Memantine: Updating a rare success story in pro-cognitive therapeutics.

The great potential for NMDA receptor modulators as druggable targets in neurodegenerative disorders has been met with limited success. Considered one of the rare exceptions, memantine has consistently demonstrated restorative and prophylactic properties in many AD models. In clinical trials memantine slows the decline in cognitive performance associated with AD. Here, we provide an overview of the basic properties including pharmacological targets, toxicology and cellular effects of memantine. Evidence demonstrating reductions in molecular, physiological and behavioural indices of AD-like impairments associated with memantine treatment are also discussed. This represents both an extension and homage to Dr. Chris Parson's considerable contributions to our fundamental understanding of a success story in the AD treatment landscape.

The Aß aggregation modulator MRZ-99030 prevents and even reverses synaptotoxic effects of Aß1-42 on LTP even following serial dilution to a 500:1 stoichiometric excess of Aß1-42, suggesting a beneficial prion-like seeding mechanism.

MRZ-99030 (GAL-101) is a small molecule that promotes the formation of off-pathway, non-toxic amorphous clusters of Aß thereby reducing the amount of toxic soluble oligomeric Aß species. MRZ-99030 clearly prevents synaptotoxic effects of Aß1-42 oligomers on synaptic plasticity and cognition. Long lasting in vivo effects indicate that MRZ-99030 seeds a beneficial self-replication of non-toxic Aß aggregates - "trigger effect". To test this, we prepared a serial dilution of MRZ-99030 starting with a 20:1 stoichiometric excess to Aß1-42. After incubating the Aß1-42/MRZ-99030 mixture for 20 min, 10% was transferred to a freshly prepared Aß1-42 solution. This dilution step was repeated 3 times finally resulting in a 500:1 stoichiometric excess of Aß1-42 over MRZ-99030. This solution was tested for its ability to impair long-term potentiation (LTP) in CA1 neurons. Even following serial dilution, MRZ-99030 prevented the synaptotoxic effect of Aß1-42 on CA1-LTP after tetanic stimulation of the Schaffer collaterals whereas incubation with MRZ-99030 (0.1 nM) without serial dilution did not prevent the synaptic deficits caused by Aß1-42 (50 nM). Time course experiments revealed that this protective effect was still evident even when the serially diluted Aß1-42/MRZ-99030 mixture was prepared up to 1 week before the LTP experiment. MRZ-99030, when serially diluted with Aß1-42, was also capable of detoxifying/reversing an already established neurotoxic process. In TEM experiments, Aß oligomers/annular protofibrils were converted to amorphous Aß clusters following incubation with serially diluted MRZ-99030 to a final concentration of MRZ-99030 (20 nM) and Aß1-42 (10 µM).

CNS repurposing - Potential new uses for old drugs: Examples of screens for Alzheimer's disease, Parkinson's disease and spasticity.

Drug repurposing is recently gaining increasing attention, not just from pharmaceutical companies but also from government agencies in an attempt to generate new medications to address increasing unmet medical needs in a cost effective and expedite manner. There are several approaches to identify novel indications for known drugs. Many are based on rational selection e.g. the known or a new mechanism of action of a drug. This review will focus rather on phenotypic or high content screening of compounds in models that are believed to be predictive of effectiveness of compounds irrespective of their mechanism of action. Three short cases studies of screens for Alzheimer's disease, Parkinson's disease and spasticity will be given as examples. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

The NMDA receptor antagonist Radiprodil reverses the synaptotoxic effects of different amyloid-beta (Aß) species on long-term potentiation (LTP).

Aß1-42 is well accepted to be a primary early pathogenic agent in Alzheimer's disease (AD). However, other amyloid peptides are now gaining considerable attention as potential key participants in AD due to their proposed higher neuronal toxicity. Impairment of the glutamatergic system is also widely accepted to be associated with pathomechanisms underlying AD. There is ample evidence that Aß1-42 affects GLUN2B subunit containing N-methyl-D-aspartate receptor function and abolishes the induction of long term potentiation (LTP). In this study we show that different ß-amyloid species, 1-42 Aß1-42 and 1-40 (Aß1-40) as well as post-translationally modified forms such as pyroglutamate-modified amyloid-(AßpE3) and nitrated Aß (3NTyr10-Aß), when applied for 90 min to murine hippocampal slices, concentration-dependently prevented the development of CA1-LTP after tetanic stimulation of the Schaffer collaterals with IC50s of 2, 9, 2 and 35 nM, respectively whilst having no effect on baseline AMPA receptor mediated fEPSPs. Aß1-43 had no effect. Interestingly, the combination of all Aß species did not result in any synergistic or additive inhibitory effect on LTP - the calculated pooled Aß species IC50 was 20 nM. A low concentration (10 nM) of the GLUN2B receptor antagonist Radiprodil restored LTP in the presence of Aß1-42, 3NTyr10-Aß, Aß1-40, but not AßpE3. In contrast to AMPA receptor mediated fEPSPs, all different ß-amyloid species tested at 50 nM supressed baseline NMDA-EPSC amplitudes. Similarly, all different Aß species tested decreased spine density. As with LTP, Radiprodil (10 nM) reversed the synaptic toxicity of Aß species but not that of AßpE3. These data do not support the enhanced toxic actions reported for some Aß species such as AßpE3, nor synergistic toxicity of the combination of different Aß species. However, whilst in our hands AßpE3-42 was actually less toxic than Aß1-42, its effects were not reversed by Radiprodil indicating that the target receptors/subunits mediating such synaptotoxicity may differ between the different Aß species tested.

Preclinical to phase II amyloid beta (Aß) peptide modulators under investigation for Alzheimer's disease.

INTRODUCTION: Alzheimer disease (AD) is the most common form of dementia and its incidence is increasing at an alarming rate all over the world. The pathophysiology of AD is characterized by chronic, progressive neurodegeneration which involves early synaptotoxicity. One of the most obvious pathological feature of AD is the accumulation of amyloid-ß (Aß) in the brain. Since current treatment options only provide symptomatic help and Aß is thought to underlie early synaptic pathology, Aß reduction or modulation in the brain may be a promising therapeutic strategy in preventing and /or reversing AD-related dysfunction. Areas covered: This paper outlines and evaluates the current landscape of preclinical and clinical studies focusing on modulating Aß pathophysiology. Data and analysis for this review were procured from PubMed, clinicaltrials.gov and Alzforum. Expert opinion: According to current knowledge, reducing Aß production offers numerous treatment options. However, targeting the initial steps by pharmacological interference with secretases is challenging due to the emergence of various side effects. The most promising approach seems to be the prevention of early Aß oligomerization. Combination approaches targeting both Aß and tau would seem to be another promising strategy that could have beneficial effects through the course of the disease.

Involvement of GluN2B subunit containing N-methyl-d-aspartate (NMDA) receptors in mediating the acute and chronic synaptotoxic effects of oligomeric amyloid-beta (Aß) in murine models of Alzheimer's disease (AD).

To elucidate whether a permanent reduction of the GluN2B subunit affects the pathology of Alzheimer's disease (AD), we cross-bred mice heterozygous for GluN2B receptors in the forebrain (hetGluN2B) with a mouse model for AD carrying a mutated amyloid precursor protein with the Swedish and Arctic mutation (mAPP) resulting in a hetGluN2B/mAPP transgenic. By means of voltage-sensitive dye imaging (VSDI) in the di-synaptic hippocampal pathway and the recording of field excitatory postsynaptic potentials (fEPSPs), hippocampal slices of all genotypes (WT, hetGluN2B, mAPP and hetGluN2B/mAPP, age 9-18 months) were tested for spatiotemporal activity propagation and long-term potentiation (LTP) induction. CA1-LTP induced by high frequency stimulation (HFS; 100 Hz/1s) was not different in all genotypes. Aß1-42 (50 nM)-application reduced potentiation of fEPSP in WT and hetGluN2B/mAPP mice, LTP in mAPP and hetGluN2B mice was not affected. For VSDI a fast depolarization signal was evoked in the granule cell layer and propagation was analysed in hippocampal CA3 and CA1 region before and after theta stimulation (100pulses/5 Hz). LTP was not significantly different between all genotypes. In mAPP mice θ-stim produced an epileptiform activity reflected in a pronounced prolongation of the FDS compared to the other genotypes. In slices of hetGluN2B/mAPP and GluN2B mice, however, these parameters were similar to WT mice indicating a reversal effect of the attenuated GluN2B expression. The induction of a hetGluN2B mutation in the mAPP reversed some pathophysiological changes on hippocampal LTP and provide further evidence for the involvement of the glutamatergic system in AD and emphasize the GluN2B subunit as a potential target for AD treatment.

Brain concentrations of mGluR5 negative allosteric modulator MTEP in relation to receptor occupancy--Comparison to MPEP.

BACKGROUND: To verify relation between brain free levels, receptor occupancy in vivo and in vitro affinity at the target for mGluR5 negative allosteric modulator (NAM) MTEP. METHODS: We evaluated plasma and brain extra-cellular fluid (ECF) concentration of MTEP at behaviourally active dose (5mg/kg) using in vivo microdialysis. These values were compared it to the affinity in vitro (receptor binding and FLIPR) and to receptor occupancy in vivo. Another, related substance, MPEP was used for comparison. RESULTS: MTEP and MPEP respectively inhibited mGluR5 receptors function in vitro with an affinity of 25.4 and 12.3 nM respectively. Accordingly peak ECF (extracellular fluid) levels were 1.3 and 0.14 µM, and peak total plasma levels were 7-11 and 2.6 µM. The ED50 for in vivo receptor occupancy was for both agents in the range of 0.8-0.7 mg/kg. CONCLUSIONS: At behaviourally active dose MTEP produced complete mGluR5 receptor occupancy but over 50 times higher ECF concentrations than affinity for mGluR5 receptor in vitro. This difference is seems lower for other mGluR5 NAM compounds such as MPEP. A possibly explanation could be different distribution in body compartments of both agents leading to errors of estimation with the microdialysis technique or different pharmacological activity at the receptor.

Patch clamp combined with voltage/concentration clamp to determine the kinetics and voltage dependency of N-methyl-D-aspartate (NMDA) receptor open channel blockers.

Electrophysiological techniques can be used to great effect to help determine the mechanism of action of a compound. However, many factors can compromise the resulting data and their analysis, such as the speed of solution exchange, expression of additional ion channel populations including other ligand-gated receptors and voltage-gated channels, compounds having multiple binding sites, and current desensitization and rundown. In this chapter, such problems and their solutions are discussed and illustrated using data from experiments involving the uncompetitive NMDA receptor antagonist memantine. Memantine differs from many other NMDA receptor channel blockers in that it is well tolerated and does not cause psychotomimetic effects at therapeutic doses. Various electrophysiological parameters of NMDA-induced current blockade by memantine have been proposed to be important in determining therapeutic tolerability; potency, onset and offset kinetics, and voltage dependency. These were all measured using whole cell patch clamp techniques using hippocampal neurons. Full results are shown here for memantine, and these are summarized and compared to those from similar experiments with other NMDA channel blockers. The interpretation of these results is discussed, as are theories concerning the tolerability of NMDA channel blockers, with the aim of illustrating how electrophysiological data can be used to form and support a physiological hypothesis.

Memantine and cholinesterase inhibitors: complementary mechanisms in the treatment of Alzheimer's disease.

This review describes the preclinical mechanisms that may underlie the increased therapeutic benefit of combination therapy-with the N-methyl-D-aspartate receptor antagonist, memantine, and an acetylcholinesterase inhibitor (AChEI)-for the treatment of Alzheimer's disease (AD). Memantine, and the AChEIs target two different aspects of AD pathology. Both drug types have shown significant efficacy as monotherapies for the treatment of AD. Furthermore, clinical observations indicate that their complementary mechanisms offer superior benefit as combination therapy. Based on the available literature, the authors have considered the preclinical mechanisms that could underlie such a combined approach. Memantine addresses dysfunction in glutamatergic transmission, while the AChEIs serve to increase pathologically lowered levels of the neurotransmitter acetylcholine. In addition, preclinical studies have shown that memantine has neuroprotective effects, acting to prevent glutamatergic over-stimulation and the resulting neurotoxicity. Interrelations between the glutamatergic and cholinergic pathways in regions of the brain that control learning and memory mean that combination treatment has the potential for a complex influence on disease pathology. Moreover, studies in animal models have shown that the combined use of memantine and the AChEIs can produce greater improvements in measures of memory than either treatment alone. As an effective approach in the clinical setting, combination therapy with memantine and an AChEI has been a welcome advance for the treatment of patients with AD. Preclinical data have shown how these drugs act via two different, but interconnected, pathological pathways, and that their complementary activity may produce greater effects than either drug individually.

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-ß-cyclodextrine (MßCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Rationale for combining glutamatergic and cholinergic approaches in the symptomatic treatment of Alzheimer's disease.

A total of 40 years of biochemical, clinical and neuropathological research have revolutionized our understanding of the pathophysiology of Alzheimer's disease, yet at the present moment the only drugs licensed for treatment are targeted essentially at symptoms. Some disease-modifying drugs remain in clinical trials, but many that have used similar approaches have failed. It is therefore of considerable interest to examine the optimal way of using existing medications for the benefit of patients. This article looks at the rationale behind the combined use of acetylcholinesterase inhibitors and the N-methyl-D-aspartate-receptor antagonist, memantine, from both preclinical and clinical perspectives.

Assessment of the effects of NS11394 and L-838417, α2/3 subunit-selective GABA(A) [corrected] receptor-positive allosteric modulators, in tests for pain, anxiety, memory and motor function.

The aim of the present paper was to study the effects of GABAA receptor-positive modulators (L-838417 and NS11394) showing a preference for α2/3 subunits of the GABAA receptor, in models of pain, anxiety, learning, memory and motor function. These compounds have been suggested to have a favourable therapeutic profile over nonselective compounds such as diazepam. In this study, we tested both compounds for their effects in rat models of formalin-induced pain, spinal nerve-ligation-induced mechanical allodynia, plus maze, open field, rotarod, balance beam walking, contextual fear conditioning and Morris water maze. Both compounds exerted analgesic, but no anxiolytic effects. However, they induced motor side-effects, and learning and memory impairment at similar doses. Therefore, the anxiolytic effect and the lack of side-effects of these compounds, as described in the literature, could not be confirmed in the present study.

Alzheimer's disease, ß-amyloid, glutamate, NMDA receptors and memantine--searching for the connections.

ß-amyloid (Aß) is widely accepted to be one of the major pathomechanisms underlying Alzheimer's disease (AD), although there is presently lively debate regarding the relative roles of particular species/forms of this peptide. Most recent evidence indicates that soluble oligomers rather than plaques are the major cause of synaptic dysfunction and ultimately neurodegeneration. Soluble oligomeric Aß has been shown to interact with several proteins, for example glutamatergic receptors of the NMDA type and proteins responsible for maintaining glutamate homeostasis such as uptake and release. As NMDA receptors are critically involved in neuronal plasticity including learning and memory, we felt that it would be valuable to provide an up to date review of the evidence connecting Aß to these receptors and related neuronal plasticity. Strong support for the clinical relevance of such interactions is provided by the NMDA receptor antagonist memantine. This substance is the only NMDA receptor antagonist used clinically in the treatment of AD and therefore offers an excellent tool to facilitate translational extrapolations from in vitro studies through in vivo animal experiments to its ultimate clinical utility.

Synthesis of a series of γ-amino alcohols comprising an N-methyl isoindoline moiety and their evaluation as NMDA receptor antagonists.

We report a series of new stereoisomeric γ-amino alcohols comprising an N-methyl isoindoline moiety as ligands for the ifenprodil binding site of the NMDA receptor. Among the four series of stereoisomers, 8a-c, 9a-c, 10a-c, and 11a-c, synthesised, the highest potencies and NMDA-NR2B subtype selectivity was found for the methyl derivative 11a and the chloro derivative 11c, both possessing the [1S,1'S] configuration. However, additional moderate potency of 11a and 11c at the hERG channel with values of 2.6 ± 2.4% and 1.6 ± 2.0%, respectively, rendered them unsuitable for medical use.

Therapeutic significance of NR2B-containing NMDA receptors and mGluR5 metabotropic glutamate receptors in mediating the synaptotoxic effects of ß-amyloid oligomers on long-term potentiation (LTP) in murine hippocampal slices.

Soluble amyloid beta (Aß) oligomers are widely accepted to be neurotoxic and lead to the memory loss and neuronal death observed in Alzheimer's disease (AD). Ample evidence suggests that impairment in glutamatergic signalling is associated with AD pathology. In particular, Aß(1-42) is thought to affect N-methyl-d-aspartate (NMDA) receptor function and abolish the induction of long-term potentiation (LTP), which is regarded to be a phenomenon relevant to memory formation. The involvement of glutamatergic signalling in the pathology of AD is underscored by the therapeutic success of memantine, an uncompetitive NMDA receptor antagonist, used to treat patients with moderate to severe AD. In this study we show that Aß(1-42) oligomers applied to acute murine hippocampal slices prevented, in a concentration-dependent manner, the development of CA1-LTP after tetanic stimulation of the Schaffer collaterals with a half maximal inhibitory concentration of around 2 nM (before oligomerization). The highest concentration of Aß(1-42) oligomers (50 nM before oligomerization) completely blocked LTP (105 ± 1% potentiation versus 141 ± 3% in control) whereas scrambled Aß(1-42) (50 nM) was without effect (144 ± 10% potentiation). Pre-incubation with memantine (1 µM) restored LTP in the presence of Aß(1-42) (50 nM; 135 ± 5% potentiation). NMDA receptors containing the NR2B subunit have been proposed to play a particularly important role in excitotoxicity, functioning as extracellular "death receptors". The metabotropic glutamate receptor 5 (mGluR5) is mechanistically coupled to postsynaptic NMDA receptors. As such, allosteric sites on both receptors offer alternative means to modulate NMDA receptor function. We therefore tested low concentrations (each 300 nM) of allosteric antagonists of NR2B (Ro 25-6981, [R-(R∗,S∗)]-α-(4-Hydroxyphenyl)-ß-methyl-4(phenylmethyl)-1-piperidine propanol hydrochloride) and mGluR5 receptors (MPEP, 2-methyl-6-(phenylethynyl)-pyridine). Both compounds restored LTP in the presence of Aß(1-42) oligomers (50 nM, fEPSPs were potentiated to 129 ± 13% and 133 ± 7% respectively). Finally, we demonstrated that slices from mice heterozygous for NR2B receptor) in the forebrain are not susceptible to the toxic effects of Aß(1-42) oligomers but express normal LTP (138 ± 6%). These experiments demonstrate that glutamate receptor antagonists delivered at concentrations which still allow physiological activities in vitro, are able to prevent Aß(1-42) oligomer-induced synaptic toxicity and further support the glutamatergic system as a target for the development of improved symptomatic/neuroprotective treatments for AD.

In vivo evidence for functional NMDA receptor blockade by memantine in rat hippocampal neurons.

Antagonising the NMDA (N-methyl-D: -aspartate) receptor complex is a widely hypothesised therapeutic approach in several neurodegenerative conditions, such as Alzheimer's disease. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist, has been in clinical use for several years and numerous experimental data support its NMDA receptor blocking effects. It has recently been reported in transfected HEK293T cells that physiological concentrations of Mg(2+) may impart partial NMDA receptor subtype selectivity and weaken the overall inhibitory actions of memantine in NMDA receptor-mediated cellular events. In the present study, we set out to investigate the effect of intravenously applied memantine on iontophoresed NMDA-evoked firing of hippocampal CA1 neurons using in vivo conditions. Cumulative doses of memantine in the rat (4, 8 and 16 mg/kg i.v.) caused the firing rate to decrease in a dose-dependent manner to 77 ± 7, 58 ± 8 and 34 ± 12% of control, respectively, while saline application had no significant effect. We show that therapeutic doses of memantine are able to antagonize NMDA receptor-mediated activity in the principal cells of the hippocampus in vivo, i.e. in the presence of physiological concentrations of Mg(2+).

Memantine improves cognition and reduces Alzheimer's-like neuropathology in transgenic mice.

Memantine is an N-methyl-d-aspartate receptor antagonist that is approved for the treatment of moderate to severe Alzheimer's disease (AD). In this study, three groups of triple-transgenic (3xTg-AD) mice with differing levels of AD-like pathology (6, 9, and 15 months of age) were treated for 3 months with doses of memantine equivalent to those used in humans. After the treatment, memantine-treated mice had restored cognition and significantly reduced the levels of insoluble amyloid-beta (Abeta), Abeta dodecamers (Abeta*56), prefibrillar soluble oligomers, and fibrillar oligomers. The effects on pathology were stronger in older, more impaired animals. Memantine treatment also was associated with a decline in the levels of total tau and hyperphosphorylated tau. Finally, memantine pre-incubation prevented Abeta-induced inhibition of long-term potentiation in hippocampal slices of cognitively normal mice. These results suggest that the effects of memantine treatment on AD brain include disease modification and prevention of synaptic dysfunction.

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-D-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors.

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC(50) around 1 microM at -70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca(2+) responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells. Memantine was confirmed to be a moderate affinity (IC(50) at -70 mV of 0.79+/-0.02 microM, Hill=0.92+/-0.02), strongly voltage-dependent (delta=0.90+/-0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 microM - onset tau(fast)=273+/-25 ms (weight 69%), onset tau(slow)=2756+/-296 ms, offset tau(fast)=415+/-82 ms (weight 38%) offset tau(slow)=5107+/-1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC(50) at -70 mV of 0.71+/-0.03 microM, Hill=0.84+/-0.02) but somewhat less pronounced voltage-dependency (delta=0.79+/-0.04), slower, single exponential kinetics (ketamine: k(on)=0.15+/-0.05 x 10(6)M(-1)s(-1), k(off)=0.22+/-0.05 s(-1)c.f. memantine following normalization k(on)=0.32+/-0.11 x 10(6)M(-1)s(-1), k(off)=0.53+/-0.10s(-1)) and was fully trapped. The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.

Blocking kinetics of memantine on NR1a/2A receptors recorded in inside-out and outside-out patches from Xenopus oocytes.

Previous experiments on primary cultures of hippocampal/cortical neurones revealed that the block and unblock of N-Methyl-D-Aspartate (NMDA) receptor channels by memantine showed double exponential kinetics and that the offset kinetics following a voltage-step were much faster than following a concentration jump. There are, however, two major problems when using such cultured primary neurones for these experiments (1) the almost certain expression of heterogeneous NMDA receptor subunits which could underlie double exponential kinetics due to different potencies at receptor subtypes and (2) slow space- and concentration-clamp due to neuronal morphology which could mask even faster kinetics. Therefore, we performed similar experiments with Xenopus oocytes exclusively expressing one NMDA receptor type (NR1a/2A) at high levels which allowed recordings from membrane patches with large currents. The use of inside-out patches for voltage-step and outside-out patches in combination with a piezo driven fast application system largely negated potential space- and concentration-clamp problems. Block and unblock of the NMDA receptor by memantine after both voltage jump and concentration jumps showed triple exponential kinetics. The fast onset kinetics of NMDA receptor channel block following both concentration-clamp and voltage jumps from +70 to -70 mV were similar. In contrast, offset kinetics after a voltage-step from -70 to +70 mV were much faster than following a concentration jump at the holding potential of -70 mV. These results provide further support for the hypothesis that rapid relief of block via strong synaptic membrane depolarisation underlies the good therapeutic profile of memantine.

Memantine does not show intracellular block of the NMDA receptor channel.

Mg2+ is known to gain access to the NMDA receptor channel from both the extra- and intracellular compartments. Memantine, being an amphiphilic substance, reaches intracellular concentrations of approximately 30 microM, which are much higher than therapeutic extracellular concentrations ( approximately 1 microM). We therefore investigated whether memantine can also block the NMDA receptor channel from the intracellular compartment. NR1a/NR2A receptors were expressed in Xenopus oocytes and in classical two electrode voltage-clamp recordings, voltage-ramps from -100 to +100 mV confirmed moderate inward rectification of NR1a/NR2A receptor control responses at positive membrane potentials above +40 mV. Patch clamp recordings from these same cells (applying 100 microM glutamate and 1 mM Mg2+) revealed similar rectification at positive potentials in cell-attached mode which disappeared after pulling an inside-out patch. Application of 1 mM Mg2+ to the intracellular side of the receptor re-introduced the rectification seen in cell-attached mode, and 5 mM Mg2+ produced much more pronounced block. In contrast, 30 microM memantine was completely unable to block the NMDA receptor from the intracellular compartment. In conclusion, intracellular block of the NMDA receptor, as reported for Mg2+, is not of significance for the therapeutic effects of memantine.