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Objective: Current pharmacologic treatments for posttraumatic stress disorder (PTSD) have shown limited efficacy, prompting a call to investigate new classes of medications. The current study investigated the efficacy of glutamate modulation with riluzole augmentation for combat-related PTSD symptoms resistant to treatment with selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs)., Methods: A randomized, double-blind, placebo-controlled, parallel trial was conducted at Walter Reed National Military Medical Center and Syracuse VA Medical Center between December 2013 and November 2017. Veterans and active duty service members with combat-related PTSD (per the Clinician Administered PTSD Scale [CAPS]) who were not responsive to SSRI or SNRI pharmacotherapy were randomized to 8-week augmentation with a starting dose of 100 mg/d of riluzole (n = 36) or placebo (n = 38) and assessed weekly for PTSD symptoms, anxiety, depression, disability, and side effects., Results: Intent-to-treat analyses (N = 74) of the primary outcome (CAPS for DSM-IV) showed no significant between-group difference in change in overall PTSD symptoms (F = 0.64, P = .422), with a small effect size (d = 0.25). There was clinically significant within-group improvement in overall PTSD symptoms in both groups, with a greater mean (SD) decrease in CAPS score in the riluzole group (-21.1 [18.9]) than in the placebo group (-16.7 [17.2]). Exploratory analyses of PTSD symptom clusters showed significantly greater improvement on hyperarousal symptoms in the riluzole group as measured by the PTSD Checklist-Specific-Subscale D (d = 0.48) and near-significant findings on the CAPS Subscale D. Riluzole augmentation was not superior to placebo on change in depression, anxiety, or disability severity., Conclusions: Although preliminary, the exploratory findings of this study offer some evidence that riluzole augmentation of an SSRI or SNRI may selectively improve PTSD hyperarousal symptoms without changes in overall PTSD symptoms, depression, anxiety, or disability. Additional investigation of the mechanism of the efficacy of riluzole for hyperarousal symptoms is warranted., Trial Registration: ClinicalTrials.gov identifier: NCT02155829., (© Copyright 2020 Physicians Postgraduate Press, Inc.)

Anti-invasive effects of riluzole and ranolazine, a neuro-protectant and an anti-anginal drug, respectively, on Mat-LyLu rat prostate cancer (PCa) cells were tested in vitro (a) at non-toxic doses and (b) under both normoxic and hypoxic conditions, the latter common to growing tumours. Tetrodotoxin (TTX) was used as a positive control. Hypoxia had no effect on cell viability but reduced growth at 48 hours. Riluzole (5 μmol/L) or ranolazine (20 μmol/L) had no effect on cell viability or growth under normoxia or hypoxia over 24 hours. Matrigel invasion was not affected by hypoxia but inhibited by TTX, ranolazine and riluzole under a range of conditions. The expression of Nav1.7 mRNA, the prevailing, pro-invasive voltage-gated sodium channel α-subunit (VGSCα), was up-regulated by hypoxia. Riluzole had no effect on Nav1.7 mRNA expression in normoxia but significantly reduced it in hypoxia. VGSCα protein expression in plasma membrane was reduced in hypoxia; riluzole increased it but only under hypoxia. It was concluded (a) that riluzole and ranolazine have anti-invasive effects on rat PCa cells and (b) that Nav1.7 mRNA and protein expression can be modulated by riluzole under hypoxia. Overall, therefore, riluzole and ranolazine may ultimately be "repurposed" as anti-metastatic drugs against PCa., (© 2020 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). Published by John Wiley & Sons Ltd.)

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (I M , KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K + channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC 50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both I M and TREK-2 currents. Likewise, inhibition of I M by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and I M inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

Manganese (Mn) is an essential element required for many biological processes and systems in the human body. Mn intoxication increases brain glutamate (Glu) levels causing neuronal damage. Recent studies have reported that ephrin-A3 regulates this glutamate transporter. However, none has explored the role of this crucial molecule in Mn-induced excitotoxicity. The present study investigated whether ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity using astrocytes and Kunming mice. The mechanisms were explored using fluoxetine (ephrin-A3 inhibitor) and riluzole (a Glu release inhibitor). Firstly, we demonstrated that Mn exposure (500 μM or 50 mg/kg MnCl 2 ) significantly increased Mn, ephrin-A3, and Glu levels, and inhibited Na + -K + ATPase activity, as well as mRNA and protein levels of GLAST and GLT-1. Secondly, we found that astrocytes and mice pretreated with fluoxetine (100 μM or 15 mg/kg) and riluzole (100 μM or 32 μmol/kg) prior to Mn exposure had lower ephrin-A3 and Glu levels, but higher Na + -K + ATPase activity, expression levels of GLAST and GLT-1 than those exposed to 500 μM or 50 mg/kg MnCl 2 . Moreover, the morphology of cells and the histomorphology of mice striatum were injured. Results showed that pretreatment with fluoxetine and riluzole attenuated the Mn-induced motor dysfunctions. Together, these results suggest that the ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity, and fluoxetine and riluzole can mitigate the Mn-induced excitotoxicity in mice brain.

Diffuse large B-cell lymphoma (DLBCL) is a leading cause of non-Hodgkin lymphomas. Existing researches have verified that long non-coding RNAs (lncRNAs) play crucial roles in the development of DLBCL, nevertheless, whether lncRNA OR3A4 has influences on the progression of DCBCL needs further exploration. In our study, it was revealed that the expression of OR3A4 was upregulated in DLBCL tumors and cell lines, and patients with high OR3A4 expression suffered from poor prognosis. Knockdown of OR3A4 suppressed cell proliferation and promoted cell apoptosis in DLBCL. Moreover, knockdown of OR3A4 inactivated Wnt/β-catenin signaling pathway, and Riluzole treatment could partially rescue the inhibitive effect of OR3A4 silencing on DLBCL cell proliferation. Furthermore, FOXM1 expression was discovered to be upregulated in DLBCL tissues, and it positively modulated the expression of OR3A4 at transcriptional leve. It was also revealed that FOXM1 knockdown inactivated Wnt/β-catenin signaling pathway. Finally, rescue assays confirmed that OR3A4 overexpression or the treatment of Riluzole could partially countervail the inhibitive effect of FOXM1 silencing on DLBCL progression. Taken together, FOXM1-induced upregulation of OR3A4 enhances the occurrence of DLBCL via Wnt/β-catenin signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that no competing interest exists in this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: 22q11.2 deletion syndrome (22q11.2DS) is associated with impaired cognitive functioning. Glutamatergic pathways have been linked with cognition and are hypothesized to be disrupted in 22q11.2DS patients, possibly 'shifting' the excitatory (glutamate)/inhibitory (GABA) balance. Hence, the glutamate/GABA balance may constitute a target for pharmacological treatment. We aimed to examine alterations of glutamate/GABA metabolites in 22q11.2DS in vivo using riluzole, a compound with glutamate/GABA-modulating action, as pharmacological challenge., Methods: Seventeen 22q11.2DS patients and 20 matched healthy controls were enrolled in this randomized double-blind placebo-controlled crossover study. Glutamate and glutamine concentrations in the anterior cingulate cortex (ACC) and striatum, as well as ACC GABA concentrations were obtained after placebo and after a single dose of 50 mg riluzole using 7-Tesla magnetic resonance spectroscopy (MRS). Within the 22q11.2DS group, the relationship between metabolite concentrations and cognition was examined., Results: No group differences were found in ACC and striatal metabolite concentrations following placebo. Riluzole numerically decreased ACC ( η 2 = 0.094 ) but not striatal glutamate concentrations as well as ACC GABA concentrations ( η 2 = 0.176 ) in all subjects. In both regions, riluzole did not alter glutamine concentration. No interaction effects were found. Although not significant after Bonferroni correction, ACC glutamate concentrations were inversely correlated with cognitive functions in 22q11.2DS patients., Discussion: We did not demonstrate altered ACC and striatal metabolite concentrations in 22q11.2DS. Nevertheless, these results suggest that glutamate and GABA can be modulated with a single dose of riluzole. Possibly, riluzole may have memory-enhancing effects in 22q11.2DS. Future studies should examine the long-term effects of riluzole on cognition.

Background: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP)., Methods: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced., Results: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca 2+ -activated K + currents and the persistent Na + current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na + channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines., Conclusions: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

Background Atrial fibrillation (AF) is a comorbidity associated with heart failure and catecholaminergic polymorphic ventricular tachycardia. Despite the Ca 2+ -dependent nature of both of these pathologies, AF often responds to Na + channel blockers. We investigated how targeting interdependent Na + /Ca 2+ dysregulation might prevent focal activity and control AF. Methods and Results We studied AF in 2 models of Ca 2+ -dependent disorders, a murine model of catecholaminergic polymorphic ventricular tachycardia and a canine model of chronic tachypacing-induced heart failure. Imaging studies revealed close association of neuronal-type Na + channels (nNa v ) with ryanodine receptors and Na + /Ca 2+ exchanger. Catecholamine stimulation induced cellular and in vivo atrial arrhythmias in wild-type mice only during pharmacological augmentation of nNa v activity. In contrast, catecholamine stimulation alone was sufficient to elicit atrial arrhythmias in catecholaminergic polymorphic ventricular tachycardia mice and failing canine atria. Importantly, these were abolished by acute nNa v inhibition (tetrodotoxin or riluzole) implicating Na + /Ca 2+ dysregulation in AF. These findings were then tested in 2 nonrandomized retrospective cohorts: an amyotrophic lateral sclerosis clinic and an academic medical center. Riluzole-treated patients adjusted for baseline characteristics evidenced significantly lower incidence of arrhythmias including new-onset AF, supporting the preclinical results. Conclusions These data suggest that nNa V s mediate Na + -Ca 2+ crosstalk within nanodomains containing Ca 2+ release machinery and, thereby, contribute to AF triggers. Disruption of this mechanism by nNa v inhibition can effectively prevent AF arising from diverse causes.

Introduction: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease involving both upper and lower motor neurons and resulting in increasing disability and death 3-5 years after onset of symptoms. Over 40 large clinical trials for ALS have been negative, except for Riluzole that offers a modest survival benefit, and Edaravone that modestly reduces disease progression in patients with specific characteristics. Thus, the discovery of efficient disease modifying therapy is an urgent need., Areas Covered: Although the cause of ALS remains unclear, many studies have demonstrated that neuroinflammation, proteinopathies, glutamate-induced excitotoxicity, microglial activation, oxidative stress, and mitochondrial dysfunction may play a key role in the pathogenesis. This review highlights recent discoveries relating to these diverse mechanisms and their implications for the development of therapy. Ongoing phase 2 clinical trials aimed to interfere with these pathophysiological mechanisms are discussed., Expert Opinion: This review describes the challenges that the discovery of an efficient drug therapy faces and how these issues may be addressed. With the continuous advances coming from basic research, we provided possible suggestions that may be considered to improve performance of clinical trials and turn ALS research into a 'fertile ground' for drug development for this devastating disease.

Our group described the oncogenic potential of a normal neuronal receptor, metabotropic glutamate receptor 1 (GRM1/mGluR1, gene/protein), when aberrantly expressed in melanocytes led to cell transformation in vitro and spontaneous metastatic tumors in vivo. Earlier, we demonstrated the accumulation of phosphorylated histone H2AX (γH2AX), a marker for DNA damage when mGluR1-expressing melanoma cells were treated with a functional inhibitor, riluzole. The precise mechanisms on how riluzole induces DNA damage in these cells are unknown. In an attempt to begin to identify possible DNA repair pathways that may be involved in riluzole-induced DNA damage, we took advantage of specific inhibitors to two well-known DNA repair pathways, homologous recombination and nonhomologous end joining (NHEJ) repair pathways. Using flow cytometry and a fluorescent antibody to γH2AX, our results demonstrate that NHEJ is likely to be the preferred DNA repair pathway to restore DNA double-stranded breaks induced by riluzole in mGluR1-expressing melanoma cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of adult life, causing weakness and wasting of voluntary muscles, associated in about 50% of cases with a cognitive impairment. Pathologically, the disease is characterized by a degeneration of upper and lower motor neurons. A hallmark of the pathological process is the aggregation of the protein TDP43 in the cytoplasm of affected neurons detected in almost 97% of cases. About 15% of cases has a family history. Currently, only two drugs have been demonstrated to be effective in ALS, riluzole and edaravone, which show only modest effects on disease progression. The quest for disease-modifying therapies in ALS has several obstacles, the most important being the sub-optimal quality of the design of clinical trials, and the clinical and pathological heterogeneity of the disease. In this paper the pathological mechanisms relevant to ALS and current and future pharmacological and non-pharmacological trials, including gene and stem cells therapies, will be presented. This article is part of the special issue entitled 'The Quest for Disease-Modifying Therapies for Neurodegenerative Disorders'., Competing Interests: Declaration of competing interest Adriano Chiò serves on scientific advisory boards for Mitsubishi Tanabe, Roche, Cytokinetics, and Biogen. Letizia Mazzini and Gabriele Mora report no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1 ) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor ( ADCYAP1R1 ) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.

We developed a microfluidic gradient device to utilize as a drug screening system with human induced pluripotent stem cell (hiPSC)-derived motoneurons. The microfluidic channel was asymmetrically designed to generate the concentration gradients and a micropillar array was used to trap and culture the motoneuron spheroids containing motoneurons for 9 days. We optimized the concentration gradients in the microfluidic device using a computational fluid dynamics (CFD) model. We also observed that the motoneuron spheroid-derived neurite network was generated in response to the concentration gradients of riluzole in the microfluidic device. Therefore, this microfluidic gradient device could be useful for screening of various drugs for neurological disease applications.

Spinal cord injury (SCI) triggers pronounced inflammatory responses that are accompanied by neuronal disruption and functional deficits. SCI treatment remains an unmet clinical need. Emerging evidence suggests that riluzole may exert a neuroprotective effect due to its anti-inflammatory properties. However, details of the underlying mechanisms remain poorly defined. The polarization of microglial/macrophages has an important role in neuroinflammation. Here, we examined whether riluzole can exert a neuroprotective effect after acute SCI, and whether this effect is associated with changes in microglia/macrophages polarization. Riluzole (4 mg/kg) or vehicle were injected intraperitoneally (i.p.) in female rats immediately following SCI and repeated for 7 consecutive days (b.i.d.). Compared with vehicle treatment, riluzole-treated SCI rats showed significant higher locomotor scores (Basso, Beattie, and Bresnahan score, Inclined Plane test score, n = 18/group). Riluzole-treated rats also developed smaller spinal cavities, showed higher levels of myelin basic protein (MBP) and neurofilament (NF)200 immunoreactivities, and lower levels of proinflammatory cytokines in the spinal cord at 7 days post-SCI. Immunofluorescence study revealed more CD206 + cells and less iNOS + cells in the injured spinal cord of riluzole-treated SCI rats, as compared to vehicle control. Using real-time PCR, we found that riluzole upregulated the mRNA levels of M2 markers, but downregulated that of M1 markers, as compared to the vehicle treatment. Current findings suggest that systemic administration of riluzole after acute SCI facilitated motor function recovery and inhibited inflammatory responses, which may be associated with polarization of M2 microglia/macrophages., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Purpose: The objective of the research undertaken was to develop the Riluzole (RIZ) nanoparticles drug delivery system using Transferrin (Tf) as a ligand in the brain. Method: RIZ-loaded chitosan nanoparticles and RIZ-Tf chitosan (CS) nanoparticles (RIZ CSNPs and RIZ-Tf CSNPs) were formulated and compared for particles size, size distribution, encapsulation efficiency, and surface morphology, respectively. The in vitro drug release, permeation, pharmacokinetic, biochemical, and pharmacodynamic experiments were done to assess the improvement in in vivo fate and efficacy of RIZ. Results: The size of optimized RIZ CSNPs was found to be 173.6 ± 2.23 nm and polydispersity index (PDI) of 0.264 ± 0.002 while that of RIZ-Tf CSNPs was 207 ± 2.49 nm and 0.406 ± 0.002. In vitro release was found to be 86.15 ± 7.316% and 91.1 ± 5.836%, respectively, while permeability coefficient was found to be 4 × 10 -2 and 4.2 × 10 -2  cm/s for RIZ CSNPs and RIZ-Tf CSNPs. The biochemical analysis studies revealed that oxidative stress was significantly decreased in case of RIZ CSNPs and RIZ-Tf CSNPs ( p  < 0.01) treated groups. The antianxiety effect and the memory restoration were evident in pharmacodynamic studies ( p  < 0.05) of the prepared formulations. Conclusion: The results of pharmacokinetic studies demonstrated the remarkable brain delivery of RIZ-Tf CSNPs through intranasal route as compared to the RIZ solution.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (I M ) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca 2+ -calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP 2 ). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the "Tandem of pore-domains in a Weakly Inward rectifying K + channel (TWIK)-related channels" (TREK) subfamily by reducing PIP 2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (I RIL ), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on I RIL was precluded by the bradykinin receptor (B 2 R) antagonist HOE-140 (d-Arg-[Hyp 3 , Thi 5 , d-Tic 7 , Oic 8 ]BK) but also by diC 8 PIP 2 which prevents PIP 2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP 3 R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of I RIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B 2 Rs resulting in PIP 2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Internal ribosome entry site (IRES)-mediated protein synthesis has been demonstrated to play an important role in resistance to mechanistic target of rapamycin (mTOR) targeted therapies. Previously, we have demonstrated that the IRES trans -acting factor (ITAF), hnRNP A1 is required to promote IRES activity and small molecule inhibitors which bind specifically to this ITAF and curtail IRES activity, leading to mTOR inhibitor sensitivity. Here we report the identification of riluzole (Rilutek ® ), an FDA-approved drug for amyotrophic lateral sclerosis (ALS), via an in silico docking analysis of FDA-approved compounds, as an inhibitor of hnRNP A1. In a riluzole-bead coupled binding assay and in surface plasmon resonance imaging analyses, riluzole was found to directly bind to hnRNP A1 and inhibited IRES activity via effects on ITAF/RNA-binding. Riluzole also demonstrated synergistic anti-glioblastoma (GBM) affects with mTOR inhibitors in vitro and in GBM xenografts in mice. These data suggest that repurposing riluzole, used in conjunction with mTOR inhibitors, may serve as an effective therapeutic option in glioblastoma.

Osteosarcomas are the most commonly occurring malignant bone cancer in young individuals. The survival rate of patients with metastatic osteosarcoma is low and has been stagnant for over two decades. We previously demonstrated that the glutamate release inhibitor, riluzole inhibits osteosarcoma cell growth. Towards the development of more effective therapy, we investigated the delivery of riluzole in human metastatic osteosarcoma xenografts in mice. We compared the efficacy of riluzole delivery by intraperitoneally injecting either free riluzole or riluzole released via two different shapes of iron oxide nanoparticles (nanocage or nanosphere) of size 15±2.5 nm. We monitored tumor size using Vernier calipers and bioluminescence assay and found a significant reduction in tumor size in the riluzole‑treated groups when injected, either in free form or via nanoparticles, compared to the control groups (PBS, nanosphere or nanocage). Importantly, nanocage‑delivered riluzole was most effective in reducing tumor size in the xenograft nude mice. While riluzole delivery induced apoptosis in tumor tissues in all three groups of riluzole‑treated animals, it was highest in tumors from the nanocage‑delivered riluzole group. Therefore, we conclude that riluzole is an effective drug to reduce tumor size in osteosarcoma and the efficacy of riluzole as a apoptotic and tumor‑reducing drug is enhanced when delivered via nanocage.

Gamma network oscillations in the brain are fast rhythmic network oscillations in the gamma frequency range (~30-100 Hz), playing key roles in the hippocampus for learning, memory, and spatial processing. There is evidence indicating that GABAergic interneurons, including parvalbumin-expressing basket cells (PVBCs), contribute to cortical gamma oscillations through synaptic interactions with excitatory cells. However, the molecular, cellular, and circuit underpinnings underlying generation and maintenance of cortical gamma oscillations are largely elusive. Recent studies demonstrated that intrinsic and synaptic properties of GABAergic interneurons and excitatory cells are regulated by a slowly inactivating or non-inactivating sodium current (i.e., persistent sodium current, I NaP ), suggesting that I NaP is involved in gamma oscillations. Here, we tested whether I NaP plays a role in hippocampal gamma oscillations using pharmacological, optogenetic, and electrophysiological approaches. We found that I NaP blockers, phenytoin (40 μM and 100 μM) and riluzole (10 μM), reduced gamma oscillations induced by optogenetic stimulation of CaMKII-expressing cells in CA1 networks. Whole-cell patch-clamp recordings further demonstrated that phenytoin (100 μM) reduced I NaP and firing frequencies in both PVBCs and pyramidal cells without altering threshold and amplitude of action potentials, but increased rheobase in both cell types. These results suggest that I NaP in pyramidal cells and PVBCs is required for hippocampal gamma oscillations, supporting a pyramidal-interneuron network gamma model. Phenytoin-mediated modulation of hippocampal gamma oscillations may be a mechanism underlying its anticonvulsant efficacy, as well as its contribution to cognitive impairments in epilepsy patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The synthesis of SCF 3 as well as SeCF 3 isosteres of two OCF 3 -containing drugs was achieved through visible light and copper-catalyzed processes. Herein, we show that chalcogen replacement modulates physicochemical and ADME properties without introducing intrinsic liabilities. The SCF 3 and SeCF 3 groups are more lipophilic than their oxygen counterpart; however, microsomal stability is unchanged, indicating that these molecular changes may be beneficial for in vivo half-life. Enabled by modern synthetic methods, we present the chalcogen-CF 3 groups as potential key players for future fluorinated pharmaceuticals., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mechanism(s) of action of most commonly used pharmacological blockers of voltage-gated ion channels are well understood; however, this knowledge is rarely considered when interpreting experimental data. Effects of blockade are often assumed to be equivalent, regardless of the mechanism of the blocker involved. Using computer simulations, we demonstrate that this assumption may not always be correct. We simulate the blockade of a persistent sodium current (INaP), proposed to underlie rhythm generation in pre-Bötzinger complex (pre-BötC) respiratory neurons, via two distinct pharmacological mechanisms: (1) pore obstruction mediated by tetrodotoxin and (2) altered inactivation dynamics mediated by riluzole. The reported effects of experimental application of tetrodotoxin and riluzole in respiratory circuits are diverse and seemingly contradictory and have led to considerable debate within the field as to the specific role of INaP in respiratory circuits. The results of our simulations match a wide array of experimental data spanning from the level of isolated pre-BötC neurons to the level of the intact respiratory network and also generate a series of experimentally testable predictions. Specifically, in this study we: (1) provide a mechanistic explanation for seemingly contradictory experimental results from in vitro studies of INaP block, (2) show that the effects of INaP block in in vitro preparations are not necessarily equivalent to those in more intact preparations, (3) demonstrate and explain why riluzole application may fail to effectively block INaP in the intact respiratory network, and (4) derive the prediction that effective block of INaP by low concentration tetrodotoxin will stop respiratory rhythm generation in the intact respiratory network. These simulations support a critical role for INaP in respiratory rhythmogenesis in vivo and illustrate the importance of considering mechanism when interpreting and simulating data relating to pharmacological blockade., Competing Interests: The authors have declared that no competing interests exist.

Study Design: A post-test design biological experiment., Objective: The aim of this study was to evaluate the osteogenic effects of riluzole on human mesenchymal stromal cells and osteoblasts., Summary of Background Data: Riluzole may benefit patients with spinal cord injury (SCI) from a neurologic perspective, but little is known about riluzole's effect on bone formation, fracture healing, or osteogenesis., Methods: Human mesenchymal stromal cells (hMSCs) and human osteoblasts (hOB) were obtained and isolated from healthy donors and cultured. The cells were treated with riluzole of different concentrations (50, 150, 450 ng/mL) for 1, 2, 3, and 4 weeks. Cytotoxicity was evaluated as was the induction of osteogenic differentiation of hMSCs. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and with Alizarin red staining. Osteogenic gene expression of type I collagen (Col1), ALP, osteocalcin (Ocn), Runx2, Sox9, Runx2/Sox9 ratio were measured by qRT-PCR., Results: No cytotoxicity or increased proliferation was observed in bone marrow derived hMSCs and primary hOBs cultured with riluzole over 7 days. ALP activity was slightly increased in hMSCs after treatment for 2 weeks with riluzole 150 ng/mL and slightly upregulated by 150% (150 ng/mL) and 90% (450 ng/mL) in hMSCs at 3 weeks. In hOBs, ALP activity almost doubled after 2 weeks of culture with riluzole 150 ng/mL (P < 0.05). More pronounced 2.6-fold upregulation was noticed after 3 weeks of culture with riluzole at both 150 ng/mL (P = 0.05) and 450 ng/mL (P = 0.05). No significant influence of riluzole on the mRNA expression of osteocalcin (OCN) was observed., Conclusion: The effect of riluzole on bone formation is mixed; low-dose riluzole has no effect on the viability or function of either hMSCs or hOBs. The activity of ALP in both cell types is upregulated by high-dose riluzole, which may indicate that high-dose riluzole can increase osteogenic metabolism and subsequently accelerate bone healing process. However, at high concentrations, riluzole leads to a decrease in osteogenic gene expression, including Runx2 and type 1 collagen., Level of Evidence: N/A.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, for which there is no effective treatment. Previously, we generated a Caenorhabditis elegans model of ALS, in which the expression of dnc-1, the homologous gene of human dynactin-1, is knocked down (KD) specifically in motor neurons. This dnc-1 KD model showed progressive motor defects together with axonal and neuronal degeneration, as observed in ALS patients. In the present study, we established a behavior-based, automated, and quantitative drug screening system using this dnc-1 KD model together with Multi-Worm Tracker (MWT), and tested whether 38 candidate neuroprotective compounds could improve the mobility of the dnc-1 KD animals. We found that 12 compounds, including riluzole, which is an approved medication for ALS patients, ameliorated the phenotype of the dnc-1 KD animals. Nifedipine, a calcium channel blocker, most robustly ameliorated the motor deficits as well as axonal degeneration of dnc-1 KD animals. Nifedipine also ameliorated the motor defects of other motor neuronal degeneration models of C. elegans, including dnc-1 mutants and human TAR DNA-binding protein of 43 kDa overexpressing worms. Our results indicate that dnc-1 KD in C. elegans is a useful model for the screening of drugs against motor neuron degeneration, and that MWT is a powerful tool for the behavior-based screening of drugs.

Chronic administration of L-DOPA, the first-line treatment of dystonic symptoms in childhood or in Parkinson's disease, often leads to the development of abnormal involuntary movements (AIMs), which represent an important clinical problem. Although it is known that Riluzole attenuates L-DOPA-induced AIMs, the molecular mechanisms underlying this effect are not understood. Therefore, we studied the behavior and performed RNA sequencing of the striatum in three groups of rats that all received a unilateral lesion with 6-hydroxydopamine in their medial forebrain bundle, followed by the administration of saline, L-DOPA, or L-DOPA combined with Riluzole. First, we provide evidence that Riluzole attenuates AIMs in this rat model. Subsequently, analysis of the transcriptomics data revealed that Riluzole is predicted to reduce the activity of CREB1, a transcription factor that regulates the expression of multiple proteins that interact in a molecular landscape involved in apoptosis. Although this mechanism underlying the beneficial effect of Riluzole on AIMs needs to be confirmed, it provides clues towards novel or existing compounds for the treatment of AIMs that modulate the activity of CREB1 and, hence, its downstream targets.

Rationale: Anterior cingulate cortex (ACC) glutamatergic abnormalities are reported in treatment-resistant schizophrenia (TRS) and implicated in functional dysconnectivity and psychopathology. Preclinical evidence indicates riluzole reduces synaptic glutamate. However, it is unknown whether riluzole can modulate glutamate metabolite levels and associated functional connectivity in TRS., Objectives: To examine the relationship between glutamatergic function and cortical connectivity and determine if riluzole can modulate glutamate metabolite levels and cortical functional connectivity in TRS., Methods: Nineteen TRS patients and 18 healthy volunteers (HV) underwent magnetic resonance imaging consisting of MR spectroscopy measuring ACC glutamate plus glutamine (Glx), fMRI measuring resting ACC-functional connectivity, and arterial spin labelling measuring regional cerebral blood flow (rCBF), and clinical measures. They then received 50 mg riluzole twice daily for 2 days when imaging was repeated., Results: Baseline (pre-riluzole) Glx levels were correlated directly with negative symptom severity (r = 0.49; p = 0.03) and inversely with verbal learning in TRS (r = - 0.63; p = 0.002), but not HV (r = - 0.24; p = 0.41). Connectivity between the ACC and anterior prefrontal cortex (aPFC) was correlated with verbal learning in TRS (r = 0.49; p = 0.04), but not HV (r = 0.28; p = 0.33). There was a significant group × time interaction effect on Glx levels (p < 0.05) and on ACC connectivity to the aPFC (p < 0.05, FWE-corrected). Riluzole decreased Glx and increased ACC-aPFC connectivity in TRS relative to HV. Change in Glx correlated inversely with change in ACC-aPFC connectivity in TRS (r = - 0.52; p = 0.02) but not HV (r = 0.01; p = 0.98). Riluzole did not alter rCBF (p > 0.05), indicating absence of a non-specific blood flow effect., Conclusion: Results indicate glutamatergic function and cortical connectivity are linked to symptoms and cognitive measures and that it is possible to pharmacologically modulate them in TRS.

Spinal cord injury (SCI) is a devastating condition that has few treatment options. Riluzole, a sodium channel blocker used to treat amyotrophic lateral sclerosis, has been initially trialed in human SCI. We performed a systematic review to critically assess the efficacy of riluzole in locomotor recovery and damage extension in SCI rat models, and the potential for clinical translation. PubMed, Embase, Cochrane Library, and Chinese databases were searched from their inception date to March 2018. Two reviewers independently selected animal studies that evaluated neurological recovery and lesion area following riluzole treatment in SCI rat models, extracted data and assessed methodological quality. Pairwise meta-analysis, subgroup analysis, and network meta-analysis were performed to assess the effects of riluzole on SCI. Ten eligible studies were included. Two studies had high methodological quality. Overall, the Basso, Beattie, and Bresnahan scores were increased in riluzole-treated animals versus controls, and effect sizes showed a gradual increase from the 1st (five studies, n = 104, mean difference = 1.24, 95% CI = 0.11 to 2.37, p = 0.03) to 6th week after treatment (five studies, n = 120, mean difference = 2.34, 95% CI = 1.26 to 3.42, p < 0.0001). Riluzole was associated with improved outcomes in the inclined plane test and the tissue preservation area. Subgroup analyses suggested an association of locomotor recovery with riluzole dose. Network meta-analysis showed that 5 mg/kg riluzole exhibited greater protection than 2.5 and 8 mg/kg riluzole. Collectively, this review suggests that riluzole has a protective effect on SCI, with good safety and a clear mechanism of action and may be suitable for future clinical trials or applications. However, animal results should be interpreted with caution given the known limitations in animal experimental design and methodological quality., (© 2019 International Society for Neurochemistry.)

The lifetime prevalence of addiction to cannabis (marijuana or cannabinoids) is among the highest of that of all illicit drugs in developed countries, and no effective treatment for cannabis addiction is currently available. Previous studies in our research group suggested that astrocyte-mediated long-term depression (LTD) in ventral tegmental area (VTA) dopamine neurons plays a pivotal role in the development of cannabis addiction. The present study was designed to investigate the effects and potential mechanisms of riluzole, a drug used for the treatment of motor neuron diseases, on cannabinoid-facilitated LTD induction. We demonstrated that riluzole significantly inhibited HU210-induced glutamate release through increased expression of glial glutamate transporter-1 (GLT-1) in cultured astrocytes, and an infusion of riluzole into the bilateral VTA in rats prevented the potent cannabinoid HU210-facilitated LTD induction in 2-month-old animals, which was blocked by bath application of dihydrokainate (DHK), a selective GLT-1 inhibitor. Our results show the effects and potential mechanism of action of riluzole in cannabinoid-facilitated LTD induction, which may provide insights into new therapeutic approaches for cannabis addiction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Environmental factors like stress affect age-related cognitive deficits and promote Alzheimer's disease (AD)-related pathology in mice. Excess glutamate has been proposed as a possible mediator underlying these effects in the hippocampus, a vulnerable brain region implicated in learning and memory., Methods: Here, we examined a) whether stress applied during a sensitive developmental period early in life affects later synaptic plasticity, learning and memory and plaque load in the APPswe/PS1dE9 mouse model for Alzheimer's disease and b) whether these effects could be rescued using long-term treatment with the glutamate modulator riluzole., Results: Our results demonstrate that ELS impairs synaptic plasticity in 6-month-old mice and increases plaque load in 12-month-old APPswe/PS1dE9 mice, while impairing flexible spatial learning in the Barnes maze at this age. Notably, spatial learning correlated well with hippocampal expression of the transporter EAAT2, which is important for extracellular glutamate uptake. The changes in LTP, plaque load and cognition after ELS were all prevented by riluzole treatment that started from post-weaning., Conclusion: These results suggest that normalising glutamate signalling may be a viable therapeutic strategy for treating vulnerable individuals at risk of developing stress-aggravated AD, particularly in relation to adverse early life experiences., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Rationale: Chronic quinpirole (QNP) sensitization is an established animal model relevant to obsessive-compulsive disorder (OCD) that has been previously shown to induce several OCD-like behavioral patterns, such as compulsive-like checking and increased locomotion., Objectives: In current study we explored the effect of antiglutamatergic drugs, memantine and riluzole, on cognitive and behavioral performance of QNP sensitized rats., Methods: During habituation phase, the rats (N = 56) were injected with QNP (0.25 mg/kg) or saline solution (every other day up to 10 injections) and placed into rotating arena without foot shocks for 50-min exploration. Active place avoidance task in rotating arena with unmarked to-be-avoided shock sector was used during acquisition phase. Rats were injected with memantine (1 mg/kg or 5 mg/kg), riluzole (1 mg/kg or 5 mg/kg) or saline solution 30 min before the trial and with QNP (0.25 mg/kg) or saline right before they were placed inside the rotating arena with 60° unmarked shock sector. Locomotion and number of entrances into the shock sector were recorded., Results: QNP sensitization led to a robust deficit in place learning. However, neither memantine nor riluzole did reverse or alleviate the deficit induced by QNP. Contrarily, memantine significantly aggravated QNP induced deficit., Conclusions: The exacerbation of cognitive deficit following antiglutamatergic agents could be mediated by decreased glutamate concentration in nucleus accumbens and decreased hippocampal activation in the QNP sensitization model., (Copyright © 2019. Published by Elsevier Inc.)

Aberrant glutamatergic signaling has been implicated in altered metabolic activity in many cancer types, including malignant melanoma. Previously, we have illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo . In this study, we showed that autocrine stimulation constitutively activates the GRM1 receptor and its downstream mitogenic signaling. GRM1-activated (GRM1 + ) melanomas exhibited significantly increased expression of glutaminase (GLS), which catalyzes the first step in the conversion of glutamine to glutamate. In cultured GRM1 + melanoma cell lines, CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, suppressed cell proliferation, while riluzole, an inhibitor of glutamate release, promoted apoptotic cell death in vitro and in vivo . Combined treatment with CB-839 and riluzole treatment proved to be superior to single-agent treatment, restricting glutamate bioavailability and leading to effective suppression of tumor cell proliferation in vitro and tumor progression in vivo . Hyperactivation of GRM1 in malignant melanoma is an oncogenic driver, which acts independently of canonical melanoma proto-oncogenes, BRAF or NRAS. Overall, these results indicate that expression of GRM1 promotes a metabolic phenotype that supports increased glutamate production and autocrine glutamatergic signaling, which can be pharmacologically targeted by decreasing glutamate bioavailability and the GLS-dependent glutamine to glutamate conversion. SIGNIFICANCE: These findings demonstrate that targeting glutaminolytic glutamate bioavailability is an effective therapeutic strategy for GRM1-activated tumors., (©2019 American Association for Cancer Research.)

Background: Riluzole is a glutamate regulator and effective in treatment of neuropsychiatric conditions., Aims: We assessed riluzole for treatment of methamphetamine dependence., Methods: In this randomized, double-blind, placebo-controlled clinical trial, male outpatients with methamphetamine dependence who were 18-65 years old received either 50 mg riluzole ( n=34) or placebo ( n=54) twice daily for 12 weeks. Patients were excluded in case of comorbid serious medical conditions or neurologic disorders, comorbid psychiatric disorders other than methamphetamine dependence requiring specific treatment interventions, simultaneous positive urine test result for substances of abuse other than methamphetamine, smoking >3 days per week, simultaneous consumption of medications which are contraindicated or have interaction with riluzole., Results: Concerning primary outcomes, the cumulative mean number of attended weekly visits was higher in the riluzole arm compared with the placebo arm approaching a statistically significant difference (riluzole, median (range)=13.00 (2.00-13.00); placebo=4.00 (2.00-13.00); Mann-Whitney U=505.00, p-value=0.073), and the weekly measured rate of positive methamphetamine urine test results was significantly lower in the riluzole arm by the end of the study (riluzole=1 (5.00%), placebo=9 (45.00%), p-value=0.004). Patients in the riluzole arm experienced significantly greater improvement on all the craving, withdrawal, and depression measures regarding mean score changes from baseline to endpoint. No significant difference was detected between the two arms in terms of incidence of adverse events., Conclusion: Future randomized clinical trials are needed to investigate proper dosing strategy in a more inclusive sample.

Background: Prostate cancer (PCa) is diagnosed at the highest rate of all non-cutaneous male cancers in the United States. The androgen-dependent (AD) transcription factor, androgen receptor (AR), drives PCa-but inhibiting AR or androgen biosynthesis induces remission for only a short time. At which point, patients acquire more aggressive castration-resistant (CR) disease with re-activated AR-dependent signaling. To combat treatment resistance, down-regulating AR protein expression has been considered as a potential treatment strategy for CR-PCa., Methods: AD- and CR-PCa cell lines were treated with the well-tolerated FDA-approved oral medicine, riluzole. Expression of full-length or wild-type AR (AR-FL) and constitutively active AR-splice variant 7 (AR-V7) was assessed by immunoblotting. AR-FL/AR-V7 activity was measured using qRT-PCR of AR-target genes. Cytoplasmic [Ca 2+ ] levels were measured using a fluorescent Ca 2+ indicator microplate assay. Markers of the endoplasmic reticulum stress (ERS) pathway and autophagy were assessed by immunoblotting. Direct interaction between AR and selective autophagy receptor p62 was demonstrated by co-immunoprecipitation., Results: We demonstrate that riluzole downregulates AR-FL, mutant ARs, and AR-V7 proteins expression by protein degradation through ERS pathway and selective autophagy. Riluzole also significantly inhibited AR transcription activity by decreasing its target genes expression (PSA, TMPRSS2, and KLK2)., Conclusions: We provide key mechanistic insights by which riluzole exerts its anti-tumorigenic effects and induces AR protein degradation via ERS pathways. Our findings support the potential utility of riluzole for treatment of PCa., (© 2018 Wiley Periodicals, Inc.)

Ischemia-related diseases are a leading cause of death worldwide, and promoting therapeutic angiogenesis is key for effective recovery from hypoxia-ischemia. Given the limited success of angiogenic factors, such as vascular endothelial growth factor, in clinical trials, it is important to find more promising angiogenic targets. Here, using both cell- and tissue-based assays and a mouse model of injury-induced ischemia, we investigated the involvement of the transient receptor potential canonical 5 (TRPC5) ion channel in angiogenesis and the effects of a TRPC5 activator, the Food and Drug Administration-approved drug riluzole, on recovery from ischemic injury. We demonstrate that TRPC5 is involved in endothelial cell sprouting, angiogenesis, and blood perfusion in an oxygen-induced retinopathy model and a hind limb ischemia model. We found a potential regulatory link between nuclear factor of activated T cell isoform c3 and angiopoietin-1 that could provide the mechanistic basis for the angiogenic function of TRPC5. Importantly, treatment with riluzole, which can activate TRPC5 in endothelial cells, improved recovery from ischemia in mice. Our study reveals TRPC5 as a potential angiogenic target and suggests riluzole as a promising drug for managing ischemic diseases., (© 2019 Zhu et al.)

Background: Riluzole is a benzothiazole anticonvulsant used in the treatment of patients with amyotrophic lateral sclerosis and it is being investigated for clinical use in patients with spinal cord injury. The present study evaluated the pharmacokinetics of riluzole in beagle dogs after oral dose administration., Methods: The oral doses (1.5, 5, 15 and 50 mg/kg) of riluzole were administered to beagle dogs and blood samples were collected from 0 h to 24 h post drug administration. Riluzole was quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)., Results: The method was sensitive, precise, accurate and selective to riluzole quantification in plasma of beagle dogs. The pharmacokinetics following oral administration was linear from 1.5 to 15 mg/kg and the t 1/2 was 2.16, 1.5, 1.8 and 3.0 h after oral administration of 1.5, 5.0, 15 and 50 mg/kg riluzole., Conclusion: The riluzole pharmacokinetics was linear up to 15 mg/kg and had a significantlyshorter t 1/2 in beagle dogs than in humans., Competing Interests: The authors have no conflict of interests., (© Georg Thieme Verlag KG Stuttgart · New York.)

Any spinal cord injury carries the potential for persistent disability affecting motor, sensory and autonomic functions. To prevent this outcome, it is highly desirable to block a chain of deleterious reactions developing in the spinal areas immediately around the primary lesion. Thus, early timing of pharmacological neuroprotection should be one major strategy whose impact may be first studied with preclinical models. Using a simple in vitro model of the rat spinal cord it is possible to mimic pathological processes like excitotoxicity that damages neurons because of excessive glutamate receptor activation due to injury, or hypoxic/dysmetabolic insult that preferentially affects glia following vascular dysfunction. While ongoing research is exploring the various components of pathways leading to cell death, current treatment principally relies on the off-label use of riluzole (RLZ) or methylprednisolone sodium succinate (MPSS). The mechanism of action of these drugs is diverse as RLZ targets mainly neurons and MPSS targets glia. Even when applied after a transient excitotoxic stimulus, RLZ can provide effective prevention of secondary excitotoxic damage to premotoneurons, although not to motoneurons that remain very vulnerable. This observation indicates persistent inability to express locomotor activity despite pharmacological treatment conferring some histological protection. MPSS can protect glia from dysmetabolic insult, yet it remains poorly effective to prevent neuronal death. In summary, it appears that these pharmacological agents can produce delayed protection for certain cell types only, and that their combined administration does not provide additional benefit. The search should continue for better, mechanism-based neuroprotective agents.

Riluzole, approved by the US Food and Drug Administration (FDA) in 1995, is the most widespread oral treatment for the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). The drug, whose mechanism of action is still obscure, mitigates progression of the illness, but unfortunately with only limited improvements. Herein we report the first demonstration, using a combination of computational and in vitro studies, that riluzole is an ATP-competitive inhibitor of the protein kinase CK1 isoform δ, with an IC 50 value of 16.1 μm. This allows us to rewrite its possible molecular mechanism of action in the treatment of ALS. The inhibition of CK1δ catalytic activity indeed links the two main pathological hallmarks of ALS: transactive response DNA-binding protein of 43 kDa (TDP-43) proteinopathy and glutamate excitotoxicity, exacerbated by the loss of expression of glial excitatory amino acid transporter-2 (EAAT2)., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Riluzole is a potent neuroprotective agent which primarily inhibits excitatory neurotransmission interfering with presynaptic release, uptake and postsynaptic actions of glutamate by mechanisms that are not well understood. Riluzole and related prodrugs with improved blood brain barrier penetrance, are shown to be effective for the treatment of amyotrophic lateral sclerosis, ataxias, epilepsy and mood disorders. Our study was undertaken to decipher molecular and subcellular mechanisms of riluzole's antiglutamatergic effect, particularly focusing on presynaptic active zone structure and function. Applying multifarious live cell imaging techniques and amperometric glutamate recordings, we measured the impact of riluzole on presynaptic activity, synaptic vesicle recycling and glutamate release. Our in vitro and in vivo data revealed a unique mechanism whereby riluzole reduces the efficacy of glutamatergic transmission by selectively lowering the size of the readily releasable pool. This effect was correlated with the inhibition of protein kinase C-dependent Munc18-1 phosphorylation which is known to interfere with neurotransmitter release., (Copyright © 2018. Published by Elsevier Ltd.)

Oxaliplatin causes acute cold hypersensitivity in most patients. We previously reported oxalate derived from oxaliplatin induced cold allodynia via overexpression of transient receptor potential melastatin 8 (TRPM8) in the dorsal root ganglion (DRG) in rats. In this study, we examined the effect of riluzole on oxaliplatin-induced cold allodynia. In cultured DRG neurons, riluzole suppressed oxalate-induced increase of the number of menthol (TRPM8 agonist)-sensitive cells. Moreover, riluzole prevented cold allodynia and increase in levels of TRPM8 mRNA in oxaliplatin-treated rats. These results suggest that riluzole prevents oxaliplatin-induced cold allodynia via inhibition of TRPM8 overexpression in the DRG., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies.

Neurotoxicity remains the most common adverse effect of oxaliplatin, limiting its clinical use. In the present study, we developed a mouse model of chronic oxaliplatin-induced neuropathy, which mimics both sensory and motor deficits observed in patients, in a clinically relevant time course. Repeated oxaliplatin administration in mice induced both cephalic and extracephalic long lasting mechanical and cold hypersensitivity after the first injection as well as delayed sensorimotor deficits and a depression-like phenotype. Using this model, we report that riluzole prevents both sensory and motor deficits induced by oxaliplatin as well as the depression-like phenotype induced by cumulative chemotherapeutic drug doses. All the beneficial effects are due to riluzole action on the TREK-1 potassium channel, which plays a central role in its therapeutic action. Riluzole has no negative effect on oxaliplatin antiproliferative capacity in human colorectal cancer cells and on its anticancer effect in a mouse model of colorectal cancer. Moreover, riluzole decreases human colorectal cancer cell line viability in vitro and inhibits polyp development in vivo. The present data in mice may support the need to clinically test riluzole in oxaliplatin-treated cancer patients and state for the important role of the TREK-1 channel in pain perception., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Riluzole is a glutamatergic modulator of particular interest in autism spectrum disorder (ASD). In this 12-week randomized, double-blind, placebo-controlled, crossover pilot study we evaluated the safety and tolerability of 5-week of adjunctive riluzole treatment (vs. 5-week of placebo, with 2-week washout period) targeting ASD-associated drug-refractory irritability in eight individuals age 12-25 years. All participants tolerated riluzole 200 mg per day, however there were no statistically significant findings for the overall treatment effect, the treatment effect by week within period of the study, or a cross-over effect across the periods of the study on the Clinical Global Impression Improvement Scale or the Aberrant Behavior Checklist Irritability subscale. The results of this trial indicate that 5-week of riluzole treatment was well tolerated, but had no significant effect on the target symptoms. Trial Registration ClinicalTrials.gov Identifier NCT02081027, Registered 5 August 2013, First participant enrolled 19 September 2013.

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1 R ) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1 R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1 R already produce GABA in E12.5 embryo, and that V1 R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1 R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents ( I Nap ). This is the first demonstration that I Nap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block I NaP , altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of I NaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks. SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1 R ) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents ( I NaP ) in driving plateau potential in V1 R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for I NaP in the excitability of V1 R and the developing SC., (Copyright © 2018 the authors 0270-6474/18/387667-16$15.00/0.)

Alzheimer's disease (AD) represents a major healthcare burden with no effective treatment. The glutamate modulator, riluzole, was shown to reverse many AD-related gene expression changes and improve cognition in aged rats. However, riluzole's effect on amyloid beta (Aβ) pathology, a major histopathological hallmark of AD, remains unclear. 5XFAD transgenic mice, which harbor amyloid β precursor protein (APP) and presenilin mutations and exhibit early Aβ accumulation, were treated with riluzole from 1 to 6 months of age. Riluzole significantly enhanced cognition and reduced Aβ42, Aβ40, Aβ oligomers levels, and Aβ plaque load in 5XFAD mice. RNA-Sequencing showed that riluzole reversed many gene expression changes observed in the hippocampus of 5XFAD mice, predominantly in expression of canonical gene markers for microglia, specifically disease-associated microglia (DAM), as well as neurons and astrocytes. Central to the cognitive improvements observed, riluzole reversed alterations in NMDA receptor subunits gene expression, which are essential for learning and memory. These data demonstrate that riluzole exerts a disease modifying effect in an Aβ mouse model of early-onset familial AD.

Background and Purpose: Chronic neuropathic pain is an important healthcare issue with significant emotional components. The amygdala is a brain region involved in pain and emotional-affective states and disorders. The central amygdala output nucleus (CeA) contains small-conductance calcium-activated potassium (SK) channels that can control neuronal activity. A clinically available therapeutic, riluzole can activate SK channels and may have antinociceptive effects through a supraspinal action. We tested the hypothesis that riluzole inhibits neuropathic pain behaviors by inhibiting pain-related changes in CeA neurons, in part at least through SK channel activation., Experimental Approach: Brain slice physiology and behavioral assays were done in adult Sprague Dawley rats. Audible and ultrasonic vocalizations and von Frey thresholds were measured in sham and neuropathic rats 4 weeks after left L5 spinal nerve ligation (SNL model). Whole cell patch-clamp recordings of regular firing CeA neurons in brain slices were used to measure synaptic transmission and neuronal excitability., Key Results: In brain slices, riluzole increased the SK channel-mediated afterhyperpolarization and synaptic inhibition, but inhibited neuronal excitability through an SK channel independent action. SNL rats had increased vocalizations and decreased withdrawal thresholds compared to sham rats, and intra-CeA administration of riluzole inhibited vocalizations and depression-like behaviors but did not affect withdrawal thresholds. Systemic riluzole administration also inhibited these changes, demonstrating the clinical utility of this strategy. SK channel blockade in the CeA attenuated the inhibitory effects of systemic riluzole on vocalizations, confirming SK channel involvement in these effects., Conclusions and Implications: The results suggest that riluzole has beneficial effects on neuropathic pain behaviors through SK channel dependent and independent mechanisms in the amygdala., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Riluzole is currently one of two approved medications for the treatment of amyotrophic lateral sclerosis (ALS). However, brain disposition of riluzole, as a substrate of P-glycoprotein (P-gp), is limited by the efflux transporters at the blood-brain barrier (BBB). We propose to develop a liposomal co-delivery system that could effectively transport riluzole to brain cells by reducing efflux pumps with a P-gp inhibitor, verapamil. Riluzole and verapamil cocktail liposomes were prepared by lipid film hydration. The average particle size of cocktail liposomes was 194.3 ± 6.0 nm and their polydispersity index (PDI) was 0.272 ± 0.017. The encapsulation efficiencies of verapamil and riluzole in the cocktail liposomes were 86.0 ± 1.4% and 85.6 ± 1.1%, respectively. The drug release from cocktail liposomes after 8 h in PBS at 37 °C was 78.4 ± 6.2% of riluzole and 76.7 ± 3.8% of verapamil. The average particle size of liposomes did not show significant changes at 4 °C after three months. Verapamil cocktail liposomes inhibited P-gp levels measured by western blotting in dose and time-dependent manners in brain endothelial bEND.3 cells. Increased drug efflux transporters were detected in bEND.3 and astrocytes C8D1A cells, promoted by tumor necrosis factor (TNF-α) or hydrogen peroxide (H 2 O 2 ). Restored accumulations of riluzole and fluorescent dye rhodamine 123 were observed in bEND.3 cells after treatments with cocktail liposomes. It indicated that inhibitory potential of co-delivery liposome system towards P-gp could mediate the transport of both P-gp substrates. Verapamil and riluzole co-loaded liposomes may be used to overcome pharmacoresistance of riluzole for improving ALS therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Leak K + currents are mediated by two-pore domain K + (K2P) channels and are involved in controlling neuronal excitability. Of 15 members of K2P channels cloned so far, TRAAK, TREK-1, and TREK-2 are temperature sensitive. In the present study, we show that strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels was present mainly in small-sized dorsal root ganglion (DRG) neurons of rats. The percentages of neurons with strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels were 27, 23, and 20%, respectively. Patch-clamp recordings were performed to examine isolated leak K + currents on acutely dissociated small-sized rat DRG neurons at room temperature of 22 °C, cool temperature of 14 °C and warm temperature of 30 °C. In majority of small-sized DRG neurons recorded (76%), large leak K + currents were observed at 22 °C and were inhibited at 14 °C and potentiated at 30 °C, suggesting the presence of temperature-sensitive K2P channels in these neurons. In a small population (18%) of small-sized DRG neurons, cool temperature of 14 °C evoked a conductance which was consistent with TRPM8 channel activation in cold-sensing DRG neurons. In these DRG neurons, leak K + currents were very small at 22 °C and were not potentiated at 30 °C, suggesting that few temperature-sensitive K2P channels was present in cold-sensing DRG neurons. For DRG neurons with temperature-sensitive leak K + currents, riluzole, norfluoxetine and prostaglandin F2α (PGE2α) inhibited the leak K + currents at both 30 °C and 22 °C degree, and did not have inhibitory effects at 14 °C. Collectively, the observed temperature-sensitive leak K + currents are consistent with the expression of temperature-sensitive K2P channels in small-sized DRG neurons.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes an impairment in both the upper and lower motor neurons. The recent description of numerous non-motor signs points to an involvement of the neocortex networks that is more complex than was previously believed. Paired associative stimulation (PAS), a combination of transcranial magnetic stimulation (TMS) and peripheral nerve stimulation, can enhance motor output in the contralateral hand through an NMDA-mediated sensorimotor mechanism., Objective: To describe the effects of PAS on ALS patients before and after Riluzole intake compared with healthy subjects., Methods: PAS was used to detect differences between 24 newly-diagnosed ALS patients and 25 age-matched healthy controls. MEP amplitude from the abductor pollicis brevis was considered before PAS, immediately after (T0) and after 10 (T10), 20 (T20), 30 (T30) and 60 (T60) minutes. Statistical significance was calculated using RM-ANOVA., Results: In healthy controls, PAS significantly increased MEP amplitude at T10, T20 and T30 (p < 0.05). In ALS patients, a significant increase in MEP amplitude was also observed after 60 min (p < 0.05), thus demonstrating NMDA-mediated enhanced facilitatory plasticity. After two weeks of riluzole intake, no MEP amplitude increase was evident after PAS at any time point. In three monomelic-onset ALS patients, a long lasting sensorimotor facilitation was evident only in the hemisphere corresponding to the affected side and appeared in the opposite hemisphere when the patients manifested contralateral symptoms., Conclusions: PAS may be considered a useful tool when investigating NMDA-mediated neocortical networks in ALS patients and the modulation of such networks after anti-glutamatergic drug intake., (Published by Elsevier Inc.)

Superficial laminae of the spinal cord possess a considerable number of neurons with spontaneous activity as reported in vivo and in vitro preparations of several species. Such neurons may play a role in the development of the nociceptive system and/or in the spinal coding of somatosensory signals. We have used electrophysiological techniques in a horizontal spinal cord slice preparation from adult mice to investigate how this activity is generated and what are the main patterns of activity that can be found. The results show the existence of neurons that fire regularly and irregularly. Within each of these main types, it was possible to distinguish patterns of spontaneous activity formed by single action potentials and different types of bursts according to intra-burst firing frequency. Activity in neurons with irregular patterns was blocked by a mixture of antagonists of the main neurotransmitter receptors present in the cord. Approximately 82% of neurons with a regular firing pattern were insensitive to synaptic antagonists but their activity was inhibited by specific ion channel blockers. It is suggested that these neurons generate endogenous activity due to the functional expression of hyperpolarisation-activated and persistent sodium currents driving the activity of irregular neurons.

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes., Competing Interests: The authors have declared that no competing interests exist.

Spinal cord injury (SCI) without radiographical abnormalities (SCIWORA) presents a significant challenge because of the loss of function despite an apparent normal anatomy. The cause of dysfunction is not understood, and specific treatment options are lacking. Some scoliosis corrective surgeries result in SCIWORA, where stretching of the spinal cord can lead to vascular compromise and hypoxia. The iatrogenic nature of this injury allows for the implantation of neuroprotective strategies that are designed to prevent damage. We utilized a model of atraumatic SCI to evaluate the efficacy of the sodium-channel blocker, riluzole, as a prophylactic neuroprotectant. As expected, the stretch injury caused a significant reduction in intraparenchymal oxygen in distraction (-53.09 ± 22.23%) and riluzole pre-treated distraction animals (-43.04 ± 22.86%). However, in contrast to the oxidative stress and metabolic impairments observed in vehicle-treated distraction animals, in which protein carbonylation increased significantly (5.88 ± 1.3 nmol/mL), riluzole kept these levels within the normal range (1.8 ± 1.0 nmol/mL). This neurprotection also prevented ventral motor neuron hypoplasia and pyknosis, characteristic features of this atraumatic SCI model, and maintained normal gait function (e.g., stride length and stance time). This study provides evidence for the use of prophylactic neuroprotective strategies in which thoracic or spine surgeries present the risk of causing atraumatic SCI.