Long-term efficacy and safety results from an open-label phase III study (UNCOVER-J) in Japanese plaque psoriasis patients: impact of treatment withdrawal and retreatment of ixekizumab.

BACKGROUND: Long-term management of moderate-to-severe psoriasis is usually discussed in terms of continuous administration; however, there are many situations in clinical practice where treatment may be withdrawn with subsequent retreatment. OBJECTIVE: To assess the clinical course after ixekizumab treatment withdrawal and retreatment, as well as the effectiveness of ixekizumab retreatment, in Japanese patients with plaque psoriasis. METHODS: This single-arm, open-label study (UNCOVER-J; NCT01624233) comprised 78 patients with plaque psoriasis. After ixekizumab treatment (160-mg loading dose, 80 mg every 2 weeks for the first 12 weeks, and then 80 mg every 4 weeks (IXE Q4W) until Week 52), 70 patients achieved a Psoriasis Area Severity Index (PASI)75 response at Week 52. These 70 patients withdrew from ixekizumab treatment from Weeks 52 to 100. Patients who relapsed (PASI ≤50) during the Treatment Withdrawal Period were retreated with IXE Q4W for 192 weeks. RESULTS: At Weeks 52, 76 and 100, PASI75 response rates were 100%, 26% and 7%; PASI90 response rates were 87%, 11% and 3%; and PASI100 response rates were 53%, 0% and 0%. After treatment withdrawal, 87% of patients relapsed; median time to relapse was 143 days. After 12 weeks of retreatment with IXE Q4W, 83% of relapsed patients achieved PASI75, 68% achieved PASI90 and 25% achieved PASI100; improvements were maintained up to 120 weeks of retreatment. Treatment-emergent adverse events and serious adverse events were reported in 56% and 4% of patients during the Treatment Withdrawal Period, and in 88% and 14% of patients during the Retreatment Period. CONCLUSION: In patients withdrawn from ixekizumab after achieving PASI75, approximately half relapsed within 5 months of withdrawal; however, most patients recaptured response within 12 weeks, and response was maintained for up to 120 weeks of retreatment.

Long-term clinical safety and efficacy of brodalumab in the treatment of Japanese patients with moderate-to-severe plaque psoriasis.

BACKGROUND: Brodalumab (KHK4827) is a human anti-interleukin-17-receptor A monoclonal antibody. In Japanese patients with moderate-to-severe plaque psoriasis, brodalumab showed rapid and robust efficacy and a favourable safety profile in a 12-week, phase 2, double-blind, randomized controlled trial. OBJECTIVES: To evaluate the long-term safety and efficacy of brodalumab, an extension of a phase 2 trial of Japanese patients with moderate-to-severe psoriasis was performed. METHODS: Patients received open-label brodalumab 210 or 140 mg subcutaneously every 2 weeks for 52 weeks. Efficacy was measured using the Psoriasis Area and Severity Index (PASI) score and the static physician global assessment (sPGA) instrument. The endpoint of psoriatic arthritis was 20% improvement in American College of Rheumatology response criteria (ACR 20). The patients were also monitored for treatment-emergent adverse events (AEs), including serious AEs (SAEs). RESULTS: Of 145 patients, 133 completed the study. The percentage of patients with ≥75% reduction of PASI scores (PASI 75), ≥90% (PASI 90) and 100% (PASI 100) at Week 52 (the last observation carried forward) were 94.4%, 87.5% and 55.6%, respectively, in the 210-mg group, and the corresponding values in the 140-mg group were 78.1%, 71.2% and 43.8%. At Week 52, 75.0% patients in 210-mg group achieved ACR 20, compared with 37.5% patients in 140-mg group. The most commonly reported AEs were nasopharyngitis (35.2%), upper respiratory tract inflammation (10.3%) and contact dermatitis (9.7%). CONCLUSION: Brodalumab showed a sustained clinical response and an acceptable safety profile through 52 weeks in Japanese patients with moderate-to-severe plaque psoriasis in this open-label extension study.

Clinical features and risk factors for developing varicella zoster virus dissemination following hematopoietic stem cell transplantation.

BACKGROUND: We retrospectively analyzed 80 instances of varicella zoster virus (VZV) disease in 72 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) and examined the clinical differences between localized and disseminated disease. Risk factors for developing VZV dissemination were also evaluated. RESULTS: Of the 80 instances, 54 instances were localized diseases and 26 were disseminated diseases. Patient characteristics did not differ significantly between the 2 groups, except for the first-line therapy and the duration from symptom onset to treatment. In the disseminated group, intravenous acyclovir was used as the first-line therapy more frequently, and more time elapsed before beginning antiviral therapy compared with the localized group. In multivariate analyses, the duration from symptom onset to treatment was identified as an independent risk factor that significantly affected the development of VZV dissemination. Gender, total body irradiation, and chronic graft-versus-host disease, of which the latter 2 factors were reported as risk factors for the development of VZV disease after HSCT, did not affect the development of VZV dissemination. CONCLUSION: Our results suggest that VZV infection or reactivation may easily progress to viremia with delayed use of antiviral agents and may result in VZV dissemination in immunocompromised patients.

Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines.

BACKGROUND: Cytokine imbalance and cellular migration to inflammatory sites are critical components of allergic diseases. Redirecting cytokine imbalance and inhibiting cell migration therefore represent important therapeutic strategies for the treatment of these disorders. OBJECTIVES: To study the in vitro effect of ebastine, a novel non-sedating H1 receptor antagonist, on cytokine secretion and migration of activated T cells, as well as production of pro-inflammatory cytokines by macrophages. METHODS: Peripheral T cells obtained from healthy volunteers were cultured in wells coated with the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA, in the presence or absence of ebastine. T cell proliferation and the production of cytokines were measured by [3H]thymidine incorporation assay and ELISA, respectively. In addition, transendothelial migration of T cells and production of pro-inflammatory cytokines by macrophages were examined. RESULTS: Ebastine inhibited T cell proliferation and the production of IL-4, IL-5, IL-6, and TNF-alpha by T cells under each co-stimulatory condition tested, whereas it exhibited no effect on the production of IL-2 or IFN-gamma. In addition, T cell migration and the production of such pro-inflammatory cytokines as TNF-alpha and IL-6 by macrophages were inhibited by ebastine. CONCLUSIONS: These results indicate that ebastine has a specific inhibitory effect on Th2-type cytokine production. Moreover, ebastine inhibited T cell migration and pro-inflammatory cytokine production by T cells and macrophages, suggesting that ebastine might be useful for the treatment of T cell-mediated allergic inflammatory disorders, including asthma, atopic dermatitis, and Th2-type autoimmune diseases.

Therapeutic guidelines for the treatment of generalized pustular psoriasis (GPP) based on a proposed classification of disease severity.

Generalized pustular psoriasis (GPP) is a rare but notoriously recalcitrant cutaneous diseases. Therefore, there have been few reports of more than ten patients with GPP who were treated at the same institution. The severity of this disease and its response to each therapeutic modality vary among patients. In some GPP is life-threatening, but in others it may show a benign, chronic course for a long period of time. Before starting treatment, a knowledge of the therapeutic efficacy and side effects of each drug used in the treatment of GPP is necessary. In our multicenter study, we compared the effectiveness of and adverse reactions to several systemically administered drugs. Following the development of a unique classification of the disease severity based on scoring the clinical symptoms and the laboratory findings, we propose here therapeutic guidelines for the treatment of GPP.

Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

Studies on the matched potential method for determining the selectivity coefficients of ion-selective electrodes based on neutral ionophores: experimental and theoretical verification.

A theory is presented that describes the matched potential method (MPM) for the determination of the potentiometric selectivity coefficients (KA,Bpot) of ion-selective electrodes for two ions with any charge. This MPM theory is based on electrical diffuse layers on both the membrane and the aqueous side of the interface, and is therefore independent of the Nicolsky-Eisenman equation. Instead, the Poisson equation is used and a Boltzmann distribution is assumed with respect to all charged species, including primary, interfering and background electrolyte ions located at the diffuse double layers. In this model, the MPM-selectivity coefficients of ions with equal charge (ZA = ZB) are expressed as the ratio of the concentrations of the primary and interfering ions in aqueous solutions at which the same amounts of the primary and interfering ions permselectively extracted into the membrane surface. For ions with unequal charge (ZA not equal to ZB), the selectivity coefficients are expressed as a function not only of the amounts of the primary and interfering ions permeated into the membrane surface, but also of the primary ion concentration in the initial reference solution and the delta EMF value. Using the measured complexation stability constants and single ion distribution coefficients for the relevant systems, the corresponding MPM selectivity coefficients can be calculated from the developed MPM theory. It was found that this MPM theory is capable of accurately and precisely predicting the MPM selectivity coefficients for a series of ion-selective electrodes (ISEs) with representative ionophore systems, which are generally in complete agreement with independently determined MPM selectivity values from the potentiometric measurements. These results also conclude that the assumption for the Boltzmann distribution was in fact valid in the theory. The recent critical papers on MPM have pointed out that because the MPM selectivity coefficients are highly concentration dependent, the determined selectivity should be used not as "coefficient", but as "factor". Contrary to such a criticism, it was shown theoretically and experimentally that the values of the MPM selectivity coefficient for ions with equal charge (ZA = ZB) never vary with the primary and interfering ion concentrations in the sample solutions even when non-Nernstian responses are observed. This paper is the first comprehensive demonstration of an electrostatics-based theory for the MPM and should be of great value theoretically and experimentally for the audience of the fundamental and applied ISE researchers.

Threshold ionic site concentrations required for Nernstian potentiometric responses of neutral ionophore-incorporated ion-selective liquid membranes.

An equation that can describe the concentrations of ionic sites required for a Nernstian potentiometric response slope of neutral ionophore-incorporated ion-selective liquid membranes is presented. This equation is derived from a model based on electrical diffuse layers on both the membrane and the aqueous sides of the interface, in which the phase boundary potential is correlated to the surface charge density as well as the salt concentrations in the bulk membrane and aqueous solution. To experimentally and accurately confirm the validity of this equation, response characteristics of field effect transistors covered by neutral ionophore-based liquid membranes with varying concentrations of a derivative of tetraphenylborate as an anionic site but free of ionic impurities were examined. The observed membrane potentials and the response slopes for membranes with various concentrations of anionic sites were in good agreement with the values calculated from the theory presented in this paper with the measured complexation stability constants for the relevant systems. This result indicates that the theoretical prediction based on the proposed equation for the anionic site concentration is accessible for the preparation of neutral ionophore-incorporated ion-selective liquid membranes, which show Nernstian response slopes for the primary ions.

Potentiometric responses of polymeric liquid membranes based on hydrophobic chelating agents to metal ions.

The effect of hydrophobicity of acidic chelating agents as sensing materials on the potentiometric responses of polymeric liquid membranes was investigated. The chelating agents tested were 8-quinolinol (HOx), dithizone (HDz), 1-(2-pyridylazo)-2-naphthol (PAN) and their alkylated analogues, 5-octyloxymethyl-8-quinolinol (HO8Q), di(phexylphenyl)thiocarbazone (C6HDz), 7-pentadecyloxy-1-(2-pyridylazo)-2-naphthol (C15PAN) and a series of N-alkylcarbonyl-N-phenylhydroxylamines (CnPHA, n = 3, 6, 9, 12). The distribution coefficients between membrane solvent and water were determined to evaluate the hydrophobicity of the agents. The potential-pH profiles of the membranes containing hydrophobic chelating agents demonstrated the generation of potentiometric responses, while less hydrophobic agents gave no response. A possible model for the generation of membrane potential is proposed. The charge separation is attained by the permselective uptake of metal cations by the chelating agent anion at membrane/solution interface, where the high hydrophobicity of the agent enables the anionic or deprotonated form of the agents to remain at the membrane/solution interface.

Detection of protein-protein interactions in vivo based on protein splicing.

In mammalian cells, protein-protein interactions constitute essential regulatory steps that modulate the activity of signaling pathways. In recent years, several approaches towards understanding the interactions have been developed. We describe herein a new method for detecting protein-protein interactions in vivo based on protein splicing and highlight some potential applications of this technique.

Influence of natural, electrically neutral lipids on the potentiometric responses of cation-selective polymeric membrane electrodes.

Ionophore-free ion exchanger electrodes were found to exhibit quite a high selectivity for the creatininium ion; however, measurements in diluted urine samples revealed large emf drifts. Potentiometric, chromatographic, NMR, and mass spectrometric evidence did not reveal any major cationic interfering agents, and anionic interfering agents cannot trivially explain the consistently positive emf drifts. Ultrafiltration of urine samples showed that the interfering agents have molecular weights below 1000 u. The drifts are apparently caused by electrically neutral lipophilic compounds of low molecular weight that are easily extracted into organic phases. Follow-up experiments showed that p-cresol and cholesterol cause no significant emf responses but that coproporphyrin, phosphatidylserine, taurocholic acid, cholic acid, phosphatidylethanolamine, and octanoic acid cause positive emf drifts of the type that was observed with the urine samples. The extent of the responses and the response time depend not only on the specific compound but also on the cation in the sample solution. These results suggest that the emf drifts are due to extraction of such natural lipids into the organic membrane phase where they interact in an ionophore-like fashion with the analyte and interfering ions. Changes in the potentiometric selectivities after contact with natural lipids support this interpretation. The same effect of natural lipids is also expected for ionophore-based electrodes. Indeed, exposure of a valinomycin-based electrode to a methylene chloride extract of urine resulted in a significant reduction of the Na+ discrimination, increasing log Kpot(K,Na) from -3.9 to -3.1.

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

Optical detection in microscopic domains. 2. Inner filter effects for monitoring nonfluorescent molecules with fluorescence.

In this research, we test whether optical detection techniques show different characteristics in microscopic solution volumes (nano-, pico-, and femtoliter range) compared to the usual macroscopic samples. In part 1 (Lu, H.; et al. Anal Chem. 2000, 72, 1569-1576.) absorption spectra of high quality were obtained, quantitatively obeying both Beer-Lambert's law and the law of superposition, despite the micrometer optical path lengths and the curvatures of the droplets studied. Addition and subtraction of absorbing molecules with diffusional microburets (DMBs), as well as more complex operations (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal resolution. Despite the unexpectedly good performance of absorption microspectrometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e.g., cell physiology). In this paper, we test whether fluorescence-based schemes can be used to indirectly measure nonfluorescent chemicals in microscopic domains. Absorption by such molecules will cause a corresponding decrease in overall fluorescence intensity of the added standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye. Its effective irradiation was absorbed by Orange G (primary IFE) or its emission by Bromophenol Blue (secondary IFE). By utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic samples by adding a standard amount of LY by a DMB, and (2) we monitored DMB delivery of nonfluorescent reagents into droplets preloaded with LY. The results prove that IFEs are sensitive indirect means of detection of absorbing molecules in microscopic domains. The techniques presented are expected to find applications in cellular studies where absorption spectrometry is usually not considered.

Relationship between lymphocyte cyclosporin sensitivity and clinical progress of psoriasis.

Cyclosporin (CYA) is a therapeutic agent used in the treatment of psoriasis vulgaris. However, the effectiveness of CYA therapy varies among patients. In the present study, due to the fact that CYA mainly acts on lymphocytes, we hypothesized that a measurement of the sensitivity of lymphocytes to CYA in vitro (IC50) could be applied to patients with psoriasis vulgaris to determine therapeutic success. We measured IC50 levels of 32 patients presenting with psoriasis prior to CYA administration, and classified them into three groups according to IC50 levels: favorable, moderate and low sensitivity. CYA sensitivity levels were correlated with the degree of improvements in psoriasis area and severity index (PASI) scores, CYA dosage and occurrence of side-effects (hepatopathy and nephropathy). Results showed the degree of improvement in PASI scores differed significantly between the favorable and low sensitivity groups (P < 0.05). Furthermore, CYA dosage was lowest in the favorable sensitivity group and highest in the low sensitivity group. Moreover, hepatopathies and nephropathies were detected in the low and moderate sensitivity groups, but not in the favorable sensitivity group. These results suggest that the effectiveness of CYA therapy as a treatment of psoriasis vulgaris is affected by the sensitivity of lymphocytes in each patient.

Scanning tunneling microscopy with chemically modified tips: discrimination of porphyrin centers based on metal coordination and hydrogen bond interactions.

STM gold tips chemically modified with 4-mercaptopyridine (4MP) were found capable of discriminating zinc(II) 5,15-bis(4-octadecyloxyphenyl)porphyrin (Por-Zn) from its metal-free porphyrin (Por-2H) and nickel(II) complexes (Por-Ni) in the mixed monolayers of these compounds, spontaneously formed at the solution/graphite interface. The porphyrin centers in STM images observed with 4MP-modified tips exhibited bright spots, while those measured with unmodified tips exhibited the porphyrin centers as dark depressions. The centers of Por-Zn were brighter than those of Por-2H and Por-Ni, thereby allowing the discrimination of Por-Zn from Por-2H or Por-Ni in mixed monolayers. The changes in the contrasts of porphyrin centers of Por-2H and Por-Zn/ Por-Ni were explained by facilitated electron tunneling due to hydrogen bond and metal coordination interactions, respectively, between porphyrin centers and the pyridyl group of 4MP on the tip.

Correlation between cytosolic calcium concentration and degranulation in RBL-2H3 cells in the presence of various concentrations of antigen-specific IgEs.

We studied the dependence of beta-hexosaminidase release from RBL-2H3 cells on the antigen-specific IgE concentrations. The cells were sensitized with DNP-specific IgE (0.5-5000 ng/ml) or OVA-specific IgE (5-50 ng/ml) and stimulated with DNP(35)-HSA (10(-2)-100 ng/ml) or OVA (10(-1) ng/ml-10 microg/ml). It was found that the beta-hexosaminidase release increased in a dose-dependent manner with the concentration of the IgEs and antigens added to the mast-cell suspension. We also studied the correlation between the intracellular calcium concentration ([Ca(2+)]i) and degranulation in RBL-2H3 cells. The percentage of beta-hexosaminidase release from the cells was well correlated with [Ca(2+)](i) increase, and the correlation coefficient was 0.88 for DNP-specific IgE and 0.99 for OVA-specific IgE. The minimum [Ca(2+)](i) required to induce the beta-hexosaminidase release was 100 nM for DNP-specific IgE, and 70 nM for OVA-specific IgE. Therefore, the [Ca(2+)](i) monitoring system is a sensitive marker of degranulation from RBL-2H3 cells and can be used to measure even low amounts of antigen-specific IgE.