salicylates and 2-hydroxy-5-(2-3-5-6-tetrafluoro-4-trifluoromethyl-benzylamino)benzoic-acid

Excess activation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors and free radicals, evoke nerve cell death following hypoxic-ischemic brain injury in various animal models. However, clinical trials in stroke patients using NMDA receptor antagonists have failed to show efficacy primarily due to the limited therapeutic time window for neuroprotection and a narrow therapeutic index. In comparison, antioxidants prolonged the time window for neuroprotection in animal models of ischemic stroke and showed greater therapeutic potential in clinical trials for ischemic stroke. Excess activation of NMDA receptors and free radicals mediate the two separate pathways of nerve cell death in stroke and a safe and multifunctional drug that can block both routes in the brain will likely provide a better therapeutic outcome in patients with stroke. Derivatives of the lead structures of sulfasalazine and aspirin have led to the discovery of a new molecule, Neu2000, that has demonstrated excellent neuroprotection against NMDA- and free radical-induced cell death. Neu2000 is an NR2B-selective, moderate NMDA receptor antagonist with potent cell-permeable, spin trapping antioxidant action even at nanomolar concentrations. Nonclinical and human phase I studies demonstrated that Neu2000 can be translated to treat patients with stroke with better efficacy and therapeutic time window.

Topics: Animals; Antioxidants; Benzoates; Brain Ischemia; Drug Delivery Systems; Fluorobenzenes; Humans; meta-Aminobenzoates; Neuroprotective Agents; Receptors, N-Methyl-D-Aspartate; Salicylates; Spin Trapping; Stroke; Time Factors

The potential of neuroprotective agents should be revisited in the era of endovascular thrombectomy (EVT) for acute large-artery occlusion because their preclinical effects have been optimized for ischemia and reperfusion injury. Neu2000, a derivative of sulfasalazine, is a multi-target neuroprotectant. It selectively blocks N-methyl-D-aspartate receptors and scavenges for free radicals. This trial aimed to determine whether neuroprotectant administration before EVT is safe and leads to a more favorable outcome.. This trial is a phase-II, multicenter, three-arm, randomized, double-blinded, placebo-controlled, blinded-endpoint drug trial that enrolled participants aged ≥ 19 years undergoing an EVT attempt less than 8 h from symptom onset, with baseline National Institutes of Health Stroke Scale (NIHSS) score ≥ 8, Alberta Stroke Program Early CT score ≥ 6, evidence of large-artery occlusion, and at least moderate collaterals on computed tomography angiography. EVT-attempted patients are randomized into control, low-dose (2.75 g), and high-dose (5.25 g) Neu2000KWL over 5 days. Seventy participants per group are enrolled for 90% power, assuming that the treatment group has a 28.4% higher proportion of participants with functional independence than the placebo group. The primary outcome, based on intention-to-treat criteria is the improvement of modified Rankin Scale (mRS) scores at 3 months using a dichotomized model. Safety outcomes include symptomatic intracranial hemorrhage within 5 days. Secondary outcomes are distributional change of mRS, mean differences in NIHSS score, proportion of NIHSS score 0-2, and Barthel Index > 90 at 1 and 4 weeks, and 3 months.. The trial results may provide information on new therapeutic options as multi-target neuroprotection might mitigate reperfusion injury in patients with acute ischemic stroke before EVT.. ClinicalTrials.gov, ID: NCT02831088 . Registered on 13 July 2016.

Topics: Brain Ischemia; Clinical Trials, Phase II as Topic; Disability Evaluation; Double-Blind Method; Endovascular Procedures; Fluorobenzenes; Humans; meta-Aminobenzoates; Multicenter Studies as Topic; Neuroprotective Agents; Prospective Studies; Randomized Controlled Trials as Topic; Recovery of Function; Republic of Korea; Salicylates; Stroke; Thrombectomy; Time Factors; Treatment Outcome

Neu2000 (NEU; 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid), a recently developed derivative of acetylsalicylic acid and sulfasalazine, potently protects against neuronal cell death following ischemic brain injury by antagonizing NMDA receptor-mediated neuronal toxicity and oxidative stress. However, it has yet to be determined whether NEU can attenuate hypoxia-induced impairment of neuronal electrical activity. In this study, we carried out extracellular recordings of hippocampal slices in order to investigate the effects of NEU on the electrical activity of neurons exposed to a hypoxic insult (oxygen and glucose deprivation). NEU prominently suppressed hypoxia-induced impairment of neuronal activity in a concentration-dependent manner. NEU, at a low dose (1 μM), competently depressed the hypoxia-induced convulsive activity in a manner similar to trolox. Furthermore, high concentrations of NEU (50 μM) markedly abolished all hypoxia-mediated impairment of neuronal activity and accelerated the slow recovery of neuronal activity more efficiently than ifenprodil and APV. These results suggest that NEU attenuates hypoxia-induced impairment of neuronal activity more potently than the antioxidant, trolox, and the NMDA receptor antagonists, ifenprodil and APV. We propose that NEU is a striking pharmacological candidate for neuroprotection against hypoxia because of its defensive action on hypoxia-mediated impairment of electrical neurotransmission as well as its neuroprotective action against neuronal cell death induced by exposure to pathological hypoxic conditions.

Topics: Animals; Antioxidants; CA3 Region, Hippocampal; Dose-Response Relationship, Drug; Fluorobenzenes; Glucose; Hypoxia; In Vitro Techniques; meta-Aminobenzoates; Microelectrodes; Neurons; Neuroprotective Agents; Oxygen; Rats; Rats, Sprague-Dawley; Salicylates; Synaptic Transmission

Neu2000, acting as an antioxidant with N-methyl-d-aspartate-receptor antagonism, demonstrates excellent protection against ischemic insults in rodents. In this study, we report that Neu2000 also dramatically enhances the activity of kainate (KA) receptors. Neu2000 non-competitively and reversibly potentiated KA-evoked responses in a voltage-independent manner, mainly by increasing the open probability of KA receptor channels.

Topics: Animals; Antioxidants; Cerebral Cortex; Evoked Potentials; Fluorobenzenes; meta-Aminobenzoates; Mice; Mice, Inbred ICR; Neurons; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Salicylates; Stimulation, Chemical

Neu2000 [2-hydroxy-5-(2,3,5,6-tetrafluoro-4 trifluoromethylbenzylamino) benzoic acid] is a dual-acting neuroprotective agent that functions both as a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist and a free radical scavenger. In the present study, we investigated the scavenging activity of Neu2000 on various classes of reactive oxygen species and reactive nitrogen species (ROS/RNS) as well as its efficacy for reducing free radicals and oxidative stress/damage induced in spinal cord mitochondrial preparations. Neu2000 exerted scavenging activity against superoxide, nitric oxide, and hydroxyl radicals, and efficiently scavenged peroxynitrite. In the mitochondrial studies, Neu2000 markedly inhibited ROS/RNS and hydrogen peroxide levels following antimycin treatment. In addition, Neu2000 effectively scavenged hydroxyl radicals generated by iron(III)-ascorbate, reduced protein carbonyl formation mediated by hydroxyl radicals and peroxynitrite, and prevented glutathione oxidation caused by tert-butyl hydroperoxide in isolated mitochondria. Interestingly, incubation of isolated mitochondria with Neu2000 followed by centrifugation and removal of the supernatant also resulted in a concentration-dependent decrease in lipid peroxidation. This observation suggests that Neu2000 enters mitochondria to target free radicals or indirectly affects mitochondrial function in a manner that promotes antioxidant activity. The results of the present study demonstrate that Neu2000 possesses potent in vitro antioxidant activity due, most likely, to its active phenoxy group.

Topics: Animals; Antioxidants; Female; Fluorobenzenes; Free Radicals; Glutathione; In Vitro Techniques; Iron; meta-Aminobenzoates; Mitochondria; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Protein Carbonylation; Rats; Rats, Long-Evans; Salicylates; Spinal Cord

The goal of the present study was to examine the neuroprotective and functional significance of targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress using a dual-acting compound, Neu2000, in rat model of moderate spinal cord injury (SCI). An initial set of experiments was conducted in uninjured rats to study the pharmacokinetic profile of Neu2000 following intraperitoneal and intravenous administration. A second experiment measured free radical production in mitochondria isolated from sham or injured spinal cords of animals receiving vehicle or Neu2000 treatment. A third set of animals was divided into three treatment groups consisting of vehicle treatment, a single dose of Neu2000 (50 mg/kg) administered at 10 min following injury, or a repeated treatment paradigm consisting of a single bolus of Neu2000 at 10 min following injury (50 mg/kg) plus a maintenance dose (25 mg/kg) administered every 24 h for an additional 6 days. Animals were tested once a week for a period of 6 weeks for evidence of locomotor recovery in an open field and kinematic analysis of fine motor control using the DigiGait Image Analysis System. At the end of the testing period, spinal cord reconstruction was performed to obtain nonbiased stereological measures of tissue sparing. The results of this study demonstrate that Neu2000 treatment significantly reduced the production of mitochondrial free radicals and improved locomotor outcomes that were associated with a significant increase in the volume of spared spinal cord tissue.

Topics: Acute Disease; Animals; Antioxidants; Benzoates; Disease Models, Animal; Drug Administration Schedule; Female; Fluorobenzenes; Free Radicals; meta-Aminobenzoates; Mitochondria; Motor Activity; Neuroprotective Agents; Neurotoxins; Oxidative Stress; Rats; Rats, Long-Evans; Recovery of Function; Salicylates; Spinal Cord; Spinal Cord Injuries; Treatment Outcome

Neu2000 [NEU, 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid], a derivative of sulfasalazine, attenuates NMDA-induced neuronal toxicity. Here we investigated the effects of NEU on the NMDA receptor (NMDAR) using whole-cell patch clamp technique to determine the molecular mechanisms underlying its neuroprotective role. NEU reversibly suppressed NMDA responses in an uncompetitive manner with fast binding kinetics. Its inhibition of NMDAR activity depended on both the concentration and the use of agonist but not on the membrane potential. NEU accelerated NMDA desensitization without affecting the binding affinity of NMDAR for its agonists and stabilized the closed state of NMDAR. Therefore, NEU should effectively alleviate disorders that are a result of glutamate excitoxicity with fewer side effects because it is a low-affinity gating modifier that antagonizes NMDAR in an uncompetitive manner. Moreover, in the presence of ifenprodil (an NR2B antagonist) but not NVP-AAM077 [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid, an NR2A antagonist], the extent of NEU block was decreased, suggesting that NEU is an NR2B-specific antagonist.

Topics: Animals; Benzoates; Biophysics; Calcium; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorobenzenes; Ion Channel Gating; Membrane Potentials; meta-Aminobenzoates; Mice; Mice, Inbred ICR; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Protein Binding; Receptors, N-Methyl-D-Aspartate; Salicylates

The Fas pathway and oxidative stress mediate neuronal death in stroke and may contribute to neurodegenerative disease. We tested the hypothesis that these two factors synergistically produce spinal motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Levels of reactive oxygen species were increased in motor neurons from ALS mice compared with wild-type mice at age 10 weeks, before symptom onset. The proapoptotic proteins Fas, Fas-associated death domain, caspase 8, and caspase 3 were also elevated. Oral administration of 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid (Neu2000), a potent antioxidant, blocked the increase in reactive oxygen species but only slightly reduced activation of proapoptotic proteins. Administration of lithium carbonate (Li(+)), a mood stabilizer that prevents apoptosis, blocked the apoptosis machinery without preventing oxidative stress. Neu2000 or Li(+) alone significantly enhanced survival time and motor function and together had an additive effect. These findings provide evidence that jointly targeting oxidative stress and Fas-mediated apoptosis can prevent neuronal loss and motor dysfunction in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Antioxidants; Apoptosis; Benzoates; Cell Survival; Disease Models, Animal; Drug Therapy, Combination; fas Receptor; Fluorobenzenes; Lithium Carbonate; meta-Aminobenzoates; Mice; Mortality; Motor Activity; Motor Neurons; Oxidative Stress; Salicylates

Excitotoxicity and oxidative stress mediate neuronal death after hypoxic-ischemic brain injury. We examined the possibility that targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress would result in enhanced neuroprotection against hypoxic-ischemia. 2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid (Neu2000) was derived from aspirin and sulfasalazine to prevent both NMDA neurotoxicity and oxidative stress. In cortical cell cultures, Neu2000 was shown to be an uncompetitive NMDA receptor antagonist and completely blocked free radical toxicity at doses as low as 0.3 micromol/L. Neu2000 showed marked neuroprotection in a masked fashion using histology and behavioral testing in two rodent models of focal cerebral ischemia without causing neurotoxic side effects. Neu2000 protected against the effects of middle cerebral artery occlusion, even when delivered 8 h after reperfusion. Single bolus administration of the drug prevented gray and white matter degeneration and spared neurologic function for over 28 days after MACO. Neu2000 may be a novel therapy for combating both NMDA receptor-mediated excitotoxicity and oxidative stress, the two major routes of neuronal death in ischemia, offering profound neuroprotection and an extended therapeutic window.

Topics: Animals; Antioxidants; Aspirin; Benzoates; Brain Ischemia; Cells, Cultured; Excitatory Amino Acid Antagonists; Fluorobenzenes; Infarction, Middle Cerebral Artery; meta-Aminobenzoates; Mice; N-Methylaspartate; Oxidative Stress; Receptors, N-Methyl-D-Aspartate; Salicylates; Sulfasalazine