Buntanetap, a Novel Translational Inhibitor of Multiple Neurotoxic Proteins, Proves to Be Safe and Promising in Both Alzheimer's and Parkinson's Patients.

BACKGROUND: Previously we reported the clinical safety and pharmacological activity of buntanetap (known as Posiphen or ANVS401) in healthy volunteers and mild cognitive impaired (MCI) patients (21). The data supported continued clinical evaluation of buntanetap for treating Alzheimer's Disease (AD). Neurodegenerative diseases such as AD and Parkinson's disease (PD) share several pathological manifestations, including increased levels of multiple neurotoxic protein aggregates. Therefore, a treatment strategy that targets toxic species common to both disorders can potentially provide better clinical outcomes than attacking one neurotoxic protein alone. To test this hypothesis, we recently completed a clinical study in early AD and early PD participants and report the data here. OBJECTIVES: We evaluated safety, pharmacokinetics, biomarkers, and efficacy of buntanetap in treating early AD and PD patients. DESIGN: Double-blind, placebo-controlled, multi-center study. SETTING: 13 sites in the US participated in this clinical trial. The registration number is NCT04524351 at ClinicalTrials.gov. PARTICIPANTS: 14 early AD patients and 54 early PD patients. INTERVENTION: AD patients were given either 80mg buntanetap or placebo QD. PD patients were given 5mg, 10mg, 20mg, 40mg, 80mg buntanetap or placebo QD. MEASUREMENTS: Primary endpoint is safety and tolerability; secondary endpoint is pharmacokinetics of buntanetap in plasma; exploratory endpoints are 1) biomarkers in cerebrospinal fluid (CSF) in both AD and PD patients 2) psychometric tests specific for AD (ADAS-Cogs and WAIS coding test) or PD (MDS-UPDRS and WAIS coding test). RESULTS: Buntanetap was safe and well tolerated. Biomarker data indicated a trend in lowering levels of neurotoxic proteins and inflammatory factors and improving axonal integrity and synaptic function in both AD and PD cohorts. Psychometric tests showed statistically significant improvements in ADAS-Cog11 and WAIS coding in AD patients and MDS-UPDRS and WAIS coding in PD patients. CONCLUSIONS: Buntanetap is well tolerated and safe at doses up to 80mg QD in both AD and PD patients. Cmax and AUC increase with dose without evidence for a plateau up to 80mg QD. The drug shows promising evidence in exploratory biomarker and efficacy measures. Further evaluation of buntanetap in larger, longer-term clinical trials for the treatment of AD and PD are warranted.

Linear and nonlinear analyses of pulsatile blood flow in a cylindrical tube.

A non-Newtonian shear-thinning constitutive relation is proposed to study pulsatile flow of whole blood in a cylindrical tube. The constitutive relation, which satisfies the principle of material frame indifference, is derived from viscometric data obtained from whole blood over a range of hematocrits. Assuming axisymmetric flow in a rigid cylindrical tube of constant diameter, a second-order, nonlinear partial differential equation governing the axial velocity component is obtained. Imposing a periodic pressure gradient, the governing equation was solved numerically using finite difference methods over a range of Stokes values and hematocrits. For a forcing frequency of 1 Hz, results are presented over tube diameters ranging between 0.1 and 2 cm and over hematocrits ranging between 10 and 80%. For a given hematocrit, velocity profiles predicted for the non-Newtonian model under sinusoidal forcing reveal attenuated volume flow rate and enhanced vorticity transport over the tube cross-section relative to a Newtonian fluid having a viscosity corresponding to the high shear-rate limit. For moderate to high Stokes numbers, consistent with flow in large arteries, our results revealed a viscosity distribution that was nearly time invariant. An analytic solution was obtained for a fluid having arbitrarily prescribed radially varying, temporally invariant viscosity and density distributions under arbitrary periodic pressure forcing. Close agreement was observed between our numerical and analytical results when the imposed viscosity distribution was chosen to approximate the time-averaged viscosity distribution predicted by the shear-thinning non-Newtonian model. For St > or approximately= 100, the disparity between our results and those of a Newtonian fluid of constant viscosity grows with a decreasing ratio of the DC to AC components of the pressure-gradient amplitude below 50%. In particular, for any purely oscillatory pressure-gradient (vanishing DC component), the Womersley solution is a particularly poor predictor of the amplitude and phase of wall shear rate for over half of the flow cycle. Under such circumstances, the analytical models presented here provide a simple and accurate means of estimating instantaneous wall shear rate, knowing only the pressure gradient and hematocrit.

An electrochemical model of the transport of charged molecules through the capillary glycocalyx.

An electrochemical theory of the glycocalyx surface layer on capillary endothelial cells is developed as a model to study the electrochemical dynamics of anionic molecular transport within capillaries. Combining a constitutive relationship for electrochemical transport, derived from Fick's and Ohm's laws, with the conservation of mass and Gauss's law from electrostatics, a system of three nonlinear, coupled, second-order, partial, integro-differential equations is obtained for the concentrations of the diffusing anionic molecules and the cations and anions in the blood. With the exception of small departures from electroneutrality that arise locally near the apical region of the glycocalyx, the model assumes that cations in the blood counterbalance the fixed negative charges bound to the macromolecular matrix of the glycocalyx in equilibrium. In the presence of anionic molecular tracers injected into the capillary lumen, the model predicts the size- and charge-dependent electrophoretic mobility of ions and tracers within the layer. In particular, the model predicts that anionic molecules are excluded from the glycocalyx at equilibrium and that the extent of this exclusion, which increases with increasing tracer and/or glycocalyx electronegativity, is a fundamental determinant of anionic molecular transport through the layer. The model equations were integrated numerically using a Crank-Nicolson finite-difference scheme and Newton-Raphson iteration. When the concentration of the anionic molecular tracer is small compared with the concentration of ions in the blood, a linearized version of the model can be obtained and solved as an eigenvalue problem. The results of the linear and nonlinear models were found to be in good agreement for this physiologically important case. Furthermore, if the fixed-charge density of the glycocalyx is of the order of the concentration of ions in the blood, or larger, or if the magnitude of the anionic molecular valence is large, a closed-form asymptotic solution for the diffusion time can be obtained from the eigenvalue problem that compares favorably with the numerical solution. In either case, if leakage of anionic molecules out of the capillary occurs, diffusion time is seen to vary exponentially with anionic valence and in inverse proportion to the steady-state anionic tracer concentration in the layer relative to the lumen. These findings suggest several methods for obtaining an estimate of the glycocalyx fixed-charge density in vivo.

A poroelastic continuum model of the cupula partition and the response dynamics of the vestibular semicircular canal.

Using mixture theory, an axisymmetric continuum model is presented describing the response dynamics of the vestibular semicircular canals to canal-centered head rotation in which the cupula partition is modeled as a poroelastic mixture of interpenetrating solid and fluid constituents. The solid matrix of the cupula is assumed to behave as a linear elastic material, whereas the fluid constituent is assumed to be Newtonian. A regular perturbation analysis of the fluid dynamics in the canal provides a dynamic boundary condition, which acts across the cupula partition. Numerical solution of the coupled system of momentum equations provides the spatio-temporal displacement fields for both the fluid and solid constituents of the cupula. Results indicate that at frequencies above 1 Hz, the fluid constituent is dynamically entrained by the solid matrix such that their motions are bound as if to exist as a single component. The resulting high-frequency response is consistent with the macromechanical response predicted by single-component viscoelastic models of the cupula. Below 1 Hz, the dynamic coupling between the fluid and solid constituents weakens and the transcupular differential pressure is sufficient to force fluid through the mixture with little deformation of the solid matrix. Results are sensitive to the precise value of the cupular permeability. One of the most important distinctions between the present analysis and previous impermeable models of the cupula arises at the micromechanical level in terms of the local fluid flow that is predicted to occur within the cupula and around the ciliary bundles and sensory hair cells. Another important result reveals that the permeation dynamics predicted below 1 Hz gives rise to the same low-frequency macromechanical response as would occur with an impermeable viscoelastic structure having a much greater stiffness. Current estimates of the mechanical stiffness of the cupula, based solely on afferent nerve data, may therefore overestimate the true value intrinsic to the solid matrix by as much as an order of magnitude.

The effect of the endothelial-cell glycocalyx on the motion of red blood cells through capillaries.

The analysis of the rheology of the blood/capillary system presented here details the complex fluid-structure interactions that arise among blood cells, plasma, and the endothelial-cell glycocalyx. The capillary is modeled as a rigid cylindrical tube lined with a biphasic poroelastic wall layer which approximates the glycocalyx. The blood is approximated as a fluid suspension of tightly fitting deformable cells driven through the tube under a pressure gradient. Using mixture theory, the wall layer is modeled as interacting fluid and solid constituents in which the fluid is assumed to be linearly viscous and the solid is assumed to be linearly elastic. Axisymmetric lubrication theory is applied to the fluid in the gap between the red cell and the glycocalyx. The analysis details the fluid-phase velocity field throughout the wall and lubrication layers and provides the Reynolds equation for the pressure gradient along the length of the cell. The shell equations of equilibrium are employed to describe the mechanics governing the axisymmetric deformation of the red-cell membrane, where it is assumed that shear stress on the surface of the cell is balanced solely by isotropic membrane tension. Making use of the analytic expressions for the Reynolds equation and shear stress distribution on the cell surface, the pressure profile, membrane tension, and red-cell shape are obtained through numerical solution of a reduced system of coupled, nonlinear, ordinary differential equations. Rheological quantities including apparent viscosity and capillary tube hematocrit are presented and compared with in vitro and in vivo experimental data. The analysis predicts that the presence of a 1/2-microm-thick glycocalyx in a 5-microm capillary results in a threefold increase in resistance and a reduction in capillary tube hematocrit of more than 30% compared with the corresponding values in a 5-microm smooth-walled tube. Results are qualitatively consistent with in vivo observations of blood flow in microvascular networks.

Variation in the velocity, deformation, and adhesion energy density of leukocytes rolling within venules.

Leukocyte rolling along the endothelium in inflammation is caused by continuous formation and breakage of bonds between selectin adhesion molecules and their ligands. We investigated trauma-induced leukocyte rolling in venules (diameter, 23 to 58 microns; wall shear stress, 1.2 to 35 dyne/cm2) of the exteriorized rat mesentery using high-resolution intravital microscopy. While rolling, the leukocytes deformed into a tear-droplike shape. Deformation continued to increase with shear stress up to the highest values observed (35 dyne/cm2). Successive leukocytes had similar rolling velocities at the same axial positions along each vessel, suggesting that heterogeneity of endothelial adhesiveness is responsible for velocity variation. Adhesion energy density varied inversely with instantaneous rolling velocity and directly with instantaneous deformation. Adhesion energy density reached a maximum of 0.36 dyne/cm, similar to values found for lymphocyte function-associated antigen-1-dependent adhesion of stimulated T cells to isolated intercellular adhesion molecule-1. We conclude that selectin-mediated adhesion during rolling produces adhesion energy densities comparable to those observed for integrin-mediated adhesion events in other experimental systems.

Human monoclonal antibody HA-1A binds to endotoxin via an epitope in the lipid A domain of lipopolysaccharide.

HA-1A, a human IgM mAb, has been shown to significantly reduce mortality in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the reported specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a solid phase; a rate nephelometry assay, which measured the ability of HA-1A to bind and aggregate lipid A in solution; and a dot-blot immunoassay, which measured the ability of HA-1A to interact with lipid A adsorbed to Immobilon-P. In all three assay systems, HA-1A bound in a dose-dependent manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to competitively inhibit HA-1A reactivity with lipid A in a dose-dependent manner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against HA-1A was also able to block the binding of HA-1A to lipid A in these assay formats. HA-1A reactivity with synthetic lipid A confirmed that HA-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assessed. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these results conclusively establish that HA-1A binds to the lipid A region of LPS by an interaction with the V region of the antibody.

Chemical modifications of the Na+-H+ antiport in Escherichia coli membrane vesicles.

The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained.

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles.

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles.

Measurements of the fluorescent properties of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) enclosed within the internal space of Escherichia coli membrane vesicles enable recordings and quantitative analysis of: (i) changes in intravesicular pH taking place during oxidation of electron donors by the membrane respiratory chain; (ii) transient alkalization of the internal aqueous space resulting from the creation of outwardly directed acetate diffusion gradients across the vesicular membrane. Quantitation of the fluorescence variations recorded during the creation of transmembrane acetate gradients shows a close correspondence between the measured shifts in internal pH value and those expected from the amplitude of the imposed acetate gradients.

Kinetic properties of Na(+) -H(+) antiport in Escherichia coli membrane vesicles: Effects of imposed electrical potential, proton gradient, and internal pH.

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.