Nagasaki-shi, Japan
Nagasaki-shi, Japan

Time filter

Source Type

Veszelka S.,Hungarian Academy of Sciences | Toth A.E.,Hungarian Academy of Sciences | Walter F.,Hungarian Academy of Sciences | Kittel A.,Hungarian Academy of Sciences | And 8 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2012

An accurate means of predicting blood-brain barrier (BBB) penetration and blood-brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r2 value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool. © 2012 Elsevier B.V. All rights reserved.


Huy N.T.,Nagasaki University | Van Giang T.,Nagasaki University | Thuy D.H.D.,PharmaCo Cell Co. | Kikuchi M.,Nagasaki University | And 3 more authors.
PLoS Neglected Tropical Diseases | Year: 2013

Background:The pathogenesis of dengue shock syndrome (DSS, grade 3 and 4) is not yet completely understood. Several factors are reportedly associated with DSS, a more severe form of dengue infection that reportedly causes 50 times higher mortality compared to that of dengue patients without DSS. However, the results from these reports remain inconclusive. To better understand the epidemiology, clinical manifestation, and pathogenesis of DSS for development of new therapy, we systematically reviewed and performed a meta-analysis of relevant studies that reported factors in both DSS and dengue hemorrhagic fever (DHF, grade 1 and 2) patients.Methods and Findings:PubMed, EMBASE, Scopus, Google Scholar, Dengue Bulletin, Cochrane Library, Virtual Health Library, and a manual search of reference lists of articles published before September 2010 were used to retrieve relevant studies. A meta-analysis using fixed- or random-effects models was used to calculate pooled odds ratios (OR) or event rate with corresponding 95% confidence intervals. Assessment of heterogeneity and publication bias, meta-regression analysis, subgroup analysis, sensitivity analysis, and analysis of factor-specific relationships were further performed. There were 198 studies constituting 203 data sets that met our eligibility criteria. Our meta-regression analysis showed a sustained reduction of DSS/dengue hemorrhagic fever (DHF) ratio over a period of 40 years in Southeast Asia, especially in Thailand. The meta-analysis revealed that age, female sex, neurological signs, nausea/vomiting, abdominal pain, gastrointestinal bleeding, hemoconcentration, ascites, pleural effusion, hypoalbuminemia, hypoproteinemia, hepatomegaly, levels of alanine transaminase and aspartate transaminase, thrombocytopenia, prothrombin time, activated partial thromboplastin time, fibrinogen level, primary/secondary infection, and dengue virus serotype-2 were significantly associated with DSS when pooling all original relevant studies.Conclusions:The results improve our knowledge of the pathogenesis of DSS by identifying the association between the epidemiology, clinical signs, and biomarkers involved in DSS. © 2013 Huy et al.


Toth A.,Hungarian Academy of Sciences | Veszelka S.,Hungarian Academy of Sciences | Nakagawa S.,PharmaCo Cell Co. | Nakagawa S.,Nagasaki University | And 4 more authors.
Recent Patents on CNS Drug Discovery | Year: 2011

The blood-brain barrier (BBB) is a regulatory interface between the circulation and the central nervous system (CNS). Therapy of neurological diseases is limited due to restricted penetration of pharmacons across the BBB. Models for screening the brain penetration of drug candidates are needed early in drug discovery. Culture-based models are useful tools for both basic research on BBB, and testing the permeability of new therapeutical molecules. This review focuses on patented in vitro BBB models and their potential application in CNS drug discovery. Cell culture models using primary and immortalized brain endothelial cells of non-human and human origin, in co-culture or mono-culture setting, in static or dynamic conditions are discussed, as well as methods to induce BBB properties in such in vitro models. The aim of these models is to reproduce as many aspects as possible of the in vivo BBB. All models should show some elements of general endothelial and specific BBB properties, like physiologically realistic cell architecture, restrictive paracellular pathway, and functional expression of transport mechanisms. Though no "ideal in vitro BBB model" has been constructed yet, the currently available models provide valuable information on BBB permeability and are useful tools in CNS drug discovery. © 2011 Bentham Science Publishers.


Horai S.,Nagasaki University | Nakagawa S.,Nagasaki University | Nakagawa S.,PharmaCo Cell Co. | Tanaka K.,Nagasaki University | And 7 more authors.
Cellular and Molecular Neurobiology | Year: 2013

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered. © 2012 Springer Science+Business Media New York.


Nishioku T.,Fukuoka University | Matsumoto J.,Fukuoka University | Dohgu S.,Fukuoka University | Sumi N.,Fukuoka University | And 7 more authors.
Journal of Pharmacological Sciences | Year: 2010

The present study was designed to elucidate the involvement of tumor necrosis factor-α (TNF-α) release from activated microglia in the induction of blood-brain barrier (BBB) dysfunction in an in vitro co-culture system with mouse brain capillary endothelial cells (MBEC4) and microglia. Lipopolysaccharide (LPS)-activated microglia increased the permeability of MBEC4 cells to sodium-fluorescein, and this hyper-permeability was blocked by a neutralizing antibody against TNF-α . LPS stimulated microglia to facilitate TNF-α release. These findings suggested that TNF-α released from activated microglia is attributable to BBB dysfunction. ©2010 The Japanese Pharmacological Society.


Matsumoto J.,Fukuoka University | Takata F.,Fukuoka University | Takata F.,PharmaCo Cell Co. | Machida T.,Fukuoka University | And 8 more authors.
Neuroscience Letters | Year: 2014

Brain pericytes are involved in neurovascular dysfunction, neurodegeneration and/or neuroinflammation. In the present study, we focused on the proinflammatory properties of brain pericytes to understand their participation in the induction of inflammation at the neurovascular unit (NVU). The NVU comprises different cell types, namely, brain microvascular endothelial cells, pericytes, astrocytes and microglia. Among these, we found pericytes to be the most sensitive to tumor necrosis factor (TNF)-α, possessing a unique cytokine and chemokine release profile. This was characterized by marked release of interleukin (IL)-6 and macrophage inflammatory protein-1α. Furthermore, TNF-α-stimulated pericytes induced expression of inducible nitric oxide synthase and IL-1β mRNAs, as an index of BV-2 microglial cell activation state, to the highest levels. Based on these findings, the possibility that brain pericytes act specifically as TNF-α-sensitive cells and as effectors of TNF-α through the release of proinflammatory factors, and that, as such, they have a role in inducing brain inflammation, should be considered. © 2014 Elsevier Ireland Ltd.


Machida T.,Fukuoka University | Takata F.,Fukuoka University | Takata F.,PharmaCo Cell Co. | Matsumoto J.,Fukuoka University | And 6 more authors.
Neuroscience Letters | Year: 2015

In the acute phase of intracerebral hemorrhage (ICH), hemorrhagic transformation and brain edema are associated with blood-brain barrier (BBB) disruption. Elevated levels of thrombin, a coagulation factor, contribute to the development of brain edema during ICH through matrix metalloproteinase (MMP)-9 production. Thrombin directly induces a variety of cellular responses through its specific receptors known as protease-activated receptors (PARs). However, it remains unclear which cell types constituting the BBB mainly produce MMP-9 in response to thrombin. Here, we compared the MMP-9 release induced by thrombin using primary cultures of rat brain microvascular endothelial cells, astrocytes, and pericytes. Brain pericytes exhibited the highest levels of MMP-9 release due to thrombin stimulation among the BBB cells. The pattern of PAR mRNA expression in pericytes was characterized by high expression of PAR1 and moderate expression of PAR4. Heat-inactivated thrombin failed to stimulate pericytes to release MMP-9. A selective PAR1 inhibitor SCH79797 blocked the thrombin-induced MMP-9 release from pericytes. These findings suggest that both PAR1 and PAR4 mediate thrombin-induced MMP-9 release from pericytes. The present study raises the possibility that brain pericytes could play a pivotal role as a highly thrombin-sensitive and MMP-9-producing cell type at the BBB in brain damage including ICH. © 2015 Elsevier Ireland Ltd.


Toyoda K.,Nagasaki University | Tanaka K.,Nagasaki University | Nakagawa S.,Nagasaki University | Nakagawa S.,PharmaCo Cell Co. | And 9 more authors.
Cellular and Molecular Neurobiology | Year: 2013

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front. © 2013 Springer Science+Business Media New York.


Takata F.,Fukuoka University | Takata F.,PharmaCo Cell Co. | Dohgu S.,Fukuoka University | Matsumoto J.,Fukuoka University | And 8 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

Blood-brain barrier (BBB) disruption occurs frequently in CNS diseases and injuries. Few drugs have been developed as therapeutic candidates for facilitating BBB functions. Here, we examined whether metformin up-regulates BBB functions using rat brain microvascular endothelial cells (RBECs). Metformin, concentration- and time-dependently increased transendothelial electrical resistance of RBEC monolayers, and decreased RBEC permeability to sodium fluorescein and Evans blue albumin. These effects of metformin were blocked by compound C, an inhibitor of AMP-activated protein kinase (AMPK). AMPK stimulation with an AMPK activator, AICAR, enhanced BBB functions. These findings indicate that metformin induces up-regulation of BBB functions via AMPK activation. © 2013 Elsevier Inc.


Sumi N.,Fukuoka University | Nishioku T.,Fukuoka University | Takata F.,Fukuoka University | Takata F.,Pharma Co Cell Co. | And 7 more authors.
Cellular and Molecular Neurobiology | Year: 2010

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase. © 2009 Springer Science+Business Media, LLC.

Loading PharmaCo Cell Co. collaborators
Loading PharmaCo Cell Co. collaborators