Mami Azuma

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a+) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages

Interferon‐inducible protein‐10 (IP‐10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon γ (Mig)/CXCL9 and interferon‐inducible T cell α chemoattractant (I‐TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP‐10/CXCL10 and I‐TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme‐linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real‐time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP‐10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I‐TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4+ lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I‐TAC/CXCL11 as well as IP‐10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.

Nerve growth factor derived from bronchial epithelium after chronic mite antigen exposure contributes to airway hyperresponsiveness by inducing hyperinnervation, and is inhibited by in vivosiRNA

Bronchial asthma is a chronic allergic airway inflammatory disease. Neurotrophins, including nerve growth factor (NGF), play an important role in the pathogenesis of asthma. However, the effects of NGF derived from epithelium on airway hyperresponsiveness (AHR) after antigen sensitization/exposure remain uncertain.

Highly stabilized amorphous 3-bis(4-methoxyphenyl)methylene-2-indolinone (TAS-301) in melt-adsorbed products with silicate compounds

Abstract 3-Bis(4-Methoxyphenyl)methylene-2-indolinone (TAS-301) is a poorly water-soluble drug showing low oral bioavailability in rats and dogs. Previously, we reported that when a physical mixture of TAS-301 and a porous calcium silicate, Florite® RE (FLR), was heated at high temperature (250°C), the drug melted and was adsorbed by the FLR in an amorphous state, and that the preparation (melt-adsorbed product) showed a significantly increased solubility and dissolution rate, and a significantly enhanced oral bioavailability of the drug. The aim of the present study was to elucidate important factors for preparing a melt-adsorbed product showing greater stability of drug in an amorphous state. We examined the effects of the kind of adsorbent, drug/adsorbent ratio, heating conditions, and drug particle size on converting drug crystal into an amorphous state, the stability of amorphous state, and chemical stability of the drug in the melt-adsorbed products under a high temperature and high humidity condition (60°C/80% RH, open). FLR, light anhydrous silicic acid and two types of hydrated silicon dioxides were tested as adsorbents. For the batch method, TAS-301 was converted into an amorphous state by heating TAS-301/adsorbents physical mixtures above the melting point of TAS-301 for more than 2 min. The amorphous state was most stabilized when FLR was used as an adsorbent and drug/FLR ratio was 1:0.5 and more. For the continuous method using the twin screw extruder that enables significantly larger scale manufacturing than batch method, TAS-301 melt-adsorbed products were able to produce when only FLR was used as adsorbent. The heating temperature was needed to be set above the melting point of TAS-301 to convert it into an amorphous state as well as batch method. The amorphous state was stabilized when drug/FLR ratio was 1:2 and more. The micronization of the drug decreased the stability of the amorphous state. These results indicate the importance of optimizing the above factors in the preparation of melt-adsorbed product.

Adenoviral interleukin‐12 gene transduction into human bronchial epithelial cells: up‐regulation of pro‐inflammatory cytokines and its prevention by corticosteroids

Background One of the potential effects of IL‐12 is to restore Th1/Th2 balance. Therefore, we investigated the possibility of developing a system for local delivery of IL‐12 into the airways by examining protein expression in a human bronchial epithelial cell line (BEAS‐2B) after adenoviral IL‐12 gene transduction. The effects of dexamethasone on the gene‐modified cells were also examined.

Long-term follow-up of pulmonary function in bronchial asthma patients treated with pranlukast

Clinical studies have shown that pranlukast, a selective cysteinyl leukotriene antagonist, is effective for bronchial asthma. In the present paper, we retrospectively analyzed long-term asthma control by pranlukast treatment in patients treated with inhaled corticosteroids. We analyzed medical records and asthma diaries of 21 patients (9 males, 12 females) (52.1 ± 3.5 years of age) with bronchial asthma who experienced increase of more than 10 L/min in peak expiratory flow in the first 4 weeks of treatment with pranlukast (450 mg/day) and were subsequently treated with pranlukast for more than 1 year. They all received inhaled corticosteroids (400–1600 µg/day of beclomethasone dipropionate or equivalent). We examined clinical control in terms of time course of self-monitored peak expiratory flow. During the analyzed period, the dose of inhaled corticosteroids was tapered in 4 patients, constant in 15 patients and increased in 2 patients. In 19 patients treated with unchanged or tapered dose of inhaled corticosteroids, improvement in the increase of mean PEF at 4-week treatment was maintained for 1 year. No difference in the add-on effect of pranlukast was observed in patients treated with less than 800 µg and more than or equal to 800 µg of inhaled corticosteroids. Four patients underwent reduction of inhaled corticosteroids in the analyzed period and PEF was well-maintained and even increased by pranlukast treatment. In 11 patients in whom data for 3 years were available, the improvement in PEF persisted for 3 years. Although the present investigation is a retrospective analysis, these data may suggest that pranlukast has no tachyphylaxis and its effect continues for more than 1 year.

Long-term regulation of catecholamine formation by ouabain in cultured bovine adrenal chromaffin cells.

The long-term effects of ouabain, an inhibitor of Na+/K+ -ATPase, on catecholamine formation in cultured bovine adrenal chromaffin cells were examined. The increase in [14C]catecholamine formation from [14C]tyrosine induced by ouabain was dependent on incubation time, and its maximal effect was observed after incubation for 8 h. The stimulatory effect of ouabain was concentration dependent (10-300 nM), causing maximal stimulation at 300 nM. The formation of [14C]catecholamines induced by ouabain was not increased by incubation with [14C]DOPA instead of [14C]tyrosine. Ouabain-induced [14C]catecholamine formation was influenced by decreases in extracellular Ca2+ concentration, but not by the presence of cycloheximide or actinomycin D. These results suggested that ouabain stimulates continuous activation of hydroxylation of tyrosine through a Ca2+ -dependent mechanism in cultured bovine adrenal chromaffin cells.

Effects of bioflavonoids on catecholamine biosynthetic activity in the adrenal gland: In vitro studies using partially purified tyrosine hydroxylase and chromaffin cell cultures

To elucidate the effects of bioflavonoids on catecholamine biosynthetic activity in the sympathoadrenergic system, the direct actions of bioflavonoids on tyrosine hydroxylase activity were first examined in vitro using the enzyme partially purified from bovine adrenal medulla. The enzyme activity was markedly and rapidly inhibited by quercetin, but not significantly affected by either apigenin or flavone. The inhibitory action of quercetin on tyrosine hydroxylase was shown to be accompanied by an alteration in kinetic properties of the enzyme. Furthermore, the effects of these flavonoid compounds on catecholamine biosynthetic activity were examined using cultured bovine adrenal chromaffin cells, and found that quercetin caused a marked decrease in the formation of [(14)C]catecholamines from [(14)C]tyrosine in the cells. In contrast, neither apigenin nor flavone caused any significant effect on the formation of [(14)C]catecholamines under the same conditions. The findings presented here suggest that quercetin may inhibit catecholamine biosynthesis through its direct action on the rate-limiting enzyme in the adrenal chromaffin cell, thus resulting in modulation of the sympathoadrenergic function.

Inhibitory action of novel arginine derivative on catecholamine secretion evoked by acetylcholine from cultured bovine adrenal chromaffin cells.

Summary: Anovel product, 4‐amino‐5‐guanidinopentanoic acid 15‐ [(4‐aminobutyl)‐3‐aminopropylcarbamoyl] pentadecyl ester (Arg‐ HSA‐Spm), was synthesized based on ptilomycalin A, which is one of the extracts from marine sponge. Arg‐HSA‐Spm contains arginine in its chemical structure. The pharmacological action of Arg‐HSASpm on catecholamine secretion from cultured bovine adrenal chromaffin cells was examined. Arg‐HSA‐Spm inhibited catecholamine secretion stimulated by the physiological secretagog acetylcholine. This inhibitory action of Arg‐HSA‐Spm on catecholamine secretion induced by 10‐4 M acetylcholine was dose‐dependent from 10‐8 M to 10‐5 M. In the presence of 3 × 10‐7 M Arg‐HSA‐Spm, the stimulation of catecholamine secretion observed by increasing acetylcholine up to 10‐3 M did not reach the maximal level observed without Arg‐ HSA‐Spm. Arg‐HSA‐Spm at 10‐5 M suppressed both the increase in intracellular free Ca2+ level and the influx of 45Ca2+ induced by 10‐4M acetylcholine. The Arg‐HSA‐Spm‐induced suppression of intracellular free Ca2+ level, the influx of 45Ca2+ and catecholamine secretion were not observed in the presence of extracellular K+ at 56 mM. The results presented in this study suggested that Arg‐HSA‐Spm may inhibit the influx of extracellular Ca2+ into the cells, probably through its blocking action related to acetylcholine receptors, resulting in the inhibition of catecholamine secretion in adrenal chromaffin cells.

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.