SAR340835, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Cardiac Function and Restores Sympathovagal Balance in Heart Failure.

In failing hearts, Na+/Ca2+ exchanger (NCX) overactivity contributes to Ca2+ depletion, leading to contractile dysfunction. Inhibition of NCX is expected to normalize Ca2+ mishandling, to limit afterdepolarization-related arrhythmias, and to improve cardiac function in heart failure (HF). SAR340835/SAR296968 is a selective NCX inhibitor for all NCX isoforms across species, including human, with no effect on the native voltage-dependent calcium and sodium currents in vitro. Additionally, it showed in vitro and in vivo antiarrhythmic properties in several models of early and delayed afterdepolarization-related arrhythmias. Its effect on cardiac function was studied under intravenous infusion at 250,750 or 1500 µg/kg per hour in dogs, which were either normal or submitted to chronic ventricular pacing at 240 bpm (HF dogs). HF dogs were infused with the reference inotrope dobutamine (10 µg/kg per minute, i.v.). In normal dogs, NCX inhibitor increased cardiac contractility (dP/dtmax) and stroke volume (SV) and tended to reduce heart rate (HR). In HF dogs, NCX inhibitor significantly and dose-dependently increased SV from the first dose (+28.5%, +48.8%, and +62% at 250, 750, and 1500 µg/kg per hour, respectively) while significantly increasing dP/dtmax only at 1500 (+33%). Furthermore, NCX inhibitor significantly restored sympathovagal balance and spontaneous baroreflex sensitivity (BRS) from the first dose and reduced HR at the highest dose. In HF dogs, dobutamine significantly increased dP/dtmax and SV (+68.8%) but did not change HR, sympathovagal balance, or BRS. Overall, SAR340835, a selective potent NCX inhibitor, displayed a unique therapeutic profile, combining antiarrhythmic properties, capacity to restore systolic function, sympathovagal balance, and BRS in HF dogs. NCX inhibitors may offer new therapeutic options for acute HF treatment. SIGNIFICANCE STATEMENT: HF is facing growing health and economic burden. Moreover, patients hospitalized for acute heart failure are at high risk of decompensation recurrence, and no current acute decompensated HF therapy definitively improved outcomes. A new potent, Na+/Ca2+ exchanger inhibitor SAR340835 with antiarrhythmic properties improved systolic function of failing hearts without creating hypotension, while reducing heart rate and restoring sympathovagal balance. SAR340835 may offer a unique and attractive pharmacological profile for patients with acute heart failure as compared with current inotrope, such as dobutamine.

Literacy Training of Kindergarten Children With Pencil, Keyboard or Tablet Stylus: The Influence of the Writing Tool on Reading and Writing Performance at the Letter and Word Level.

During the last years, digital writing devices are increasingly replacing handwriting with pencil and paper. As reading and writing skills are central for education, it is important to know, which writing tool is optimal for initial literacy education. The present training study was therefore set up to test the influence of the writing tool on the acquisition of literacy skills at the letter and word level with various tests in a large sample of kindergarten children (n = 147). Using closely matched letter learning games, children were trained with 16 letters by handwriting with a pencil on a sheet of paper, by writing with a stylus on a tablet computer, or by typing letters using a virtual keyboard on a tablet across 7 weeks. Training using a stylus on a touchscreen is an interesting comparison condition for traditional handwriting, because the slippery surface of a touchscreen has lower friction than paper and thus increases difficulty of motor control. Before training, immediately after training and four to five weeks after training, we assessed reading and writing performance using standardized tests. We also assessed visuo-spatial skills before and after training, in order to test, whether the different training regimens affected cognitive domains other than written language. Children of the pencil group showed superior performance in letter recognition and improved visuo-spatial skills compared with keyboard training. The performance of the stylus group did not differ significantly neither from the keyboard nor from the pencil group. Keyboard training, however, resulted in superior performance in word writing and reading compared with handwriting training with a stylus on the tablet, but not compared with the pencil group. Our results suggest that handwriting with pencil fosters acquisition of letter knowledge and improves visuo-spatial skills compared with keyboarding. At least given the current technological state, writing with a stylus on a touchscreen seems to be the least favorable writing tool, possibly because of increased demands on motor control. Future training studies covering a more extended observation period over years are needed to allow conclusions about long-term effects of writing tools on literacy acquisition as well as on general cognitive development.

Discovery and Optimization of 1-Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na+/H+ Exchanger Type 3 (NHE3).

The design, synthesis, and structure-activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na+/H+ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with 13d as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of 13d then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally 13d, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas.

Stopping eyes and hands: evidence for non-independence of stop and go processes and for a separation of central and peripheral inhibition.

In the stop-signal paradigm, participants perform a primary reaction task, for example a visual or auditory discrimination task, and have to react to a go stimulus as quickly as possible with a specified motor response. In a certain percentage of trials, after presentation of the stimulus (go signal), another stimulus (stop signal) is presented with a variable stop-signal delay. Whenever a stop signal occurs, the participant is asked to inhibit the execution of the response. Here, an extended test of the popular horse race model for this task (Logan and Cowan, 1984) is presented. Responses for eye and hand movements in both single-task and dual-task conditions were collected. Saccadic reaction times revealed some significant violations of the model's basic assumption of independent go and inhibition processes for all six participants. Saccades that escaped an early stop signal were systematically slower and had smaller amplitudes compared to saccades without a stop signal. Moreover, the analysis of concomitant electromyographic responses recorded from the upper arm suggests the existence of two separate inhibitory mechanisms: a slow, selective, central inhibitory mechanism and a faster, highly efficient, peripheral one, which is probably ineffective for saccades.

Antihypertensive and laxative effects by pharmacological inhibition of sodium-proton-exchanger subtype 3-mediated sodium absorption in the gut.

High intestinal sodium absorption is one mechanism of hypertension and constipation. The sodium-proton-exchanger subtype 3 (NHE3) is an important mediator of sodium absorption in the gut. SAR218034 (SAR) is an orally nonabsorbable specific NHE3 inhibitor. The effect of SAR (1 mg/kg per day in chow) on feces sodium excretion, systolic blood pressure via tail cuff, and gene expression of NHE3 in the gut were studied in senescent lean hypertensive rats (spontaneously hypertensive rats-lean, loaded with NaCl 0.7% in drinking water) and in hypertensive, obese, and hyperinsulinemic rats (spontaneously hypertensive rats-obese, not loaded with NaCl). In spontaneously hypertensive rats-lean, inhibition of intestinal NHE3 by SAR increased feces sodium excretion and reduced urinary sodium excretion, whereas absolute sodium balance and serum sodium concentration were not changed. This suggests reduced intestinal sodium absorption in SAR-treated animals and was associated with increased feces water content (58% versus 42% in placebo treated animals; P=0.0001) and reduction in systolic blood pressure from 222 ± 7 to 198 ± 2 mm Hg (P=0.0001). Angiotensin-converting enzyme inhibition by ramipril plus NHE3 inhibition resulted in an additive blood pressure-lowering effect. In spontaneously hypertensive rats-obese, SAR lowered systolic blood pressure but did not modify serum insulin or cholesterol levels. Gene expression of NHE3 was upregulated in the ileum and colon but not in the jejunum of SAR-treated rats. Reduction of intestinal sodium absorption by selective NHE3 inhibition in the gut reduces high blood pressure and increases feces water excretion. Intestinal NHE3 blockade could be a new treatment strategy for elderly patients suffering from high blood pressure and constipation.

Transport of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] and high-affinity interaction of nucleoside reverse transcriptase inhibitors with human organic cation transporters 1, 2, and 3.

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1-3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [(3)H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3'-azido-3'-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (K(d1) = 12.3-15.4 pM) and a low-affinity site (K(d2) = 1.9-3.4 mM). Measuring direct uptake of [(3)H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (K(m) = 1.25 +/- 0.1 mM; V(max) = 10.40 +/- 0.32 nmol/mg protein/min; V(max)/K(m) = 8.32 +/- 0.40 microl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [(3)H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (K(i) = 22-500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Polyspecific cation transporters mediate luminal release of acetylcholine from bronchial epithelium.

In airway epithelia, non-neuronal cholinergic regulations have been described; however, the route for acetylcholine (ACh) release has not been verified. To investigate whether organic cation transporters (OCTs) serve this function, we studied the expression of OCTs in airway epithelia and their capability to translocate ACh. Using immunohistochemistry in rats and humans, OCT1, OCT2, and OCT3 were localized to the luminal membrane of ciliated epithelial cells. In humans, OCT2 showed the strongest expression in the luminal membrane. We expressed the OCT isoforms in oocytes of Xenopus laevis and measured uptake and efflux of ACh. Tracer flux measurements showed that ACh is transported by OCT1 and OCT2 but not by OCT3. Two-electrode-voltage-clamp measurements revealed that OCT2 mediates electrogenic uptake and efflux of ACh. For ACh uptake by human OCT2, a K(M) value of approximately 0.15 mM was determined. At -50 mV, ACh efflux by human OCT2 was trans-inhibited by micromolar concentrations of the inhalational glucocorticoid budesonide, which is used in treatment of asthma (K(i) approximately 2.7 microM). The data show that OCT1 and OCT2 mediate luminal ACh release in human airways and suggest that ACh release is blocked after inhalation of budesonide.

Regulation of the human organic cation transporter hOCT1.

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO-hOCT1) and in human embryonic kidney cells (HEK293-hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)) as substrate. hOCT1 expressed in CHO-cells was inhibited by protein kinase A (PKA) activation (1 microM forskolin, -58 +/- 6%, n = 12), calmodulin inhibition (0.1 microM calmidazolium, -68 +/- 3%, n = 6; 10 microM ophiobolin A, -48 +/- 10%, n = 7), calmodulin-dependent kinase II inhibition (1 microM KN62, -78 +/- 4%, n = 12), and inhibition of p56(lck) tyrosine kinase (10 microM aminogenistein, -35 +/- 7%, n = 12). The apparent affinities for TEA(+) were lower in CHO-hOCT1 than in HEK293-hOCT1, while those for TPA(+) and quinine were almost identical; the rank order of EC(50) values (TPA(+) > quinine > TEA(+)) was independent of the expression system. EC(50) values for TEA(+) in CHO-hOCT1 or HEK293-hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin-dependent kinase II, and p56(lck) tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.

Countermanding saccades: evidence against independent processing of go and stop signals.

In a stop signal paradigm, subjects were instructed to make a saccade to a visual target appearing left or right of the fixation point. In 25% of the trials, an auditory stop signal was presented after a variable delay that required the subject to inhibit the saccade. Observed saccadic response times in stop failure trials were longer than predicted by Logan and Cowan's (1984) race model. Saccadic response time and amplitude decreased with the time between stop signal presentation and saccade execution, suggesting an inhibitory effect between the stop signal and the go signal processes that is not compatible with an independent race assumption. Moreover, countermanding a saccade was more difficult when stop and go signals appeared at the same location.

Two stages in crossmodal saccadic integration: evidence from a visual-auditory focused attention task.

Saccadic reaction time (SRT) toward a visual target stimulus was measured under simultaneous presentation of an auditory non-target (accessory stimulus). Horizontal position of the target was varied (25 degrees left and right of fixation) as well as position and intensity of the auditory accessory. SRT was reduced under the presence of the accessory, and it decreased both with increasing intensity of the auditory accessory and with decreasing distance between target and accessory. The absence of a significant interaction between distance and auditory intensity suggests (1) that the intensity of the accessory stimulus has no direct influence on the process of crossmodal integration, and (2) that spatial position and intensity of the accessory are processed in separate stages. This was supported by a probability inequality test showing that the amount of neural coactivation depends on spatial distance but not on auditory intensity. The results are discussed in the framework of a two-stage model assuming separate processing of unimodal and bimodal characteristics of the stimuli. These results are related to several recent neurophysiological findings.

Structure and Function of Renal Organic Cation Transporters.

Polyspecific transport systems in the kidney mediate the excretion and reabsorption of organic cations. Electrogenic import systems and electroneutral export systems in the basolateral and luminal plasma membranes of proximal renal tubules are involved. Two subtypes of electrogenic import systems have been cloned from rats and humans and functionally characterized.