Hiroaki Mitsuhashi

Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells

Human airway trypsin‐like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease‐activated receptor‐2 (PAR‐2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR‐epidermal growth factor receptor pathway in the airway epithelial cell line NCI‐H292. In this study, the mechanisms by which HAT‐induced AR release can occur were investigated. HAT‐induced AR gene expression was mediated by extracellular signal‐regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR‐2 agonist peptide (PAR‐2 AP) induced ERK phosphorylation; further, desensitization of PAR‐2 with a brief exposure of cells to PAR‐2 AP resulted in inhibition of HAT‐induced ERK phosphorylation, suggesting that HAT activates ERK through PAR‐2. Moreover, PAR‐2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR‐2‐mediated activation of ERK is essential for HAT‐induced AR production. However, in contrast to HAT, PAR‐2 AP could not cause AR release into extracellular space; it appears that activation of PAR‐2 is not sufficient for HAT‐induced AR release. Finally, HAT‐induced AR release was eliminated by blockade of tumour necrosis factor α‐converting enzyme (TACE) by the TAPI‐1 and RNA interference, suggesting that TACE activity is necessary for HAT‐induced AR release. These observations show that HAT induces AR production through the PAR‐2 mediated ERK pathway, and then causes AR release by a TACE‐dependent mechanism.

Pharmacological activities of TEI-8362, a novel inhibitor of human neutrophil elastase.

TEI‐8362, 4‐(N‐(3‐((3‐carboxypropyl)amino)‐8‐methyl‐1‐oxo‐4‐azaisochromen‐6‐yl)carbamoyl)‐4‐((phenylmethoxy)carbonylamino)butanoic acid (C26H28N4O9) is a novel inhibitor of human neutrophil elastase (HNE). We evaluated its pharmacological profile in vitro and in vivo. TEI‐8362 demonstrated potent inhibition of HNE with a Ki value of 1.38×10−9u2003M. Its selectivity for HNE among a variety of proteases ranged from 163 fold to 68,000 fold in favour of HNE. The pulmonary haemorrhage that occurred after i.t. instillation of HNE to hamsters was inhibited by either i.t., i.v., or inhalant administration of TEI‐8362. Intratracheal administration of lipopolysaccharide induced pulmonary neutrophilia. Twenty‐four hours after lipopolysaccharide administration, the additional treatment with formyl‐methionyl‐leucyl‐phenylalanine resulted in a specific neutrophil‐dependent acute lung injury. In this model, lung injury was significantly attenuated by i.t., i.v., or inhalant administration of TEI‐8362. These pharmacological actions of TEI‐8362 suggest that this drug has therapeutic value in the treatment of destructive lung diseases due to neutrophils.

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.