Hirotsugu Komatsu

Identification of a Potent and Orally Active Non-peptide C5a Receptor Antagonist

The anaphylatoxin C5a is a potent chemotactic factor for neutrophils and other leukocytes, and functions as an important inflammatory mediator. Through a high capacity screening followed by chemical optimization, we identified a novel non-peptide C5a receptor antagonist,N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1- carboxamide hydrochloride (W-54011). W-54011 inhibited the binding of125I-labeled C5a to human neutrophils with aK i value of 2.2 nm. W-54011 also inhibited C5a-induced intracellular Ca2+ mobilization, chemotaxis, and generation of reactive super oxide species in human neutrophils with IC50 values of 3.1, 2.7, and 1.6 nm, respectively. In C5a-induced intracellular Ca2+ mobilization assay with human neutrophils, W-54011 did not show agonistic activity at up to 10 μm and shifted rightward the concentration-response curves to C5a without depressing the maximal responses. Examination on the species specificity of W-54011 revealed that it was able to inhibit C5a-induced intracellular Ca2+ mobilization in neutrophils of cynomolgus monkeys and gerbils but not mice, rats, guinea pigs, rabbits, and dogs. In gerbils, oral administration of W-54011 (3–30 mg/kg) inhibited C5a-induced neutropenia in a dose-dependent manner. The present report is the first description of an orally active non-peptide C5a receptor antagonist that could contribute to the treatment of inflammatory diseases mediated by C5a.

Reactive oxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells

We previously reported the induction of apoptotic DNA fragmentation by nonsteroidal anti-inflammatory drugs (NSAIDs) in cultured rat gastric cells, and indicated that prostaglandin-synthesis is only marginally involved in the apoptotic process. In the present study, we examined whether the generation of reactive oxygen species is critically involved in NSAID-induced apoptosis. Indomethacin, sodium diclofenac, flurbiprofen, zaltoprofen, etodolac, but not mofezolac, enhanced apoptotic DNA fragmentation and mRNA expression for cyclooxygenase-2 in AGS cells, a cell line derived from human gastric epithelium. The apoptotic effect of indomethacin was then confirmed by fluorescent staining of the cells with annexin V. Apoptotic DNA fragmentation induced by indomethacin and flurbiprofen was suppressed by incubation of the cells with the anti-oxidants pyrrolidine dithiocarbamate, diphenyleneiodonium chloride, and N-acetyl-L-cysteine. These two NSAIDs also enhanced release from the cells of 8-isoprostane, a nonenzymatic product by free-radical-mediated peroxidation of arachidonic acid. Further, lucigenin chemiluminescence showed that the intracellular production of reactive oxygen species increased in cells treated with indomethacin. The present data thus indicate a crucial association between the generation of reactive oxygen species and NSAID-induced apoptosis in gastric epithelial cells.

Inhibition of eosinophil activation in bronchoalveolar lavage fluid from atopic asthmatics by Y-24180, an antagonist to platelet-activating factor

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the expression of adhesion molecules in peripheral blood and bronchoalveolar lavage fluid (BALF) eosinophils from atopic asthmatics. Y-24180 (20 mg/day) was administered to 4 atopic asthmatics for 8 weeks. The number of eosinophils, the level of eosinophil cationic protein (ECP), the bindings of soluble intercellular adhesion molecule-1 (sICAM-1) and fibronectin (FN), and the expressions of CD11b (alpha chain of Mac-1) and CD49d (alpha chain of VLA-4) on eosinophils were evaluated in peripheral blood (n=4) and BALF (n=3) before and after the administration of Y-24180. The infiltration of eosinophils into the bronchial wall was also examined by taking biopsies. Eosinophil count, sICAM-1 and FN binding to eosinophils in BALF significantly decreased after the administration of Y-24180 (p<0.05). The level of CD11b expression also decreased remarkably after the administration (n=2). In peripheral blood, eosinophil count and ECP level did not change. The binding of sICAM-1 and FN, and expression of CD11b on eosinophils in peripheral blood showed a tendency to decrease after the administration. The level of CD49d expression on eosinophils changed neither in BALF nor in blood. Eosinophil infiltration into the bronchial wall markedly decreased in one out of 3 cases after the administration. These results suggest that Y-24180 inhibits the activation of eosinophils by antagonizing the actions of PAF in atopic asthmatics.

Treatment with Y‐40138, a multiple cytokine production modulator, inhibits lipopolysaccharide‐ or tumour necrosis factor‐α‐induced production of pro‐inflammatory cytokines and augments interleukin‐10

N‐[1‐(4‐[4‐(pyrimidin‐2‐yl)piperazin‐1‐yl]methyl phenyl)cyclopropyl] acetamide.HCl (Y‐40138) suppresses liver injury in concanavalin A‐ and d‐galactosamine/lipopolysaccharide (LPS)‐induced mouse hepatitis models. However, the mechanism of action of Y‐40138 has not been fully investigated. In this study, we examined the effect of Y‐40138 on cytokine production in mice. Cytokine production was induced by intraperitoneal injection of LPS (0.5 mg kg−1) or intravenous injection of recombinant mouse tumour necrosis factor (TNF)‐α (10μg mouse−1) in BALB/c mice. TNF‐α and interleukin (IL)‐10 reached maximum levels 1.5 h after the LPS injection. IL‐12 and interferon‐γ (IFN‐γ) reached maximum levels 3 to 9 h after the injection. When Y‐40138 was orally administered 30 min prior to the injection, it inhibited TNF‐α, IL‐12 and IFN‐γ production and augmented IL‐10 production. Y‐40138 also inhibited IL‐12 production and augmented IL‐10 production in TNF‐α‐stimulated mice. In IL‐10 knockout mice, Y‐40138 inhibited TNF‐α and IL‐12 production 1.5h after the LPS injection but not after 3 h or later, unlike in wild mice. In addition, TNF‐α production was inhibited by Y‐40138 at concentrations that could not augment IL‐10 production. These data suggest that Y‐40138 modulates pro‐inflammatory cytokine production by both IL‐10‐dependent and ‐independent mechanisms.

Effects of Y-24180, a receptor antagonist to platelet-activating factor, on allergic cutaneous eosinophilia in mice

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF), on allergic cutaneous eosinophilia and cytokine production in the skin of mice. Mice sensitized actively with ovalbumin (OA) were challenged by an intradermal injection of OA solution. The number of inflammatory cells, including eosinophils and eosinophil peroxidase (EPO) activity reflecting eosinophil infiltration into the tissue increased in OA-challenged skin 12 hr after the challenge. The levels of interleukin-4 (IL-4) and IL-5 also increased significantly in the challenged skin 12 hr and 3-24 hr, respectively, but that of interferon-gamma (IFN-gamma) did not change. Then, we evaluated the effects of Y-24180, ketotifen, suplatast and prednisolone on the increase in EPO activity, IL-4 and IL-5. These drugs were orally administered once a day for 5 days beginning 4 days before the challenge. Y-24180 (10 mg/kg) and prednisolone (5 mg/kg) significantly suppressed these parameters. Suplatast did not affect EPO activity, but significantly decreased the levels of IL-4 and IL-5. Ketotifen had no effect on them. These results indicate that the inhibition of IL-4, IL-5 and PAF are required to suppress the cutaneous eosinophilia and Y-24180 contributes to the treatment of allergic cutaneous eosinophilia.

( + )-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4-b]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1β and IL-6 at the level of messenger RNA expression in cultured human monocytes

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

Inhibition of leukotriene B4-induced increase in intracellular calcium ion level of human peripheral blood polymorphonuclear leukocytes by Y-24180, an antagonist of platelet-activating factor receptor.

Y-24180 ((+/-)-4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dim eth yl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a] [1,4] diazepine), an antagonist of platelet-activating factor (PAF) receptor, has already been reported to inhibit leukotriene B4 (LTB4)-induced activation of polymorphonuclear leukocytes. In this article, to clarify the mechanism of inhibition of LTB4-induced activation by Y-24180, we examined the effect of Y-24180 on LTB4-induced increase in intracellular calcium ion ([Ca2+]i) level of human polymorphonuclear leukocytes at a single cell level using a laser scanning confocal microscope. Preincubation with Y-24180 significantly inhibited the increase in [Ca2+]i level at 0.3-3 microM, while WEB 2086 (4-[3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a ][1,4]diazepin-2-yl]propionyl]morpholine), another PAF receptor antagonist, did not show the inhibitory effect at 3-30 microM, indicating that the difference in potency between these compounds was more than 100-fold. The LTB4-induced increase in [Ca2+]i level was suppressed by microinjection of anti-PAF antibody, but not control antibody, into leukocytes. Microinjection of Y-24180 at 0.003-0.03 microM or WEB 2086 at 0.03-0.3 microM into leukocytes also inhibited the LTB4-induced increase. The microinjection of WEB 2086 at the 10-fold greater concentrations than those of Y-24180 inhibited to the almost equal level to that obtained by Y-24180. In addition, microinjection of PAF into leukocytes induced the increase in [Ca2+]i level. Preincubation with Y-24180 at 1 or 3 microM significantly attenuated the PAF microinjection-induced increase, but WEB 2086 showed little effect at 3-30 microM. These results indicate that Y-24180 inhibits LTB4-induced activation of leukocytes by suppressing the increase in [Ca2+]i level and the inhibitory effect is mediated by antagonistic action against intracellular PAF-induced up-regulation of [Ca2+]i level. The difference in inhibitory activity for the increase in [Ca2+]i level between Y-24180 and WEB 2086 when these are added in the culture medium may depend upon their aptitude for the transmembrane influx.

Inhibition of activation of human peripheral blood eosinophils by Y-24180, an antagonist to platelet-activating factor receptor.

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the PAF- or leukotriene B4 (LTB4)-induced activation of eosinophils using human peripheral blood in vitro. As activation markers, CD11b expression level and soluble intercellular adhesion molecule-1 (sICAM-1)-binding activity were analyzed by flow cytometry. Y-24180 significantly inhibited PAF-induced increase in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding at 0.01 microM or more. WEB 2086, another PAF receptor antagonist, also inhibited the increase significantly at 1 microM or more. LTB4-induced increases in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding were inhibited by Y-24180 at 1 microM, but not WEB 2086 up to 10 microM. These results indicate that Y-24180 inhibits the PAF- or LTB4-induced activation of eosinophils in human peripheral blood more potently than WEB 2086.