Safety of Repeated-Dose Intratympanic Injections with AM-101 in Acute Inner Ear Tinnitus.

Objective To evaluate the safety and tolerability of repeated intratympanic administration of the gel-formulated NMDA receptor antagonist AM-101 in acute patients with inner ear tinnitus. Study Design Prospective, double-blind, randomized, placebo-controlled study. Setting Sixty-nine secondary and tertiary sites in North America, Europe, and Asia.

MEK5 is activated by shear stress, activates ERK5 and induces KLF4 to modulate TNF responses in human dermal microvascular endothelial cells.

Objective: ECs lining arteries respond to LSS by suppressing pro-inflammatory changes, in part through the activation of MEK5, ERK5 and induction of KLF4. We examined if this anti-inflammatory pathway operates in human ECs lining microvessels, the principal site of inflammatory responses.

Methods: We used immunofluorescence microscopy of human skin to assess ERK5 activation and KLF4 expression in HDMECs in situ.

Inhibition of pro-inflammatory cytokine production by the dual p38/JNK2 inhibitor BIRB796 correlates with the inhibition of p38 signaling.

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.

The determination and correlation of molecular and cellular equilibrium Kd and kinetic k(off) values for small molecule allosteric antagonists of LFA-1.

Molecular K(d) and k(off) parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1).

Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors.

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity).

Statistics of assay validation in high throughput cell imaging of nuclear factor kappaB nuclear translocation.

This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis.

Top 1 in 10,000: a 10-year follow-up of the profoundly gifted.

Adolescents identified before the age of 13 (N = 320) as having exceptional mathematical or verbal reasoning abilities (top 1 in 10,000) were tracked over 10 years. They pursued doctoral degrees at rates over 50 times base-rate expectations, with several participants having created noteworthy literary, scientific, or technical products by their early 20s. Early observed distinctions in intellectual strength (viz.

Molecular regulation of the interaction between leukocyte function-associated antigen-1 and soluble ICAM-1 by divalent metal cations.

Surface plasmon resonance (SPR) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). Full-length LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. The binding of sICAM-1 to LFA-1 was specific and dependent upon Mg2+; Abs to both sICAM-1 and LFA-1 blocked the interaction, and EDTA abolished all binding.

Molecular comparison of soluble intercellular adhesion molecule (sICAM)-1 and sICAM-3 binding to lymphocyte function-associated antigen-1.

The interactions of intercellular adhesion molecules-1 and -3 (ICAM-1 and ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) have been characterized and compared on the molecular and cellular level. Enzyme-linked immunosorbent-based molecular assays have been utilized to calculate the binding affinities of soluble ICAM-1 (sICAM-1) and soluble ICAM-3 (sICAM-3) for LFA-1. Consistent with previously published data, we found that sICAM-1 binds to LFA-1 with an affinity of approximately 60 nM.

Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity.

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores.

Characterization of molecular interactions between intercellular adhesion molecule-1 and leukocyte function- associated antigen-1.

The ability of soluble ICAM-1 (sICAM-1) to inhibit ICAM-1/LFA-1 adhesion events has been reported previously by numerous investigators. sICAM-1 has been demonstrated to inhibit various in vitro assays at concentrations ranging from 2 nM to greater than 40 microM. Given the hypothesis that circulating ICAM-1 modulates immune functions, the ability of sICAM-1 to inhibit cellular functions may have significant ramifications.

Binding affinities of the SH2 domains of ZAP-70, p56lck and Shc to the zeta chain ITAMs of the T-cell receptor determined by surface plasmon resonance.

The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated.

Kinetic characterization of human immunodeficiency virus type 1 protease: determination of inhibitor rate constants during dynamic monomer-dimer interconversion.

A numerical method was applied to a system of differential rate equations describing the monomer-dimer-inhibitor (M-D-I) interaction involving human immunodeficiency virus type 1 protease and a peptidomimetic, competitive inhibitor. Two pairs of progress curves were obtained, one involving the M-D interaction and the other the M-D-I interaction. Each pair of reactions was designed to begin with extreme conditions and end at the identical equilibrium position.

Time-resolved ligand exchange reactions: kinetic models for competitive inhibitors with recombinant human renin.

The on and off rate constants (kon and koff) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the sample inhibitor and initiating the reaction by addition of probe.

Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2.

Determination of kinetic rate constants for the binding of inhibitors to HIV-1 protease and for the association and dissociation of active homodimer.

Association and dissociation rate constants for a competitive inhibitor of HIV-1 protease were determined by a novel method employing a pair of integrated rate equations. This method, termed the paired progress curve method, is both rapid and reproducible. Progress curves, taken at a single concentration of inhibitor, are analyzed simultaneously to determine association and dissociation rate constants, the concentration of active sites, and the catalytic rate constant.

Estimation and correlation of drug water solubility with pharmacological parameters required for biological activity.

A procedure for estimating the molar water solubility (S) for a series of structurally related drug compounds is presented. HPLC methods for the determination of partition coefficients (P) are combined with semiempirical calculations for S. Multidimensional plots are developed with the physical constants S and P along the x and y axes and with a biological response, e.

Isotype choice for chimeric antibodies affects binding properties.

Construction of a series of chimeric antibodies (murine variable region and human constant region) derived from the murine antibody BIRR1, which recognizes intercellular adhesion molecule 1 (ICAM-1), has revealed differences in the relative binding abilities of the chimeric antibody to antigen. The chimeric antibodies show a ranking of their ability to compete with BIRR1 for antigen on the surface of cells with the order BIRR1 = cIgG1 (100%) > cIgG4 (30%) > cIgG2 (10%) as demonstrated by solid-phase competitive enzyme-linked immunosorbent assay. Papain digestion yielded Fab fragments that were purified to homogeneity.

Metallothionein: a bifunctional chelator for the radiolabeling of biologically active molecules.

Cysteine-rich metallothionein has been shown to bind as many as seven TcO3+ cores in TcO (Cys) units which adopt square-base pyramidal geometry. The Tc atoms in 99mTc,Zn-MT and 99mTc,Zn-MT-MAb species formed in the exchange reaction with the labile 99mTcO (glucoheptonate) complex are stable to oxidation and transchelation in vivo. The kinetics of labeling with the carrier-free radionuclide 99mTc are diffusion-limited at MT concentrations below 10(-5) M.

Physical and enzymatic properties of a class II alcohol dehydrogenase isozyme of human liver: pi-ADH.

Homogeneous class II alcohol dehydrogenase (pi-ADH) has been isolated from human liver homogenates by chromatography on DE-52 cellulose, 4-[3-[N-(6-amino-caproyl)amino]propyl]pyrazole-Sepharose, SP-Sephadex C-50, and agarose-hexane-AMP, yielding an enzyme that has a significantly higher specific activity and is markedly more stable than that isolated by an earlier procedure. pi-ADH is composed of two identical 40 000-dalton subunits, contains 4 mol of zinc/dimer, and is readily inhibited by metal-chelating agents. The purified enzyme binds two molecules of coenzyme per dimer, exhibits an absorption maximum at 280 nm, epsilon 280 = 57 000, and exhibits an isoelectric point of 8.