Efficacy of flupirtine on cognitive function in patients with CJD: A double-blind study.

BACKGROUND: In cell culture experiments, flupirtine maleate (FLU), a triaminopyridine compound, was able to protect neuronal cells from apoptotic cell death induced by prion protein fragments and beta-amyloid peptides. As FLU is a clinically safe drug, the authors started a double-blind placebo-controlled study in patients with Creutzfeldt-Jakob disease (CJD). METHODS: Twenty-eight patients with CJD were randomized to an oral treatment with either FLU (n = 13) or matching placebo (PLA; n = 15). For inclusion and continuing the study, the patients had to achieve at least 50% in two of the subscales of the dementia tests employed. A battery of standardized questionnaires was employed to monitor the progression of the disease. The main outcome variable was the cognitive part of the Alzheimer's Disease Assessment Scale (ADAS-Cog); the difference between baseline and the best score under treatment was defined as the primary efficacy variable for hypothesis testing. RESULTS: CJD types were homogeneously distributed among the treatment groups. Patients treated with FLU showed significantly less deterioration in the dementia tests than patients treated with PLA. The mean change in ADAS-Cog (baseline to best) was +8.4 (+/-15.3) in the FLU group and +20.6 (+/-15.1) in the PLA group (p = 0.02, one-sided t-test). CONCLUSIONS: FLU has beneficial effects on cognitive function in patients with CJD. These positive results also may suggest a treatment potential of FLU in other neurodegenerative disorders. However, further studies are necessary.

SELEDO: a 5-year long-term trial on the effect of selegiline in early Parkinsonian patients treated with levodopa.

The SELEDO (from selegiline plus levodopa) study was carried out as a randomized, prospective, placebo-controlled, double- blind, multicenter long-term, 5-year trial to evaluate the possible advantages of combining selegiline and levodopa in the early treatment of Parkinson's disease. One-hundred-and-sixteen patients were randomized either to selegiline or placebo. Before starting the study medication, the levodopa dose was titrated to the individual requirements of each patient. The primary study end point (time when levodopa had to be increased by >50% of the titrated dose) was reached in 23 of 59 patients in the selegiline group and 26 of 48 patients in the placebo group. At the end of the 5 years' treatment period the rates derived from a life-table analysis were 50.4% in the selegiline group and 74.1% in the placebo group (P = 0.027, log-rank test). The median time to reach the primary end point was 4.9 years in the selegiline group and 2.6 years in the placebo group. In patients treated with selegiline, the mean levodopa dose changed only slightly over the 5 years of treatment compared to the initially titrated dose, but rose markedly in the placebo group, where the dose of levodopa had to be adjusted earlier than in the selegiline group. At the same time, the lower levodopa dosage in the selegiline group was accompanied by at least equal therapeutic efficacy (which is necessary for an unambiguous interpretation). Subgroup analyses showed greater benefit for selegiline treated) patients in the earlier stages. Long-term side effects appeared later in the selegiline group, although the difference was not significant. The early combination of selegiline and levodopa proved to be clearly superior to levodopa monotherapy.

[Reduction of spastic increased muscle tone in multiple sclerosis by the nonopioid analgesic flupirtine]. / Reduktion der spastischen Muskeltonuserhöhung bei Multipler Sklerose durch das Nonopioidanalgetikum Flupirtin. Eine randomisierte, doppelblinde Cross-over-Studie.

OBJECTIVES: A reduction of muscle tone in rats can be found after intraperitoneal or intrathecal injection of flupirtine in analgetic doses. However, a muscle-relaxing effect in patients with spasticity has not yet been shown. In this study we investigated whether flupirtine can reduce the increased muscle tone in patients with spasticity. METHODS: The muscle-relaxing effect of flupirtine was compared to that of tetrazepam and of placebo in a randomised, double-blind, cross-over design. Altogether 9 patients with spastically increased muscle tone of the lower limbs were investigated. The EMG activity integral of the rectus femoris muscle was chosen as quantitative parameter for degree and change of muscle tone. The EMG activity integral was measured using a dynamic bicycle ergometer 60, 120 and 180 min after administration of the tested drugs. The degree of spasticity was measured as the quotient of the EMG activity integral of the active and passive rotation phase. This quotient is inversely proportional to the spastic muscle activity. RESULTS: With flupirtine a significant increase in the EMG activity integral was found after 60 min (maximum effect). From 120 min it showed a slow decline. With tetrazepam the maximal effect was reached after 180 min. Placebo did not change the EMG activity integral during the experiment. CONCLUSION: These results support the thesis that flupirtine has a muscle-relaxing effect in patients with pathologically increased muscle tone.

Antioxidant properties of the triaminopyridine, flupirtine.

Flupirtine is a triaminopyridine-derived centrally acting analgesic, which interacts with mechanisms of noradrenergic pain modulation. Recently, it has been found to display neuroprotective effects in various models of excitotoxic cell damage, global and focal ischemia. Although this profile suggests that flupirtine acts as an antagonist of the N-methyl-D-aspartate (NMDA) and glutamate-triggered Ca2+ channel, there is no direct interaction with the receptor. In this paper, we examined whether flupirtine can act as an antioxidant and prevent free radical-mediated structural damage. Flupirtine at 5-30 microM inhibited ascorbate/ Fe2+ (1-10 microM)-stimulated formation of thiobarbituric reactive substances, an indicator of lipid peroxidation, in rat brain mitochondria. Interestingly, we found an increasing effectiveness of the drug at higher iron concentrations. Additionally, higher concentrations of flupirtine also provided protection against protein oxidation, as demonstrated by a decrease in protein carbonyls formed after treatment of rat brain homogenates with ascorbate/Fe2+. In PC12 cell culture, flupirtine at 10-100 microM was able to attenuate H2O2-stimulated cell death and improve the survival by 33%.

Pharmacological intervention in age-associated brain disorders by Flupirtine: Alzheimer's and prion diseases.

Alzheimer's disease, a major form of dementia in the elderly has become an increasingly important health problem in developed countries. In vitro studies on primary neurons demonstrate that Flupirtine (Katadolon) at a concentration of 1 microg/ml, significantly reduces the neurotoxic (apoptotic) effect displayed by A beta25-35, a segment of the amyloid beta-protein precursor the etiologic agent of Alzheimer's disease. Flupirtine, which has been in clinical use since 10 years ago, prevents the toxic effect of PrP, the presumed etiologic agent of the Creutzfeldt-Jakob disease as well as the excitatory amino acid glutamate on cortical neurons. Flupirtine displays a bimodal activity. Its strongest cytoprotective effect against glutamate-induced neurotoxicity was measured if administered at least 120 min prior to the addition of the glutamate. A likewise potent anti-apoptotic activity was measured if cells were simultaneously incubated with Flupirtine and the apoptotic inducers. Administration of Flupirtine during postincubation time in the experiments with glutamate did not result in neuroprotection. In parallel with the determination of the effect of Flupirtine on the toxin (A beta, PrP or glutamate)-induced neuronal death the effect of the drug on the intracellular Ca2+ level [Ca2+]i, was measured. It is well established that incubation of neurons with glutamate causes an increase in [Ca2+]i. It was found that a simultaneous administration of Flupirtine and glutamate did not reduce the glutamate-induced high Ca2+ level. Only if the cells had been preincubated for approximately 30 min with the drug the intracellular Ca2+ level was significantly lower. Experimental evidence given here shows that the molecular basis for the antiapoptotic effect of Flupirtine against glutamate, triggered during pre-incubation, is an increased expression of the protooncogene bcl-2. The neuroprotective effect determined during coincubation with the inducer is attributed to a normalization of the glutathione level which dropped in the presence of the inducers. It is concluded that Flupirtine is a promising drug to treat neurodegenerative disorders occurring with age, e.g. Alzheimer's disease and prion based diseases, like Creutzfeldt-Jakob disease. This conclusion is corroborated by the favourable pharmacokinetic profile of Flupirtine.

Effect of the triaminopyridine flupirtine on calcium uptake, membrane potential and ATP synthesis in rat heart mitochondria.

1. Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular. 2. The findings show that flupirtine increases Ca2+ uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg(-1) mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg(-1) mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria. 3. Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg(-1) mitochondrial protein and a return to control values at 20 nmol mg(-1) protein. 4. The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume. 5. Calcium at 80 and 160 nmol mg(-1) mitochondrial protein decreased ATP synthesis by 20-25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg(-1) protein was added before the addition of calcium. 6. Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg(-1) mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level.

Flupirtine, a nonopioid centrally acting analgesic, acts as an NMDA antagonist.

1. Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties. 2. In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain. 3. Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-D-aspartate (NMDA) antagonist with little evidence that it acts on AMPA-kainate type glutamate receptors. 4. No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor. 5. Studies on cultured cortical neurons show that the NMDA-induced influx of 45Ca2+ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N'-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of 45Ca2+ is produced by flupirtine. 6. Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress. 7. To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.

Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species.

Flupirtine (KATADOLON), known as a nonopiate centrally acting analgesic drug, was tested as to its potential to prevent apoptosis of human endothelial cells induced by reactive oxygen species (ROS). It was found that Flupirtine displayed no effect on viability and cell proliferation of human umbilical vein endothelial cells (HUVEC) up to a concentration of 10 microg/mL. Apoptosis, induced by ROS and generated by hypoxanthine/xanthine oxidase (EC 1.1.3.22) (HX/XOD) or t-butyl hydroperoxide, was reduced after preincubation with Flupirtine for 3 hr by 35% and 41%, respectively. The maximal cytoprotective effect against apoptosis was observed at a drug concentration of 1 to 3 microg/mL. Flow cytometric studies revealed that Flupirtine was able to decrease the number of necrotic cells as well as of apoptotic cells. Neither the simultaneous administration of Flupirtine with the apoptosis-inducing agent nor the preincubation of HUVEC with Flupirtine influenced the increase in the intracellular Ca2+ concentration [Ca2+]i caused by the production of ROS.

Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine.

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 micro M - 1 mM) was 100 micro M. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.

Effect of flupirtine on Bcl-2 and glutathione level in neuronal cells treated in vitro with the prion protein fragment (PrP106-126).

Flupirtine, trade name Katadolon, is a centrally acting nonopioid analgesic that has recently been found to display cytoprotective activity in vitro and in vivo on neurons induced to undergo apoptosis. This report shows that the PrP106-126 fragment of the prion protein, which is the likely etiological agent for a series of encephalopathies, is toxic to cortical neurons in vitro. Simultaneously, PrP106-126 influences the molecular GSH content and the bcl-2 expression in neurons. Significant toxicity (32% reduction in cell viability) was observed at a concentration of 50 microM of the peptide after 9 days of incubation, while at higher concentrations toxicity increased to 70%. Neurotoxicity was greatly reduced following coincubation with 1 to 3 microg/ml flupirtine. Concomitant with PrP106-126-mediated cytotoxicity, glutathione (GSH) content fell by > 70% with respect to untreated controls. This decrease in GSH level was strongly blocked by flupirtine under incubation conditions that reduce cell toxicity. In addition to normalizing GSH content, flupirtine induced the expression of the anti-apoptotically acting proto-oncogene bcl-2. Based on these in vitro data and on the favorable pharmacokinetic profile of the drug, we strongly suggest that flupirtine may prove useful for treatment of patients with prion disease.

Protection of flupirtine on beta-amyloid-induced apoptosis in neuronal cells in vitro: prevention of amyloid-induced glutathione depletion.

Effective drugs are not available to protect against beta-amyloid peptide (A beta)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment A beta25-35 at 1 microM in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 microM A beta25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 microg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with A beta25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like DNA fragmentation pattern and by the TUNEL technique. A beta25-35-induced DNA fragmentation could be abolished by preincubation of the cells with 1 microg/ml flupirtine. Incubation with A beta25-35 reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/10(6) cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where A beta accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.

Induction of apoptosis in cultured human retinal pigment epithelial cells is counteracted by flupirtine.

PURPOSE: The aim of the study was to determine whether flupirtine can counteract the induction of apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures were subjected to experimental ischemia (medium free of serum, glucose, and oxygen) with or without various substances for specific periods. The cells were then examined for breakdown of DNA by the TUNEL procedure and agarose gel electrophoresis. Moreover cells were processed for the localization of oncogene proteins (bcl-2, TIAR, ICH-1t) associated with apoptosis. The effect of flupirtine on reactive oxygen species also was determined. RESULTS: When RPE cells were subjected to ischemia for 72 hours approximately 65% of cells remained attached to the coverslips and approximately 65% of their nuclei showed clear fragmentation of DNA by TUNEL. Most of the cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells given ischemia for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of flupirtine (flupirtine gluconate, 100 microM) or 10% fetal calf serum in the medium prevented ischemia-induced apoptosis occurring after 72 hours. Neither N-methyl-D-aspartate (NMDA) (100 microM) nondeferoxamine (100 microM) nor the NMDA antagonists dextromethorphan (100 microM), memantine (100 microM), and MK-801 (10 microM) had a similar effect. NMDA, and to a lesser extent memantine, induced apoptosis independently. Treatment of RPE cells in serum-free medium with flupirtine (flupirtine gluconate, 100 microM) for 72 hours caused an upregulation of bcl-2 protein. In contrast, the oncogene proteins for TIAR and ICH-1t, were lower in flupirtine-treated cells than in control cells. Flupirtine, like deferoxamine, prevents iron-ascorbate-induced reactive oxygen species formation in retinal cells, but only flupirtine prevents ischemia-induced apoptosis in RPE cells. CONCLUSIONS: The combined data demonstrate that flupirtine is an effective agent in preventing death by apoptosis. Flupirtine reduces formation of reactive oxygen species in retinal dissociates and causes changes in various oncogene products in RPE cultures, which may explain its action in preventing apoptosis induced by ischemia. The current results also suggest that NMDA receptors are not involved in the induction of ischemia-induced apoptosis in RPE, cells.

Antiparkinsonian and other motor effects of flupirtine alone and in combination with dopaminergic drugs.

In this study we attempted to specify the behavioural profile of the analgesic flupirtine (1, 10 and 20 mg/kg p.o.) in the rat with respect to (i) its antiparkinsonian potential alone and as an adjunct to L-dihydroxyphenylalanine (L-DOPA) in the haloperidol-induced catalepsy (0.5 mg/kg haloperidol i.p.), (ii) locomotion and exploratory behaviour in the open field with holeboard, and (iii) possible psychomotor stimulating effects in the experimental chamber. In the two latter tests, behaviour was additionally challenged by D-amphetamine (2 mg/kg i.p.). In the catalepsy tests (horizontal bar, podium, grid) flupirtine alone was anticataleptic at doses of 10 and 20 mg/kg p.o., and the antiparkinsonian potential of a subthreshold dose of L-DOPA (50 mg/kg p.o.) was potentiated by 1 and 10 mg/kg p.o. flupirtine. On spontaneous forward locomotion in the open field with holeboard, flupirtine (1 and 10 mg/kg p.o.) had no marked effect but increased the frequency and duration of head dips, indicative for augmenting exploratory behaviour. Spontaneous rearing was reduced and D-amphetamine-induced rearing was enhanced by 1 mg/kg p.o. flupirtine. Grooming was reduced by 1 and 10 mg/kg p.o. flupirtine. In contrast, turning and grooming behaviour (spontaneous as well as D-amphetamine-induced) was not markedly influenced by flupirtine in the experimental chamber. Sniffing was increased in this test by 1 mg/kg p.o. flupirtine but not by the higher dose. Flupirtine is highly effective in antagonising neuroleptic-induced catalepsy as well as in potentiating L-DOPA treatment in the rat, suggesting it is a prospective new candidate for the therapy of Parkinson's disease.

Flupirtine reduces functional deficits and neuronal damage after global ischemia in rats.

Global cerebral ischemia leads to selective neuronal damage in the CA1 sector of the hippocampus and in the dorsolateral striatum. In addition, it results in deficits in spatial learning and memory as shown by an increase in escape latency and swim distance during the escape trials and a reduction of time spent in the quadrant of the former platform position during the probe trial of the water maze. Flupirtine is a non-opioid, centrally acting analgesic which has been shown to be neuroprotective against N-methyl-D-aspartate (NMDA)-mediated toxicity in vitro. The purpose of the present study was to investigate the potential protective effect of flupirtine in vivo with both behavioural and histological measures of global cerebral ischemia. Global ischemia was induced by four-vessel-occlusion (4VO) for 20 min in rats. Flupirtine was administered at a dose of 5 mg/kg i.p. either 20 min before and 50 min after occlusion (pre-treatment) or directly and 70 min after occlusion (post-treatment). 1 week after surgery, spatial learning and memory was tested in the Morris water maze. Pre-treatment with flupirtine reduced the increase in escape latency and in swim distance induced by 4VO. It also diminished the deficit in spatial memory as revealed by an increase in time spent in the quadrant of the former platform position during the probe trial which was reduced by 4VO. Post-treatment with flupirtine had no effect on the deficits in spatial learning and memory induced by 4VO. Neuronal damage in the CA1 sector of the hippocampus and in the striatum produced by 4VO was significantly attenuated with pre-treatment of flupirtine whereas post-treatment did not affect this neuronal damage. The present data demonstrate that pre-treatment with flupirtine exerts a protective effect on hippocampal and striatal neuronal damage and on deficits in spatial learning induced by 4VO.

Flupirtine protects both neuronal cells and lymphocytes against induced apoptosis in vitro: implications for treatment of AIDS patients.

In the present study we demonstrate that flupirtine, an already clinically used, centrally acting, non-opiate analgesic agent, protects rat cortical neurons against HIV-gp120 induced apoptotic cell death. The drug was active at concentrations between 1 and 10 microg/ml. Furthermore we show inhibition of in vitro induced apoptosis in human blood mononuclear cells, using flupirtine. Induced apoptosis in peripheral blood mononuclear cells from healthy individuals and HIV-1 infected patients was reduced to approximately 50% after in vitro preincubation with flupirtine at concentrations between 0.1 and 10 microg/ml. The anti-apoptotic effect of flupirtine was restricted to CD3+ lymphocytes and in particular to CD4+ cells. Flupirtine does not affect uninduced apoptosis in human lymphocytes in vitro. The selective potential of flupirtine to reduce apoptosis without influencing uninduced apoptosis may qualify this compound as a potential drug in the therapy of HIV-1 infected patients.

Flupirtine increases the levels of glutathione and Bc1-2 in hNT (human Ntera/D1) neurons: mode of action of the drug-mediated anti-apoptotic effect.

Flupirtine is a triaminopyridine analogue which has been successfully applied in clinics as a non-opiate analgesic drug. Previously we described that flupirtine acts like an N-methyl-D-aspartate (NMDA) receptor antagonist in neuronal cells both in vitro and in vivo. Here we show that flupirtine displays its anti-apoptotic effect also in hNT (human Ntera/D1) neurons. hNT neurons were induced to apoptosis applying glutamate (Glu; at concentrations > or = 1 mM) or NMDA (> or = 1 mM). During Glu/NMDA-mediated apoptosis the levels of the intracellular anti-apoptotic agents Bc1-2 and glutathione dropped by more than 50%. Flupirtine completely abolished this reduction of Bc1-2 and glutathione level at a concentration of 10 microM. In the presence of 3 microM flupirtine a > 6-fold increase of the Bc1-2 (B-cell leukemia/lymphoma-2) level was observed in hNT neurons. At the same concentration, the intracellular level of glutathione increased to 200%. We conclude that the Glu/NMDA-mediated neuronal cell death in vitro is controlled at least partially by Bc1-2 and glutathione. Neuronal cell death by Glu or NMDA in vitro can be overcome applying the drug flupirtine which is in clinical use.

[Flupirtine in comparison with chlormezanone in chronic musculoskeletal back pain. Results of a multicenter randomized double-blind study]. / Flupirtin im Vergleich zu Chlormezanon und Plazebo bei chronischen muskuloskelettalen Rückenschmerzen. Ergebnisse einer multizentrischen randomisierten Doppelblindstudie.

METHOD: The analgesic and muscle-relaxing properties of flupirtine maleate, chlormezanone and placebo were compared in a total of 184 patients. Of these patients, 164 met the criteria of the treatment plan (intention to treat), and the data of 140 patients were finally evaluated in accordance with the test protocol. A positive response was defined as a reduction in pain intensity and muscle tension by 2 categories on the 5-category verbal scale "very severe/severe/moderate/mild/ none" on the seventh day of treatment. RESULTS: In the per-protocol-analysis the responder rate was 60.9% for flupirtine, 47.8% for chlormezanone, and 43.8% for placebo, the difference between drugs and placebo not being significant. The overall assessment of the physicians involved was very good/ good in 47.8% and satisfactory in 37.0% of the flupirtine group, very good/good in 45.6% and satisfactory in 17.4% of the chlormezanone group, the corresponding figures for the placebo group being 33.4% and 20.6%, respectively. Flupirtine was thus superior to placebo (p = 0.007). The incidence of adverse drug reactions was 14.8% (8/54) for flupirtine, 19.3% (11/57) for chlormezanone, and 7.3% (4/55) for placebo.

Protection of rabbit retina from ischemic injury by flupirtine.

PURPOSE: The aim of this study was to determine wether flupirtine can slow down the changes seen in the rabbit retina after ischemia--reperfusion. METHODS: A suction-cup procedure, which raises intraocular pressure, was used to give an ischemic insult to the rabbit retina. Electroretinograms were recorded before ischemia and at different periods after ischemia. In some instances, flupirtine was injected into the eye before ischemia. Immunohistochemistry was used to study the effect of ischemia-reperfusion on the gamma-aminobutyric acid (GABA) immunoreactivity and uptake of serotonin by the retina. The effect of flupirtine and ischemia on retinal adenosine triphosphate (ATP) levels were determined in in vivo and in vitro experiments. RESULTS: Ischemia for 75 minutes causes a change in the nature of normal GABA immunoreactivity and reduction in the b-wave of the electroretinogram. When flupirtine is injected into the vitreous humor at the onset of ischemic insult, the changes in GABA immunoreactivity are reduced and the recovery of the reduced b-wave of the electroretinogram after defined reperfusion times is enhanced significantly. Rat retinas incubated in vitro in physiological solution containing flupirtine caused a significant rise in the tissues' ATP content compared with control samples. However, incubation of the tissue in physiological solution saturated with nitrogen caused a drop in retinal ATP levels. Addition of flupirtine prevented this decrease from taking place. Serotonin injected into the vitreous humor of the rabbit eye is taken up by certain amacrine cells. The amount of serotonin taken up is reduced greatly in retinas, as judged by immunohistochemistry when tissues are subjected to ischemia. Because the ischemia was shown to cause a drop in the tissue ATP level, it is concluded that this is the cause of the reduced uptake of exogenous serotonin. Injection of flupirtine into the vitreous humor during ischemia enhanced the uptake of serotonin. CONCLUSIONS: Combined data show that flupirtine is a neuroprotective agent in retinal ischemia and that one mode of its mechanism of action is to influence ATP levels. Flupirtine may lower the activity of NMDA receptors, thus causing ATP levels to be less affected in the presence of the drug as a secondary effect.

Antiparkinsonian effect of flupirtine in monoamine-depleted rats.

Excitatory amino acid receptor antagonists lead to marked suppression of parkinsonian-like symptoms in rodent and primate models of Parkinson's disease and are able to potentiate the ability of L-DOPA to reverse akinesia and ameliorate muscular rigidity displayed in these animal models. Flupirtine, which is clinically used as a non-opioid analgesic agent, has some N-methyl-D-aspartate (NMDA) antagonistic properties in several in vivo and in vitro experiments. We now report that in monoamine depleted rats (pretreated with reserpine, 5 mg/kg, and alpha-methyl-para-tyrosine, 250 mg/ kg i.p.) flupirtine dose-dependently (1-20 mg/kg i.p.) suppressed rigidity, measured as tonic EMG activity in the gastrocnemius muscle, but had no effect on akinesia, measured as locomotor activity. In addition, it potentiated the antiparkinsonian effect of L-DOPA on akinesia and rigidity in this rodent model of Parkinson's disease. These effects of flupirtine are of particular clinical relevance, since flupirtine is devoid of the typical side effects of NMDA-receptor antagonists.

Neurotoxicity in rat cortical cells caused by N-methyl-D-aspartate (NMDA) and gp120 of HIV-1: induction and pharmacological intervention.

Incubation of highly enriched neurons from rat cerebral cortex with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 18 h results in fragmentation of DNA at internucleosomal linkers, a feature of apoptosis. We report that neurons respond to exposure to gp120 with an increased release of arachidonic acid via activation of phospholipase A2. This process is not inhibited by antagonists of the N-methyl-D-aspartate (NMDA) receptor channels. To investigate the influence of arachidonic acid on the sensitivity of NMDA receptor towards its against, low concentrations of NMDA were coadministered with arachidonic acid. Under these conditions the NMDA-mediated cytotoxicity was enhanced. We conclude that gp120 causes an activation of phospholipase A2, resulting in an increased release of arachidonic acid which in turn sensitizes the NMDA receptor. Two compounds were found to act cytoprotectively against the deleterious effect caused by gp120 on neurons: Memantine [1-amino-3,5-dimethyladamantane] and Flupirtine [2-amino-3-ethoxycarbonylamino-6-(4-fluoro-benzyl-amino)-pyridine maleate]. Both compounds have been found to display a potent cytoprotective effect on neurons treated with the excitatory amino acid NMDA or with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. The NMDA antagonist Memantine, a drug currently used in the therapy of spasticity and Parkinson's disease, prevented the effects of gp120 at micromolar concentrations. Flupirtine was previously found to be a centrally acting, nonopiate analgesic agent which additionally possesses anticonvulsant and muscle-relaxant activity at doses similar to those producing analgesia. The cytoprotective effect of Flupirtine in vitro was significant (above 10 microM). Considering the fact that both Memantine and Flupirtine display almost no clinical side effects, these drugs may prove useful both in preventing primary infection of brain cells with the HIV virus, as well as in treating the neurological disorders often associated with the immunodeficiency syndrome such as AIDS-related dementia.