Masao Takei

S1319: A novel β2-adrenoceptor agonist from a marine sponge Dysidea sp.

In the course of screening of potential leads for beta2-receptor agonists, we found a novel beta2-adrenoceptor selective agonist, S1319, from a marine sponge Dysidea sp. The active compound was isolated and structurally characterized as 4-hydroxy-7-[1-(1-hydroxy-2-methylamino)ethyl]-1,3-benzothiazole-2(3H)-o ne, a new member of the beta2-adrenoceptor agonist. This is the first example of a sponge-derived beta2-adrenoceptor agonist.

The effects of S1319, a novel marine sponge-derived β2-adrenoceptor agonist, on IgE-mediated activation of human cultured mast cells

Abstract.Objective: This study aimed to evaluate the abil-ity of S1319 (4-hydroxy-7-[1-(1-hydroxy-2-methylamino) ethyl]-1,3-benzothiazol-2(3H)-one acetate), a novel β2-adrenoceptor selective agonist derived from marine sponge, to inhibit IgE-mediated activation of human cultured mast cells (HCMC) in vitro.¶Materials and Methods: We examined the effect of S1319 (racemate) on tryptase release and tumor necrosis factor- α (TNF-α) production in HCMC generated from human cord blood cells, after cross-linking of high affinity immunoglobulin E receptors (FcεRI), compared with those of the non-selective β-adrenoceptor agonist, isoproterenol (R-isomer), the selective β2-adrenoceptor agonist, salbutamol (racemate), and the selective and long-acting β2-adrenoceptor agonist, formoterol (racemate). We also evaluated the effect of S1319 on the intracellular cAMP level, inositol phosphate production and protein tyrosine phosphorylation in HCMC.¶Results: S1319 and β-adrenoceptor agonists inhibited the IgE-mediated release of tryptase. Approximate IC50 values of S1319, formoterol, isoproterenol and albuterol for the inhibition of tryptase release were 0.51 ± 0.12, 0.15 ± 0.1, 0.80 ± 0.09, and 28 ± 32.4 nM, respectively. S1319 and β-adrenoceptor agonists also inhibited TNF-α production by HCMC in a concentration-dependent manner. Approximate IC50 values of S1319, formoterol and isoproterenol for the inhibition of TNF-α production were 0.19 ± 0.03, 0.28 ± 0.02 and 0.32 ± 0.03 nM, respectively. S1319 caused a concentration-dependent increase in total cell cyclic AMP levels in HCMC. On the other hand, S1319 inhibited the accumulation of inositol 1,4,5-triphosphate and IgE-mediated protein tyrosine phosphorylation of 42-kDa protein, p42 mitogen activated protein (MAP) kinase (ERK-2).¶Conclusion: These results indicate that S1319 and β-adrenoceptor agonists are potent inhibitors of the IgE-mediated release of mediators from HCMC.

Ilexosides E, F, G, H and I, novel 18,19-seco-ursane glycosides from fruit of ilex crenata☆

Abstract From the fresh fruits of Ilex crenata has been isolated five new saponins named ilexosides E, F, G, H and I, respectively. Their structures were established on the basis of spectral and chemical evidence. They exhibited anti-allergic activities.

Inhibitory effect of anti-allergic agent NCO-650 on histamine release induced by various secretagogues

Histamine release from rat peritoneal mast cells induced by antigen and anti-IgE was essentially complete within 2 min and 3 min, respectively, but that due to Concanavalin A (Con A) was complete only within 9 min. An anti-allergic agent NCO-650 [trans-4-Guanidinomethylcyclohexanecarboxylic acid p-tert-butylphenyl ester hydrochloride], which is a strong inhibitor of trypsin, dose-dependently inhibited anti-IgE-induced histamine release from rat peritoneal mast cells. Moreover, the rate and extent of histamine release from rat peritoneal mast cells induced by various histamine liberators such as antigen, concanavalin A, ionophore A 23187 and compound 48/80 are significantly diminished in samples incubated with NCO-650. The IC50 values of NCO-650 on histamine release induced by antigen, anti-IgE, Concanavalin A, A23187 and compound 48/80 were in the order of micromolar range, i.e. 1.9, 3.6, 4.6, 2.9 and 6.1 μM, respectively. On a molecular basis, NCO-650 is 1000-fold more potent than DSCG, an anti-allergic drug, in inhibiting the antigen-induced histamine release. The present results suggest that the effect of NCO-650 might be due to the inhibition of a common process underlying the release of histamine by various histamine liberators.

Mast cell activation by pedicellarial toxin of sea urchin, Toxopneustes pileolus

Pedicellarial toxin, partially purified from the sea urchin Toxopneustes pileolus, dose‐dependently and time‐dependently caused histamine release from rat peritoneal mast cells. Pedicellarial toxin induced a rapid initial rise in [Ca2+]i within several seconds which was followed by a further slower increase of [Ca2+] (second rise). The toxin induced a dose‐dependent formation of inositol 1,4,5‐triphosphate (IP3) as well as the histamine release in mast cells. Furthermore, the toxin stimulated phosphoinositide‐specific phospholipase C (PI‐PLC) activity in mast cell membranes. 2‐Nitro‐4‐carboxyphenyl‐N,N. ‐diphenylcarbamate (NCDC), a PLC inhibitor, inhibited the activation of PI‐PCL induced by pedicellarial toxin. Cholera toxin inhibited pedicellarial toxin‐induced histamine release, whereas pretreatment of pertussis toxin failed to inhibit it. These results suggest that pedicellarial toxin from T. pileolus activates PI‐PCL and the stimulation of PI turnover may lead to the release of IP3 into the cytoplasm, resulting in histamine release from rat mast cells.

Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast cells induced by anti-IgE

NCDC dose-dependently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE. Moreover, NCDC inhibited Ca(2+)-mobilization from intracellular Ca(2+)-stores as well as histamine release in mast cells activated by anti IgE, the effect on both of these phenomena being closely correlated. Anti-IgE induced a rapid increase in IP3 production from phosphoinositides in mast cells, with its production in 15 sec, followed to baseline levels within 1 min. Anti-IgE stimulated PLC activity on mast cells membrane preparation. NCDC dose-dependently inhibited the generation of IP3. These results suggest that the inhibitory effect of NCDC on the release of histamine induced by anti-IgE is due to, in part at least, the inhibition of PI-specific PLC and that the inhibitory effects of NCDC are involved in intracellular calcium store.

Dendritic Cells Promoted by Ginseng Saponins Drive a Potent Th1 Polarization

Dendritic cells (DC) play a pivotal role in the initiation of T-cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. The interaction of T cells with DC is crucial for directing T cell differentiation towards the Th1, Th2 or Th17 type, and several factors determining the direction of the T cell polarization. IL-12 plays a central role in the immune system, not only by augmenting the cytotoxic activity of T cells and NK cells and regulating IFN-γ production, but also by the capacity of IL-12 to promote the development of Th1 cells. Therefore, it is important to identify factors that might affect the differentiation, maturation and function of DC. Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. Moreover, T-cadinol and calamenene are sesquterpenes isolated from the heartwood of Cryptomeria japonica being pharmacologically active substances. We investigated whether M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, as well as T-cadinol and calamenene can drive DC maturation from human monocytes in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days under standard conditions, followed by another 2 days in the presence of M1, M4, T-cadinol or calamenene. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC were enhanced with a concomitant decrease in endocytic activity. M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC enhanced the T cell stimulatory capacity in an allo MLR (allogeneic mixed lymphocyte reaction). Naïve T cells co-cultured with allogeneic M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC turned into typical Th1 cells, which produced large quantities of IFN-γ and released small amounts of IL-4 depending on IL-12 secretion. In the CTL assay (cytotoxic T-lymphocyte assay), the production of IFN-γ and 51Cr release on M4-primed DC was more augmented than of immature DC or TNF-α-primed DC. These results suggest that M1, M4, T-cadinol and calamenene appear to be a good factor to induce DC maturation, or even better in some respect, for the use in clinical DC therapy to induce strong Th1 type immune responses.

Diterpene, 16-phyllocladanol enhances Th1 polarization induced by LPS-primed DC, but not TNF-α-primed DC

16-Phyllocladanol is diterpene isolated form the heartwood of Cryptomeria japonica. We demonstrate that the effect of 16-phyllocladanol on the phenotypic and functional maturation of human monocytes-derived DC in vitro. Human monocytes were exposed to 16-phyllocladanol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD83 and HLA-DR on LPS-primed DC were enhanced by 16-phyllocladanol. 16-Phyllocladanol dose-dependently augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC and the production of IL-12p70 by LPS-primed DC. The cytokine production by 16-phyllocladanol-primed DC was not inhibited by anti-TLR2 and 4 mAbs. IFN-gamma secretion from naïve T cells co-cultured with DC differentiated with LPS was also augmented by 16-phyllocladanol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by 16-phyllocladanol via the activation of IL-12p70 and independent on TLR2 or TLR4.

Tracheal relaxing effects and β2 adrenoceptor selectivity of S1319, a novel sponge-derived bronchodilator agent, in isolated guinea-pig tissues

S1319 (4‐hydroxy‐7‐[1‐(1‐hydroxy‐2‐methylamino)ethyl]‐1,3‐benzothiazol‐2(3H)‐one acetate), a novel non‐catecholamine β‐adrenoceptor agonist, has been compared with isoprenaline, salbutamol and formoterol for activity in vitro on a range of β‐adrenoceptor containing preparations from guinea‐pig. S1319, like isoprenaline, salbutamol and formoterol, relaxed preparations of guinea‐pig trachea (contracted by histamine) in a concentration‐dependent manner. The relaxing activity of S1319 appeared to be more potent than that of isoprenaline and salbutamol, and similar to that of formoterol (pD2 values of 10.58±0.03 vs 7.60±0.01, 7.50±0.01 and 10.52±0.04, respectively), and was blocked by the β2‐adrenoceptor selective antagonist (ICI 118,551). The intrinsic activity of S1319 was close to 1.0. In the β1‐adrenoceptor containing preparations, guinea‐pig right and left atria, a monophasic inotropic response of S1319 was observed. The pD2 value of S1319 for left atrial and right atrial inotropism was 6.70±0.15 and 7.81±0.01, respectively. The selectivity ratio (trachea/left atrial inotropism) of S1319, formoterol, salbutamol and isoprenaline was 8523, 284, 4.8 and 0.45, respectively. The relative selectivity ratio of S1319 was 18743, 1858 and 30 times greater than that of isoprenaline, salbutamol and formoterol, respectively. Relaxant responses of guinea‐pig trachea to S1319 declined rapidly when the agonist was washed from the tissues, with complete recovery within 30 min. The duration of action of S1319 was similar to that of isoprenaline and less than that of salbutamol and formoterol. In summary, S1319, a sponge‐derived β‐adrenoceptor agonist, is a potent and selective β2‐adrenoceptor agonist with a short‐duration of action in isolated guinea‐pig tracheas.

Effect of interleukin-3, interleukin 5 and hyaluronic acid on cultured eosinophils derived from human umbilical cord blood mononuclear cells.

Background: Several studies have shown that cultured eosinophils can be generated from human umbilical cord blood mononuclear cells (UCMC) in the presence of interleukin (IL)–3 and IL–5 in vitro. Other reports have indicated that cellular adhesion to hyaluronic acid (HA) enhances the proliferation of cultured eosinophils derived from CD34+ cells purified from UCMC. The aim of this study was to obtain large numbers of mature eosinophils from UCMC using IL–3, IL–5 and HA, and to investigate their functions. Methods: We examined several combinations of IL–3 and IL–5 and their effect on eosinophil development from UCMC in HA–coated on non–coated flasks. We also examined whether cultured eosinophils degranulated eosinophil–derived neurotoxin (EDN) induced by secretory immunoglobulin A conjugated to sepharose beads (sIgA–beads) and responded to eotaxin. Results: Culture with HA–coated flasks for 35 days (in the presence of IL–3 and IL–5, with IL–3 omitted after day 14 of culture) caused a 11.2–fold augmentation in the proliferation of UCMC. On day 35 of the culture, 98% of cultured cells were eosinophils judging from May–Grünwald and Giemsa staining and transmission electron micrographs. The EDN content of the cultured eosinophils on day 35 was 156 ng/105 cells. Cultured eosinophils degranulated EDN induced by sIgA–beads and responded to eotaxin by chemotaxis and intracellular Ca2+ mobilization. Conclusion: We found a useful culture system to obtain large numbers of eosinophils derived from UCMC, which may facilitate the investigation of eosinophil function, since there was no significant difference in response to sIgA–beads and eotaxin between cultured and peripheral eosinophils.