A study of riluzole in the treatment of advanced stage or elderly patients with amyotrophic lateral sclerosis.

Treatment with the neuroprotective drug riluzole has previously been shown to increase the probability of survival in patients with amyotrophic lateral sclerosis. This report describes a placebo-controlled, double-blind randomised clinical trial of riluzole carried out in ALS patients with advanced stage disease or aged over 75 years. The primary objective was to enable access to treatment to patients excluded from the pivotal trial which was run in parallel. Another goal was to assess the safety of riluzole in patients with advanced-stage disease. One hundred and sixty-eight patients were included, randomised to either riluzole 50 mg b. i. d. or to placebo, and treated for eighteen months. Riluzole was well-tolerated in this patient population, and the adverse events observed were similar in nature and frequency to those observed in previously published clinical trials in patients included in pivotal trials. The study could not include enough patients to reach adequate power to detect differences in survival between the two treatment groups, and no such difference was in fact observed. In conclusion, riluzole is well-tolerated in ALS patients with advanced stage disease.

Efficacy and tolerability of entacapone as adjunctive therapy to levodopa in patients with Parkinson's disease and end-of-dose deterioration in daily medical practice: an open, multicenter study.

Entacapone is a potent, reversible and orally active inhibitor of catechol-O-methyltransferase. This open multicenter study evaluated the efficacy, safety and tolerability of entacapone as adjunct therapy to levodopa/dopa decarboxylase inhibitor (> or = 3 daily doses) in patients with idiopathic Parkinson's disease and end-of-dose motor fluctuations. The 8-week study included 489 patients under conditions of typical daily medical practice. Patients were treated with a 200-mg fixed dose of entacapone administered with each scheduled dose of levodopa to a maximum of 10 doses per day. Other antiparkinsonian medication should have been stable for at least 1 month. The primary efficacy criteria were: (1) Part II (activities of daily living, ADL) of the Unified Parkinson's Disease Rating Scale (UPDRS), (2) the reduction of 'off' time during the daily waking period as assessed by the percentage of patients improving by at least one category at Item 39 of Part IV of the UPDRS. Secondary outcome measures included: (1) the investigator's global assessment of change, (2) quality of life (QoL) was assessed using the Parkinson's Disease Questionnaire (PDQ-39). Adverse events, vital signs and liver enzymes were monitored at weeks 2 and 8. The baseline mean score for ADL was 10.5 (+/-7.04), which decreased to 8.5 (+/-6.37) at the end of the study (p < 0.0001). Compared to baseline, 40.8% of patients experienced a reduction in 'off' time during the waking period; this improvement was highly significant (p < 0.0001). A reduction in the daily dose of levodopa was observed in 35.8% of patients (mean decrease 209 +/- 149 mg). QoL was improved by a mean of 10% in all categories of the PDQ-39 (p < 0.001), except social support and cognition. This improvement was statistically significant (p < 0.001). The dyskinesia score (UPDRS Item 32) was decreased significantly from 2.3 to 2.1 from baseline to end of study (p < 0.001), although 52.7% of patients reported levodopa-induced dyskinesia as an adverse event. There was no case of increased liver enzymes. The study results confirm that the excellent risk/benefit ratio seen in phase III controlled studies can be seen in daily neurological practice. Moreover, the study suggests that the benefits of entacapone are associated with a significant improvement in QoL.

Population pharmacokinetics of riluzole in patients with amyotrophic lateral sclerosis.

OBJECTIVES: To characterize the population pharmacokinetic of riluzole in patients with amyotrophic lateral sclerosis (ALS). METHODS: One hundred patients with ALS who were participating in a multicenter phase III dose-ranging trial of riluzole were sampled on 179 visits. The sampling strategy (two samples per visit) was varied across patients to define the population kinetic profile (full screen). Riluzole plasma levels were determined by HPLC, and the data were analyzed by nonlinear mixed-effect modeling (NONMEM program) with use of a one-compartment structural model. The model incorporated interoccasion (visit-to visit) variability. RESULTS: In the basic one-compartment pharmacokinetic model, interindividual variability in plasma clearance (51.4%) was higher than intraindividual (visit-to-visit) variability (28.0%), indicating uniform pharmacokinetic behavior during long-term therapy. Riluzole clearance was independent of dosage (25 to 100 mg twice daily), treatment duration (up to 10 months), age, and renal function; gender and smoking were the most important patient covariates, with hepatic function having lesser influence. Typical value of clearance was 51.4 L/hr for a nonsmoking male patient. It was 32% lower in women than in men and 36% lower in nonsmokers than in smokers. Gender- and smoking-related variations in riluzole exposure at the recommended dosage (50 mg twice daily) were within the range of exposures achieved (with no untoward effect) in this dose-ranging study. CONCLUSION: The pharmacokinetics of riluzole has been characterized in patients during long-term therapy. Riluzole clearance is independent of dose and treatment duration. Within-patient variability is low. Gender and smoking status are the main covariates to explain interpatient variability.

A confirmatory dose-ranging study of riluzole in ALS. ALS/Riluzole Study Group-II.

ALS is a progressive motor neuron disease with no effective treatment. The anti-excitotoxic drug riluzole (100 mg/day) has been shown to decrease mortality and muscular deterioration in ALS patients. To confirm and extend the therapeutic effect of riluzole, we performed a double-blind, placebo-controlled, multicenter, international, dose-ranging (50, 100, 200 mg/day), stratified study in 959 ALS outpatients treated for up to 18 months. Primary efficacy criterion was survival and the effect of treatment was analyzed before (Wilcoxon and log rank tests) and after adjustment on prognostic factors (Cox model). Secondary efficacy criterion was disease progression assessed through change in functional measures. Tracheostomy-free survival rates were: 50.4% (placebo), 55.3% (50 mg riluzole) (p = 0.23, Wilcoxon test; p = 0.25, log-rank test), 56.8% (100 mg riluzole) (p = 0.05, Wilcoxon test; p = 0.076, log-rank test), and 57.8% (200 mg riluzole) (p = 0.061, Wilcoxon test; p = 0.075, log-rank test). At the end of the 18-month study, there was a significant dose-related decrease in risk of death or tracheostomy (p = 0.04). Adjustment for baseline prognostic factors showed a 35% decreased risk of death with the 100-mg dose compared with placebo (p = 0.002). No significant treatment effects were detected for the functional assessments. The most frequent dose-related adverse events included nausea, asthenia, and elevated liver enzyme levels. This study confirms the therapeutic effect of riluzole in a large representative ALS sample, over an 18-month period. Riluzole is well tolerated and decreases the risk of death or tracheostomy in ALS patients.

Antagonism by riluzole of entry of calcium evoked by NMDA and veratridine in rat cultured granule cells: evidence for a dual mechanism of action.

1. Intracellular calcium levels were measured in cultured cerebellar granule cells of the rat by use of the fluorescent dye, indo-1/AM. 2. Intracellular calcium levels were increased by depolarizing stimuli such as N-methyl-D-aspartate (NMDA) (100 microM), glutamic acid (20 microM), and veratridine (10 microM). This increase was essentially due to entry of external calcium. 3. Riluzole (10 microM) blocked responses to all the depolarizing agents. 4. Riluzole could still block the increase in intracellular calcium evoked by NMDA or glutamic acid when sodium channels were blocked by tetrodotoxin, suggesting that this effect is not mediated by a direct action of riluzole on the voltage-dependent sodium channel. 5. Pretreatment of the cells with pertussis toxin (0.1 micrograms ml-1) did not modify the increases in intracellular calcium evoked by NMDA, glutamic acid or veratridine. 6. In pertussis toxin-treated cells, riluzole could no longer block responses to excitatory amino acids, but still blocked responses to veratridine. 7. It is concluded that riluzole has a dual action on cerebellar granule cells, both blocking voltage-dependent sodium channels and interfering with NMDA receptor-mediated responses via a pertussis toxin-sensitive mechanism. Furthermore, these two processes have been shown to be independent.

Differential effects of propofol and ketamine on cytosolic calcium concentrations of astrocytes in primary culture.

Propofol has been shown recently to alter cellular communication mediated by gap junctions between astrocytes (a glial cell subpopulation involved in major brain functions). As marked increases in concentrations of cytosolic calcium ([Ca2+]i) produce closure of the gap junction, we have investigated the effects of both propofol and ketamine on resting [Ca2+]i concentrations in mouse cultured astrocytes using microfluorimetry with the indo-1 fluorescent probe. Propofol 10(-5) and 10(-4) mol litre-1 induced a monophasic transitory Ca2+ peak in a large subpopulation of the cells tested. This response exhibited characteristics close to those of the peak elicited by [L-Pro9] substance P (10(-7) mol litre-1), an activator of phospholipase C in astrocytes. In both cases, it possibly corresponded to mobilization of Ca2+ from endogenous stores. In a few cases, however, administration of propofol induced a moderate, but sustained increase in [Ca2+]i corresponding to the entry of external Ca2+ into the cells. In contrast, ketamine 10(-5) and 10(-4) mol litre-1 failed to affect [Ca2+]i resting concentrations. These findings indicate that clinically relevant concentrations of propofol, but not ketamine, increased [Ca2+]i concentration in astrocytes.

Monoamine oxidase inhibitors, cognitive functions and neurodegenerative diseases.

Recent data obtained in animals and in humans suggest that both MAO-A and MAO-B inhibitors present cognitive enhancing properties of possible interest in the treatment of cognitive disorders. In addition, the rational for using selegiline as a neuroprotector in Parkinson's disease may also be applicable in Alzheimer's disease in which a dramatic increase in the MAO-B activity has been reported. It seems then worthwhile to investigate the neuroprotective effect of MAOIs in humans and to assess, furthermore, the real therapeutical benefit of their cognitive enhancing properties.

2-Chloroadenosine potentiates the alpha 1-adrenergic activation of phospholipase C through a mechanism involving arachidonic acid and glutamate in striatal astrocytes.

In cultured striatal astrocytes, 2-chloroadenosine, an adenosine analog resistant to adenosine deaminase, although inactive alone, markedly potentiated the activation of phospholipase C induced by methoxamine, an alpha 1-adrenergic agonist. This effect was suppressed by antagonists of either A1 adenosine or alpha 1-adrenergic receptors. An influx of calcium and two distinct G-proteins are involved in this phenomenon since the potentiating effect of 2-chloradenosine was suppressed in the absence of external calcium or when cells were pretreated with pertussis toxin. In addition, arachidonic acid is likely involved in this potentiating effect. This was shown first by examining the effects of inhibitors of phospholipase A2 or arachidonic metabolism, then by examining the action of arachidonic acid on the production of inositol phosphates in either the presence or absence of methoxamine, and finally by measuring the release of arachidonic acid. The sequential activation of phospholipase C and of protein kinase C is required for the 2-chloroadenosine-induced activation of phospholipase A2 since 2-chloroadenosine markedly stimulated phospholipase C activity in the absence of methoxamine when protein kinase C was activated by a diacylglycerol analog. Finally, the enhancing effect of 2-chloroadenosine on the methoxamine-evoked response seems to result from an inhibition of glutamate reuptake into astrocytes by arachidonic acid. Indeed, the potentiating effect of 2-chloroadenosine was suppressed when external glutamate was removed enzymatically and mimicked by either selective inhibitors of the glutamate reuptake process or direct application of glutamate.

Synergistic regulation of cytosolic Ca2+ concentration by somatostatin and alpha 1-adrenergic agonists in mouse astrocytes.

The effects of somatostatin and alpha 1-adrenergic receptor agonists on cytosolic Ca2+ in striatal astrocytes from the embryonic mouse in primary culture have been investigated by microfluorimetry. Methoxamine or somatostatin induced a transitory increase in cytosolic Ca2+, but their combined addition led to a sustained increase in cytosolic Ca2+ which seems to be due to a Ca2+ influx since it was not observed in the absence of external Ca2+. Voltage-independent Ca2+ channels contribute to this process. Indeed, voltage-operated calcium channels are not involved since neither dihydropyridines nor La3+ were effective in suppressing the sustained cytosolic Ca2+ elevation. Moreover, depolarization by 50 mM KCl, which was ineffective alone, suppressed the effect of somatostatin observed in the presence of the alpha 1 agonist, methoxamine. The implication of arachidonic acid in the observed potentiation is suggested by the following observations: 1) arachidonic acid induced a sustained elevation of cytosolic Ca2+ similar to that evoked by the co-application of methoxamine and somatostatin; 2) the addition of ETYA, an inactive and non-metabolizable analogue of arachidonic acid suppressed the calcium plateau produced by the agonists. In addition, direct activation of PKC by an exogeneous diacylglycerol analogue allowed somatostatin alone to evoke a sustained elevation of cytosolic Ca2+. Therefore, methoxamine through the successive activation of PLC and PKC could allow a lipase, probably PLA2, to be stimulated by somatostatin. Since arachidonic acid has already been shown to trigger the opening of K+ channels and the formation of inositol phosphates, somatostatin, through the arachidonic acid-mediated hyperpolarization could increase the Ca2+ driving force and thus improve Ca2+ influx through the inositol phosphate gated channels.

Synergistic regulation of cytosolic Ca2+ concentration in mouse astrocytes by NK1 tachykinin and adenosine agonists.

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.

Somatostatin potentiates the alpha 1-adrenergic activation of phospholipase C in striatal astrocytes through a mechanism involving arachidonic acid and glutamate.

As previously shown with adenosine, somatostatin, which is ineffective alone, enhanced the alpha 1-adrenergic-agonist-stimulated production of inositol phosphates in cultured striatal astrocytes. This effect was suppressed in cells pretreated with pertussis toxin. It required external calcium and was selectively antagonized by both mepacrine, an inhibitor of phospholipase A2, and 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of arachidonic acid. In addition, a long-lasting elevation of cytosolic calcium and a release of arachidonic acid were observed only under the combined stimulation of somatostatin and alpha 1-adrenergic receptors. Arachidonic acid could in turn inhibit glutamate uptake into astrocytes, and the resulting external accumulation of glutamate could account for the somatostatin-evoked amplification of the alpha 1-adrenergic-agonist-stimulated hydrolysis of inositol-phospholipids. The effect of somatostatin was indeed reproduced by glutamate or glutamate uptake inhibitors and suppressed by enzymatic removal of external glutamate. Thus, astrocytes may contribute to long-term plasticity events in glutamatergic synapses through regulation of external glutamate levels.

Synergistic effects in the alpha 1- and beta 1-adrenergic regulations of intracellular calcium levels in striatal astrocytes.

1. Using indo-1 as a calcium fluorescent probe, we have observed the following in striatal astrocytes in primary culture. 2. The stimulation of alpha-adrenoceptors induces a rapid rise in cytosolic calcium resulting from an internal calcium mobilization followed by an external calcium influx (4-min duration). 3. The stimulation of beta 1-adrenoceptors evokes only a slight internal calcium mobilization (90-sec duration). 4. The simultaneous stimulation of beta 1- and alpha 1-adrenoceptors induces a more prolonged calcium influx (10 min). The latter phenomenon could explain the calcium-dependent synergistic effects of alpha 1 and beta stimulation on cAMP production already described in the brain.

Are several G proteins involved in the different effects of endothelin-1 in mouse striatal astrocytes?

High-affinity specific receptors of endothelin (ET-1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I-ET-1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET-1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide-binding proteins.

Both Astrocytes and Neurons Contribute to the Potentiation Mediated by alpha1-Adrenoceptors of the beta-Adrenergic-Stimulated Cyclic AMP Production in Brain.

Using primary neuronal or astrocyte cultures from the striatum of the embryonic mouse, we have observed that the beta-adrenergic agonist isoprenaline (10-5 M) induced a more pronounced accumulation of cAMP in astrocytes than in neurons. In both cell types, the alpha-adrenergic selective agonist methoxamine (10-4 M), which alone did not affect the production of cAMP, potentiated the isoprenaline-evoked response. In support of these observations, when associated alpha2-noradrenergic and D1-dopaminergic responses were prevented, the mixed alpha1- and beta-adrenergic agonist noradrenaline (10-5 M) induced a production of cAMP which was totally blocked by propranolol (10-6 M) and partially abolished by prazosin (10-6 M). Since experiments were made in the presence of 3-isobutyl-1-methylxanthine (1 mM), the observed effects of cAMP accumulation were not related to a modulation of phosphodiesterase activities. In addition, both in astrocytes and in neurons, the potentiation by alpha1-adrenergic agonists of the beta-adrenergic-evoked response required external calcium. Using INDo 1 as a fluorescent probe, methoxamine (25 microM) was shown to induce in astrocytes an increase in cytosolic calcium concentration which was prolonged by isoprenaline (10-5 M) only in the presence of external calcium. These results suggest that the prolonged increase in cytosolic calcium concentration linked to the activation of alpha1- and beta-adrenergic receptors is responsible for the potentiation of the beta-adrenergic-induced production of cAMP, which is partially dependent on external calcium.

Clinical neuropharmacology of calcium antagonists.

Experimental and clinical data clearly demonstrate that calcium antagonists (CA) may have an action on the central nervous system (CNS). The cerebrovascular action of CA justifies their use in cerebral ischaemia, vasospasm and hypoxia. Several clinical trials have demonstrated such beneficial effects. On the other hand a number of reports indicate that CA may have a direct neuronal effect, although most of such trials have not been verified or are mere case reports. In addition, the large number of conditions susceptible to being corrected by CA is impressive: epilepsy, pain, dystonia, dyskinesia, psychiatric conditions, etc. Other papers are disconcerting that report extrapyramidal disorders induced by flunarizine and cinnarizine in the elderly, whereas nicardipine does not produce such side effects and may even alleviate some parkinsonian symptoms. In various experimental models (e.g. stroke, oedema), pharmacological effects have been shown to vary from one compound to the other. Two main questions are yet to be answered: 1) has the direct neuronal effect of CA been clearly established? 2) are the multiple clinical effects on the CNS really linked to calcium antagonism?