Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (gCl) with an IC50 value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC gCl after oral administration to normal mice (ED50 = 25 mg/kg). Although gCl, at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease

CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate1

Dopamine (DA) containing midbrain neurons play critical roles in several psychiatric and neurological diseases, including schizophrenia and attention deficit hyperactivity disorder, and the substantia nigra pars compacta neurons selectively degenerate in Parkinson’s disease. Pharmacological modulation of DA receptors and transporters are well established approaches for treatment of DA-related disorders. Direct modulation of the DA system by influencing the discharge pattern of these autonomously firing neurons has yet to be exploited as a potential therapeutic strategy. Small conductance Ca2+-activated K+ channels (SK channels), in particular the SK3 subtype, are important in the physiology of DA neurons, and agents modifying SK channel activity could potentially affect DA signaling and DA-related behaviors. Here we show that cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), a subtype-selective positive modulator of SK channels (SK3 > SK2 > > > SK1, IK), decreased spontaneous firing rate, increased the duration of the apamin-sensitive afterhyperpolarization, and caused an activity-dependent inhibition of current-evoked action potentials in DA neurons from both mouse and rat midbrain slices. Using an immunocytochemically and pharmacologically validated DA release assay employing cultured DA neurons from rats, we show that CyPPA repressed DA release in a concentration-dependent manner with a maximal effect equal to the D2 receptor agonist quinpirole. In vivo studies revealed that systemic administration of CyPPA attenuated methylphenidate-induced hyperactivity and stereotypic behaviors in mice. Taken together, the data accentuate the important role played by SK3 channels in the physiology of DA neurons, and indicate that their facilitation by CyPPA profoundly influences physiological as well as pharmacologically induced hyperdopaminergic behavior

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory/protective state of microglia for the development of novel PET tracers.Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues.Results: Here we provide evidence that P2RYI2 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-II labeled tracer targeting P2RYI2, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient.Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.</div

Pharmacological gating modulation of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2.x and KCa3.1).

This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2 and KCa3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite KCa channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative KCa2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective KCa modulators entering into drug development for these indications

Pharmacological gating modulation of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2.x and KCa3.1).

This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2 and KCa3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite KCa channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative KCa2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective KCa modulators entering into drug development for these indications