Katsushi Takahashi

Hypouricemic effect of the novel xanthine oxidase inhibitor, TEI-6720, in rodents.

We investigated the xanthine oxidase/xanthine dehydrogenase inhibitory activity and hypouricemic effect of a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazole-carboxylic acid, and compared its effects with those of allopurinol in rodents. TEI-6720 was found to inhibit bovine milk xanthine oxidase, and mouse liver and rat liver xanthine oxidase/xanthine dehydrogenase with IC50 values of 1.4, 1.8 and 2.2 nM, respectively. On bovine milk xanthine oxidase, TEI-6720 exhibited mixed-type inhibition and the Ki value was 0.7 nM. TEI-6720 displayed prolonged urate-lowering activity in normal mice and rats. We evaluated the hypouricemic effect of TEI-6720 on hyperuricemia induced by the uricase inhibitor, potassium oxonate (250 mg/kg s.c., 1 h before the test drugs), and measured the total molarity of both serum allantoin and urate in rats. Oral TEI-6720 and allopurinol had a hypouricemic effect 2 h after their administration to oxonate-pretreated rats with ED50 values of 1.5 and 5.0 mg/kg, respectively. Both compounds also reduced the combined molarity of uric acid and allantoin in rats. The ED50 values of TEI-6720 and allopurinol were 2.1 and 6.9 mg/kg p.o., respectively. These results suggest that TEI-6720 may be useful for the treatment of hyperuricemia.

HUMAN NEUTROPHILS EXPRESS MESSENGER RNA OF VITAMIN D RECEPTOR AND RESPOND TO 1α,25-DIHYDROXYVITAMIN D3

ABSTRACT 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to modulate the production of various cytokines or the expression of certain differentiation markers in human T cells or monocytes. Its effects on neutrophils, however, are poorly understood. In this paper, we show several lines of evidence indicating that neutrophils express functional vitamin D receptors (VDR). Sort-purified neutrophils from human peripheral blood expressed VDR mRNA at a level comparable to that of monocytes. As reported to occur in monocytes, protein expression of CD14 on the cell surface of neutrophils was augmented when the cells were incubated with 1,25(OH)2D3. To investigate the physiological roles for VDR in neutrophils, we investigated possible modulating effects of 1,25(OH)2D3 on the expression of several genes in lipopolysaccharide-stimulated neutrophils by using differential display analysis. Of the genes we identified, trappin-2/elafin/SKALP, which was originally reported to be an inhibitor of elastase, was induced in neutrophils by lipopolysaccharide, but was suppressed significantly in the presence of 1,25(OH)2D3. Under the same conditions, interleukin-1β expression was also inhibited. These findings suggest that 1,25(OH)2D3 has a potential to affect the inflammatory process by modulating the expression of neutrophil genes.

Effect of interleukin-10 on Anti-CD40- and interleukin-4-induced immunoglobulin E production by human lymphocytes

We studied the effect of IL-10 on the in vitro synthesis of IgE and IgG by human peripheral blood mononuclear cells (PBMC) following stimulation with anti-CD40 monoclonal antibody and IL-4. Anti-CD40- and IL-4-stimulated PBMC showed an increase in IL-10 synthesis together with increases in IgE and IgG production. Addition of anti-IL-10 antibody to this system suppressed IgE as well as IgG production without affecting the proliferation of PBMC. Addition of IL-10 enhanced IgE and IgG production if PBMC were activated with anti-CD40 and IL-4. PBMC costimulated with anti-CD40, IL-4 and IL-10 showed a remarkable increase in IL-6 production, but had no effect on IFN-gamma production. Addition of IL-10 to purified human tonsillar B cells stimulated with anti-CD40 and IL-4 enhanced B cell proliferation and IgG production, but not IgE production. These results suggest that IL-10 accelerates IgE production by anti-CD40- and IL-4-stimulated PBMC by enhancing IL-6 production through activation of T lymphocytes.

Effect of TEI-9874, an inhibitor of immunoglobulin E production, on allergen-induced asthmatic model in rats.

As TEI-9874, 2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)ben zoic acid reduces allergen-specific immunoglobulin E (IgE) production by human peripheral blood mononuclear cells in vitro, we evaluated its potency on an allergen-induced asthmatic model in Brown-Norway rats. Inhaled ovalbumin induced the immediate-phase asthmatic response, the late-phase asthmatic response, the infiltration of leukocytes into bronchoalveolar lavage fluid, and an increase of serum anti-ovalbumin IgE. These parameters were suppressed by the treatment with TEI-9874 (3, 10, and 30 mg/kg p.o.). The ovalbumin-induced airway hyperresponsiveness was prevented by TEI-9874 (30 mg/kg p.o.). Furthermore, the suppression of the immediate-phase asthmatic response and the late-phase asthmatic response by TEI-9874 was almost completely extinguished by the exogenous administration of rat anti-ovalbumin antiserum. These results indicate that the efficacy of TEI-9874 on the asthmatic response is mainly mediated by the suppression of allergen-specific IgE production and TEI-9874 appears to be a good candidate as therapy for IgE-mediated allergic asthma.

TEI-A00114: A new vitamin D3 analogue that inhibits neutrophil recruitment in an acute lung injury hamster model while showing reduced hypercalcemic activity

While searching for vitamin D(3) analogues which inhibit neutrophil recruitment in the lung without elevating plasma calcium level, we found that (5Z,7E)-(1S,3R)-20(R)-[(5E)-(2S)-2-hydroxy-2-methyl-cyclopentanone-5-ylidene]methyl-9,10-secopregna-5,7,10(19)-triene-1,3-diol (TEI-A00114) had the best efficacy and calcemic action. TEI-A00114 has a vitamin D receptor affinity 2.5-fold weaker and a vitamin D binding protein affinity 330.9-fold weaker than those of 1α,25(OH)(2)D(3). The estimated effective doses for 40% inhibition (ED(40)) via peroral and intratracheal administration are 7.6 and 0.4 μg/kg, respectively. TEI-A00114 was also tested by inhaled administration, and its ED(40) was calculated as 0.2 μg/kg. The estimated 40% inhibitory concentration (IC(40)) of TEI-A00114 on interleukin (IL)-8 production induced by lipopolysaccharide and on IL-1β in human whole blood cells in vitro were 9.8 × 10(-8) or 1.8 × 10(-9)M, respectively. These levels of TEI-A00114s activities are equal to those of 1α,25(OH)(2)D(3). On the other hand, the calcemic action of TEI-A00114, which was evaluated at day 14 after sequential peroral quaque die administration, was 89-fold weaker (molar ratio) than that of 1α,25(OH)(2)D(3). These results indicate that TEI-A00114 has a dissociated profile between inhibition of neutrophil recruitment in the lung and calcemic action, suggesting its suitability over 1α,25(OH)(2)D(3) as a candidate for the treatment of acute lung injury.

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC50) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.

Suppression of human immunoglobulin E antibody production by a new naphthalene derivative

A new naphthalene derivative, (E)-2-(7-(2-naphthyl)-6-heptenamide)benzoic acid (TEI-8364) was assessed for its effect on interleukin (IL)-4- and anti-CD40 monoclonal antibody-induced immunoglobulin E (IgE) production by cultured human lymphocytes. TEI-8364 preferentially suppressed the production of IgE by peripheral blood mononuclear cells (PBMC) in a dose-dependent manner, without inhibiting PBMC proliferation. In addition, TEI-8364, at a concentration of 10 microM, completely inhibited IL-4- and anti-CD40-induced IgE production by purified tonsillar B lymphocytes, suggesting that TEI-8364 affects B cells by interfering with signals provided by IL-4 or through CD40 and IL-4. TEI-8364 also had a profound inhibiting effect on the in vitro production of specific antibody to a T cell-dependent antigen by PBMC from an immunized volunteer, cultured in the presence of antigen. Furthermore, TEI-8364 at a dose of 1 mg/mouse/day selectively inhibited IgE production by severe combined immunodeficiency mice engrafted with human PBMC, if the drug was administered subcutaneously for five consecutive days. These findings suggest that TEI-8364 is a potent therapeutic agent that may be useful in the treatment of IgE-mediated allergic disorders.