The metabotropic glutamate receptor subtype 5 antagonist MPEP and the Na+ channel blocker riluzole show different neuroprotective profiles in reversing behavioral deficits induced by excitotoxic prefrontal cortex lesions.

Overactivation of excitatory amino acid receptors has been involved in several neurodegenerative diseases. The present study aims at investigating the potential neuroprotective action of 2-methyl-6-(phenylethylnyl)-pyridine (MPEP), a selective non-competitive antagonist of metabotropic glutamate receptor subtype 5, and 2-amino-6-trifluoro methoxy-benzothiole (riluzole), a Na+ channel blocker exhibiting anti-glutamatergic properties, on the ibotenate-induced damage to the rat medial prefrontal cortex. The neuroprotective efficacy of these compounds was assessed on the recovery from behavioral deficits induced by prefrontal cortical excitotoxic lesions in a reaction time task. MPEP (3, 10 or 30 mg/kg) or riluzole (2, 4 or 8 mg/kg) was administered i.p. 30 min before and after medial prefrontal cortex lesions. As previously found, lesions to the medial prefrontal cortex significantly altered the motor preparatory processes involved in the reaction time task. These deficits were prevented by MPEP 3 mg/kg and riluzole 2 mg/kg while higher doses of either compound were ineffective. Furthermore, the neuron-specific nuclear protein immunostaining of the lesioned cortical area in animals treated with the efficient dose of either compound revealed that MPEP reduced the volume of the lesion whereas riluzole reversed the decrease of neuronal density within the lesioned area. Altogether, these results suggest a neuroprotective action of MPEP as well as riluzole at both behavioral and cellular levels on excitatory amino acid-induced toxicity.

Unraveling substantia nigra sequential gene expression in a progressive MPTP-lesioned macaque model of Parkinson's disease.

Taking advantage of a progressive nonhuman primate model mimicking Parkinson's disease (PD) evolution, we monitored transcriptional fluctuations in the substantia nigra using Affymetrix microarrays in control (normal), saline-treated (normal), 6 days-treated (asymptomatic with 20% cell loss), 12 days-treated (asymptomatic with 40% cell loss) and 25 days-treated animals (fully parkinsonian with 85% cell loss). Two statistical methods were used to ascertain the regulation and real-time quantitative PCR was used to confirm their regulation. Surprisingly, the number of deregulated transcripts is limited at all time points and five clusters exhibiting different profiles were defined using a hierarchical clustering algorithm. Such profiles are likely to represent activation/deactivation of mechanisms of different nature. We briefly speculate about (i) the existence of yet unknown compensatory mechanisms is unraveled, (ii) the putative triggering of a developmental program in the mature brain in reaction to progressing degeneration and finally, (iii) the activation of mechanisms leading eventually to death in final stage. These data should help development of new therapeutic approaches either aimed at enhancing existing compensatory mechanisms or at protecting dopamine neurons.

Assessment of sensorimotor and cognitive deficits induced by a moderate traumatic injury in the right parietal cortex of the rat.

The purpose of this study was to set-up a battery of behavioral tests to assess sensorimotor and cognitive deficits following a moderate traumatic brain injury (TBI) in rats. Coordinated walking ability was evaluated in an accelerated rotarod test. Vestibulomotor function and fine motor coordination were assessed by using a beam-walking task. Rotarod and beam-walking performances were both altered in injured rats compared to sham-operated and control rats. A more pronounced and longer-lasting deficit was measured in the beam-walking test. Cognitive function was studied by using the Lashley maze paradigm. A spatial localization deficit was significant for 4 weeks posttrauma in TBI rats. The beam-walking task and the Lashley maze are robust and sensitive methods in detecting sensorimotor and cognitive impairment after TBI in rats, respectively. These tests are proposed for evaluating the ability of new pharmacological agents to improve the functional recovery after a TBI in rats.

Riluzole reduces hyperactivity of subthalamic neurons induced by unilateral 6-OHDA lesion in the rat brain.

An abnormal increase in the activity of neurons of the subthalamic nucleus is a key pathophysiological feature of Parkinson's disease. We sought to determine whether riluzole, a sodium channel inhibitor that interferes with glutamatergic neurotransmission, affects neuronal activity in this brain region. Intravenous administration of riluzole reduced the discharge rate of subthalamic neurons in rats with 6-OHDA-induced lesions of the midbrain. By contrast, no effect was observed in nonlesioned control animals. This property may contribute to the neuroprotective effects of riluzole in animal models of PD through the modulation of the glutamatergic inputs these neurons feedback to nigral dopaminergic neurons.

RPR 119990, a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid antagonist: synthesis, pharmacological properties, and activity in an animal model of amyotrophic lateral sclerosis.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonists are of potential interest for the treatment of certain acute and chronic neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe the synthesis and pharmacological properties of 9-carboxymethyl-4-oxo-5H,10H-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-phosphonic acid (RPR 119990). The compound displaced [3H]AMPA from rat cortex membranes with a K(i) of 107 nM. In oocytes expressing human recombinant AMPA receptors, RPR 119990 depressed ion flux with a K(B) of 71 nM. The antagonist properties of this compound were confirmed on rat native AMPA receptors in cerebella granule neurons in culture and in hippocampal slices where it antagonized electrophysiological responses with IC50 values of 50 and 93 nM, respectively. RPR 119990 antagonized hippocampal evoked responses in vivo, demonstrating brain penetration at active concentrations. RPR 119990 is a potent anticonvulsant in the supramaximal electroshock in the mouse with an ED50 of 2.3 mg/kg 1 h post s.c. administration, giving it a workably long action. Pharmacokinetic studies show good passage into the plasma after subcutaneous administration, whereas brain penetration is low but with slow elimination. This compound was found active in a transgenic mouse model of familial amyotrophic lateral sclerosis (SOD1-G93A) where it was able to improve grip muscle strength and glutamate uptake from spinal synaptosomal preparations, and prolong survival with a daily dose of 3 mg/kg s.c.

Synthesis of anticonvulsive AMPA antagonists: 4-oxo-10-substituted-imidaz.

The overstimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been implicated in the physiopathogenesis of epilepsy as well as in acute and chronic neurodegenerative disorders. An original series of readily water soluble 4-oxo-10-substituted-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives was synthesized. The most potent derivative 6a exhibited nanomolar binding affinity (IC50 = 35nM) and antagonist activity (IC50 = 6nM) at ionotropic AMPA receptor. This compound also demonstrated potent anticonvulsant properties in MES in mice and rats with long durations of action with ED50 values in the 1-3 mg/kg dose range following ip and iv administration.

Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity.

BACKGROUND AND PURPOSE: Heparin and heparinoids have long been proposed for stroke treatment. This study investigates the effect of enoxaparin (Lovenox, Clexane), a low-molecular-weight heparin, on functional outcome (neuroscore) and lesion size in stroke models with reversible and irreversible cerebral ischemia using middle cerebral artery occlusion (MCAO) in the rat. METHODS: Ischemia was induced in rats by transient occlusion for 2 hours or by permanent electrocoagulation of the left MCA. Forty-eight hours after ischemia, neurological deficit was evaluated by scoring sensorimotor functions and ischemic damage was quantified by histological evaluation of lesion volumes. RESULTS: After transient MCAO, enoxaparin at 2x1.5 mg/kg IV (2 and 24 hours after insult) significantly reduced lesion size by 30% (P<0.05) and improved neuroscore (P<0.01). This significant effect on lesion size and neuroscore was still evident when treatment was started 5 hours after insult. Administered under the same protocol with a 5 hours delay post permanent MCAO, enoxaparin reduced lesion size by 49% (P<0.05) and improved neuroscore (P<0.01). CONCLUSIONS: This study indicates that standard nonhemorrhagic doses of enoxaparin reduce ischemic damage with a wide therapeutic window. In addition to its anticoagulant properties, other properties of enoxaparin could act in synergy to explain its neuroprotective profile in ischemia. Thus clinical application of enoxaparin treatment in stroke warrants serious consideration.

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action.

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.

Enoxaparin reduces brain edema, cerebral lesions, and improves motor and cognitive impairments induced by a traumatic brain injury in rats.

Traumatic brain injury (TBI) is often accompanied by secondary ischemia due, in part, to edema-induced blood vessel compression. Enoxaparin, a low-molecular weight heparin, which is efficacious in models of myocardial and brain ischemia was studied in lateral fluid percussion-induced TBI in rats. Enoxaparin was administered 2 h post-TBI at 0.5 mg/kg i.v. followed by 4 x 0.5, 4 x 1, or 4 x 2 mg/kg s.c. over 30 h. Brain edema was measured in the hippocampus, temporal cortex and parietal cortex. Edema was reduced by enoxaparin (0.5 + 4 x 0.5 mg/kg) in the hippocampus (-53%, p = 0.07) and the parietal cortex (-39%, ns). At 0.5 + 4 x 1 mg/kg edema was reduced in the hippocampus (-63%, p < 0.05) and the parietal cortex (-47%, p = 0.06). At 0.5 + 4 x 2 mg/kg, the reduction was more important in the hippocampus (-69%, p < 0.01) and in the parietal cortex (-50%, p < 0.05). No reduction was seen in the temporal cortex. The lesion size was reduced by enoxaparin at 0.5 + 4 x 1 mg/kg (-50%, p < 0.05), and at 0.5 + 4 x 2 mg/kg (-35%, ns). The neurological deficit evaluated with a 9-point scale was also improved with enoxaparin at 0.5 + 4 x 1 mg/kg 1 week post-TBI (p < 0.05). The cognitive impairment evaluated with a Lashley maze task was improved with enoxaparin (0.5 + 4 x 1 mg/kg) from 48 h (p < 0.05) to 2 weeks post-TBI (p < 0.01). Our results demonstrate for the first time that enoxaparin significantly reduces the brain contusion and edema, and improves the functional outcomes induced by a TBI. Therefore, enoxaparin could be a candidate drug to treat acute brain-injured patients.

Riluzole prolongs survival and delays muscle strength deterioration in mice with progressive motor neuronopathy (pmn).

The neuroprotective drug riluzole (Rilutek) is a sodium channel blocker and anti-excitotoxic drug which is marketed for the treatment of amyotrophic lateral sclerosis (ALS). Previous studies have shown that riluzole prolongs survival of transgenic mice harboring the mutated form of Cu,Zn-superoxide dismutase found in familial forms of the human disease. In this study we have examined the effect of treatment with riluzole in mice suffering from progressive motor neuronopathy (pmn), a hereditary autosomal recessive wasting disease which shares some symptoms of ALS. These mutants display hind limb weakness starting during the 3rd week of life and leading to paralysis and death during the 7th week of life. Daily treatment with 8 mg/kg of riluzole by oral route significantly retarded the appearance of paralysis, increased life span and improved motor performance on grip test and electromyographic results in the early stage of the disease. There was no effect of riluzole on weight gain. These data demonstrate that riluzole significantly prolongs life span, retards the onset of paralysis and slows the evolution of functional parameters connected with muscle strength in the pmn mouse model of motor neuron disease.

8-Methylureido-10-amino-10-methyl-imidazo[1,2-a]indeno[1,2-e] pyrazine-4-ones: highly in vivo potent and selective AMPA receptor antagonists.

Water soluble 8-methylureido-10-amino-10-methyl-imidazo[1,2-a]indeno[1,2-e]pyraz ine-4-one 4 represents a novel class of highly potent and selective AMPA receptors antagonists with in vivo activity. The dextrorotatory isomer (+)-4 was found to display the highest affinity with an IC50 of 10 nM. It also exhibited very good anticonvulsant effects after i.p., s.c. and i.v. administration in mice subjected to electrical convulsions (MES) and i.p. in audiogenic seizure-e in DBA/2 mice (ED50's < or = 10 mg/kg).

Indeno[1,2-b]pyrazin-2,3-diones: a new class of antagonists at the glycine site of the NMDA receptor with potent in vivo activity.

Indeno¿1,2-bpyrazin-2,3-diones have been identified as a novel series of potent ligands on the glycine site of the NMDA receptor. To improve their in vivo activities, an acetic acid-type side chain was introduced to the 5-position, giving water-soluble compounds when formulated as the sodium salt (>10 mg/mL). Introduction of a chlorine atom in the 8-position led to a dramatic improvement of anticonvulsant activity and this was surprising since this change did not improve binding affinity. A plausible explanation is a reduced recognition by a Na(+),K(+)-ATPase active transport system responsible for the excretion of these compounds from the brain and kidney. This promising new chemical series led to the optically active isomer (-)-10i (RPR 118723), a glycine/NMDA antagonist with nanomolar binding affinity and in vivo activity in animal model of convulsions and electrophysiology at doses in the range of 2-3 mg/kg following iv administration.

4,10-Dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives: highly potent and selective AMPA receptors antagonists with in vivo activity.

A novel series of 2-substituted-4,5-dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazine derivatives was synthesised. One of them, 4e-a highly water soluble compound exhibited a nanomolar affinity and demonstrated competitive antagonist properties at the ionotropic AMPA receptors. This compound also displayed potent anticonvulsant properties against electrically or sound-induced convulsions in mice after systemic administration, thus suggesting adequate brain penetration.

Effects of riluzole on the electrophysiological activity of pallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkey.

The effect of riluzole administration, an antiglutamatergic compound, on the electrophysiological activity of the pallidal complex of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys is compared with those induced by two dosages of levodopa (L-DOPA), the first affording the best clinical alleviation, the second sufficient to induce dyskinesias. Both dosages of L-DOPA reduced sharply the firing frequency of globus pallidus pars internalis (GPi) neurons (respectively, 43.8+/-23.0 and 27.4+/-20.2 vs. 111. 2+/-31.4 Hz), decreased the percentage of bursting cells (respectively, 60.7 and 50.0 vs. 80.3%) and augmented the number of regular cells (respectively, 6.5 and 33.0 vs. 4.8%). Riluzole restored the firing frequency (75.0+/-26.9 Hz) and the firing pattern of the GPi (39.7% bursting, 9.5% regular and 50.8% irregular). These results suggest that the emergence of dyskinesia may well be due to a modification of the neuronal messages transmitted from the GPi to the motor nuclei of the thalamus. Riluzole would represent an interesting alternative to dopamine therapy in Parkinson's disease since it regularizes firing but does not cause dyskinesia.

8-Methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]pyrazines: highly potent in vivo AMPA antagonists.

A novel series of readily water soluble 8-methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]++ +pyrazines were synthesized. The -10-yl acetic acid ((+)-3) and -10-carboxylidene (4) derivatives exhibit potent affinities (IC50=4 and 19 nM, respectively) and antagonist properties (IC50 = 2 and 3 nM, respectively) at the ionotropic AMPA receptor. These compounds also display anticonvulsant properties against both electrically and sound-induced convulsions in mice after ip, sc and iv administration with ED50 values between 0.9 and 11 mg/kg, thus suggesting adequate brain penetration.

Synthesis and potent anticonvulsant activities of 4-oxo-imidazo[1,2-a]inden.

The over-stimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been suggested to be associated with neurodegenerative disorders. Here we describe an original series of readily water soluble 4-oxo-imidazo[1,2-a] indeno[1,2-e]pyrazin-8- and -9-carboxylic (acetic) acid derivatives. One of these compounds, 4f, exhibited nanomolar binding affinity, potent competitive antagonism at the ionotropic AMPA receptor and a long duration of anticonvulsant activity after administration by parenteral route in vivo.

Spiro-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one derivatives are mixed AMPA and NMDA glycine-site antagonists active in vivo.

Original spiro-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one derivatives were synthesised and led to the identification of 3e which showed good affinities for both the AMPA and the NMDA glycine-site receptors, and displayed good anticonvulsant effects after i.p. and i.v. administrations in the electroshock-induced convulsion assay in mice. The corresponding dextrorotatory isomer (+)-3e was notably more potent than the levorotatory isomer (-)-3e in in vitro and in vivo assays.

The effect of riluzole and mannitol on cerebral oedema after cryogenic injury in the mouse.

A cryogenic lesion was produced under halothane anaesthesia in the mouse by placing a cotton swab soaked in liquid nitrogen onto the surface of the cranium. This provoked an oedematous lesion which developed within the hour after the insult and evolved over the following week. Treatment with mannitol at 3 g/kg i.v. caused a significant 22% reduction in oedema 1 h later, when administered immediately after lesion, but not when administered 23-h post lesion. Likewise riluzole (16 mg/kg, i.v.) significantly reduced oedema by 17% when administered immediately after lesion, or 13% (P < 0.05) when administered 23 h after lesion. Repeated doses (2 x 16 mg/kg, i.p.) of riluzole were also able to reduce oedema significantly (24%, P < 0.05) at 24 h post lesion. Riluzole, in four repeated doses of 8 mg/kg i.p. was also able to reduce lesion surface size by 16% (P < 0.05) 48 h after lesion.

Neuroprotective effects of riluzole in neurotrauma models: a review.

Physical injury to the central nervous system (CNS) remains one of the main causes of mortality and disability in young adults. Numerous therapies have been successfully evaluated in experimental traumatic brain or spinal cord injuries (TBI, SCI) and, although some of them are currently under clinical trials for these indications, no drug therapy is at present available. Thus, an interesting approach to reduce the CNS injury-induced damage could be the blockade of Na(+)-channels by drugs such as riluzole which is neuroprotective in models of TBI or SCI as summarized in this review. Repeated doses ranging from 2 to 8 mg/kg were administered between 24 h to 10 days post-injury, with a first administration given either at 15 min or up to 6 h post-injury. In these models riluzole was found to reduce both the size of spinal cord and brain lesions as well as brain edema, and to restore the neurological, motor and cognitive impairments consequent of these injuries. The largest therapeutic time window obtained was 1 to 6 h in TBI. This such a compound should be considered as an interesting candidate for the treatment or SCI or TBI.

Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury.

1. The aim of this study was to investigate the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium-independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. 2. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg(-1)) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium-independent NOS) were evaluated 3 days after ischaemia. 3. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 4. 1400W attenuated the calcium-independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. 5. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.