The attenuating effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in human neuroblastoma SH-SY5Y cells

Memantine, a clinically used N-methyl-D-aspartate (NMDA)-receptor antagonist, has been shown to prevent apoptotic neuronal damage connected with the over-activity of NMDA receptors. In the present study, we examined the effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in the SH-SY5Y cell line which does not possess functional NMDA receptors. Electrophysiological recordings and toxicity studies showed no response to NMDA-evoked currents in this cell line, irrespective of the stage of its neuronal differentiation. Memantine (0.1-2 microM) attenuated staurosporine-induced apoptosis as evidenced by reversal of the changes in mitochondrial membrane potential (DeltaPsi(m)) and decreased caspase-3 activity, lactate dehydrogenase (LDH) release and DNA fragmentation. Wortmannin (10 nM) and LY 294002 (10 microM) (inhibitors of phosphatidylinositol-3-kinase, PI3-K) reversed the inhibitory effect of memantine on the staurosporine-induced LDH release, suggesting that the PI3-K/Akt prosurvival pathway is a possible target for antiapoptotic action of memantine. Memantine at low micromolar concentrations also attenuated salsolinol- and doxorubicin-induced LDH release and DNA fragmentation, but only in the case of salsolinol was this effect accompanied by a decrease in caspase-3 activity. The present data indicate that memantine attenuates the toxic effects of various proapoptotic agents and the cytoprotective effect of memantine does not seem to be connected with its action on NMDA receptor but rather with its influence on intracellular pathways engaged in cellular survival/apoptotic processes.     

<Emphasis Type="Italic">N</Emphasis>-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate Receptor Antagonists Have Variable Affect in 3-Nitropropionic Acid Toxicity

There is accumulating evidence that excitotoxicity and oxidative stress resulting from excessive activation of glutamate (N-methyl-d-aspartate) NMDA receptors are major participants in striatal degeneration associated with 3-nitropropionic acid (3NP) administration. Although excitotoxic and oxidative mechanisms are implicated in 3NP toxicity, there are conflicting reports as to whether NMDA receptor antagonists attenuate or exacerbate the 3NP-induced neurodegeneration. In the present study, we investigated the involvement of NMDA receptors in striatal degeneration, protein oxidation and motor impairment following systemic 3NP administration. We examined whether NMDA receptor antagonists, memantine and ifenprodil, influence the neurotoxicity of 3NP. The development of striatal lesion and protein oxidation following 3NP administration is delayed by memantine but not affected by ifenprodil. However, in behavioral experiments, memantine failed to improve and ifenprodil exacerbated the motor deficits associated with 3NP toxicity. Together, these findings suggest caution in the application of NMDA receptor antagonists as a neuroprotective agent in neurodegenerative disorders associated with metabolic impairment.     

Increase of desensitization as an additional mode of action for classical NMDA receptor channel blockers

It is well known that not all effects of such classical NMDA receptor channel blockers as spermine and phylantotoxins are madiated via channel blockade. To determine if these side effects have any functional role we studied inhibition of NMDA receptors by derivatives of phenylcyclohexyl and clinically approved memantine. We showed that these drugs not only blocked the channel pore but also potentiated desensitization of NMDA receptors. Apparently, these effects are not interrelated because there was no correlation between a degree of “trapping” and a level of channel desensitization induced by these blockers. Yet, in contrast to channel blockade, which was strongly voltage-dependent, these compounds affected NMDA receptor desensitization in a voltage-independent manner. Based on these facts, we suppose the existence of two different binding sites for memantine and functionally related compounds, one being responsible for channel blockade and another being coupled to channel desensitization. This possibility should be taken into account when interpreting experimental and clinical effects of these channel blockers.     

No interaction of memantine with acetylcholinesterase inhibitors approved for clinical use

The loss of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may underlie aspects of the dementia. Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie the degeneration of cholinergic cells. New drug therapies have been designed to either enhance cholinergic function by inhibition acetylcholinesterase (AChE), e.g. galanthamine, tetrahydroaminoacridine or donepezil, or by attenuation of NMDA receptor function, e.g. memantine. A combination of these two therapeutic approaches may be more beneficial at slowing the progression of the AD. The current study investigated whether memantine would attenuate the inhibition of AChE produced by these three drugs. The results indicate that these AChE inhibitors do not lose their therapeutic efficacy in combination with memantine. Our in vitro data suggest that the clinical combination of memantine with a reversible AChE inhibitor should be a valuable pharmacotherapeutic approach to dementia.     

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and γ-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [³H]MK-801 with a K_i value of 0.27 μm, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.     

Design, synthesis, and biological evaluation of tricyclic heterocycle-tetraamine conjugates as potent NMDA channel blockers

We have developed a new class of N-methyl-d-aspartate (NMDA) channel blockers having a conjugate structure that consists of a nitrogenous heterocyclic head and a tetraamine tail. Among them, dihydrodibenzazepine-homospermine conjugate (8) exhibited potent antagonistic activity at NR1/NR2A or NR1/NR2B NMDA subtype receptors compared with the lead compound, AQ343 (1), or memantine, as well as weak cytotoxicity. Its superior biological profiles compared with known compounds point to its potential use as therapeutic agents for neurological disorders.     

Anticataleptic potencies of glutamate-antagonists

Summary The anticataleptic effects of non-competitive and competitive NMDA antagonists as well as those of an agonist at the allosteric glycine binding site of the NMDA receptor were tested in the catalepsy model. Some of these drugs were further tested in a reaction time task demanding rapid locomotor initiation. The results show that the non-competitive NMDA antagonists dizocilpine and memantine as well as the competitive antagonists CGP 39551, CGP 37849 and CPPene antagonized dopamine D2 receptor mediated catalepsy induced by haloperidol. D-cycloserine, a partial glycine agonist per se had no effects, but it enhanced the anticataleptic effects of dizocilpine when coadministered. However, the effects of CGP 37849 were abolished. Dopamine D1 receptor mediated catalepsy induced by SCH 23390 was antagonized by dizocilpine, memantine, CPPene, but not by CGP 37849. In the reaction time task dizocilpine, memantine and CGP 37849 were tested for their anti-akinetic and anti-bradykinetic potencies. All these compounds improved haloperidolinduced slowing of reaction time. However, they acted differentially on haloperidol-induced slowing of movement execution and decreased initial acceleration. Thus, antagonists at the NMDA receptor may have a therapeutic potential in the treatment of Parkinson's disease. Their potency can be manipulated specifically at the glycine binding site.     

An involvement of BDNF and PI3-K/Akt in the anti-apoptotic effect of memantine on staurosporine-evoked cell death in primary cortical neurons

Memantine, a clinically used NMDA receptor antagonist possesses neuroprotective properties, but the exact mechanisms of its beneficial action on neuronal survival are poorly recognized. In the present study, some intracellular mechanisms of memantine effects on staurosporine-evoked cell death were investigated in primary cortical neurons. Memantine (0.1–2 μM) suppressed neuronal apoptosis evoked by staurosporine in 7 DIV cortical neurons, whereas other antagonists of NMDA receptor, MK-801 (1 μM) and AP-5 (100 μM) were ineffective. The anti-apoptotic effects of memantine were not connected with any changes in cytoplasmic calcium concentration or reactive oxygen species level. The immunoblot analysis showed that the staurosporine induced a decrease in p-Akt protein kinase level and that this effect was reversed by memantine treatment. Moreover, the PI3-K inhibitors, wortmannin and LY 294002 attenuated the anti-apoptotic action of memantine on staurosporine-induced cell damage. Furthermore, the ELISA studies showed increased cellular and released BDNF protein level after combined treatment with memantine and staurosporine. There was no effect of memantine on the activation and expression of other protein kinases involved in the mechanism of cellular survival, i.e. ERK1/2, JNK and GSK3-β. The obtained data suggest an NMDAR-independent action of memantine in attenuation of neuronal apoptosis and point to the engagement of BDNF and PI3-K/Akt pathway in these processes.     

NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA-Evoked Striatal Acetylcholine but Not Spermidine Release

Abstract: NMDA receptor stimulation concomitantly increases the release of [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The NMDA-induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µ M ). However, certain other channel blockers, including dextromethorphan (1–100 µ M ), which antagonized NMDA-evoked acetylcholine release without affecting NMDA-evoked spermidine release, and dextrorphan (1–100 µ M ) and memantine (1–100 µ M ), which block NMDA-evoked acetylcholine release more potently than NMDA-evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µ M ) blocked NMDA-evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µ M ), philanthotoxin₃₄₃, and argiotoxin₆₃₆ (10 µ M ) and was also noted for desipramine (1–100 µ M ). The NMDA-induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.     

NMDA channel blockers: memantine and amino-aklylcyclohexanes –In vivo characterization

The previous overviews provided the basis for better therapeutic efficacy/tolerability of low to moderate affinity NMDA channel blockers. This prediction finds support in in vitro studies comparing protective and plasticity impairing effects (therapeutic vs. side-effect) of memantine and (+)MK-801. In fact it turned out that memantine had a far better therapeutic index. But can it be confirmed in the in vivo situation?     

Memantine inhibits and reverses the Alzheimer type abnormal hyperphosphorylation of tau and associated neurodegeneration

Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, reduces the clinical deterioration in moderate-to-severe Alzheimer disease (AD) for which other treatments are not available. The activity of protein phosphatase (PP)-2A is compromised in AD brain and is believed to be a cause of the abnormal hyperphosphorylation of tau and the consequent neurofibrillary degeneration. Here we show that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices. Such restorative effects of memantine were not detected either with 5,7-dichlorokynurenic acid or with D(-)-2-amino-5-phosphopentanoic acid, NMDA receptor antagonists active at the glycine binding site and at the glutamate binding site, respectively. These findings show (1) that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation of tau/neurofibrillary degeneration and (2) that this drug might be useful for the treatment of AD and related tauopathies.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

Restorative effects of glutamate antagonists in experimental parkinsonism

Several compounds with antagonistic actions on N-methyl-D-aspartate (NMDA) receptors were tested for an antiakinesic action in hypoactive MPTP-treated C57 BL/6 mice rendered tolerant to the motor activity enhancing effects of the 20 mg/kg, s.c., dose of L-Dopa; each compound was administered 60 min before the administration of the dopamine precursor. The classes of compounds studied included the noncompetitive NMDA antagonists, memantine, amantadine and MK-801, the competitive NMDA antagonist, CGP 40116, the anticonvulsive and putative anticonvulsive agents, lamotrigine and FCE 26743, with a partial glutamatergic antagonistic action. All six compounds elevated locomotor, rearing and total activity counts of L-Dopa-tolerant mice in co-administration with L-Dopa in dose-specific or dose-dependent manners but only memantine and MK-801 affected motor activity in the control mice, that also received chronic L-Dopa treatment. Thus, the restorative actions of those compounds in suprathreshold L-Dopa-tolerant MPTP-treated mice subjected to "wearing-off" of L-Dopa efficacy were assessed in a series of experiments. Within each class of potentially therapeutic agents a differential restorative efficacy of the motor activity-stimulating effects of hypoactive MPTP mice was obtained, confirming the putative antiparkinsonian applications of compounds with glutamate antagonistic actions.     

Novel benzopolycyclic amines with NMDA receptor antagonist activity

A new series of benzopolycyclic amines active as NMDA receptor antagonists were synthesized. Most of them exhibited increased activity compared with related analogues previously published. All the tested compounds were more potent than clinically approved amantadine and one of them displayed a lower IC₅₀ value than memantine, an anti-Alzheimer’s approved drug.     

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials.     

Ocular-Motor Profile and Effects of Memantine in a Familial Form of Adult Cerebellar Ataxia with Slow Saccades and Square Wave Saccadic Intrusions

Fixation instability due to saccadic intrusions is a feature of autosomal recessive spinocerebellar ataxias, and includes square wave intrusions (SWI) and macrosaccadic oscillations (MSO). A recent report suggested that the non-competitive antagonist of NMDA receptors, memantine, could decrease MSO and improve fixation in patients with spinocerebellar ataxia with saccadic intrusions (SCASI). We similarly tested two sisters, respectively of 58 and 60 years, with an unrecognized form of recessive, adult-onset cerebellar ataxia, peripheral neuropathy and slow saccades, who showed prominent SWI and also complained with difficulty in reading. We tested horizontal visually guided saccades (10°–18°) and three minutes of steady fixation in each patient and in thirty healthy controls. Both patients showed a significant reduction of peak and mean velocity compared with control subjects. Large SWI interrupting steady fixation were prominent during steady fixation and especially following visually guided saccades. Eye movements were recorded before and during the treatment with memantine, 20 mg/daily for 6 months. The treatment with memantine reduced both the magnitude and frequency of SWI (the former significantly), but did not modified neurological conditions or saccade parameters. Thus, our report suggests that memantine may have some general suppressive effect on saccadic intrusions, including both SWI and MSO, thereby restoring the capacity of reading and visual attention in these and in other recessive forms of ataxia, including Friedreich’s, in which saccadic intrusions are prominent.     

Memantine attenuates cognitive impairments after status epilepticus induced in a lithium–pilocarpine model

The capability of memantine, a noncompetitive antagonist of the NMDA receptors, to prevent impairments of cognitive functions in rats was investigated in the lithium–pilocarpine model of epilepsy. After status epilepticus, rats exhibited impaired exploratory behavior and spatial memory, and a decline of extinction of orienting behavior. Memantine administration prevented these disturbances. Thus, the blockade of the NMDA receptors immediately after status epilepticus allowed prevention of the development of the possible cognitive impairments.     

In vitro and in vivo evaluation of polymethylene tetraamine derivatives as NMDA receptor channel blockers

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1–4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-d-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at ?70 mV, only compound 2 (0.4 mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10 mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke.     

Fluorine-18 radiolabelling, biodistribution studies and preliminary PET evaluation of a new memantine derivative for imaging the NMDA receptor

A synthetic method has been established for preparing [18F]1-amino-3-fluoromethyl-5-methyl-adamantane ([18F]AFA). Biodistribution of the radiotracer in mice showed high brain uptake. The peak uptake (3.7% I.D/g organ) for the brain occurred at 30 min after injection. Accumulation of radioactivity in mouse brain was consistent with the known distribution of the NMDA receptors. The binding of [18F]AFA to the phencyclidine (PCP) binding sites of the NMDA receptor complex and the sigma recognition sites in a Rhesus monkey was also examined using positron emission tomography (PET). The regional brain distribution of [18F]AFA was changed by memantine and by (+)-MK-801, indicating competition for the same binding sites. Treatment with haloperidol caused a marked reduction of radioactivity uptake in all the brain regions examined. (-)-Butaclamol, which has pharmacological specificity for sigma sites, did not have any significant effects.     

Acute liver failure-induced death of rats is delayed or prevented by blocking NMDA receptors in brain

Developing procedures to delay the mechanisms of acute liver failure-induced death would increase patients' survival by allowing time for liver regeneration or to receive a liver for transplantation. Hyperammonemia is a main contributor to brain herniation and mortality in acute liver failure (ALF). Acute ammonia intoxication in rats leads to N-methyl-D-aspartate (NMDA) receptor activation in brain. Blocking these receptors prevents ammonia-induced death. Ammonia-induced activation of NMDA receptors could contribute to ALF-induced death. If this were the case, blocking NMDA receptors could prevent or delay ALF-induced death. The aim of this work was to assess 1) whether ALF leads to NMDA receptors activation in brain in vivo and 2) whether blocking NMDA receptors prevents or delays ALF-induced death of rats. It is shown, by in vivo brain microdialysis, that galactosamine-induced ALF leads to NMDA receptors activation in brain. Blocking NMDA receptors by continuous administration of MK-801 or memantine through miniosmotic pumps affords significant protection against ALF-induced death, increasing the survival time approximately twofold. Also, when liver injury is not 100% lethal (1.5 g/kg galactosamine), blocking NMDA receptors increases the survival rate from 23 to 62%. This supports that blocking NMDA receptors could have therapeutic utility to improve survival of patients with ALF.