IL)-10 production from thioglycollate-induced peritoneal macrophages stimulated with lipopolysaccharide (LPS) in vitro. In addition, the oral administration of TC-G 1008 enhanced serum IL-10 concentrations in an LPS-induced murine model of sepsis. The ablation of G protein-coupled receptor 39 significantly reduced IL-10 production by TC-G 1008 in thioglycollate-induced peritoneal macrophages stimulated with LPS and in the LPS-induced murine model of sepsis. Moreover, the oral administration of TC-G 1008 significantly improved the survival rate in the LPS-induced murine model of sepsis. Taken together, our data suggest that G protein-coupled receptor 39 exhibits an anti-inflammatory activity by enhancing IL-10 production from macrophages.

Heterogeneity of auto-antibodies against nAChR in myasthenic serum and their pathogenic roles in experimental autoimmune myasthenia gravis.

Many myasthenia gravis (MG) patients have auto-antibodies against the nicotinic acetylcholine receptor (nAChR), and monoclonal antibodies against the main immunogenic region (MIR) of nAChR can induce experimental autoimmune MG (EAMG). We investigated whether Fab fragment of MIR antibody (Fab35) could block the pathogenicity of polyclonal antibodies. Fab35 partially inhibited nAChR downmodulation, blocked EAMG serum-induced binding of polyclonal antibodies and complement deposition in vitro. Moreover, Fab35 did not ameliorate the EAMG serum-induced EAMG phenotype in rats. These results suggested that the EAMG serum possessed several different pathogenic antibodies that might be sufficient to induce the EAMG phenotype.

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools.

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.

Design, synthesis, and biological evaluation of 3-(1-Aryl-1H-indol-5-yl)propanoic acids as new indole-based cytosolic phospholipase A2α inhibitors.

This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A2α (cPLA2α, a group IVA phospholipase A2) inhibitors. A screening-hit compound from our library, (E)-3-{4-[(4-chlorophenyl)thio]-3-nitrophenyl}acrylic acid (5), was used to design a class of 3-(1-aryl-1H-indol-5-yl)propanoic acids as new small molecule inhibitors. The resultant structure-activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(p-O-substituted)phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA2α. Optimization of the p-substituents on the N1 phenyl group led to the discovery of 56n (ASB14780), which was shown to be a potent inhibitor of cPLA2α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models.

Pharmacokinetic/pharmacodynamic analyses of chymase inhibitor SUN13834 in NC/Nga mice and prediction of effective dosage for atopic dermatitis patients.

A chymase inhibitor SUN13834 has been shown to improve skin condition in animal models for atopic dermatitis. In the present study, effective dosages of SUN13834 for atopic dermatitis patients were predicted by pharmacokinetic/pharmacodynamic (PK/PD) analyses of SUN13834 in NC/Nga mice, which spontaneously develop atopic dermatitis-like skin lesions. For the PK/PD analyses, we utilized the minimum effective plasma concentration of unbound SUN13834 in late-phase reaction of trinitrochlorobenzene (TNCB)-induced biphasic dermatitis in mice, based on the assumption that the minimum effective plasma concentrations are the same among the two animal models. In late-phase reaction of biphasic dermatitis, SUN13834 was most effective when its plasma concentration was highest at the elicitation, and the minimum effective plasma concentration of unbound SUN13834 at the elicitation was calculated to be 0.13-0.2 ng/mL. Oral administration of SUN13834 improved dermatitis in NC/Nga mice at 15 mg/kg (twice a day; bid) and 30 mg/kg (once a day; qd), but not at 60 mg/kg (every other day; eod). At the three dosages, the duration times over the plasma level of 0.13-0.2 ng/mL were 16.1-20.3, 10.7-12.2 and 7.8-8.8h, respectively, suggesting an importance of maintenance of the minimum effective plasma concentration for at least about 10-12h. The clinical effective dosage predicted in this paper is also discussed in relation to a recently conducted Phase 2a study.

Analysis of signal transduction pathways involved in anti-CD30 mAb-induced human eosinophil apoptosis.

BACKGROUND: Activation of cell surface CD30 by immobilized anti-CD30 monoclonal antibodies (mAbs) induces extremely rapid and intense apoptosis in human eosinophils in vitro. This anti-CD30 mAb-induced eosinophil apoptosis was inhibited by the addition of inhibitors of p38 and ERK1/2 mitogen-activated protein kinases (MAPKs). However, the signal transduction pathways other than for MAPKs remain unclear. In the present study, we tried to clarify the molecules involved in the induction of apoptosis after cross-linking of CD30. METHODS: Purified human eosinophils were suspended in Iscove's minimal essential medium supplemented with 10% FCS and 1 ng/ml IL-5 and then cultured for 4 h in plates precoated with anti-CD30 mAb (clone Ber-H8) in the presence or absence of various signal transduction inhibitors. Eosinophil apoptosis was assessed using annexin V and flow cytometry. RESULTS: The addition of inhibitors of caspase-3, caspase-8, pan-caspases, nuclear factor kappaB and protein kinase C at reasonable concentrations did not alter anti-CD30 mAb-induced eosinophil apoptosis in vitro. However, apoptosis was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors: wortmannin at very high concentrations (>1 microM) significantly and LY294002 at a reasonable concentration partially inhibited that apoptosis. CONCLUSIONS: Anti-CD30 mAb-induced eosinophil apoptosis is likely to be mediated mainly through MAPKs and partially through PI3K, but independent of caspase activation. Downstream signaling molecules of MAPK activation in eosinophils have to be clarified in future studies.

Oral chymase inhibitor SUN13834 ameliorates skin inflammation as well as pruritus in mouse model for atopic dermatitis.

Chymase is a chymotrypsin-like serine protease exclusively stored in secretory granules of mast cells and has been thought to participate in allergic diseases. It has already been shown that chymase inhibitor SUN13834 improves dermatitis in NC/Nga mice that spontaneously develop dermatitis resembling atopic dermatitis. In the present study, effect of chymase inhibitor SUN13834 on itch, the major feature of atopic dermatitis, was examined using a mouse dermatitis model induced by repeated topical application of 2,4-dinitrofluorobenzene (DNFB). Oral administration of SUN13834 once a day for 5 weeks inhibited not only skin swelling but accumulation of inflammatory cells including mast cells and eosinophils in the skin of the mice. In addition, SUN13834 also decreased significantly at 10 and 50 mg/kg the amount of scratching behavior induced by the DNFB challenge. This result indicates for the first time that mast cell chymase may be involved in itch induction. In conclusion, SUN13834 is thought to be useful as therapeutic agent for atopic dermatitis.

Rapid and strong induction of apoptosis in human eosinophils by anti-CD30 mAb-coated microspheres and phagocytosis by macrophages.

BACKGROUND: Eosinophils represent a potential therapeutic target in allergic diseases. We previously reported that two clones of anti-CD30 mAbs (HRS-4 and Ber-H8) induced extremely rapid and intense apoptosis in human eosinophils in vitro, but only when the mAbs were immobilized on plates [Matsumoto K, J Immunol 2004;172:2186]. As the initial step towards clinical application of these anti-CD30 mAbs in the treatment of allergic diseases, we made an attempt to clarify two issues; first, whether or not anti-CD30 mAb-coated microspheres can efficiently induce apoptosis in human eosinophils, and second, whether or not these apoptotic eosinophils can be phagocytosed by macrophages without the release of granular proteins. METHODS: Purified human eosinophils were treated with anti-CD30 mAb-coated polystyrene microspheres (diameter, 1.44 mum). Apoptosis was determined by annexin V-binding. For the phagocytosis assay, eosinophils were co-cultured with monocyte-derived human macrophages or PMA-pretreated U-937 cells. Phagocytosis was determined by light microscopy and by the eosinophil-derived neurotoxin (EDN) concentration in the supernatant. RESULTS: Anti-CD30 mAb-coated, but not control IgG1-coated microspheres significantly reduced eosinophil survival in a dose-dependent manner. Marked phagocytosis of the apoptotic eosinophils by macrophages was also observed when the eosinophils were treated with anti-CD30 mAb-coated microspheres. The apoptotic eosinophils released large amounts of EDN in the absence of macrophages; however, the EDN levels were significantly decreased when the eosinophils were co-cultured with macrophages. CONCLUSIONS: Anti-CD30 mAb-coated microspheres are capable of inducing rapid and strong apoptosis in human eosinophils. Furthermore, the apoptotic eosinophils were also phagocytosed by macrophages with minimal release of the granular proteins.

CpG oligodeoxynucleotide prolongs eosinophil survival through activation of contaminating B cells and plasmacytoid dendritic cells in vitro.

BACKGROUND: In our previous study, oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) significantly prolonged eosinophil survival without inducing active release of eosinophil-derived neurotoxin or interleukin 8. In addition, this survival-promoting activity was nuclear factor-kappaB dependent. However, some eosinophil preparations from different donors hardly responded to CpG ODNs at all. To clarify why CpG ODN-induced nuclear factor-kB activation in eosinophils does not cause eosinophil-derived neurotoxin or interleukin 8 release and why the survival-promoting activity of CpG ODNs was not found in some eosinophil preparations, we determined the effect of extensive removal of contaminating B cells and plasmacytoid dendritic cells from human eosinophil preparations. METHODS: Eosinophils were purified from the peripheral blood of healthy or slightly allergic donors by gradient sedimentation and negative selection with anti-CD16 alone or a combination of anti-CD16, anti-CD19 and anti-blood dendritic cell antigen 4 (BDCA4) immunomagnetic beads. Eosinophil survival was measured with FITC-conjugated annexin V and propidium iodide by FACS after incubation with synthetic CpG 2006(CpG-B), CpG 2216 (CpG-A) or their GpC control ODNs for 24 h. RESULTS: The addition of anti-CD19 and anti-BDCA4 immunomagnetic beads reduced the number of contaminating CD19+ cells and CD123+ BDCA2+ cells in eosinophil preparations. CpG 2006 and CpG 2216, but not their GpC control ODNs, significantly prolonged survival of eosinophils purified with anti-CD16 immunomagnetic beads alone but not eosinophils purified with a combination of anti-CD16, anti-CD19 and anti-BDCA4 beads. CONCLUSIONS: These results strongly suggest that contaminating B cells or plasmacytoid dendritic cells in eosinophil preparations critically regulate CpG ODN-mediated prolongation of eosinophil survival and that CpG ODNs do not activate eosinophils directly.

Mast cell chymase induces expression of chemokines for neutrophils in eosinophilic EoL-1 cells and mouse peritonitis eosinophils.

Human chymase induced release of interleukin-8 (IL-8) in human EoL-1 cells that had been differentiated into eosinophil-like cells with butyric acid. The chymase-induced IL-8 production was specific in that other cytokines/chemokines examined were not induced. Human chymase also increased mRNA for IL-8 in the differentiated EoL-1 cells, showing involvement of mRNA synthesis. The chymase-induced IL-8 release was inhibited by pertussis toxin as well as U0126 (an inhibitor for extracellular signal-regulated kinase pathway) and SB203580 (p38 inhibitor), suggesting that the chymase-induced IL-8 production is mediated by G protein-coupled receptor and mitogen-activated protein kinases. Mouse mast cell protease-4 (mMCP-4), a mouse chymase, induced macrophage-inflammatory protein-2 (MIP-2), a mouse homologue for IL-8, in mouse eosinophils in vitro. Intradermal injection of mMCP-4 not only induced skin edema but increased MIP-2 content and neutrophil number at the injection site. Taken together, our findings demonstrate that mast cell chymase may contribute to the interaction between eosinophils and neutrophils by inducing IL-8/MIP-2 in eosinophils at allergic inflamed sites.

NR4A orphan nuclear receptor family in peripheral blood eosinophils from patients with atopic dermatitis and apoptotic eosinophils in vitro.

To identify novel genes related to the clinical signs of atopic dermatitis (AD), differentially expressed genes were sought in peripheral blood eosinophils from both AD patients and healthy volunteers. RNA was prepared from eosinophils, expression of various genes was monitored using the Affymetrix GeneChip, and expression was quantified by real-time RT-PCR. Two genes, Nur77 and NOR1, members of NR4A orphan nuclear receptor family, were expressed at a significantly higher level in AD patients than in healthy volunteers. Expression of another gene in the NR4A receptor family, Nurr1, was also higher in AD patients than in healthy volunteers. When peripheral blood leukocytes from healthy volunteers were fractionated, NOR1 expression was highest in eosinophils, but expression of Nur77 and Nurr1 genes was not eosinophil-specific. Extremely intense apoptosis was induced in both eosinophils and an eosinophil cell line, AML14.3D10, by treatment with antibody (Ab) to both CD30 and Fas. Rapid expression of the genes for the NR4A receptor family was observed with anti-CD30 Ab treatment but not with anti-Fas Ab. The NR4A orphan nuclear receptor family gene expression and the subsequent eosinophil apoptosis were downregulated by the MAPK inhibitor, U0126. These results suggest that the expression of the NR4A receptor family genes through CD30 signaling may regulate eosinophil apoptosis in allergic conditions such as AD.

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway.

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.

Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro.

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab')(2) of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-kappaB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.