piperidines and lubeluzole

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Na

Topics: Animals; Humans; Myotonia; Piperidines; Riluzole; Thiazoles; Voltage-Gated Sodium Channels

The administration of oral aspirin within 48 h and tissue plasminogen activator within 3 h of ischaemic stroke onset remain the only treatments that have been shown to have clinical benefit. There has been much excitement about neuroprotection over the last two decades, as it may minimise the harmful effects of ischaemic neuronal damage. Although each step along the ischaemic cascade offers a potential target for therapeutic intervention, and neuroprotection has shown benefit in animal studies, this has been difficult to translate to humans. Some hope has been offered by the recent finding that the free radical scavenger NXY-059 may improve outcomes in patients presenting within 6 h of onset of ischaemic stroke. There is logic to the idea that neuroprotection may be most effective when reperfusion has occurred with thrombolysis, as the neuroprotectant will have greater access to ischaemic tissue and the opportunity is presented to minimise free radical-mediated reperfusion injury. Numerous studies in animal models support this view, but the concept has not, as yet, been rigorously tested in humans.

Topics: Animals; Benzenesulfonates; Brain; Brain Ischemia; Drug Therapy, Combination; Fibrinolytic Agents; Free Radical Scavengers; GABA Agonists; Humans; Neuroprotective Agents; Nitrogen Oxides; Piperidines; Randomized Controlled Trials as Topic; Sodium Channel Blockers; Stroke; Thiazoles; Thrombolytic Therapy; Time Factors; Tissue Plasminogen Activator

Lubeluzole was developed as a neuroprotective substance for use in acute ischaemic stroke. Its biological function is related to its ability to alter the biochemical cascade that leads to irreversible neural damage in the penumbra by preventing an increase in extracellular glutamate and normalizing neuronal excitability in the peri-infarct region. Phase II and early phase III trials indicated an improvement in neurological outcome, whereas a final large randomized and placebo controlled trial did not show superiority versus placebo.

Topics: Aged; Brain Ischemia; Humans; Neuroprotective Agents; Nitrous Oxide; Piperidines; Stroke; Thiazoles

Experimental studies have shown that ischaemic insults cause excess release of excitatory amino acid (EAA) neurotransmitters, particularly glutamate. Glutamate re-uptake is impaired under ischaemic conditions. In preclinical models of stroke, antagonists of excitatory amino acids or of glutamate release protect against ischaemic injury, even when administered after the ischaemic insult. Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in different experimental models inhibiting glutamate release, nitric oxide (NO) synthesis and blocking voltage-gated Na+ and Ca2+ ion channels.. The objective of this review is to assess the effectiveness and safety of lubeluzole given in the acute phase of acute ischaemic stroke.. The Cochrane Stroke Group trials register was searched. Additional searches of Cochrane Controlled Trials Register (CENTRAL/CCTR), Medline, Embase, Pascal BioMed (1996-2001) and Current Contents CCSearch reg 7 Editions (1996-2001) were made to supplement the Stroke Group general strategy. We contacted Janssen Research Foundation to identify further studies.. All randomised unconfounded trials comparing intravenous lubeluzole with placebo or open control in patients with a clinical syndrome definitely considered as an acute stroke in whom CT scanning showed an infarct or was normal.. Two reviewers independently selected trials for inclusion, assessed trial quality and extracted the data.. Five trials involving a total of 3,510 patients were included. The quality of the trials did not vary considerably. Sensitivity/subgroups analysis was not completely performed because of lack of data. Lubeluzole given at the doses of 5, 10 and 20 mg/day for 5 days was tested against a placebo-control group. There was no evidence that Lubeluzole given at any dose either reduced the odds of death from all causes (OR=0.93, 95% CI 0.79-1.09) or reduced the odds of being dead or dependent at the end of follow-up (OR=1.04, 95% CI 0.91-1.19). On the other hand, given at any dose, Lubeluzole was associated with a significant excess of heart-conduction disorders (Q-T prolonged > 450 msec) at the end of follow-up (OR=1.43, 95% CI 1.09-1.87).. Lubeluzole, given in the acute phase of ischaemic stroke, is not associated with a significant reduction of death or dependency at the end of scheduled follow-up period but seems to be associated with a significant increase of heart-conduction disorders (Q-T prolonged >450 msec).

Topics: Acute Disease; Brain Ischemia; Humans; Neuroprotective Agents; Piperidines; Thiazoles

The concept of neuroprotection relies on the principle that delayed neuronal injury occurs after ischemia. The phenomenon of the "ischemic cascade" has been described, and each step along this cascade provides a target for therapeutic intervention. A wide variety of drugs have been studied in humans. Ten classes of neuroprotective agents have reached phase III efficacy trials but have shown mixed results. They included calcium channel antagonists, NMDA receptor antagonists, lubeluzole, CDP-choline, the free radical scavenger tirilazad and ebselen, enlimomab, GABA agonist clomethiazole, the sodium channel antagonist fosphenytoin, magnesium, glycine site antagonist GV150526 and piracetam. Furthermore, the mechanisms that underlie the development of focal ischemic injury continue to be discovered, opening new therapeutic perspective for neuroprotection that might clinically be applicable in the future.

Topics: Acute Disease; Adult; Aged; Animals; Antioxidants; Calcium Channel Blockers; Chlormethiazole; Clinical Trials as Topic; Clinical Trials, Phase III as Topic; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Forecasting; GABA Modulators; Guanidines; Humans; Imidazoles; Middle Aged; Naltrexone; Narcotic Antagonists; Neuroprotective Agents; Pipecolic Acids; Piperidines; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Stroke; Thiazoles

Topics: Acute Disease; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Antibodies; Brain Ischemia; Calcium Channel Blockers; Cardiovascular Agents; Cell Adhesion Molecules; Clinical Trials as Topic; Cytidine Diphosphate Choline; Cytokines; Drug Therapy, Combination; Fibrinolytic Agents; Free Radical Scavengers; gamma-Aminobutyric Acid; Growth Substances; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; N-Methylaspartate; Neuroprotective Agents; Nootropic Agents; Piperidines; Stroke; Thiazoles; Thrombolytic Therapy

The concept of neuroprotection relies on the principle that delayed neuronal injury occurs after ischemia. The phenomenon of the "ischemic cascade" has been described, and each step along this cascade provides a target for therapeutic intervention. In animal models of global and focal cerebral ischemia, numerous preclinical studies have demonstrated various agents to be neuroprotective at different steps along this cascade. A wide variety of drugs has also been studied in humans. Ten classes of neuroprotective agents have reached phase III efficacy trials but have shown mixed results. They include calcium channel antagonists, NMDA receptor antagonists, lubeluzole, CDP-choline, the free radical scavenger tirilizad, anti-intercellular adhesion molecule-1 (ICAM-1) antibody, GM-1 ganglioside, clomethiazole, the sodium channel antagonist fosphenytoin, and piracetam. In the future, clinicians may have an armamentarium of treatments for acute ischemic stroke at their disposal, with a combination of agents directed at different sites in the ischemic cascade being the ultimate goal.

Topics: Brain Ischemia; Calcium Channel Blockers; Clinical Trials as Topic; Cytoprotection; Excitatory Amino Acid Antagonists; Humans; Neuroprotective Agents; Piperidines; Thiazoles

Although stroke is a major cause of morbidity and mortality, it is only relatively recently that a concerted effort has been made to develop acute treatments. Thrombolytics, such as recombinant tissue plasminogen activator (rt-PA), may benefit selected patients within 3 h of cerebral infarction. CUrrently, rt-PA is only licensed for use in the United States. Many potential strategies for neuroprotection exist and are currently under investigation. Because the mechanisms of neurotoxicity involve numerous interdependent processes, it may be that the interpretation of a single site in the cascade of events is insufficient to provide effective neuroprotection. Drugs acting at several sites in the neurotoxic cascade may be more effective, and the results of Phase III studies with the novel neoroprotectant lubeluzole are anticipated.

Topics: Cerebrovascular Disorders; Disease Models, Animal; Humans; Neuroprotective Agents; Piperidines; Plasminogen Activators; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Thiazoles; Thrombolytic Therapy; Tissue Plasminogen Activator

Hyperglycaemia in acute ischaemic stroke is traditionally associated with a worsened outcome. However, it is unclear whether the impact of hyperglycaemia on stroke outcome is similar in lacunar and non-lacunar infarctions. The relation between serum glucose measured within 6 h after stroke onset and functional outcome was investigated in 1375 ischaemic stroke patients who had been included in two placebo-controlled trials with lubeluzole. The endpoint was a favourable outcome, defined as a modified Rankin Scale score < or =2 at 3 months. Classification into lacunar (n = 168) and non-lacunar (n = 1207) strokes was based on clinical criteria according to the Oxfordshire Community Stroke Project and findings on brain CT scan. Hyperglycaemia was defined as blood glucose >8 mmol/l. A possible concentration-dependent effect of glucose on outcome was investigated in both lacunar and non-lacunar stroke. Multivariate analysis showed that hyperglycaemia was associated with decreased odds of a favourable outcome in non-lacunar stroke (OR 0.60; 95% CI 0.41-0.88, P = 0.009), but with increased odds of a favourable outcome in lacunar stroke (multivariate OR for glucose >8 mmol/l: 2.70; 95% CI 1.01-7.13, P = 0.048). In non-lacunar stroke, there appeared to be a concentration-effect relation, as the odds of favourable outcome gradually decreased with increasing glucose levels. In lacunar stroke, an association with favourable outcome was observed with glucose levels >8 mmol/l, but this beneficial effect diminished with more severe hyperglycaemia >12 mmol/l. In conclusion, hyperglycaemia has a detrimental effect in non-lacunar stroke, but moderate hyperglycaemia may be beneficial in lacunar stroke.

Topics: Acute Disease; Aged; Aged, 80 and over; Blood Glucose; Brain Infarction; Female; Humans; Hyperglycemia; Male; Middle Aged; Neuroprotective Agents; Piperidines; Prognosis; Severity of Illness Index; Stroke; Thiazoles; Treatment Outcome

Topics: Drug Therapy, Combination; Feasibility Studies; Humans; Neuroprotective Agents; Piperidines; Stroke; Thiazoles

A neuroprotective drug may be safe and effective if given very early and in combination with recombinant tissue-type plasminogen activator (rt-PA) to acute stroke patients. No clinical trial has yet tested this hypothesis.. To assess the feasibility, safety and efficacy of simultaneously combining the neuroprotective drug lubeluzole with rt-PA.. Patients who qualified for and received rt-PA within 3 h of symptom onset were randomly allocated 1:1 to lubeluzole (7.5 mg i.v. over 1 h, then continuous 5-day infusion of 10 mg/day) or placebo. Infusion of the study medication was started before the end of the 1-hour rt-PA infusion. Inclusion criteria were the same as those of the FDA-approved guidelines for rt-PA, plus National Institutes of Health Stroke Scale (NIHSS) >5 and absence of serious ventricular arrhythmia, atrioventricular block or Q-T >450 ms. EKG was continuously monitored until 48 h after treatment. The primary outcomes were adverse events, especially hemorrhage and severe arrhythmia, and functionality as determined by the Barthel Index divided into >70,0-70 and dead.. 89 patients were randomized at 34 centers over 8 months. The study was terminated by the sponsor before the planned enrollment of 200 patients when a concurrent phase 3 trial of lubeluzole versus placebo given up to 8 h after stroke was negative. In our study, the mean NIHSS was 14.5, and the mean time from symptom onset to rt-PA was 2.5 h and to randomization to lubeluzole or placebo 3.2 h. Mortality was 26%, intracerebral hemorrhage occurred in 10% and serious adverse events in 51%. There were no differences between the two treatment groups in any of these variables, outcomes or in the Barthel Index or other measures of functionality.. Combining neuroprotective drugs such as lubeluzole simultaneously with rt-PA is feasible and safe. The efficacy of this strategy, using a potentially more effective neuroprotective agent, should be evaluated in an adequately powered clinical trial.

Topics: Activities of Daily Living; Aged; Cerebral Hemorrhage; Double-Blind Method; Feasibility Studies; Female; Glasgow Coma Scale; Humans; Male; Neuroprotective Agents; Piperidines; Plasminogen Activators; Prospective Studies; Recombinant Proteins; Stroke; Thiazoles; Tissue Plasminogen Activator

This trial was a double-blind, placebo-controlled, phase III trial with an 8-hour inclusion window to assess the efficacy and safety of an intravenous loading dose of 7.5 mg followed by a daily intravenous dose of 10 mg lubeluzole for 5 days in acute ischemic stroke patients.. A total of 1786 patients were randomized: 901 to lubeluzole and 885 to placebo. Overall, 212 patients (23.5%) from the lubeluzole group and 213 (24.1%) from the placebo group discontinued the trial prematurely. In the lubeluzole group 201 patients (22.3%) discontinued because of adverse events compared with 193 patients (21.8%) in the placebo group.. The primary population for the efficacy analysis comprised the core stroke patients (exclusion of older patients aged >75 years with severe stroke) in the 0- to 6-hour inclusion time window. The primary efficacy parameter was a 3-category functional status (Barthel Index 70 to 100/0 to 70/vegetative, dead) at week 12. In the lubeluzole group 207 patients (47.8%) were classified as mildly dependent/independent at week 12, 131 (30.3%) were moderately/severely dependent, and 95 (21.9%) were vegetative/dead. In the placebo group these numbers were 221 (54.4%), 112 (27.6%), and 73 (18.0%), respectively. Logistic regression analysis showed no statistically significant difference between the treatment groups (P:=0.162). Additionally, for none of the secondary efficacy parameters (mortality at week 12, modified Rankin score, total Barthel score) was a statistically significant difference between the lubeluzole and placebo groups obtained. There were no statistically significant differences between the 2 treatments for all treated patients, patients included within the 6- to 8-hour window, and patients with severe strokes aged >75 years. Overall, of all treated patients, 401 (22.5%) died: 203 (22.5%) in the lubeluzole group and 198 (22.4%) with placebo. Of all subjects treated, 853 (95%) on lubeluzole and 826 (93%) on placebo reported an adverse event during their treatment period or within the next 2 days after discontinuation of treatment. The most frequently observed adverse events were fever (25.9% lubeluzole; 23.4% placebo), constipation (20.2%; 19.7%), and headache (17.6%; 21.2%). Imbalances were found for atrial fibrillation (1.8% lubeluzole; 1.1% placebo) and QT prolongation (0.9%; 0.2%).. This study failed to show an efficacy of lubeluzole in the treatment of acute stroke. On the other hand, lubeluzole treatment by the current dosage schedule was not associated with a significant safety problem.

Topics: Acute Disease; Aged; Double-Blind Method; Drug Administration Schedule; Female; Humans; Infusions, Intravenous; Male; Neuroprotective Agents; Piperidines; Placebos; Severity of Illness Index; Stroke; Thiazoles; Treatment Outcome

It has been suggested that subtle signs of early cerebral infarction on CT are important indicators of outcome and of the effect of thrombolytic treatment in acute ischaemic stroke. We studied these signs prospectively, in 260 patients with an anterior circulation stroke from a European-Australian randomised trial of lubeluzole in acute ischaemic stroke. Interobserver reliability was assessed by means of the chi statistic. The validity of the early signs was assessed by comparing the assessments of the first CT with another CT at 1 week after the onset of stroke, and with stroke outcome at 12 weeks. Each initial CT study was assessed by two of a group of five reviewers, who were blinded to each other's assessments and to the findings on the follow-up CT. The images were assessed twice, once without clinical information and again after disclosure of the side (left or right hemisphere) of the lesion. All reviewers were experienced clinicians with a special interest and training in vascular neurology and CT. The median time between stroke onset and the first CT was 3.2 h; 59% of the patients were imaged within 3 h and 77% within 6 h. More than half of the patients (52%) had a large middle cerebral artery territory (MCA) infarct on follow-up CT. Chance-adjusted interobserver agreement (chi) for any early infarct was 0.27 (95% confidence interval (CI): 0.15 to 0.39). Agreement (chi) on the extent of a middle cerebral artery (MCA) infarct and on the indication for treatment with recombinant tissue plasminogen activator (rt-PA) was fair: 0.37 and 0.35, respectively. Patients with early signs of an infarct of more than 1/3 of the MCA territory were more likely to have a large MCA infarct on follow-up CT (odds ratio 5.7, 95% confidence interval 2.8-11.5); the positive and negative predictive value of these signs was 81% and 57%, respectively. Chance-adjusted interobserver agreement on early, subtle signs of a large MCA territory infarct on CT by neurologists was thus no more than fair, and the accuracy of prediction of actual infarct size on the basis of these signs only moderate, under circumstances which resemble everyday clinical practice.

Topics: Adult; Aged; Aged, 80 and over; Cerebral Infarction; Female; Fibrinolytic Agents; Humans; Male; Middle Aged; Neuroprotective Agents; Observer Variation; Piperidines; Predictive Value of Tests; Randomized Controlled Trials as Topic; Reproducibility of Results; Thiazoles; Thrombolytic Therapy; Time Factors; Tissue Plasminogen Activator; Tomography, X-Ray Computed

Early identification of acute stroke patients at risk of fatal brain swelling is necessary to facilitate implementation of aggressive therapies. Initial clinical, laboratory, and CT characteristics that may be used as selection criteria were analyzed to determine predictors of herniation and neurological death.. Data from the placebo arm of the Lubeluzole-International-9 trial were reviewed to identify patients with fatal brain edema. Early clinical, laboratory, and radiographic parameters were evaluated in a case-control design. Initial CT scans were analyzed for early ischemic abnormalities by 2 blinded investigators.. Twenty-three patients died from brain swelling, with minimum baseline National Institutes of Health Stroke Scale (NIHSS) scores of 20 (n=12; mean, 23.2+/-1.8) with left and 15 (n=11; mean, 17.6+/-2.2) with right hemispheric infarctions (P=0. 0001). A sample of 112 subjects with comparably severe strokes, but who did not die from brain swelling, was selected from the remaining population according to the same NIHSS scores. Among clinical and laboratory characteristics, nausea/vomiting within 24 hours after onset (odds ratio [OR], 5.1; 95% CI, 1.7 to 15.3; P=0.003) and 12-hour systolic blood pressure >/=180 mm Hg (OR, 4.2; 95% CI, 1.4 to 12.9; P=0.01) were independently associated with fatal brain swelling. Among radiographic factors, only hypodensity of >50% of the middle cerebral artery territory on initial CT scan was an independent predictor (OR, 6.1; 95% CI, 2.3 to 16.6; P=0.0004).. Patients with baseline NIHSS score >/=20 with left or >/=15 with right hemispheric infarctions within 6 hours of symptom onset who also have nausea/vomiting or >50% middle cerebral artery territory hypodensity are at high risk for developing fatal brain swelling.

Topics: Acute Disease; Aged; Brain Edema; Brain Ischemia; Case-Control Studies; Cerebral Arteries; Female; Hospital Mortality; Humans; Male; Middle Aged; Neuroprotective Agents; Piperidines; Prognosis; Risk Factors; Severity of Illness Index; Thiazoles; Tomography, X-Ray Computed

The single-dose pharmacokinetics of lubeluzole were investigated in 2 single-blind, placebo-controlled, dose-escalation studies in healthy male subjects. In the first study, 6 subjects received an intravenous infusion of 2.5, 5, and 10 mg lubeluzole. In the second study, a 15 mg dose of lubeluzole was administered to 6 subjects, of whom 5 also received 20 mg and 2 also 25 mg lubeluzole. Following the infusion, plasma lubeluzole concentrations decayed biphasically, with a mean distribution half-life (t1/2alpha) of 30 to 65 minutes and a mean terminal half-life (t1/2beta) of 15 to 24 hours. The results of the 2 studies indicate that lubeluzole exhibits linear kinetics over the dose range tested in healthy male subjects.

Topics: Adult; Humans; Infusions, Intravenous; Male; Middle Aged; Neuroprotective Agents; Piperidines; Single-Blind Method; Thiazoles

The safety and tolerability of single escalating doses of lubeluzole were evaluated in healthy male volunteers in 2 studies. In the first of 2 randomized, single-blind, placebo-controlled, dose-escalation studies, 6 subjects received single 30-minute infusions of 2.5, 5, and 10 mg of lubeluzole, and 2 additional subjects received placebo. In the second study 6 different subjects received a 1-hour infusion of 15 mg of lubeluzole, 5 of whom received the 20-mg dose, and 2 received 25 mg of lubeluzole. Two additional subjects received placebo. Small increases and decreases in PQ, QRS, QT, QTc, and QTm intervals were noted after infusion of all lubeluzole doses and placebo, however, these changes were within the normal ranges for these values except for the QTc for the 25-mg dose of lubeluzole. Significant prolongation of the QTc interval was observed at the end of the 1-hour infusion in both subjects receiving the 25-mg dose of lubeluzole. No clinically relevant changes in systolic time intervals, heart rate, blood pressure, and clinical laboratory values were noted in subjects receiving 2.5-25 mg of lubeluzole or placebo. Adverse experiences, predominantly lightheadedness and dizziness, were reported by subjects receiving doses of lubeluzole greater than or equal to 10 mg. Lubeluzole, administered as single intravenous doses of 2.5-15 mg, is safe and well tolerated in healthy male volunteers.

Topics: Adult; Cardiovascular Agents; Dose-Response Relationship, Drug; Electrocardiography; Hemodynamics; Humans; Injections, Intravenous; Male; Piperidines; Single-Blind Method; Thiazoles

Lubeluzole is a novel benzothiazole compound that has shown neuroprotective activity in preclinical models of ischemic stroke. The present multicenter, double-blind, placebo-controlled study was conducted to assess the efficacy and safety of lubeluzole in the treatment of ischemic stroke.. Seven hundred twenty-one patients with clinical symptoms of acute ischemic stroke were randomized to receive either lubeluzole (7.5 mg over 1 hour, followed by a continuous daily infusion of 10 mg for up to 5 days) or placebo. Treatment was initiated within 6 hours of symptom onset. Mortality at 12 weeks was the primary efficacy end point. Secondary efficacy end points included neurological recovery (based on the National Institutes of Health Stroke Scale [NIHSS]), functional status (based on the Barthel Index), and level of disability (based on the Rankin Scale). Safety assessments included standard and continuous electrocardiographic monitoring, physical examination, measurements of vital signs, clinical laboratory evaluation, and adverse events reports.. The overall mortality rate at 12 weeks for lubeluzole-treated patients was 20.7% compared to 25.2% for placebo-treated patients (NS). Controlling for relevant covariates, the degree of neurological recovery (NIHSS) at week 12 significantly favored lubeluzole over placebo (P = .033). Lubeluzole treatment similarly resulted in significantly greater improvements in functional status (Barthel Index) (P = .038) and overall disability (Rankin Scale) (P = .034) after 12 weeks. A global test statistic confirmed that lubeluzole-treated patients had a more favorable clinical outcome at 12 weeks (P = .041). The safety profile of lubeluzole resembled that of placebo.. Treatment with lubeluzole within 6 hours of the onset of ischemic stroke had a nonsignificant effect on mortality and resulted in improved clinical outcome compared with placebo, with no safety concerns.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Brain Ischemia; Cerebrovascular Disorders; Double-Blind Method; Female; Heart Diseases; Humans; Male; Middle Aged; Mortality; Neuroprotective Agents; Piperidines; Survival Analysis; Thiazoles; Treatment Outcome

We aimed to assess the safety and efficacy of lubeluzole in patients with a clinical diagnosis of acute (< 6 hours) ischemic stroke in the carotid artery territory.. A randomized, double-blind, placebo-controlled multicenter trial was conducted in 232 patients. Because treatment was administered within 6 hours and a CT scan was not mandatory before the start of treatment, 39 patients with either an intracerebral hemorrhage or ischemic stroke in the vertebrobasilar circulation were excluded from the primary efficacy analysis as prespecified in the protocol. Of the 193 patients with acute ischemic stroke in the carotid artery territory (target population), 61 received placebo, 66 lubeluzole 7.5 mg over 1 hour followed by 10 mg/d for 5 days, and 66 lubeluzole 15 mg over 1 hour followed by 20 mg/d for 5 days.. The trial, initially aimed at a patient inclusion of 270, was terminated prematurely according to the advice of the Safety Committee because of an imbalance in mortality between the treatment groups. Mortality rates at the final follow-up of 28 days for placebo, lubeluzole 10 mg/d, and lubeluzole 20 mg/d were, respectively, 18%, 6%, and 35% in the target population, results that were confirmed in the intent-to-treat population. Multivariate logistic regression analysis showed that the lower mortality in the lubeluzole 10 mg/d group was significantly in favor of the 10 mg/d treatment (P = .019). The higher mortality rate in the 20 mg/d group could be explained, at least in part, by an imbalance at randomization that led to a higher number of patients in that group with severe ischemic stroke. A total of 26 of 66 patients (39%) who received lubeluzole 10 mg/d had a score on the Barthel Index of > 70 at day 28, indicating no or mild disability, compared with 21 of 61 (34%) in the placebo group and 19 of 66 (29%) in the lubeluzole 20 mg/d group (P = NS).. In patients with acute ischemic stroke, the dosage regimen of 7.5 mg over 1 hour followed by 10 mg/d of intravenous lubeluzole is safe and statistically significantly reduced mortality. Further clinical trials in a larger number of patients are ongoing to confirm efficacy.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Brain Ischemia; Cardiovascular Agents; Carotid Artery Diseases; Cerebrovascular Disorders; Double-Blind Method; Drug Administration Schedule; Female; Follow-Up Studies; Humans; Infusions, Intravenous; Injections, Intravenous; Logistic Models; Male; Middle Aged; Multivariate Analysis; Piperidines; Placebos; Safety; Thiazoles

Acute ischemic stroke continues to devastate millions of individuals worldwide. Current treatments work to restore blood flow but not rescue affected tissue. Our goal was to develop a combination of neuroprotective agents administered intra-arterially following recanalization to target ischemic tissue. Using C57Bl/6J male mice, we performed tandem transient ipsilateral middle cerebral/common carotid artery occlusion, followed by immediate intra-arterial pharmacotherapy administration through a standardized protocol. Two pharmacotherapy agents, verapamil and lubeluzole, were selected based on their potential to modulate different aspects of the ischemic cascade; verapamil, a calcium channel blocker, works in an acute fashion blocking L-type calcium channels, whereas lubeluzole, an N-methyl-D-aspartate modulator, works in a delayed fashion blocking intracellular glutamate trafficking. We hypothesized that combination therapy would provide complimentary and potentially synergistic benefit treating brain tissue undergoing various stages of injury. Physiological measurements for heart rate and pulse distention (blood pressure) demonstrated no detrimental effects between groups, suggesting that the combination drug administration is safe. Tissue analysis demonstrated a significant difference between combination and control (saline) groups in infarct volume, neuronal health, and astrogliosis. Although a significant difference in functional outcome was not observed, we did note that the combination treatment group had a greater percent change from baseline in forced motor movement as compared with controls. This study demonstrates the safety and feasibility of intra-arterial combination therapy following successful recanalization and warrants further study.

Topics: Animals; Calcium Channel Blockers; Combined Modality Therapy; Infusions, Intra-Arterial; Ischemic Stroke; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Piperidines; Thiazoles; Treatment Outcome; Verapamil

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy.

Topics: Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Doxorubicin; Female; Humans; Ovarian Neoplasms; Piperidines

Lubeluzole, a neuroprotective anti-ischemic drug, was tested for its ability to act as both antibiotic chemosensitizing and antipropulsive agent for the treatment of infectious diarrhea.. In the present report, the effect of lubeluzole against antidiarrheal target was tested. The antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated together with its ability to affect ileum and colon contractility.. Concerning the antimicrobial activity, lubeluzole showed synergistic effects when used in combination with minocycline against four common Gram-positive and Gram-negative bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922), although relatively high doses of lubeluzole were required. In ex vivo experiments on sections of gut smooth muscles, lubeluzole reduced the intestinal contractility in a dose-dependent manner, with greater effects observed on colon than on ileum, and being more potent than reference compounds otilonium bromide and loperamide.. All above results identify lubeluzole as a possible starting compound for the development of a novel class of antibacterial adjuvants endowed with spasmolytic activity.

Topics: Animals; Anti-Bacterial Agents; Antidiarrheals; Bacteria; Colon; Diarrhea; Dose-Response Relationship, Drug; Gram-Negative Bacterial Infections; Gram-Positive Bacterial Infections; Guinea Pigs; Ileum; Loperamide; Male; Microbial Sensitivity Tests; Muscle Contraction; Neuroprotective Agents; Piperidines; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Thiazoles

Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking.. We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding.. We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution.. This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Topics: Animals; CHO Cells; Cricetulus; Ether-A-Go-Go Potassium Channels; HEK293 Cells; Humans; Molecular Docking Simulation; Neuroprotective Agents; Patch-Clamp Techniques; Piperidines; Point Mutation; Protein Binding; Protein Conformation, alpha-Helical; Thiazoles

An affinity capillary electrophoresis (ACE) method to estimate apparent dissociation constants between bovine brain calmodulin (CaM) and non-peptidic ligands was developed. The method was validated reproducing the dissociation constants of a number of well-known CaM ligands. In particular, the potent antagonist 125-C9 was ad hoc synthesized through an improved synthetic procedure. The ACE method was successfully applied to verify CaM affinity for lubeluzole, a well-known neuroprotective agent recently proved useful to potentiate the activity of anti-cancer drugs. Lubeluzole was slightly less potent than 125-C9 (Kd = 2.9 ± 0.7 and 0.47 ± 0.06 μM, respectively) and displayed Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibition (IC50 = 40 ± 1 μM). Possible binding modes of lubeluzole to CaM were explored by docking studies based on the X-ray crystal structures of several trifluoperazine-CaM complexes. An estimated dissociation constant in good agreement with the experimental one was found and the main aminoacidic residues and interactions contributing to complex formation were highlighted. The possibility that interference with Ca(2+) pathways may contribute to the previously observed chemosensitizing effects of lubeluzole on human ovarian adenocarcinoma and lung carcinoma cells are discussed.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Cattle; Humans; Molecular Docking Simulation; Piperidines; Protein Conformation; Protein Kinase Inhibitors; Thiazoles

Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.

Topics: Animals; Carbamazepine; Disease Models, Animal; Flecainide; HEK293 Cells; Humans; Mexiletine; Muscle, Skeletal; Myotonia Congenita; Orphenadrine; Piperidines; Propafenone; Rats; Rats, Wistar; Riluzole; Sodium Channel Blockers; Thiazoles

Three new, concise, and protecting group-free synthetic routes for (RS)- and (S)-lubeluzole are reported in higher (46-62%) overall yields compared to the reported procedures (6-35%). The key steps involve C-N bond formation via epoxide aminolysis and nucleophilic substitution of 2-chlorobenzothiazole with suitably designed precursor amines and are performed in aqueous medium. Water offers an advantage in promoting the reactions compared to organic solvents and its role is envisaged as hydrogen-bond mediated electrophile-nucleophile dual activation.

Topics: Hydrogen Bonding; Molecular Structure; Piperidines; Solvents; Stereoisomerism; Thiazoles; Water

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combination with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear prevalence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for both drugs were observed over a wide concentration window (0.005-5 μM), the lowest limit being at least 40 times lower than human plasma concentrations previously reported as causing serious side effects.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Doxorubicin; Drug Synergism; Female; Humans; Lung Neoplasms; Ovarian Neoplasms; Paclitaxel; Piperidines; Stereoisomerism; Thiazoles

Lubeluzole, which acts on various targets in vitro, including voltage-gated sodium channels, was initially proposed as a neuroprotectant. The lubeluzole structure contains a benzothiazole moiety [N-methyl-1,3-benzothiazole-2-amine (R-like)] related to riluzole and a phenoxy-propranol-amine moiety [(RS)-1-(3,4-difluorophenoxy)-3-(piperidin-1-yl)propan-2-ol (A-core)] recalling propranolol. Both riluzole and propranolol are efficient sodium channel blockers. We studied in detail the effects of lubeluzole (racemic mixture and single isomers), the aforementioned lubeluzole moieties, and riluzole on sodium channels to increase our knowledge of drug-channel molecular interactions. Compounds were tested on hNav1.4 sodium channels, and on F1586C or Y1593C mutants functionally expressed in human embryonic kidney 293 cells, using the patch-clamp technique. Lubeluzole blocked sodium channels with a remarkable effectiveness. No stereoselectivity was found. Compared with mexiletine, the dissociation constant for inactivated channels was ~600 times lower (~11 nM), conferring to lubeluzole a huge use-dependence of great therapeutic value. The F1586C mutation only partially impaired the use-dependent block, suggesting that additional amino acids are critically involved in high-affinity binding. Lubeluzole moieties were modest sodium channel blockers. Riluzole blocked sodium channels efficiently but lacked use dependence, similar to R-like. F1586C fully abolished A-core use dependence, suggesting that A-core binds to the local anesthetic receptor. Thus, lubeluzole likely binds to the local anesthetic receptor through its phenoxy-propranol-amine moiety, with consequent use-dependent behavior. Nevertheless, compared with other known sodium channel blockers, lubeluzole adds a third pharmacophoric point through its benzothiazole moiety, which greatly enhances high-affinity binding and use-dependent block. If sufficient isoform specificity can be attained, the huge use-dependent block may help in the development of new sodium channel inhibitors to provide pharmacotherapy for membrane excitability disorders, such as myotonia, epilepsy, or chronic pain.

Topics: Anesthetics, Local; Binding Sites; Cell Line; HEK293 Cells; Humans; Membrane Potentials; Membrane Proteins; Mutation; NAV1.4 Voltage-Gated Sodium Channel; Neuroprotective Agents; Piperidines; Protein Binding; Protein Interaction Domains and Motifs; Protein Isoforms; Riluzole; Sodium Channel Blockers; Thiazoles

Lubeluzole [(S)-9] has been synthesized by a convergent synthesis, alkylation of N-methyl-N-piperidin-4-yl-1,3-benzothiazol-2-amine (4) with (+)-(R)-1-chloro-3-(3,4-difluorophenoxy)propan-2-ol [(+)-(R)-8] being the key step. Alcohol (+)-(R)-8 was obtained from commercially available (R)-epichlorohydrin [(R)-6], while the thiazole derivative 4 was easily obtained starting from N-protected piperidin-4-one (1) in a three-step procedure. The same method was used in order to obtain both the (R)-stereoisomer of lubeluzole [(R)-9] and its racemate [(RS)-9]. Overall yields ranged from 20% to 35%. The enantiomeric excess values for (S)-9 and (R)-9 were 97% and 94% respectively, as analyzed by chiral HPLC.

Topics: Cardiovascular Agents; Molecular Structure; Piperidines; Stereoisomerism; Thiazoles

Traumatic brain injury (TBI) involves direct mechanical damage, which may be aggravated by secondary insults such as ischemia. We utilized an in vitro model of stretch-induced injury to investigate the effects of mechanical and combined mechanical/ischemic insults to cultured mouse cortical cells. Stretch injury alone caused significant neuronal loss and increased uptake of the dye, propidium iodide, suggesting cellular membrane damage to both glia and neurons. Exposure of cultures to ischemic conditions for 24h, or a combination of stretch and 24h of ischemia, caused greater neuronal loss compared to stretch injury alone. Next, we tested the neuroprotective effects of superoxide dismutase (SOD), and the nitric oxide (NO) synthase inhibitors 7-nitroindazole (7-NINA) and lubeluzole. In general, these agents decreased neuronal loss following stretch injury alone, but were relatively ineffective against the combined injury paradigm. A combination of SOD with 7-NINA or lubeluzole offered no additional protection than single drug treatment against stretch alone or combined injury. These results suggest that the effects of primary mechanical damage and secondary ischemia to cortical neurons are cumulative, and drugs that scavenge superoxide or reduce NO production may not be effective for treating the secondary ischemia that often accompanies TBI.

Topics: Animals; Brain Ischemia; Cell Count; Cell Survival; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Immunohistochemistry; Indazoles; Mice; Mitogen-Activated Protein Kinase 1; Neurons; Neuroprotective Agents; Nitric Oxide Synthase Type I; Physical Stimulation; Piperidines; Stress, Mechanical; Superoxide Dismutase; Thiazoles

Lubeluzole [S-4-(2-benzothiazolylmethylamino)-alpha-((3,4-difluorophenoxy)methyl)-1-piperidineethanol] reduces the severity of cerebral injury in animal models of brain ischemia. Its beneficial effects may include decreased concentration of extracellular glutamate, blockade of sodium and calcium channels, and attenuation of nitric oxide-mediated neuronal death. Previous studies have shown that global cerebral ischemia in rabbits impaired the subsequent acquisition of a trace-conditioned eyeblink reflex. Here, we examined the effect of preischemic treatment with lubeluzole on the acquisition of a trace-conditioned eyeblink response after 6.5 min of global cerebral ischemia. Three groups of rabbits underwent cerebral ischemia: one group underwent ischemia alone (I) and two groups underwent ischemia and also received lubeluzole (L(1), 1.25 mg/kg, and L(2), 2.5 mg/kg). All animals were subsequently trained using classical trace conditioning. Each training session consisted of the presentation of the conditioned stimulus (an 85-dB, 6-kHz auditory tone lasting for 100 ms) followed by a trace interval (a period of 300 ms during which no external stimulus was delivered) followed finally by the delivery of the unconditioned stimulus (a 150-ms puff of air directed at the cornea). We found that animals receiving preischemic administration of 1.25 mg/kg of lubeluzole demonstrated a significantly improved acquisition of the trace-conditioned reflex as compared to animals that did not receive lubeluzole. This finding demonstrates improved long-term neurobehavioral outcome with preischemic administration of 1.25 mg/kg of lubeluzole.

Topics: Animals; Brain Ischemia; Conditioning, Psychological; Learning; Male; Piperidines; Rabbits; Thiazoles

The effects of lubeluzole on sodium currents were examined in guinea-pig isolated cardiac myocytes by use of the whole-cell patch clamp technique. Lubeluzole (0.01-100 microM) reduced peak Na+ current (INa) obtained at a holding potential of -80 mV with an IC50 value of 9.5 (3.5-21.9) microM and a Hill coefficient of 1.1. These effects were rapid and reversible. Lubeluzole (10 microM) produced a shift in the inactivation curve to hyperpolarized potentials (by -9.7 mV, P < 0.05), but produced no change in the voltage-dependence of activation. Lubeluzole (10 microM) produced significant tonic block of INa obtained at a holding potential of -120 mV (2.7 +/- 1.4% and 27.5 +/- 5.8% for control and lubeluzole, respectively; n = 6; P < 0.05). Use-dependent block of INa was also observed. Recovery from block was delayed by lubeluzole (10 microM; tau1=4.4 +/- 6.2, tau2=22.7 +/- 1.5 milliseconds for control and tau1=311 +/- 144, tau2 = 672 +/- 23 milliseconds for lubeluzole; n = 6; P < 0.001) confirming use-dependency of block. The results indicate that lubeluzole produces both tonic and use-dependent block of cardiac sodium channels at concentrations similar to those that block neuronal sodium channels, due mainly to interaction of the drug with channels in the inactivated state.

Topics: Animals; Dose-Response Relationship, Drug; Guinea Pigs; Male; Membrane Potentials; Myocardium; Piperidines; Sodium Channels; Thiazoles

The aim of the present study was to test the neuroprotective effect of the novel benzothiazol compound lubeluzole on neuronal cell damage in fetal sheep arising from global cerebral ischemia. Thirteen fetal sheep were prepared at a mean gestational age of 127 +/- 1 d (term is at 147 d). Six fetuses were treated with lubeluzole (0.33 mg/kg estimated body weight) before induction of global cerebral ischemia (-90, -60, and -30 min), while the remainder (n = 7) received solvent. Cerebral ischemia was induced by occluding both carotid arteries for 30 min. Cerebral blood flow was measured by injecting radio-labeled microspheres before (-90 min), during (+3 min and +27 min), and after (+40 min, +3 h, and +72 h) cerebral ischemia. Neuronal cell damage was assessed in the cerebrum and deeper brain structures by light microscopy. Values are given as means +/- SD. In control fetuses, blood flow to the cerebrum was reduced from 100 +/- 25 mL.100 g(-1) min(-1) to less than 20 mL.100 g(-1) min(-1) during ischemia. Shortly after ischemia, hyperperfusion occurred (217 +/- 66 mL.100 g(-1)min(-1)) followed by a tendency toward hypoperfusion (72 +/- 17 mL.100 g(-1) min(-1)) later on (+3 h). Significant differences in blood flow to the various brain structures between the control and study groups could not be observed. Neuronal cell damage was concentrated in the parasagittal regions of the cerebrum. Preischemic application of lubeluzole did not have any effect on the extent of neuronal cell damage. From these results, we conclude that pretreatment with lubeluzole fails to protect the brain of fetal sheep near term from injury after transient global cerebral ischemia. However, because the observation period lasted only 3 d, a possible effect of lubeluzole on pathophysiological mechanisms inducing delayed neuronal cell death cannot be fully excluded.

Topics: Acid-Base Equilibrium; Animals; Blood Gas Analysis; Brain; Brain Ischemia; Cerebrovascular Circulation; Female; Fetal Diseases; Gestational Age; Humans; Neuroprotective Agents; Piperidines; Pregnancy; Sheep; Thiazoles

The administration of a neurotoxic regimen of methamphetamine (MA) produces an acute elevation in the extracellular concentrations of dopamine and glutamate in the striatum and a long-term depletion of striatal dopamine content in rats. The intent of the present study was to determine whether attenuation of the MA-induced increase in extracellular glutamate would prevent the depletion of striatal dopamine. Male rats were treated with MA (10 mg/kg, i.p.) or vehicle every 2 h for four injections and concomitantly perfused intrastriatally with either artificial cerebrospinal fluid or lubeluzole (300 microM), a novel neuroprotectant that has been shown to prevent the increase in extracellular glutamate after the induction of neocortical infarct in rats. Lubeluzole significantly attenuated the MA-induced increase in extracellular glutamate in the striatum without affecting the MA-induced increase in extracellular dopamine or the MA-induced hyperthermic response. Nevertheless, lubeluzole did not prevent the long-term depletion of striatal dopamine produced by a neurotoxic regimen of MA. These results suggest that the MA-induced depletion of striatal dopamine may not be dependent on the increased extracellular concentration of striatal glutamate.

Topics: Animals; Body Temperature; Dopamine; Drug Interactions; Extracellular Space; Glutamic Acid; Male; Methamphetamine; Neostriatum; Neurons; Neuroprotective Agents; Piperidines; Rats; Rats, Sprague-Dawley; Sodium Channels; Thiazoles; Veratridine

1-Chloro-3-(3,4-difluorophenoxy)-2-propanol was kinetically resolved by lipase-catalyzed esterification with vinyl butanoate in organic medium to yield the (S)-butanoate and the (R)-alcohol as the remaining substrate. In an enantioconvergent synthesis the mixture was subject to Mitsunobu esterification in one pot which converted the (R)-alcohol to the (S)-ester. The (S)-butanoate was hydrolyzed by lipase catalysis to give (S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol. The two enantiopure chiral building blocks were used for synthesis of Lubeluzole and its enantiomer respectively.

Topics: Cardiovascular Agents; Catalysis; Lipase; Piperidines; Propanols; Stereoisomerism; Substrate Specificity; Thiazoles

Increases in extracellular glutamate during cerebral ischemia may play an important role in neuronal injury. Lubeluzole is a novel neuroprotective drug, which in previous in vitro and focal ischemia studies has been shown to inhibit nitric oxide synthesis, to block voltage-gated Na+-ion channels, and to inhibit glutamate release. In this study, we investigated the ability of lubeluzole to inhibit glutamate accumulation during episodes of transient global cerebral ischemia. Twenty-five New Zealand white rabbits were randomized to one of four groups: a normothermic control group; a hypothermic group; a 1.25 mg/kg lubeluzole group; or a 2.5 mg/kg lubeluzole group. The animals were anesthetized, intubated, and ventilated before microdialysis probes were placed in the hippocampus. Lubeluzole was given intravenously 90 min before the onset of ischemia. Esophageal temperature was maintained at 38 degrees C in the control, and lubeluzole treated groups, while the animals in the hypothermia group were cooled to 30 degrees C. A 15-min period of global cerebral ischemia was produced by inflating a neck tourniquet. Glutamate concentrations in the microdialysate were determined using high-performance liquid chromatography (HPLC). During ischemia and early reperfusion, glutamate concentrations increased significantly in the control group and returned to baseline after 15 min of reperfusion. In the lubleuzole 2.5 mg/kg and hypothermia groups, glutamate levels were significantly lower (P<0.05) than in the control group and there was no significant change from baseline levels during the entire experiment. This study suggests that lubeluzole is effective in inhibiting extracellular glutamate accumulation during global cerebral ischemia, and has the potential to produce potent neuroprotection when instituted prior to an ischemic event.

Topics: Action Potentials; Animals; Body Temperature; Brain Ischemia; Cardiovascular Physiological Phenomena; Extracellular Space; Glutamic Acid; Hippocampus; Microdialysis; Nerve Degeneration; Neurons; Neuroprotective Agents; Piperidines; Rabbits; Reperfusion Injury; Respiratory Physiological Phenomena; Thiazoles

Lubeluzole is a newly designed neuroprotectant which has proved effective in the treatment of experimental stroke in rats, mainly by inhibition of the glutamate-activated NO pathway, but also by counteracting osmotic stress by a mechanism associated with the release of the osmotically active amino acid taurine (Tau). Here we show that lubeluzole administered i.p. decreases by 25% the high (50 mM) K+-evoked accumulation of Tau in striatal microdialysates of healthy rats and by 34% in rats with thioacetamide-induced hepatic failure, where the increased extracellular accumulation of Tau signifies ongoing hepatic encephalopathy. Lubeluzole does not affect the nonstimulated accumulation of Tau in either group of rats. The results indicate that lubeluzole may be effective in ameliorating ionic or osmotic stress in a range of pathological conditions involving the rise of extracellular K+, and also in decreasing the vulnerability to stress in rats with hepatic failure.

Topics: Animals; Extracellular Space; Glutamic Acid; Hepatic Encephalopathy; Male; Microdialysis; Neostriatum; Neurons; Neuroprotective Agents; Osmotic Pressure; Piperidines; Potassium Chloride; Rats; Rats, Wistar; Taurine; Thiazoles; Thioacetamide

The ability to elucidate the molecular mechanisms that modulate programmed cell death (PCD) may provide the crucial clues to unravel the cellular basis of neurodegenerative disorders. Employing both a novel assay to follow serially PCD in individual living neurons and the neuroprotective agent lubeluzole as an investigative tool, we examined the development of nitric oxide (NO)-induced PCD over time through the reversible annexin V labelling of membrane phosphatidylserine (PS) exposure and the electron microscopy of genomic DNA in primary rat hippocampal neurons. Exposure to the NO generators SNP (300 microM) or NOC-9 (300 microM) alone increased annexin V-positive neurons in the population from 7% +/- 4% in untreated cultures to 13% +/- 4% at 1 hr and to 61% +/- 5% at 24 hr. Administration of a neuroprotective concentration of lubeluzole (750 nM) at the time of NO exposure initially prevented the exposure of PS residues, but consistently maintained DNA integrity over a 24 hr period. During posttreatment paradigms of lubeluzole (750 nM) at 2, 4, and 6 hr following NO exposure, progression of membrane PS inversion was reversed and subsequently suppressed over a 24 hr course. Our work illustrates that neuronal PCD is composed of at least two physiologically distinct and separate pathways that consist of the externalization of membrane PS residues and the independent maintenance of genomic DNA integrity. In addition, neuronal injury is fluid and reversible in nature, suggesting a "window of opportunity" for the repair and reversal of neurons yet to be committed to PCD.

Topics: Animals; Animals, Newborn; Annexin A5; Apoptosis; Biomarkers; Cell Membrane; Cell Nucleus; DNA; Enzyme Inhibitors; Free Radical Scavengers; Hippocampus; Neurons; Neuroprotective Agents; Nitric Oxide; Phosphatidylserines; Piperidines; Rats; Rats, Sprague-Dawley; Thiazoles; Triazenes

We studied the neuroprotective effect of lubeluzole, a NOS (nitric oxide synthase) pathway modulator, on the development of ischemic damage within the first six hours after a photochemically induced neocortical infarct in rats using diffusion-weighted MRI and Apparent Diffusion Coefficient (ADC) maps. A unilateral photochemical infarct was induced in the hindlimb sensorimotor neocortex of Wistar rats. One hour after infarction, rats received either vehicle (n=10) or lubeluzole (n=11; a 0.31 mg/kg i.v. bolus followed by a one-hour 0.31 mg/kg i.v. infusion). During the first six hours after infarct induction, multislice T2- and Diffusion-Weighted magnetic resonance images (MRI) were obtained to measure percent change of volume of ischemic damage, whereas regional ADC maps were used to measure time-dependent density of ischemic damage. Lubeluzole reduced the percent increase of volume of ischemic damage relative to baseline (at 1 h after infarct induction just before drug treatment), by 18% at 5 and 6 hrs after infarct induction. Lubeluzole attenuated the ADC decreases in the peripheral rim of the infarct, but left the ADC values in the core unaffected. In conclusion, the neuroprotectant lubeluzole attenuates growth of ischemic damage as well as its density in the periphery of a photochemically induced neocortical infarct in rats.

Topics: Animals; Cerebral Infarction; Diffusion; Magnetic Resonance Imaging; Male; Motor Cortex; Neocortex; Neuroprotective Agents; Photochemistry; Piperidines; Rats; Rats, Wistar; Somatosensory Cortex; Thiazoles

1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD). 2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 microM) or SIN-1 (300 microM) directly induced PCD. 3. Neurons positive for PCD increased from 22+/-3% (untreated) to 72+/-3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72+/-3% (NO only) to 25+/-3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure. 4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.

Topics: Animals; Annexin A5; Apoptosis; Biomarkers; Cell Membrane; Cell Survival; DNA Fragmentation; Hippocampus; Molsidomine; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Phosphatidylserines; Piperidines; Rats; Rats, Sprague-Dawley; Thiazoles; Vasodilator Agents

Besides other pharmacological effects, the neuroprotective compound lubeluzole blocks low-voltage-activated (iLVA) and high-voltage-activated (iHVA) calcium channel currents. We investigated the site of action of lubeluzole on Ca(2+) channels in isolated dorsal root ganglion cells of the rat, using whole-cell voltage clamp. Experiments with extracellular application of 3 microM lubeluzole (pK(a) = 7.6) at different values of extracellular pH suggest that the protonated form of lubeluzole contributes to the block of iLVA and iHVA from the extracellular side. The partial block of iLVA and iHVA by 3 microM lubeluzole at extracellular pH 9 and intracellular pH (pH(i)) 9 indicates that the uncharged form of lubeluzole (L) may contribute to the block as well. The voltage-dependent acceleration of the apparent inactivation of iHVA by lubeluzole was much more pronounced at lower pH(i), which is consistent with membrane penetration of L and an open channel block of iHVA by the prononated form of lubeluzole acting from the intracellular side. Decreasing pH(i) induced a negative shift of the half-inactivation potential of iLVA and increased the lubeluzole-induced block of iLVA. Experiments with extracellular or intracellular application of a quaternary ammonium derivative of lubeluzole (R133121), which was less potent than lubeluzole, support the above conclusions on the side of action of lubeluzole. Application of lubeluzole via the patch pipette affected iLVA and iHVA only minimally compared with extracellular application, probably partly due to efflux of L through the cell membrane. These experiments suggest that lubeluzole blocks Ca(2+) channels from both the extracellular and the intracellular side.

Topics: Animals; Benzothiazoles; Calcium Channel Blockers; Calcium Channels; Electric Stimulation; Ganglia, Spinal; Hydrogen-Ion Concentration; Kinetics; Male; Neurons, Afferent; Neuroprotective Agents; Patch-Clamp Techniques; Piperidines; Rats; Rats, Wistar; Thiazoles

The effect of the neuroprotective drug lubeluzole on cortical receptor binding was investigated in animals with photothrombotic ischemic lesions. Control animals were treated with the inactive stereoisomer of the drug. Lubeluzole was applied intravenously as a single bolus (0.31 mg/kg) followed by a 1-h infusion of 0.31 mg/kg. Lubeluzole selectively increased gamma-amino-butyric acid(A) (GABA(A)) receptor binding but had no significant and/or consistent effects on N-methyl-D-aspartate (NMDA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainate receptors. Lubeluzole caused a significant up-regulation of GABA(A) receptor binding in the lesioned area as well as in unimpaired cortical areas of both hemispheres. This effect appeared in the hours following the lesion and peaked at 24 h. Our findings suggest that reduced cortical excitability brought about by increased binding capacities of GABA(A) receptors may contribute to the neuroprotective effect of lubeluzole.

Topics: Animals; Brain Ischemia; Male; Muscimol; Neocortex; Neuroprotective Agents; Photochemistry; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, AMPA; Receptors, GABA-A; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Rose Bengal; Thiazoles; Tritium; Up-Regulation

The neuroprotective effects of a novel synthetic compound, M50463, have been determined by using embryonic rat neocortical neurons in various culture conditions. M50463 was initially characterized as a potent specific ligand for a voltage-dependent sodium channel by radioligand binding studies. In fact, M50463 inhibited neuronal cell death induced by veratrine and inhibited an increase of the intracellular calcium level in neurons evoked by veratrine. In addition to such expected effects, M50463 had the ability to prevent glutamate neurotoxicity, to promote the neuronal survival in serum-deprived medium and to prevent nitric oxide-induced neurotoxicity. These results suggested that M50463 is not a simple sodium channel blocker, but a neuroprotective agent which has some crucial mechanism of action on neuronal death occurring in various situations, and it is a novel, innovative candidate for neuroprotective therapy for various neurodegenerative disorders.

Topics: Acetylcysteine; Animals; Antihypertensive Agents; Batrachotoxins; Binding Sites; Binding, Competitive; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cell Death; Cells, Cultured; Culture Media, Serum-Free; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fetus; Glutamic Acid; Indoles; Neurons; Neuroprotective Agents; Neurotoxins; Nicardipine; Nitroprusside; Piperidines; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Thiazoles; Veratrine; Vitamin E

Lubeluzole, a novel nitric oxide synthase (NOS) pathway modulator, was shown to be neuroprotective in cerebral ischemia as studied in animal models and clinical trials. The present study investigated the effect of lubeluzole on contusion volume and brain edema following traumatic brain injury. Sprague-Dawley rats (n = 36) were subjected to cortical impact injury. Lubeluzole (0.8 mg/kg i.v.; n = 18) or a corresponding volume of vehicle (n = 18) was injected 15 and 75 minutes following trauma. Animals were sacrificed 24 hours following trauma. Contusion volume was measured planimetrically from coronal slices stained with hematoxylin and eosin. In this group, T2-weighted magnetic resonance imaging (MRI) was also performed 90 minutes and 6 and 24 hours after trauma. Hemispheric swelling and water content were determined gravimetrically 24 hours after trauma. In this group, intracranial pressure (ICP), mean arterial blood pressure (MABP), and cerebral perfusion pressure (CPP) were monitored for 30 minutes before sacrifice. Lubeluzole did not reduce contusion volume, hemispheric swelling, or water content. ICP, MABP, and the resulting CPP did not differ between treated and untreated rats 24 hours after injury. T2-weighted MRI revealed a higher volume of edema at 90 minutes after trauma in treated rats. However, at 6 and 24 hours after trauma, no significant difference was discernible. Under these experimental conditions, lubeluzole fails to exert beneficial effects following experimental traumatic brain injury (TBI).

Topics: Animals; Blood Gas Analysis; Blood Pressure; Body Water; Brain Edema; Brain Injuries; Enzyme Inhibitors; Intracranial Pressure; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Piperidines; Rats; Rats, Sprague-Dawley; Thiazoles; Time Factors

In this study, the isolated chicken retina was used as an "in-vitro"-model for investigation of neuronal lesions to show the neuroprotective effects of lubeluzole. Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in photochemical stroke models in rats. In the retina, the typical cell swelling of a developing lesion is accompanied by a very strong intrinsic optical signal (IOS), occurring simultaneous with the electrical signal which is based on changes in light scattering. In the presented model, lesions were elicited electrically with a tungsten microelectrode (0.1 MOmega). The degree of damage was evaluated with optical methods by measuring area and brightness of the affected tissue. Lubeluzole was much more effective in reducing the growth of the lesions than its R-isomer. However, both compounds enhanced the possibility of the neuronal tissue to recover after excitotoxic stimuli.

Topics: Animals; Chickens; Electric Stimulation; Electrophysiology; In Vitro Techniques; Neurons; Neuroprotective Agents; Piperidines; Retina; Stereoisomerism; Thiazoles

Topics: Acute Disease; Brain Ischemia; Drugs, Investigational; Humans; Neuroprotective Agents; Piperidines; Stroke; Thiazoles

Topics: Animals; Chlormethiazole; Cytidine Diphosphate Choline; Drugs, Investigational; Humans; Neuroprotective Agents; Nootropic Agents; Piperidines; Stroke; Thiazoles

This study uses a new strategy to investigate the hypothesis that, of the various Ca2+ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca2+ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca2+ concentrations ([Ca2+]o). So, at low [Ca2+]o the K+-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60-70% by 2 microM omega-agatoxin IVA (P/Q-type Ca2+ channel blockade), by 3 microM omega-conotoxin MVIIC (N/P/Q-type Ca2+ channel blockade), or by 3 microM lubeluzole (N/P/Q-type Ca2+ channel blockade); in high [Ca2+]o these blockers inhibited the responses by only 20-35%. At 1-3 microM omega-conotoxin GVIA (N-type Ca2+ channel blockade) or 3 microM furnidipine (L-type Ca2+ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca2+]o. Combined furnidipine plus omega-conotoxin MVIIC, omega-agatoxin IVA or omega-conotoxin GVIA exhibited additive blocking effects at both Ca2+ concentrations. The results suggest that Q-type Ca2+ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca2+ that enters the cell through a Ca2+ channel located near to chromaffin vesicles will saturate the K+ secretory response at both [Ca2+]o, i.e. 0.5 mM and 5 mM. In contrast, Ca2+ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca2+]o.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Catecholamines; Cattle; Chromaffin Cells; Dihydropyridines; In Vitro Techniques; Neurosecretory Systems; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Piperidines; Potassium; Spider Venoms; Thiazoles

Previously reported effects of lubeluzole, such as inhibition of glutamate release, inhibition of nitric oxide (NO) synthesis and blockage of voltage-gated Na+- and Ca2+-ion channels, suggest a neuroprotective action of this drug. Here we report about the effects of lubeluzole and its R-isomer on glutamate-induced neuronal cell death in mixed hippocampal cultures. In addition, we studied the effect of lubeluzole in focal cerebral ischemia models in mice and rats. In hippocampal cultures exposed to 500 nM glutamate for 1 h, lubeluzole (0.1-100 nM), but not the R-isomer (1-100 nM), reduced the percentage of damaged neurons from 42 +/- 8% to 18 +/- 7% (P < 0.01). In mice and rats, lubeluzole reduced ischemic brain damage, when administered immediately after middle cerebral artery occlusion. Interestingly, the protective effect (reduction of the infarct volume in rats to 77% of control; P < 0.01) was also found when the lubeluzole treatment (2.5 mg/kg) was started 3 h after ischemia. Especially this latter effect suggests that lubeluzole will be a useful drug for stroke therapy.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Ischemia; Cells, Cultured; Excitatory Amino Acids; Glutamic Acid; Hippocampus; Male; Mice; Neurons; Neuroprotective Agents; Piperidines; Rats; Rats, Inbred F344; Stereoisomerism; Thiazoles

Seizures are one of the most frequent complications after cerebral ischemia in patients. Up to now it is unknown which mechanisms are responsible for this. As shown previously photothrombotic infarction in rat neocortex leads to a sweeping suppression of GABAergic inhibition. In this study we investigated whether and to what extent epileptiform discharges can be observed in this ischemia model. In neocortical slices from lesioned animals we did not find spontaneous epileptic activity or paroxysmal depolarisation shifts. However, ipsi- and contralateral to a photothrombotic lesion the frequency of double and multiple discharges was markedly increased when compared to unlesioned controls. Surprisingly, neither the drug lubeluzole which was has been shown to prevent the GABAergic disinhibition observed after photothrombotic lesioning of rat neocortex, nor the prevention of spreading depressions by the NMDA-receptor antagonist MK-801 during lesion induction significantly affected the frequency of epileptiform discharges. This indicates that the epileptiform discharges are probably caused by functional alterations of glutamatergic receptors.

Topics: Animals; Cerebral Infarction; Dizocilpine Maleate; Electric Stimulation; Electrophysiology; Epilepsy; In Vitro Techniques; Intracranial Embolism and Thrombosis; Light; Male; Neocortex; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Reaction Time; Thiazoles

Topics: Acute Disease; Brain Ischemia; Cardiovascular Agents; Cerebrovascular Disorders; Humans; Piperidines; Thiazoles

Topics: Cerebrovascular Disorders; Dose-Response Relationship, Drug; Double-Blind Method; Humans; Neuroprotective Agents; Piperidines; Randomized Controlled Trials as Topic; Research Design; Thiazoles; Thrombolytic Therapy; Time Factors; Treatment Outcome

Lubeluzole is neuroprotective in a photochemical stroke model, whereas the (R)-enantiomer of the same molecule is not [De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Janssen M and Van Reet G (1996) J Pharmacol Exp Ther 279:748-758]. We investigated the effects of lubeluzole and the (R)-enantiomer on voltage-sensitive Ca++ channels of isolated rat dorsal root ganglion cells, using whole-cell voltage-clamp, with Ba++ as the charge carrier. Both compounds blocked the low-voltage-activated Ba++ current (iLVA or T current) with an IC50 value of 1.2 microM. Lubeluzole and the (R)-enantiomer also blocked the high-voltage-activated calcium channel current (iHVA), with IC50 values of 2.6 and 3.5 microM, respectively, and accelerated the apparent inactivation of iHVA. This acceleration was more pronounced with lubeluzole than with the (R)-enantiomer at 3 and 10 microM. Both compounds produced a clear tonic block of iLVA and iHVA, even in the absence of previous stimulation. Lubeluzole and the (R)-enantiomer induced a negative shift of the inactivation curve of iLVA and showed down the recovery from inactivation. This resulted in a stronger inhibition of iLVA at more depolarized conditioning potentials and higher stimulation frequencies. The block of iHVA was voltage and frequency dependent. Lubeluzole and the (R)-enantiomer also blocked iHVA in isolated rat superior cervical ganglion cells and cerebellar Purkinje cells. The Ca++ channel-blocking properties of lubeluzole may contribute to its neuroprotective effect. However, the small difference between the two enantiomers in inhibition of Ca++ channel currents does not explain the stereospecificity of the neuroprotective properties of lubeluzole in vitro and in vivo.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Dose-Response Relationship, Drug; Ganglia, Spinal; Male; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Stereoisomerism; Thiazoles

1. Incubation of bovine adrenal chromaffin cells with veratridine (10-100 microM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(-) enantiomer, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3-10 microM) decreased the veratridine-induced LDH release. 2. Penfluridol did not increase LDH release at concentrations 0.003-1 microM; 3-10 microM increased LDH release to 50-60%, after 24 h exposure. Penfluridol (0.03-0.3 microM) did not protect against the cytotoxic effects of veratridine; at 1 microM, 15% protection was produced. Higher concentrations (3-10 microM) enhanced the cytotoxic effects of veratridine. 3. Ba2+ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 microM lubeluzole and fully counteracted by 1 microM penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+. 4. Ouabain (10 microM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3-10 microM) and the lower concentrations of penfluridol (0.003-0.3 microM) prevented the ouabain cytotoxic effects. At higher concentrations (3 microM), penfluridol increased drastically the ouabain cytotoxic effects. 5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 microM. Lubeluzole (3-10 microM) or penfluridol (0.03-0.3 microM) had no cytoprotective effects against 6-OHDA. 6. Lubeluzole (3 microM), R91154 (3 microM) and penfluridol (1 microM) blocked the current through Na+ channels in voltage-clamped chromaffin cells (I(Na)) by around 20-30%. Ca2+ current through Ca2+ channels (I(Ca)) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversible. 7. Lubeluzole (3 microM) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura-2-loaded cells exposed to 30 or 100 microM veratridine. Penfluridol (1 microM) inhibited those oscillations in an irreversible manner. 8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as well as the [Ca2+]i oscillations. The Ba2+ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blockin

Topics: Adrenal Glands; Animals; Barium; Calcium; Cattle; Chromaffin Cells; Cytosol; Neuroprotective Agents; Ouabain; Oxidopamine; Penfluridol; Piperidines; Sodium; Thiazoles; Veratridine

Tau is a microtubule-associated protein which is regulated by phosphorylation. Highly phosphorylated tau does not bind microtubules and is the main component of the paired helical filaments seen in Alzheimer's and related neurodegenerative diseases. Recent reports suggested that patterns of tau phosphorylation changed following ischemia and/or reperfusion in vivo. We used an in vitro model employing rat and human neocortical slices to investigate changes in tau phosphorylation which accompany oxygen and glucose deprivation. Western blotting with polyclonal and phosphorylation-sensitive Tau-1 monoclonal antisera was used to monitor changes in tau which accompanied conditions of oxygen and glucose deprivation and reestablishment of these nutrients. In vitro hypoglycemia/hypoxia caused tau to undergo significant dephosphorylation in both rat and human neocortical slices after 30 and 60 min of deprivation. This dephosphorylation was confirmed using immunoprecipitation experiments after radiolabeling tau and other proteins with 32Pi. Okadaic acid, a phosphatase inhibitor, was able to prevent tau dephosphorylation in both control and ischemic slices. Lubeluzole, a benzothiazole derivative with in vivo neuroprotective activity, did not significantly alter patterns of tau phosphorylation. Restoration of oxygen and glucose following varied periods of in vitro hypoxia/hypoglycemia (15-60 min) led to an apparent recovery in phosphorylated tau. These data suggest that tau undergoes a rapid, but reversible dephosphorylation following brief periods of in vitro hypoxia/hypoglycemia in brain slices and that changes in tau phosphorylation help determine the extent of recovery following oxygen and glucose deprivation.

Topics: Animals; Brain Ischemia; Cell Hypoxia; Enzyme Inhibitors; Glucose; Humans; Hypoglycemia; Hypoxia, Brain; Male; Neocortex; Neuroprotective Agents; Okadaic Acid; Organ Culture Techniques; Phosphorylation; Piperidines; Precipitin Tests; Rats; Rats, Sprague-Dawley; tau Proteins; Temporal Lobe; Thiazoles

Neuronal survival after ischemic injury is determined through the induction of several biological pathways. We examined whether lubeluzole, an agent efficacious in both clinical and experimental models of cerebral ischemia, modulated the signal transduction mechanisms of nitric oxide (NO), a downstream mediator of anoxic neurodegeneration. Both pretreatment [NO survival = 23 +/- 3%, NO/lubeluzole (750 nM) survival = 63 +/- 2%, p < 0.001] and coadministration [NO survival = 25 +/- 3%, NO/lubeluzole (750 nM) survival = 59 +/- 3%, p < 0.001] of lubeluzole with NO generators equally protected cultured hippocampal neurons in a dose-dependent manner against the toxic effects of NO, suggesting that the agent protects by acutely modifying toxic cellular pathways rather than preconditioning the neuron before injury. The protection observed with lubeluzole was stereospecific but was not limited to pre- or coadministration. Lubeluzole also was found to significantly protect against the toxicity of NO for a period of 4-6 h after NO exposure [NO survival = 31 +/- 2%, NO/lubeluzole (750 nM) survival at 6 h = 56 +/- 3%, p < 0.001]. We conclude that the neuroprotective ability of lubeluzole is unique and involves the direct modulation of the NO pathway. In addition, the mechanisms of NO toxicity are dynamic and reversible processes that, if left unaltered, will lead to neuronal injury. Further investigation of the downstream signal transduction mechanisms below the level of NO generation may elucidate the specific cellular events responsible for neurodegeneration.

Topics: Animals; Cell Death; Ischemic Preconditioning; Neurons; Neuroprotective Agents; Nitric Oxide; Piperidines; Rats; Rats, Sprague-Dawley; Signal Transduction; Thiazoles

Veratridine blocks Na(+)-channel inactivation and causes a persistant Na(+)-influx. Exposure of hippocampal slices to 10 microM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca(+)-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K+ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanisms of action includes Na(+)-channel modulation were neuroprotective (IC50-values in microM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine > or = 10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and PH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitor (nitro-L-arginine and L-NAME) Ca(2+)-channel blockers (cadmium, nimodipine) and a K(+)-channel blocker (TEA). Lubeluzole significantly delayed in time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca2+ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC50-value 0.54 microM). A concentration of lubeluzole, which was 40 x higher than its IC50-value for neuroprotection against veratridine, had no effect on repetitive Na(+)-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na(+)-influx, without altering normal Na(+)- channel function may be of benefit in stroke.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Cerebral Infarction; Cerebrovascular Circulation; Extracellular Space; Guinea Pigs; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Microelectrodes; Neuroprotective Agents; Piperidines; Pyramidal Cells; Sodium Channels; Thiazoles; Veratridine

Cerebral ischemia may lead to glutamate-induced excitotoxic damage in vulnerable brain areas. Lubeluzole is not an N-methyl-D-aspartate antagonist but prevents postischemic increase in extracellular glutamate concentrations. The present study examined whether lubeluzole, administered after global incomplete ischemia in rats, is capable of preserving the structural integrity of CA1 hippocampus.. Ischemia was induced by bilateral carotid artery occlusion and severe hypotension for a duration of 9 minutes. Delayed neuronal cell death was histologically evaluated 7 days later. This was done by scoring acidophilic cell change and coagulative necrosis and by counting the number of surviving neurons in the CA1 subfield. Experiments were performed according to a paired design (13 animals per treatment group).. Posttreatment with lubeluzole (0.31 mg/kg i.v. bolus at 5 minutes and 0.31 mg/kg i.v. infusion during 1 hour) resulted in significant neuroprotection. Whereas in the untreated rats there were 42 (median) viable neurons per millimeter CA1 layer in the left and 69 in the right hemisphere, in the drug-treated rats 99 viable neurons per millimeter were found in the left (P = .002) and 113 in the right hemisphere (P = .013). Histological scores, reflecting altered staining properties of the hippocampal cells, correlated strongly with the quantitative data, reflecting the structural integrity of CA1 pyramidal neurons.. Lubeluzole, when administered after an ischemic insult in rats, protects vulnerable brain regions against delayed structural injury. The results support the potential clinical use of this new drug in stroke treatment.

Topics: Animals; Brain Damage, Chronic; Brain Ischemia; Carotid Stenosis; Cell Death; Drug Evaluation, Preclinical; Hippocampus; Ligation; Male; Neurons; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Shock; Thiazoles; Time Factors

Focal cerebral lesions in the rat brain induced by photothrombosis cause hyperexcitability of the surrounding brain. This can be demonstrated in brain slices taken from animals several days after lesioning, by analysis of field potential responses to paired-pulse stimulation. We now investigated whether and how these remote effects of a cortical lesion can be modified pharmacologically. Application of the NMDA receptor antagonist, MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibnzo[a,d]cyclohepten-5,10-imine), was shown to block induction of immediate early genes and activation of astrocytes as evidenced by glial fibrillary acidic protein (GFAP) staining in the photothrombosis model. However, MK-801 did not affect the hyperexcitability that had been demonstrated by field potential recordings in brain slices. In another series of experiments, lubeluzole ((+)-(S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoroph enoxy) methyl]-1-piperidineethanol), which inhibits the glutamate-activated nitric oxide pathway as evidenced by down-regulation of intracellular cyclic GMP, was given immediately after induction of the insult. This reduced hyperexcitability as investigated 7 days later. In the light of these data one can suggest that a nitric oxide-cyclic GMP-related mechanism may be responsible for functional alterations in the surround of photothrombotic brain lesions.

Topics: Animals; Cerebral Cortex; Dermatitis, Phototoxic; Disease Models, Animal; Dizocilpine Maleate; Electrophysiology; Evoked Potentials; Excitatory Amino Acid Antagonists; Intracranial Embolism and Thrombosis; Ischemic Attack, Transient; Male; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Thiazoles

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (-) enantiomer R91154 on whole-cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of -80 mV, at 10 s intervals had an average magnitude of 1 nA. 2. Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration-dependent manner; their IC50s were 1.94 microM for lubeluzole and 2.54 microM for R91154. In a current-voltage protocol, lubeluzole (3 microM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I-V curve were detected. 3. After 10 min exposure to 3 microM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered. 4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at -110 mV and 87% at -50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at -110 mV, decreased at -80 mV and did not occur at -50 mV. 5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 microM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 microM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa. 6. The effects of lubeluzole (3 microM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At -80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole). 7. In mouse chromaffin cells approximately half of the whole-cell IBa was sensitive to 3 microM nifedipine (L-type Ca2+ channels) and the other half to 3 microM omega-conotoxin MVIIC (non-L-type Ca2+ channels). In omega-conotoxin MVIIC-treated cells, 3 microM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound. 8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca2+ channels expressed by bovine and mouse chromaffin ce

Topics: Animals; Barium; Calcium Channel Blockers; Cattle; Chromaffin Cells; Mice; Neuroprotective Agents; Patch-Clamp Techniques; Piperidines; Stereoisomerism; Thiazoles

Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in a photochemical stroke model in rats. Tissue swelling, which occurs in the peri-infarct zone, is accompanied by a compensatory taurine release. Therefore, using a microdialysis technique, we aimed at measuring changes of extracellular concentrations of taurine in the peri-infarct zone and the effects of lubeluzole and its R-isomer. Lubeluzole blocked the increase of taurine in tissue immediately surrounding a photochemically induced thrombotic neocortical infarct. By contrast, the R-isomer was completely inactive. We hypothesize that lubeluzole may reduce osmoregulatory stress in peri-infarct tissue.

Topics: Animals; Cerebral Infarction; Male; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Stereoisomerism; Taurine; Thiazoles

A microdialysis probe was positioned inside the peri-infarct zone of a photochemically induced neocortical infarct in rats. Extracellular glutamate rose within 20 min after the start of infarct induction and continued to increase during the 5 h observation period to 5.5-fold the pre-infarct baseline value of 0.8 +/- 0.4 micromol/l. Glutamine increased only 1.4-fold. Changes in peri-infarct glutamate were preceded by steep rises in taurine (a 3.9-fold increase from the baseline value of 2.8 +/- 0.7 micromol/l), which coincided with spreading depressions during infarct induction. Post-treatment with lubeluzole ((S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoro-phenoxy) methyl]-1-piperidineethanol, 1.25 mg/kg i.v.), a new cerebroprotective drug, blocked the peri-infarct increases of glutamate and taurine, whereas the R-enantiomer was ineffective. Since lubeluzole has previously been shown to stereospecifically decrease glutamate-activated nitric oxide (NO) toxicity in vitro, the present in vivo stereospecific effect of lubeluzole may be related to modulation of the cascade of NO toxicity, thus preventing NO toxicity-mediated increases in extracellular glutamate. Blockade of the peri-infarct taurine response suggests that lubeluzole also may have reduced cellular osmotic stress in the peri-infarct zone.

Topics: Animals; Blood Pressure; Cerebral Infarction; Cortical Spreading Depression; Glutamic Acid; Glutamine; Heart Rate; Male; Neocortex; Neuroprotective Agents; Piperidines; Rats; Rats, Wistar; Taurine; Thiazoles

The cardiovascular effects of the antihypoxic and neuroprotective drug, lubeluzole, were investigated using beagle dogs anesthetized with halothane. Endocardial-contact electrode catheter was used for continuous monitoring of monophasic action potential (MAP), which could provide a precise information of repolarization phase. Intravenous administration of an efficacious dose of lubeluzole (0.63 mg/kg, n = 6) slightly decreased both the heart rate and the blood pressure. It did not change PQ interval and QRS width, while it significantly prolonged QT interval, corrected QT (QTc) and the duration of the MAP during the observation period over 60 min. The effects of drug on repolarization phase were late-onset and long-lasting compared with the time course of plasma drug concentrations, which changed as predicted by the two-compartment theory of pharmacokinetics. Additional injection of lubeluzole (2.5 mg/kg, n = 6) showed qualitatively similar changes to those of lower dose, and did not induce the cardiovascular collapse in any dog. Neither afterdepolarization nor ventricular escaped beat was detected during the observation period. The drug concentration in cardiac tissue was correlated linearly with the plasma drug concentration at 60 min after the second drug administration. These results indicate that lubeluzole exerts only minor cardiovascular effects except the prolongation of the repolarization period. The monitoring of plasma drug concentration may be helpful to estimate the steady-state distribution of drug to the heart, but less helpful to predict the QT prolongation. In future clinical trials, care must be taken with patients, especially those at risk to have prolonged repolarization.

Topics: Action Potentials; Animals; Blood Pressure; Cardiac Catheterization; Cardiovascular Agents; Disease Models, Animal; Dogs; Electrocardiography; Heart; Heart Rate; Injections, Intravenous; Microelectrodes; Myocardium; Neuroprotective Agents; Piperidines; Rats; Thiazoles

In search of a better treatment for acute ischemic stroke, we evaluated the use of lubeluzole and hemodilution with diaspirin cross-linked hemoglobin (DCLHb) therapy to test whether treatment with two complementary acting compounds provides more potent protection than either treatment alone.. We used unilateral reversible middle cerebral artery (MCA) and common carotid artery (CCA) occlusion of various durations in Long-Evans rats to produce ischemic cortical lesions. We calculated the average maximal lesion volume (Volmax) and the time required to produce half maximal lesion size (T50) in control animals (n = 31) and evaluated the effects on cerebral perfusion and infarct size of treatment with lubeluzole (n = 23), hemodilution (to 30% hematocrit) with albumin (n = 17) or DCLHb (n = 23), and combined lubeluzole + DCLHb therapy initiated 15 minutes after MCA/CCA occlusion.. The Volmax produced by MCA/CCA occlusion in control animals was 138.5 +/- 7.7 mm3, and T50 was 98.5 +/- 10.2 minutes. Lubeluzole alone reduced Volmax by 53% with no significant effect on T50. In contrast to lubeluzole, DCLHb hemodilution prolonged T50 by 68% with no significant effect on Volmax. Prolongation of T50 by DCLHb was not due to hemodilution itself, since a similar degree of hemodilution with albumin had no effect. Finally, combined lubeluzole+DCLHb rescued 72% of the tissue and augmented the effect of lubeluzole alone by 40% (Volmax, 66.3 +/- 13.0 versus 39.4 +/- 12.2 mm3) while prolonging T50 by 31%.. Combination therapy for acute stroke using compounds with complementary action can result in more complete attenuation of neuronal damage and demonstrates the possible clinical utility of combined neuroprotective and reperfusion therapies.

Topics: Animals; Aspirin; Cardiovascular Agents; Cerebral Infarction; Cerebrovascular Disorders; Drug Therapy, Combination; Hemodilution; Hemoglobins; Male; Neuroprotective Agents; Piperidines; Rats; Rats, Inbred Strains; Reperfusion; Thiazoles

Lubeluzole is a neuroprotective compound in the final stages of clinical evaluation. We evaluated the effects of intravenous followed by intraperitoneal doses of lubeluzole on histological outcome after reversible tandem middle cerebral/common carotid artery occlusion in Long-Evans rats, with particular emphasis on the time window of efficacy. Lubeluzole, started 15 min after the onset of ischemia, had no adverse physiological or behavioral effects and reduce maximal infarct volume produced by 120 min or more of arterial occlusion by approximately 50%, from 143.2 +/- 11.8 mm3 (p < 0.05). Lubeluzole did not prolong the duration of middle cerebral artery occlusion which could be tolerated before histological damage occurred. Lubeluzole was still effective if started 30 min after the onset of ischemia (34% reduction of maximal infarct volume; p < 0.05), but not after delays of 60 or 120 min. we conclude that lubeluzole has promise as a neuroprotective drug, particularly for more severe strokes, but must be started very rapidly after the onset of ischemia to be effective.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Neuroprotective Agents; Piperidines; Rats; Thiazoles

Posttreatment with lubeluzole, the S-isomer of a novel 3,4-difluoro benzothiazole, potently rescued tactile/proprioceptive hindlimb placing reactions contralateral to unilateral thrombotic infarcts in the hindlimb area of the parietal sensorimotor neocortex of rats. Administered at 5 min postinfarct, a single i.v. bolus of lubeluzole was three times as potent as the racemate, whereas the R-isomer was inactive. Neurological protection was near-maximal for treatment delays through 1 hr postinfarct, but declined with longer delays. However, when administered at 6 hr, 1.25 mg/kg i.v. still protected 60% of infarcted rats. An i.v. bolus followed by a 1-hr i.v. infusion produced equieffective neurologic protection at both 6- and 3-hr delays. This optimal lubeluzole regimen, started at 5 min postinfarct, reduced infarct volume by 22 to 24% at 4 hr postinfarct and by 28% at 7 days postinfarct. Again, the R-isomer was inactive. Down-regulation of the glutamate-activated nitric oxide synthase pathway leading to neurotoxicity and neuronal death may constitute a neuroprotective mechanism of action for lubeluzole.

Topics: Animals; Brain; Cerebral Cortex; Cerebral Infarction; Dizocilpine Maleate; Dose-Response Relationship, Drug; Male; Neuroprotective Agents; Nimodipine; Nitric Oxide Synthase; Photochemistry; Piperidines; Rats; Rats, Wistar; Stereoisomerism; Thiazoles

The novel drug lubeluzole, but not its (-)-R-isomer, protects against sensorimotor deficits provoked by photochemical stroke in rats. We studied the mechanism of protection of lubeluzole against glutamate toxicity in primary hippocampal cell cultures. In a model for glutamate antagonism, i.e., treatment of the cultures with compound during the glutamate trigger, lubeluzole was not protective. In contrast, after prolonged pretreatment, i.e., administration of compound to the culture for 7 days before glutamate, lubeluzole was neuroprotective. It had an IC50 of 48 nM and its R-isomer was nine times less active. Under these conditions, lubeluzole inhibited glutamate-stimulated guanosine 3',5'-cyclic monophosphate production (IC50 37 nM). Again the R-isomer was seven times less active. The compounds did not affect nitric oxide synthase activity, guanylate cyclase activity or arginine uptake. After prolonged pretreatment, lubeluzole attenuated citrulline production in the culture, which could not be compensated for by excess arginine. Because prolonged lubeluzole treatment does not inhibit glutamate-activated [Ca+2]i rise in these cultures, the findings may indicate that expression of nitric oxide synthase or levels of its cofactors were reduced. Treatment of C6 glioma cells with lubeluzole did not affect lipopolysaccharide/gamma interferon induced guanosine 3',5'-cyclic monophosphate levels, suggesting that lubeluzole does not inhibit the glial nitric oxide synthase pathway. In conclusion, the long-term neuroprotective property of lubeluzole against glutamate toxicity in hippocampal cultures is reflected by the fact of interference with the glutamateactivated nitric oxide synthase pathway. Prolonged treatment may reduce expression of nitric oxide synthase or levels of its cofactors.

Topics: Animals; Arginine; Cells, Cultured; Cyclic GMP; Glutamic Acid; Guanylate Cyclase; Hippocampus; Interferon-gamma; Lipopolysaccharides; Neuroprotective Agents; Nitric Oxide Synthase; Piperidines; Rats; Stereoisomerism; Thiazoles