Effective Neuroprotective Agent Research Articles

Multiple system atrophy: an update.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder that usually manifests in the early sixth decade of life and progresses relentlessly with a mean survival of 9 years. Clinically, MSA is dominated by autonomic/urogenital failure, which may be associated with either levodopa (L-dopa) -unresponsive parkinsonism in 80% of cases (MSA-P subtype) or with cerebellar ataxia in 20% of cases (MSA-C subtype). Pathologically, MSA is characterized by a neuronal multisystem degeneration and abnormal glial cytoplasmic inclusions containing alpha-synuclein aggregates. Pharmacological treatment of motor features is disappointing except for a transient L-dopa response in a minority of MSA-P patients. In contrast, autonomic and urogenital features of MSA should be identified early on, because they can be treated effectively in many instances. Neuroprotective strategies are presently unavailable, however, two multicentre European trials have been launched to evaluate the effects of riluzole and human recombinant growth hormone on disease progression in MSA. Clearly, further randomised, controlled trials are required to identify effective symptomatic or neuroprotective agents in MSA. Several in vivo models have become available to allow a careful preselection of candidate agents. Several research groups have been formed in Europe (EMSA-SG, NNIPPS) and United States (NAMSA-SG), providing a framework for coordinated trial activity in MSA.

Functional and Histological Evidence for the Protective Effect of NXY-059 in a Primate Model of Stroke When Given 4 Hours After Occlusion

NXY-059 has substantial protective effects when administered immediately after the onset of ischemia in a primate model of stroke. This study examined the efficacy of this drug when administered 4 hours after onset, a more clinically relevant time point. Before surgery, marmosets were trained and tested on a number of neurological tests, which assessed general neurological function, motor ability, and spatial awareness. Four hours after permanent middle cerebral artery occlusion (pMCAO), marmosets received a bolus of saline (n=13) or NXY-059 (n=13), and osmotic minipumps were implanted, providing 48-hour saline or drug (85 micromol/kg per hour) infusion. The monkeys were retested 3 and 10 weeks after surgery. Finally, infarct size was evaluated with histological analysis. The unbound plasma NXY-059 concentration was 200+/-9 micromol/L after 24-hour infusion, a concentration well tolerated in stroke patients. Drug treatment ameliorated the long-term motor impairment produced by pMCAO; the marmosets were better at using their contralesional, stroke-affected arm than controls at both 3 and 10 weeks. Saline-treated animals had a debilitating spatial neglect at 3 weeks with residual signs evident at 10 weeks. NXY-059 treatment substantially attenuated neglect at 3 weeks, with no deficit being seen at 10 weeks. NXY-059 reduced the overall infarct size by 28% (saline, 324+/-46 mm3; NXY-059, 234+/-30 mm3) with protection to the cortex, white matter, and subcortical structures. NXY-059 is an effective neuroprotective agent when administered 4 hours after pMCAO in a primate species, attenuating both motor and spatial neglect. The compound also substantially lessened the volume of cerebral damage.

Animal models of stroke: do they have value for discovering neuroprotective agents?

There has been a series of high-profile failures of drugs in clinical trials of acute ischaemic stroke that were designed to meet criteria necessary for drug regulatory approval. This has, again, called into question the value of animal models for identifying effective neuroprotective agents. Here, we review evidence that physiological changes (reperfusion, hyperglycaemia, hypothermia and blood pressure) produce comparable changes in outcome in both animal models and human stroke patients, which indicates that the models should identify clinically effective neuroprotective agents. We suggest that most clinical failures have occurred because compounds were administered differently in animal and clinical studies. We review earlier guidelines on the information that is necessary from preclinical studies before a compound enters clinical trials, and propose modifications to these guidelines.

Fibroblast growth factor-18 reduced infarct volumes and behavioral deficits after transient occlusion of the middle cerebral artery in rats.

Fibroblast growth factor 18 (FGF18) is expressed in rodent brain and is a trophic factor for neuron-derived cells in culture. The purpose of the present study was to evaluate whether FGF18 was neuroprotective in a rat model of cerebral ischemia and to compare the results with those obtained with FGF2. Cerebral ischemia was produced in rats by a transient 2-hour occlusion of the middle cerebral artery (MCAo) with an intraluminal filament followed by 22-hour reperfusion. Starting 15 minutes after MCAo, FGF18 or FGF2 was administered by a 3-hour intravenous infusion. Infarct volumes and behavioral deficits were measured 24 hours after MCAo. Infusion of FGF18 produced dose-dependent reductions in infarct volumes and improvements in tests of reference and working memory, motor ability, and exploratory behavior. FGF18 was more efficacious than FGF2 on virtually all measures examined. The reductions in infarct volume and behavioral deficit were associated with FGF-mediated increases in regional cerebral blood flow. These results demonstrate that FGF18 is an effective neuroprotective agent in a rat model of transient MCAo.

Comparison of the neuroprotective effect of clomethiazole, AR-R15896AR and NXY-059 in a primate model of stroke using histological and behavioural measures

Three experimental neuroprotective agents (clomethiazole, AR-R15896AR and NXY-059) have recently been tested in a primate model of acute ischaemic stroke. As the experimental techniques used in all three studies were similar and the compounds were administered at clinically relevant doses, a comparative analysis of the functional benefits of these drug-treatments has now been performed. Furthermore a more detailed histological analysis of the neuroprotection afforded by the drugs has also been made. NXY-059 produced almost twice the degree of neuroprotection than that seen following clomethiazole or AR-R15896AR. Protection by NXY-059 was seen in measurements of damage to cortex and white matter. Clomethiazole and AR-R15896AR provided less protection of cortex and white matter than NXY-059. Conspicuously, AR-R15896AR was without effect in sub-cortical regions. NXY-059 was the only compound to produce a major, statistically significant improvement in the motor deficit induced by the stroke. All three drugs also reduced the degree of spatial neglect 3 weeks after pMCAO, and 10 weeks later only NXY-059 still provided significant additional functional benefit to the spontaneous improvement seen in stroked control animals not receiving treatment. The overview of the behavioural effects and these new histological findings suggest that NXY-059 was by far the most effective neuroprotective agent of the three examined.

Thrombolysis in stroke

The ability to use an effective treatment constitutes a main concern in the management of acute ischemic stroke. Randomized trials have so far failed to demonstrate any beneficial effect of neuroprotective agents on neurologic outcome after an acute stroke. Thrombolytic agents can achieve early recanalization of an occluded intracerebral artery. The benefit of their use in ischemic stroke has been assessed in several randomized trials involving more than 5,000 patients. Among them, a single randomized trial, the National Institute of Neurological Diseases and Stroke Trial, showed a beneficial effect of thrombolysis on outcome after an acute ischemic stroke with a significant increase in the number of patients with no or minimal disability at 3 months in the group receiving intravenous recombinant tissue plasminogen activator (rt-PA) compared with placebo. However, there was no reduction in mortality and there was, as in all other trials, a significant increase in the risk of symptomatic intracerebral hemorrhage (ICH).

In vitro blood–brain barrier permeability and cerebral endothelial cell uptake of the neuroprotective nitrone compound NXY-059 in normoxic, hypoxic and ischemic conditions

The free radical trapping nitrone compounds α-phenyl- N- tert-butylnitrone (PBN), 2-sulfophenyl- N- tert-butylnitrone (S-PBN) and disodium 2,4-disulfophenyl- N- tert-butyl nitrone (NXY-059) are effective neuroprotective agents in experimental models of both transient and permanent focal ischemia. A recent in vivo study suggested that NXY-059 had poor brain uptake in a transient ischemia model. We have now examined its blood–brain barrier permeability and cerebral endothelial uptake during hypoxic and ischemic conditions using an in vitro model of the blood–brain barrier. The in vitro blood–brain barrier permeability and cerebral endothelial uptake of NXY-059 and S-PBN were low during normoxic conditions. In contrast, PBN had very high blood–brain barrier penetration in vitro which confirmed earlier in vivo results. The permeability of [ 14C]NXY-059 increased 3.5 times after 9 h of hypoxia or 3 h of ischemia. There was, respectively, a 5-fold and more than 10-fold increase, after 6 and 9 h of ischemia. The control molecule [ 3H]inulin ( M r≈5000) showed a similar increase in permeability under the same experimental conditions indicating a major change in the transport properties of the endothelium. There was a 60% reduction in the ATP levels of astrocytes after 3 h of ischemia and a 90% reduction after 9 h. The reduction in ATP levels in endothelial cells was somewhat lower. The uptake of NXY-059 in cerebral endothelial cells under normoxic, hypoxic or 9 h of ischemic conditions was negligible. NXY-059, S-PBN and PBN showed no effects on vesicular transport or the integrity of the blood–brain barrier in normoxic or ischemic conditions, nor did the compounds induce any change in the ATP levels of the cells. In conclusion, it is possible that the increase in blood–brain barrier permeability of [ 14C]NXY-059 which occurs during prolonged ischemia in vitro reflects a change which may be of importance to the neuroprotective effects of this nitrone free radical trapping agent.

Why do neuroprotective drugs that are so promising in animals fail in the clinic? An industry perspective.

1. No neuroprotective drug has yet been shown to be effective in treating acute ischaemic stroke in the clinic, despite evidence of efficacy in animal models. 2. An academic/industry round-table group recently published guidelines to be met if a drug was to be progressed to clinical trial. 3. Major points included obtaining full dose-response evaluation and measurement of the therapeutic time window for efficacy, functional behavioural testing in addition to measurement of infarct volume, measurement of physiological parameters, use of appropriate models (transient and permanent focal ischaemia) and reproducibility of data by external laboratories. 4. The present paper examines both failed compounds and disodium 4-[(tert-butylimino) methyl] benzene-1, 3-disulphonate N-oxide (NXY-059), a nitrone radical-trapping agent currently in clinical development. It aims to determine whether these guidelines were met by compounds that have failed and, thus, determine whether following the guidelines, as is being done with NXY-059, will increase the chances of developing efficacious drugs for treating acute ischaemic stroke. 5. It is concluded that we will only achieve the goal of producing a clinically effective neuroprotective agent if the guidelines have been met by the novel compound under investigation.

Neuroprotective actions in vivo and electrophysiological actions in vitro of 202W92

202W92 ( R-(−)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) is a novel compound in the same chemical series as the antiepileptic drug lamotrigine and the neuroprotective sipatrigine. Here 202W92 was quantitatively assessed as a neuroprotective agent in focal cerebral ischaemia, and as an inhibitor of sodium and calcium channels and of synaptic transmission. In the rat permanent middle cerebral artery occlusion (MCAO) model of acute focal ischaemia, 202W92 reduced infarct volume by 75% in cortex and by 80% in basal ganglia, with ED 50 approximately 2 mg/kg (single i.v. dose, 10 min post-occlusion). In whole-cell current recordings from single cells, 202W92 completely and reversibly inhibited voltage gated sodium channels (IC 50 3×10 −6 M) in rat freshly-isolated cortical neurons and in the GH 3 pituitary cell line. 202W92 also inhibited a nifedipine-sensitive fraction (approximately 35%) of native high-voltage-activated (HVA) calcium current in rat cortical neurons (IC 50 15×10 −6 M) and weakly inhibited low-voltage-activated (LVA) calcium currents of the recombinant α1I-mediated T-type (IC 50>100×10 −6 M). The drug inhibited the amplitude and frequency of 4-aminopyridine-evoked glutamatergic excitatory post-synaptic currents (EPSCs). In conclusion, 202W92 is an effective neuroprotective agent when administered post-ischaemia and a potent sodium channel inhibitor in vitro.

Combination Therapy Stroke Trial: Recombinant Tissue-Type Plasminogen Activator with/without Lubeluzole

Background: 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. Objective: To assess the feasibility, safety and efficacy of simultaneously combining the neuroprotective drug lubeluzole with rt-PA. Method: 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. Results: 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. Conclusion: 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.

Animal models of traumatic brain injury: a review.

To review the main types of animal models of traumatic brain injury (TBI), their desirable characteristics and limitations, the major structural changes in the brain modelled, and special features of paediatric TBI modelling. Animal models have contributed substantially to our understanding of the mechanisms of TBI in humans, but many aspects of closed head injury remain to be elucidated and effective neuroprotective agents are few. Many of the advances in the pathogenesis of human TBI also have application in veterinary medicine and there is ample opportunity for veterinarians to contribute to the selection, development and characterisation of potential animal models of neurotrauma and other disorders and ensure that animal welfare standards are maintained.

The failure of neuronal protective agents versus the success of thrombolysis in the treatment of ischemic stroke. The predictive value of animal models.

Agents claimed to be neuroprotective in animal stroke models have all failed in human trials. Thrombolysis has been reported as beneficial in animal and human stroke. We explore the reasons for this disparity, using a review of published results of agents tested both in animal stroke models and in human stroke trials. In animals the effect of neuroprotective agents and of thrombolytic agents on infarct size is time-dependent: early initiation of treatment works best; and benefit is progressively--and eventually totally--lost with increasing delay of time of first treatment. The animal data also show that, overall, the beneficial effects of the neuroprotective agents are weaker, and are totally lost sooner, than those of thrombolytics. The human data show that the failed trials of the neuroprotective agents had entry windows that went far beyond the windows of (any) success seen in tests of these agents in animals. By contrast, human thrombolysis trials uniformly restricted time of entry to windows in which these agents have shown beneficial effect in animals. In clinical stroke trials, neuroprotective agents failed to produce benefit because their effects at best are too weak, and they were used at times predictable from the animal models as too late. Thrombolytic therapy, which has a stronger effect than neuroprotective agents in animal models, was used clinically during the early window of optimal effectiveness, and produced beneficial results. "Too little/too late" is the recipe for failure in the treatment of ischemic stroke.

Intrathecal cyclosporin prolongs survival of late-stage ALS mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood–brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.

Special Article Pharmacological Treatment Of Acute Spinal Cord Injury: How Do We Build On Past Success?

Most acute spinal cord injuries (SCI) do not involve complete transection of the spinal cord; typically, a rim of white matter survives. The potential for neurological recovery depends on optimal preservation of the ascending and descending white matter axons and their normal myelination. Pharmacologic strategies focus on the control of secondary injury processes, primarily lipid peroxidation (LP), and the salvage of as much white matter as possible. The first effective neuroprotective agent was methylprednisolone (MP), a glucocorticosteroid that in high doses improves neurological recovery in animals and humans following acute SCI. Tirilazad is a more targeted non-glucocorticoid LP inhibitor that has been shown to be neuroprotective and has fewer side effects than MP. Future SCI therapy is likely to encompass various neuroprotective agents, including inhibitors of LP, inhibitors of the nitric oxidederived reactive oxygen species peroxynitrite, inhibitors of calpain (which is responsible for degrading the spinal cord cytoskeleton), and inhibitors of post-traumatic apoptosis of neurons and myelin-forming oligodendroglia. In addition, neuroprotective strategies will eventually be followed by neurorestorative agents that stimulate the plasticity of surviving neural pathways, and will be used in conjunction with other neurorestorative therapies like cell transplantation and gene therapy techniques.J Spinal Cord Med. 2001 ;24:142–146

Neuroprotective effects of citicoline on brain edema and blood-brain barrier breakdown after traumatic brain injury.

Cytidine 5'-diphosphocholine (CDPC), or citicoline, is a naturally occurring endogenous compound that has been reported to provide neuroprotective effects after experimental cerebral ischemia. However, in no study has such protection been shown after traumatic brain injury (TBI). In this study the authors examined the effect of CDPC on secondary injury factors, brain edema and blood-brain barrier (BBB) breakdown, after TBI. After anesthesia had been induced in Sprague-Dawley rats by using 1.5% halothane, an experimental TBI was created using a controlled cortical impact (CCI) device with a velocity of 3 m/second, resulting in a 2-mm deformation. Four sham-operated control animals used for brain edema and BBB breakdown studies underwent the same surgical procedure, but received no injury. Brain edema was evaluated using the wet-dry method 24 hours postinjury, and BBB breakdown was evaluated by measuring Evans blue dye (EBD) extravasation with fluorescein 6 hours after TBI. The animals received intraperitoneal injections of CDPC (50, 100, or 400 mg/kg two times after TBI [eight-10 animals in each group]) or saline (eight animals) after TBI. Traumatic brain injury induced an increase in the percentage of water content and in EBD extravasation in the injured cortex and the ipsilateral hippocampus. No significant benefit from CDPC treatment was observed at a dose of 50 mg/kg. Cytidine 5'-diphosphocholine at a dose of 100 mg/kg attenuated EBD extravasation in both regions, although it reduced brain edema only in the injured cortex. In both regions, 400 mg/ kg of CDPC significantly decreased brain edema and BBB breakdown. This is the first report in which dose-dependent neuroprotective effects of CDPC have been demonstrated in the injured cortex as well as in the hippocampus, a brain region known to be vulnerable to injury, after experimental TBI. The results of this study suggest that CDPC is an effective neuroprotective agent on secondary injuries that appear following TBI.

Neuroprotection in optic neuropathy: assessment of effect

Assessment of the therapeutic effect of neuroprotective agents requires knowledge of the natural history and structural and functional effects of individual optic neuropathies. New technologies are now available for a more accurate quantification of optic nerve and retinal nerve fiber layer topography (confocal scanning laser ophthalmoscopy and polarimetry), surface vasculature (laser Doppler flowmetry), visual acuity,visual field (SITA, high-pass resolution, SWAP, frequency doubling technology), and electrophysiologic responses (multifocal ERG, VEP).

The development of new drugs for acute stroke

Stroke represents the third common cause of death and hospitalization. However, there are yet no drugs that have reliable effects on acute stroke in Japan. Therefore, the development of new drugs that can support patients is required. There are various candidate drugs for acute stroke such as antithrombotic agents, anticoagulants, thrombolytic agents, neuroprotectants, and so on. Recently clinical trials suggest that aspirin may improve outcome, although these studies demonstrated a modest benefit of aspirin. Abciximab (ReoPro) is a human/mouse monoclonal antibody directed against the platelet receptor glycoprotein IIb/IIIa. It appears to be safe and might improve functional outcome. The large randomized trails were started to test the hypothesis that thrombolysis by an intravenous administration of a recombinant tissue type plasminogen activator (rtPA) could restore cerebral blood flow and improve patient outcome in acute ischemic stroke. These results can support the use of intravenous rtPA for stroke treatment within 3 h after onset, but not beyond 3 h. Development of an effective neuroprotective agent for the treatment of acute stroke remains problematic. Antioxidants, MCI-186 and ebselen, have finished phase III of clinical trials in Japan and were effective. We hope that efficacious drugs for acute stroke can be used for patients.

AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+],. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA- induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca2+-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents. [Neurol Res 1999; 21: 524–528]

Increased funding for stroke research.

It is an exciting time for stroke research. Over the last couple of years with the use of tPA, and now at the most recent International Stroke and Cerebral Circulation Conference meeting, the announcement of 2 promising new thrombolytic agents (Ancrod and Pro-urokinase) for acute stroke treatment, it is an exciting time indeed. Neuroprotective agents are still being investigated, and while an effective neuroprotective agent has not yet been identified in humans, almost assuredly one will come to light soon. The American Heart Association/American Stroke Association (AHA/ASA) is making significant efforts to provide dollars for stroke research for both clinical and basic science areas. The AHA/ASA for 1997–98 provided some $6 million for stroke-specific research, with a projection for the year 2001 of $11.3 million. Another $33.7 million was provided for research that was related in some way to stroke. In addition, there is a very exciting new commitment …

In vivo evidence against clomethiazole being neuroprotective against MDMA (‘ecstasy’)-induced degeneration of rat brain 5-HT nerve terminals by a free radical scavenging mechanism

Clomethiazole is an effective neuroprotective agent against the degeneration of 5-HT neurones that follows administration of 3,4-methylenedioxymethamphetamine (MDMA or ‘ecstasy’). Since there is good evidence that free radical formation resulting from auto-oxidation of MDMA metabolites is responsible for the degeneration we have examined whether clomethiazole is a free radical scavenger. MDMA (15 mg/kg i.p.) increased the formation of 2,3- and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) from salicylic acid perfused through a microdialysis tube implanted in the hippocampus, indicating increased free radical formation. Clomethiazole (50 mg/kg i.p.) administered 5 min prior and 55 min post MDMA prevented both the acute MDMA-induced hyperthermia and the rise in 2,3- and 2,5-DHBA. However, when the temperature of the MDMA+clomethiazole treated rats was kept elevated to that of the MDMA treated rats with a homeothermic blanket there was no inhibition of the MDMA-induced increase in 2,3-DHBA or 2,5-DHBA. These data suggest firstly that free radical formation is inhibited when the acute MDMA-induced hyperthermia is prevented. Secondly the data further indicate that clomethiazole has no free radical scavenging activity since the drug produces substantial neuroprotection when MDMA+clomethiazole treated rats are kept hyperthermic. This conclusion was strengthened by our observation that clomethiazole is a weak inhibitor (IC 50>1 mM) of lipid peroxidation in synaptosomes when it had been induced by addition of FeCl 2+ascorbic acid.