Chemical and photonic interactions in vitro and in vivo between fluorescent tracer and nanoparticle-based scavenger for enhanced molecular imaging.

We hereby present a concept of scavenging excess imaging agent prior to a diagnostic imaging session, consequently allowing for enhanced contrast of signals originating from the tissue area of interest to the signals originating from systemic imaging agent residues. In our study, a prospective silica core-shell nanoparticle-based scavenger was designed and explored for its feasibility to scavenge a specific imaging agent (tracer) in the bloodstream. The developed tracer-scavenger system was first investigated under in vitro conditions to ensure proper binding between tracer and scavenger is taking place, as confirmed by Förster/fluorescence resonance energy transfer studies. In vivo, two-photon imaging was utilized to directly study the interaction of the scavenger particles and the tracer molecules in the vasculature of mice. To our knowledge, a methodological solution for in vivo differentiation between signals, originating from tissue and blood, has not been presented elsewhere.

A novel CGRP-neutralizing Spiegelmer attenuates neurogenic plasma protein extravasation.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP-mediated processes as a new therapeutic option in migraine. In this study, we describe the generation of NOX-L41, a CGRP-neutralizing mirror-image (L-)aptamer (Spiegelmer) and investigate its in vitro and in vivo function. EXPERIMENTAL APPROACH: A CGRP-binding Spiegelmer was identified by in vitro selection. Binding studies were performed using surface plasmon resonance (SPR), and the inhibitory activity was determined in cell-based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two-photon microscopy was employed to follow extravasation from meningeal vessels. Finally, in vivo efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (PPE) in rats. KEY RESULTS: We identified NOX-L41, a novel CGRP-neutralizing Spiegelmer. SPR studies showed that NOX-L41 binds to human and rat/mouse CGRP with sub-nanomolar affinities and is highly selective against related peptides such as amylin. In vitro, NOX-L41 effectively inhibited CGRP-induced cAMP formation in SK-N-MC cells. In rats, NOX-L41 had a plasma half-life of 8 h. Pharmacodynamic studies showed that NOX-L41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal PPE for at least 18 h after single dosing. CONCLUSIONS AND IMPLICATIONS: This is the first description of the CGRP-neutralizing Spiegelmer NOX-L41. Preclinical studies confirmed a role for CGRP in neurogenic PPE and provided proof-of-concept for the potential use of this new drug candidate for the treatment or prevention of migraine.

The mechanisms of inhibition of frog endplate currents with homologous derivatives of the 1,1-dimethyl-3-oxybutyl phosphonic acid.

The mode of inhibition of endplate currents by four esters of 1,1-dimethyl-3-oxybutyl phosphonic acid with different lipophilicities and molecule lengths were estimated by mathematical modeling based on previous electrophysiological data supplemented by several experiments with rhythmic stimulation. The aim was to discriminate between their receptor and non-receptor effects. It was shown that all esters have a two-component mechanism of depression: inhibition of the receptor open channel and allosteric modulation of the receptor-channel complex. The ratio of both functional components depends on the length and lipophilicity of the esters. Short and less lipophilic esters mostly act as open channel inhibitors and the rate of inhibition substantially depends on the rate of stimulation, i. e. probability of the receptor-channel opening. As the length of the ester radicals and their lipophilicity increased, these compounds were more active as allosteric receptor inhibitors, probably hindering the function of nAChRs from the lipid annulus.

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane.

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q(10) ∼4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q(10) ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q(10) ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q(10) ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.

Mechanisms of the effect of 1,1-dimethyl-3-hydroxybutyl phosphonic acid derivatives on synaptic transmission in neuromuscular junction.

We studied the effect of homologues derivatives of 1,1-dimethyl-3-hydroxybutyl phosphonic acid on synaptic transmission in frog neuromuscular junction. Here we reviewed general mechanisms of inhibition of the postsynaptic current.