Biocompatibility of Polysulfone Hemodialysis Membranes and Its Mechanisms: Involvement of Fibrinogen and Its Integrin Receptors in Activation of Platelets and Neutrophils.

Activation of blood cells during hemodialysis is considered to be a significant determinant of biocompatibility of the hemodialysis membrane because it may affect patient health adversely through microvascular inflammation and oxidative stress. This study found very different cell activation among various polysulfone (PSf) hemodialysis membranes. For example, CX-U, a conventional PSf membrane, induced marked adhesion of platelets to its surface and increased surface expression of activated CD11b and production of reactive oxygen species (ROS) by neutrophils; while NV-U, a hydrophilic polymer-immobilized PSf membrane, caused little platelet adhesion and slight CD11b expression and ROS production by neutrophils. Analysis of the molecular mechanisms of the above phenomena on CX-U and NV-U indicated that anti-integrin GPIIb/IIIa antibody blocked platelet adhesion, and that the combination of anti-CD11b (integrin α subunit of Mac-1) and anti-integrin αvß3 antibodies blocked ROS production by neutrophils. Plasma-derived fibrinogen, a major ligand of GPIIb/IIIa, Mac-1, and αvß3 on membranes, was thus analyzed and found to be more adsorbed to CX-U than to NV-U. Moreover, comparison between five PSf membranes showed that the number of adherent platelets and neutrophil ROS production increased with increasing fibrinogen adsorption. These results suggested that fibrinogen, adsorbed on membranes, induced GPIIb/IIIa-mediated platelet activation and Mac-1/αvß3-mediated neutrophil activation, depending on the amount of adsorption. In conclusion, the use of biocompatible membranes like NV-U, which show lower adsorption of fibrinogen, is expected to reduce hemodialysis-induced inflammation and oxidative stress by minimizing cell activation.

Development of a Novel Site-Specific Pegylated Interferon Beta for Antiviral Therapy of Chronic Hepatitis B Virus.

Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-α2a) can suppress hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from hepatocytes. A novel site-specific pegylated recombinant human IFN-ß (TRK-560) was recently developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were performed. TRK-560 significantly suppressed the production of intracellular HBV replication intermediates and extracellular HBV surface antigen (HBsAg) (P < 0.001 and P < 0.001, respectively), and the antiviral effects of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was significantly higher than that by PEG-IFN-α2a treatment both in vitro and in vivo (P = 0.004 and P = 0.046, respectively), and intracellular HBV covalently closed circular DNA (cccDNA) reduction by TRK-560 treatment was also significantly higher than that by PEG-IFN-α2a treatment in vivo (P = 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-α2a in the induction potency of interferon-stimulated genes. TRK-560 shows a stronger antiviral potency via higher induction of interferon-stimulated genes and stronger stimulation of immune cell chemotaxis than PEG-IFN-α2a. As HBsAg loss and HBV cccDNA eradication are important clinical goals, these results suggest a potential role for TRK-560 in the development of more effective treatment for chronic hepatitis B infection.

Nalfurafine hydrochloride, a selective κ opioid receptor agonist, has no reinforcing effect on intravenous self-administration in rhesus monkeys.

Nalfurafine hydrochloride [(E)-N-[17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxymorphinan-6ß-yl]-3-(furan-3-yl)-N-methylprop-2-enamide monohydrochloride; nalfurafine] is used in Japan as an antipruritic for the treatment of intractable pruritus in patients undergoing hemodialysis or with chronic liver disease. It is a potent and selective agonist at the κ opioid receptor, but also has weak and partial agonist activity at µ opioid receptors. Opioids, especially those acting at µ receptors, carry a risk of abuse. This is an important factor in the consideration of therapeutic risk vs. benefit in clinical use and the potential for misuse as a public health problem. It is therefore necessary to carefully evaluate the reinforcing effects of nalfurafine. To this end, we investigated intravenous self-administration of nalfurafine in rhesus monkeys. The number of self-administration of nalfurafine at doses of 0.0625, 0.125 and 0.25 µg/kg/infusion was not higher than that of saline in rhesus monkeys that frequently self-administered pentazocine (0.25 mg/kg/infusion). These results indicate that nalfurafine has no reinforcing effect in rhesus monkeys in the intravenous self-administration paradigm.

A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice.

The mitochondrial outer membrane protein mitoNEET is a binding protein of the insulin sensitizer pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) and is considered a novel target for the treatment of type II diabetes. Several small-molecule compounds have been identified as mitoNEET ligands using structure-based design or virtual docking studies. However, there are no reports about their therapeutic potential in animal models. Recently, we synthesized a novel small molecule, TT01001 [ethyl-4-(3-(3,5-dichlorophenyl)thioureido)piperidine-1-carboxylate], designed on the basis of pioglitazone structure. In this study, we assessed the pharmacological properties of TT01001 in both in vitro and in vivo studies. We found that TT01001 bound to mitoNEET without peroxisome proliferator-activated receptor-γ activation effect. In type II diabetes model db/db mice, TT01001 improved hyperglycemia, hyperlipidemia, and glucose intolerance, and its efficacy was equivalent to that of pioglitazone, without the pioglitazone-associated weight gain. Mitochondrial complex II + III activity of the skeletal muscle was significantly increased in db/db mice. We found that TT01001 significantly suppressed the elevated activity of the complex II + III. These results suggest that TT01001 improved type II diabetes without causing weight gain and ameliorated mitochondrial function of db/db mice. This is the first study that demonstrates the effects of a mitoNEET ligand on glucose metabolism and mitochondrial function in an animal disease model. These findings support targeting mitoNEET as a potential therapeutic approach for the treatment of type II diabetes.

Analgesic effect of GT-0198, a structurally novel glycine transporter 2 inhibitor, in a mouse model of neuropathic pain.

This study was conducted to identify the characteristic pharmacological features of GT-0198 that is phenoxymethylbenzamide derivatives. GT-0198 inhibited the function of glycine transporter 2 (GlyT2) in human GlyT2-expressing HEK293 cells and did not bind various major transporters or receptors of neurotransmitters in a competitive manner. Thus, GT-0198 is considered to be a comparatively selective GlyT2 inhibitor. Intravenous, oral, and intrathecal injections of GT-0198 decreased the pain-related response in a model of neuropathic pain with partial sciatic nerve ligation. This result suggests that GT-0198 has an analgesic effect. The analgesic effect of GT-0198 was abolished by the intrathecal injection of strychnine, a glycine receptor antagonist. Therefore, GT-0198 is considered to exhibit its analgesic effect via the activation of a glycine receptor by glycine following presynaptic GlyT2 inhibition in the spinal cord. In summary, GT-0198 is a structurally novel GlyT2 inhibitor bearing a phenoxymethylbenzamide moiety with in vivo efficacy in behavioral models of neuropathic pain.

HIS-388, a novel orally active and long-acting 11ß-hydroxysteroid dehydrogenase type 1 inhibitor, ameliorates insulin sensitivity and glucose intolerance in diet-induced obesity and nongenetic type 2 diabetic murine models.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is considered a potential therapeutic target in the treatment of type 2 diabetes mellitus. In this study, we investigated the pharmacological properties of HIS-388 (N-[(1R,2s,3S,5s,7s)-5-hydroxyadamantan-2-yl]-3-(pyridin-2-yl) isoxazole-4-carboxamide), a newly synthesized 11ß-HSD1 inhibitor, using several mouse models. In cortisone pellet-implanted mice in which hypercortisolism and hyperinsulinemia occur, single administration of HIS-388 exhibited potent and prolonged suppression of plasma cortisol and lowered plasma insulin levels. These effects were more potent than those achieved using the same dose of other 11ß-HSD1 inhibitors (carbenoxolone and compound 544 [3-[(1s,3s)-adamantan-1-yl]-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine]), indicating that HIS-388 potently and continuously suppresses 11ß-HSD1 enzyme activity in vivo. In diet-induced obese mice, HIS-388 significantly decreased fasting blood glucose, plasma insulin concentration, and homeostasis model assessment-insulin resistance score, and ameliorated insulin sensitivity. In addition, HIS-388 significantly reduced body weight and suppressed the elevation of blood glucose during the pyruvate tolerance test. In nongenetic type 2 diabetic mice with disease induced by a high-fat diet and low-dose streptozotocin, HIS-388 also significantly decreased postprandial blood glucose and plasma insulin levels and improved glucose intolerance. The effects of HIS-388 on glucose metabolism were indistinguishable from those of an insulin sensitizer, pioglitazone. Our results suggest that HIS-388 is a potent agent against type 2 diabetes. Moreover, amelioration of diabetic symptoms by HIS-388 was at least in part attributable to an antiobesity effect or improvement of hepatic insulin resistance. Therefore, potent and long-lasting inhibition of 11ß-HSD1 enzyme activity may be an effective approach for the treatment of type 2 diabetes and obesity-associated disease.

Design and synthesis of novel p38α MAP kinase inhibitors: discovery of pyrazole-benzyl ureas bearing 2-molpholinopyrimidine moiety.

The discovery that pyrazole-benzyl urea derivatives bearing a 2-molpholinopyrimidine moiety are novel p38α inhibitors is described. A comparative view of the binding modes of SB-203580 and BIRB-796 by structural alignment of two X-ray co-crystal structures was utilized to identify this novel series. Modification of the benzyl group led to compound 2b, a highly potent p38α inhibitor. In in vivo studies, 2b inhibited the production of tumor necrosis factor-alpha in lipopolysaccharide-treated mouse in a dose-dependent manner. Furthermore, the results of a 5-day repeated oral dose toxicity study suggest that 2b has low hepatotoxicity.

Anti-pruritic effect of isothiocyanates: Potential involvement of toll-like receptor 3 signaling.

The innate immune system has an emerging role as a mediator of neuro-immune communication and a therapeutic target for itch. Toll-like receptor 3 (TLR3) plays an important role in itch, as shown in TLR3 knock-out mice. In this study, to evaluate effects of TLR3 inhibitors on histamine-independent itch, we used two kinds of isothiocyanate (ITC). Both phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) inhibited Poly I:C (PIC)-induced signaling in the RAW264.7 cell line. We then investigated the anti-pruritic effect of these compounds on PIC- and chloroquine (CQ)-induced scratching behavior. PEITC and SFN both suppressed PIC-evoked scratching behavior in mice, and PEITC also inhibited CQ-induced acute itch. Finally, we examined the oxazolone-induced chronic itch model in mice. Surprisingly, oral dosing of both compounds suppressed scratching behaviors that were observed in mice. Our findings demonstrate that TLR3 is a critical mediator in acute and chronic itch transduction in mice and may be a promising therapeutic target for pruritus in human skin disorders. It is noteworthy that SFN has potential for use as an antipruritic as it is a phytochemical that is used as a supplement.

A new hydrophilic polysulfone hemodialysis membrane can prevent platelet-neutrophil interactions and successive neutrophil activation.

PURPOSE:: Microaggregates have often been observed during hemodialysis and are clearly associated with complications of hemodialysis therapy. In this study, we aimed to clarify the effects of two polysulfone membranes, with different abilities to activate blood cells, on the formation of these microaggregates; we also investigated their molecular mechanisms. METHODS:: Human whole blood was circulated through a mini-module dialyzer using the membranes in vitro; platelet-neutrophil complexes in blood were determined by flow cytometry. Isolated human neutrophils were incubated with the membranes in plasma, in the presence or absence of platelets, followed by flow cytometric analysis of intracellular reactive oxygen species and cell-surface activated CD11b on neutrophils. RESULTS:: CX-U, a conventional polysulfone membrane with remarkable cell activation, induced the formation of platelet-neutrophil complexes; however, NV-U, a new hydrophilic polysulfone membrane with slight or no cell activation, did not cause complex formation. Moreover, CX-U-induced reactive oxygen species production and the increase in activated CD11b expression on neutrophils were enhanced by platelets. On the other hand, NV-U hardly affected neutrophil activation, regardless of whether platelets were present or not. The enhancement of CX-U-induced neutrophil activations by platelets was greatly inhibited by anti-CD62P antibody. CONCLUSION:: The ability of polysulfone membranes to activate blood cells is closely related to platelet-neutrophil interaction. Therefore, a biocompatible membrane, like NV-U, can be expected to prevent microaggregate formation during hemodialysis and avoid subsequent cell activation.

Salvianolic acid B inhibits platelet adhesion under conditions of flow by a mechanism involving the collagen receptor alpha2beta1.

Salvianolic acid B (SAB) is a component of Danshen, a herb widely used in Chinese medicine, and was previously shown to exert a number of biological activities including inhibition of platelet function, but the exact mechanisms involved are unclear. SAB dose-dependently inhibited platelet deposition from flowing, anticoagulated whole blood to immobilized collagen at both venous and arterial shear rate, whereas platelet deposition to immobilized fibrinogen was not affected. The inhibitory effect of SAB on platelet adhesion to collagen was independent of alphaIIbbeta3, since SAB still inhibited platelet deposition in the presence of a alphaIIbbeta3-blocking peptide. SAB inhibited static platelet adhesion to a synthetic peptide specific for the collagen receptor alpha2beta1, whereas platelet adhesion to a glycoprotein VI-specific peptide was not affected. SAB inhibited binding of an antibody against alpha2beta1 to platelets as studied by flow cytometry, and inhibited the interaction of soluble alpha2beta1 to immobilized collagen in a solid phase binding assay. These combined results indicate that SAB inhibits platelet adhesion to immobilized collagen by interfering with the collagen receptor alpha2beta1.

Anti-IL-12/IL-23p40 antibody ameliorates dermatitis and skin barrier dysfunction in mice with imiquimod-induced psoriasis-like dermatitis.

Psoriasis is a chronic inflammatory skin disease characterized by erythema, skin hyperplasia, scales, and keratinocyte hyperproliferation. While the cause of psoriasis is not clearly understood, a dysregulated immune system, especially activation of IL-23/IL-17 axis, has been strongly implicated in the pathogenesis of psoriasis. For example, anti-IL-23 therapy is effective in psoriasis patients, and thus IL-23 is considered as a potential therapeutic target for the treatment of psoriasis. The skin barrier provides protection of the human body against infection from external pathogens. Dysfunction of the skin barrier is also one of the characteristics in psoriasis and is correlated with disease severity. However, there have been no reports regarding the effectiveness of antipsoriatic agents on the skin barrier dysfunction of psoriasis. In this study, we examined the effect of anti-IL-12/IL-23p40 monoclonal antibody (p40 mAb) on dermatitis symptoms and skin barrier dysfunction in mice with imiquimod-induced psoriasis-like dermatitis. We found that p40 mAb suppressed epidermal thickness and increased transepidermal water loss (TEWL) as indicator for skin barrier function with accompanying suppression of IL-23p19, IL-17A, IL-22, and keratin 16 gene expression. These results suggest that p40 mAb is not only effective against dermatitis symptoms but also skin barrier dysfunction in mice with imiquimod-induced psoriasis-like dermatitis. This is the first report on the effect of p40 mAb on skin barrier dysfunction related to psoriasis. Taken together, our results indicate the possibility of new insights as well as the therapeutic potential of anti-IL-23 for the treatment of psoriasis.

Interferon-ß Mediates Signaling Pathways Uniquely Regulated in Hepatic Stellate Cells and Attenuates the Progression of Hepatic Fibrosis in a Dietary Mouse Model.

The results of clinical and experimental studies suggest that type I interferons (IFNs) may have direct antifibrotic activity in addition to their antiviral properties. However, the mechanisms are still unclear; in particular, little is known about the antifibrotic activity of IFN-ß and how its activity is distinct from that of IFN-α. Using DNA microarrays, we demonstrated that gene expression in TWNT-4 cells, an activated human hepatic stellate cell line, was remarkably altered by IFN-ß more than by IFN-α. Integrated pathway enrichment analyses revealed that a variety of IFN-ß-mediated signaling pathways are uniquely regulated in TWNT-4 cells, including those related to cell cycle and Toll-like receptor 4 (TLR4) signaling. To investigate the antifibrotic activity of IFN-ß and the involvement of TLR4 signaling in vivo, we used mice fed a choline-deficient l-amino acid-defined diet as a model of nonalcoholic steatohepatitis-related hepatic fibrosis. In this model, the administration of IFN-ß significantly attenuated augmentation of the area of liver fibrosis, with accompanying transcriptional downregulation of the TLR4 adaptor molecule MyD88. Our results provide important clues for understanding the mechanisms of the preferential antifibrotic activity of IFN-ß and suggest that IFN-ß itself, as well as being a modulator of its unique signaling pathway, may be a potential treatment for patients with hepatic fibrosis in a pathogenesis-independent manner.

Pharmacologically distinct phenotypes of α1B -adrenoceptors: variation in binding and functional affinities for antagonists.

BACKGROUND AND PURPOSE: The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B -adrenoceptor subtype in various tissue preparations and under various conditions. EXPERIMENTAL APPROACH: [(3) H]-prazosin binding to α1B -adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B -adrenoceptors. KEY RESULTS: In association and saturation-binding experiments with rat liver, binding affinity for [(3) H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B -adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B -adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B -adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B -adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B -adrenoceptor profile. CONCLUSIONS AND IMPLICATIONS: In contrast to the consistent profile of recombinant α1B -adrenoceptors, the pharmacological profile of native α1B -adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional 'subtype' characterization, 'phenotype' pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.

Production of soluble integrin alpha2beta1 heterodimer complex functionally active in vitro and in vivo.

Integrin alpha2beta1, which is a membrane protein consisting of noncovalently bound alpha2 and beta1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each alpha2 and beta1 chain was fused to hinge and Fc regions of human IgG(1)gamma chain were cotransfected into CHO cells. Soluble integrin alpha2beta1 (salpha2beta1) in which alpha2 and beta1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. salpha2beta1 maintained functional characteristics of cell surface alpha2beta1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered salpha2beta1 in rats colocalized with collagen in inflamed microvessels. Moreover, salpha2beta1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that salpha2beta1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute.

Reconstitution of adhesive properties of human platelets in liposomes carrying both recombinant glycoproteins Ia/IIa and Ib alpha under flow conditions: specific synergy of receptor-ligand interactions.

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ib alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-liposomes) instantaneously and irreversibly adhered to the collagen surface in the presence of soluble von Willebrand factor (VWF) at high shear rates, in marked contrast with translocation of liposomes carrying rGPIb alpha alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface decreased with increasing shear rates, similar to liposomes carrying rGPIa/IIa alone. While adhesion of liposomes with exofacial rGPIa/IIa and rGPIb alpha densities of 2.17 x 10(3) and 1.00 x 10(4) molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIb alpha density to 5.27 x 10(3) molecules per particle resulted in decreased adhesion even in the presence of soluble VWF. A 50% reduction in the exofacial rGPIa/IIa density resulted in a marked decrease in the adhesive ability of the liposomes at all shear rates tested. The inhibitory effect of antibody against GPIb alpha (GUR83-35) on liposome adhesion was greater at higher shear rates. Further, the anti-GPIa antibody (Gi9) inhibited liposome adhesion more than GUR83-35 at all shear rates tested. These results suggest that the rGPIa/IIa-collagen interaction dominates the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa-collagen and rGPIb alpha-VWF interaction complements each other, and they synergistically provide the needed functional integration required for liposome adhesion at high shear rates. This study thus has confirmed for the first time the proposed mechanisms of platelet adhesion to the collagen surface under flow conditions using the liposome system.

Hemostatic effects of polymerized albumin particles bearing rGPIa/IIa in thrombocytopenic mice.

The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa-polyAlb to thrombocytopenic mice ([platelet] = 2.1+/-0.3 x 10(5) particles/ microL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11)particles/kg, respectively, significantly reduced their bleeding time to 426+/-71, 378+/-101, and 337+/-46 s, respectively, whereas that of the control groups (PBS) was 730+/-198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11)particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet substitute.