Pharmacodynamics of memantine: an update.

Memantine received marketing authorization from the European Agency for the Evaluation of Medicinal Products (EMEA) for the treatment of moderately severe to severe Alzheimer s disease (AD) in Europe on 17(th) May 2002 and shortly thereafter was also approved by the FDA for use in the same indication in the USA. Memantine is a moderate affinity, uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist with strong voltage-dependency and fast kinetics. Due to this mechanism of action (MOA), there is a wealth of other possible therapeutic indications for memantine and numerous preclinical data in animal models support this assumption. This review is intended to provide an update on preclinical studies on the pharmacodynamics of memantine, with an additional focus on animal models of diseases aside from the approved indication. For most studies prior to 1999, the reader is referred to a previous review [196].In general, since 1999, considerable additional preclinical evidence has accumulated supporting the use of memantine in AD (both symptomatic and neuroprotective). In addition, there has been further confirmation of the MOA of memantine as an uncompetitive NMDA receptor antagonist and essentially no data contradicting our understanding of the benign side effect profile of memantine.

Agonist concentration dependency of blocking kinetics but not equilibrium block of N-methyl-D-aspartate receptors by memantine.

Memantine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist which is registered in both Europe and the USA for the treatment of Alzheimer's disease (AD). Cultured rat hippocampal neurons were used to evaluate the potency and blocking kinetics of this therapeutically very well-tolerated agent in the presence of various concentrations of the synthetic agonist NMDA and a constant, saturating concentration of the co-agonist D-serine (10 microM). Whole-cell patch-clamp experiments at -70 mV revealed that the degree of "equilibrium" blockade of NMDA-induced currents by memantine was largely unaffected by the concentration of the agonist NMDA. The IC50 values for NMDA at 300, 100, 30 and 10 microM were 0.80+/-0.12, 1.01+/-0.08, 0.92+/-0.13 and 1.31+/-0.09 microM, respectively, giving an average IC(50) for all agonists concentrations tested of 1.01+/-0.11 microM. In contrast, and as expected, the onset and offset kinetics of blockade were clearly dependent on agonist concentration. For NMDA 300, 100, 30 and 10 microM, kon values were 10.55+/-1.41, 8.60+/-0.17, 4.90+/-0.20 and 3.22+/-0.08x10(4) M(-1) s(-1), respectively; 1/tauon values at the IC50 concentration of memantine-i.e. 1 microM-were 0.58+/-0.11, 0.28+/-0.05, 0.15+/-0.02 and 0.11+/-0.03 s(-1), respectively and koff values were 0.24+/-0.01, 0.19+/-0.01, 0.14+/-0.00 and 0.09+/-0.01 s(-1), respectively. It therefore appears that the kinetics, but not the equilibrium potency, of memantine are agonist concentration-dependent. These fast agonist concentration-dependent kinetic properties, in addition to the clear voltage-dependence of memantine, are proposed to be important for the therapeutic tolerability of this compound in the treatment of AD.

Excitatory amino acid neurotransmission.

In recent years great progress has been made in understanding the function of ionotropic and metabotropic glutamate receptors; their pharmacology and potential therapeutic applications. It should be stressed that there are already N-methyl-D-aspartate (NMDA) antagonists in clinical use, such as memantine, which proves the feasibility of their therapeutic potential. It seems unlikely that competitive NMDA receptor antagonists and high-affinity channel blockers will find therapeutic use due to limiting side-effects, whereas agents acting at the glycineB site, NMDA receptor subtype-selective agents and moderate-affinity channel blockers are far more promising. This is supported by the fact that there are several glycineB antagonists, NMDA moderate-affinity channel blockers and NR2B-selective agents under development. Positive and negative modulators of AMPA receptors such as the AMPAkines and 2,3-benzodiazepines also show more promise than e.g. competitive antagonists. Great progress has also been made in the field of metabotropic glutamate receptors since the discovery of novel, allosteric modulatory sites for these receptors. Selective agents acting at these transmembrane sites have been developed that are more drug-like and have a much better access to the central nervous system than their competitive counterparts. The chapter will critically review preclinical and scarce clinical experience in the development of new ionotropic and metabotropic glutamate receptor modulators according to the following scheme: rational, preclinical findings in animal models and finally clinical experience, where available.

Aminoadamantanes as NMDA receptor antagonists and antiparkinsonian agents--preclinical studies.

Aminoadamantanes such as 1-aminoadamantane (amantadine) and 1-amino-3,5-dimethyladamantane (memantine) are N-methyl-D-aspartate (NMDA) receptor antagonists which show antiparkinsonian-like activity in animal models and in Parkinson's patients. The issue of whether NMDA antagonism plays a role in the symptomatological antiparkinsonian activity of amantadine and memantine is addressed by comparing: behaviourally effective doses, serum/brain levels, and their potency as NMDA receptor antagonists. In the case of memantine, blockade of NMDA receptors is probably the only mechanism responsible for antiparkinsonian activity, whereas for amantadine the situation is clearly far more complex. There are a number of differences between memantine and amantadine both in vitro and in vivo, and although NMDA receptor antagonism certainly participates in the antiparkinsonian activity of amantadine, other effects, some of which are elusive, also play a role. Moreover, it has been suggested that the pathomechanism of Parkinson's disease involves excitotoxic processes and that treatment with NMDA receptor antagonists might also slow the progression of neurodegeneration. If this claim is true, such an effect could be achieved with amantadine and memantine which show neuroprotective activity in animals at therapeutically relevant doses.

Amino-alkyl-cyclohexanes as a novel class of uncompetitive NMDA receptor antagonists.

Because of its widespread involvement in the physiology and pathology of the CNS, the glutamatergic system has gained considerable attention as a potential target for development of new agents for a number of therapeutic indications. In this respect, the glutamate receptor subtype of the NMDA type has been most intensively studied. The present review describes the rational for developing amino-alkyl-cyclohexanes, as new uncompetitive NMDA receptor antagonists based on our positive experience with memantine which has been used clinically for many years for the treatment of neurodegenerative dementia. Many amino-alkyl-cyclohexane derivatives have been evaluated in vitro and in animal models, and in turn, one structure, namely neramexane HCl (MRZ 2/579) was selected for further development. This agent shows some similarity to memantine e.g. channel blocking kinetics, voltage dependency, and affinity. Preclinical tests indicated particularly good activity in animal models of alcoholism (self-administration, withdrawal-induced audiogenic seizures etc.) and pain (chronic pain, inhibition of tolerance to the analgesic effects of morphine). It turn, this agent has recently entered phase II of clinical trials in alcoholism after a favourable profile seen in phase I studies.

Memantine restores long term potentiation impaired by tonic N-methyl-D-aspartate (NMDA) receptor activation following reduction of Mg2+ in hippocampal slices.

This study compared the ability of memantine and (+)MK-801 to counteract deficits in the induction of long term potentiation (LTP) following reduction of Mg2+ in hippocampal slices--a model of increased synaptic noise due to tonic N-methyl-D-aspartate (NMDA) receptor activation. Decreasing Mg2+ from 1 mM to 10 microM for 60 min enhanced baseline field excitatory post-synaptic potential (fEPSP) slopes (87.2 +/- 10.6% above control) and impaired LTP (-4.1 +/- 9.8% compared to pre-tetanic levels). Long pre-incubations with memantine (1 microM), a concentration achieved in the CSF of dementia patients, almost fully restored the induction of LTP (to 43.4 +/- 8.4%) without changing the enhancement of baseline fEPSP slopes (84.1 +/- 11.6%). Memantine (10 microM) fully restored the induction of LTP (61.5 +/- 5.3%) and also decreased the enhancement of baseline fEPSP slopes (30.1 +/- 4.9%). In contrast, although (+)MK-801 (0.01, 0.1 and 1 microM) caused a concentration-dependent reduction in the low Mg2+ -induced enhancement of baseline fEPSP slopes, it was not able to restore the induction of LTP (3.0 +/- 9.8%, 16.3 +/- 5.7% and 4.8 +/- 6.7% respectively). These data indicate that memantine could produce symptomatological improvement in learning under conditions of tonic NMDA receptor activation such as those occurring in chronic neurodegenerative diseases whereas (+)MK-801 is likely to have only negative effects.

Differential effects of NMDA-receptor antagonists on long-term potentiation and hypoxic/hypoglycaemic excitotoxicity in hippocampal slices.

Whole-cell patch clamp recording from cultured hippocampal neurones was used to investigate the NMDA antagonistic effects of the glycineB antagonist 5,7-DCKA and the competitive antagonist CGP 37849. Extracellular field potential recording from area CA1 of hippocampal slices was used to investigate their effects on the induction of LTP and hypoxia/hypoglycaemia-induced suppression of fEPSPs. Additionally, memantine and (+)MK-801 were tested in the later model. 5,7-DCKA inhibited NMDA-induced plateau currents (IC50=0.24+/-0.02 microM) with around nine times higher potency than against peak (IC50=2.14+/-0.17 microM). In contrast, CGP 37849 slowed the onset of NMDA-induced currents considerably and antagonized currents at the time point when the peak component occurred in control responses (IC50=0.18+/-0.01 microM) with around seven times higher potency than against plateau (IC50=1.26+/-0.19 microM). Both 5,7-DCKA and CGP 37849 inhibited the induction of LTP (IC50s=2.53+/-0.13 and 0.37+/-0.04 microM respectively) with potencies close to those inhibiting peak currents in patch clamp studies. 5,7-DCKA and CGP 37849 also blocked the hypoxia/hypoglycaemia-induced suppression of fEPSPs but CGP 37849 (EC50=4.3+/-0.33 microM) was far less potent than against the induction of LTP whilst 5,7-DCKA (EC50=1.47+/-0.04 microM) had similar potency in these two models. Memantine and (+)MK-801 also blocked hypoxia/hypoglycaemia-induced suppression of fEPSPs with EC50s of 14.1+/-0.52 and 0.53+/-0.02 microM respectively. Whereas memantine blocked this effect with similar potency as we previously reported for LTP, (+)MK-801 was four time less potent in this model. The calculated relative therapeutic indices (IC50 LTP over EC50 hypoxia/hypoglycaemia) for 5,7-DCKA, CGP 37849, memantine and (+)MK-801 were 1.72, 0.09, 0.82 and 0.24 respectively. These results show that even in a severe model of hypoxia/hypoglycaemia, glycineB site antagonists and moderate affinity channel blockers exhibit a better therapeutic index than competitive antagonists and high affinity channel blockers. It is likely that in milder forms of pathology the observed differences in therapeutic indices remain the same but the absolute values are expected to be higher.

Comparative patch clamp studies on the kinetics and selectivity of glutamate receptor antagonism by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine (GYKI 52466).

The glutamate antagonistic effects of NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] and GYKI 52466 [1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine] were compared on inward current responses of cultured superior collicular and hippocampal neurones with the whole cell patch clamp technique. Both NBQX (8 microM) and GYKI 52466 (33 microM) selectively reduced responses to AMPA [(S)-alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid, 50 microM] and kainate (50 microM) whilst having little effect on responses to NMDA (N-methyl-D-aspartate, 100 microM). The effects of the two antagonists on the kinetics of AMPA (50 microM) responses were, however, very different--NBQX dramatically slowed the rise time of responses so that peak currents (IC50 60.4 +/- 4.2 nM) were markedly more effected than desensitized plateau currents (IC50 706 +/- 99 nM) whereas GYKI 52466 antagonized plateau responses (IC50 4.44 +/- 0.21 microM) somewhat more than peak responses (IC50 6.87 +/- 0.46 microM) and had only marginal effects on kinetics. In fact, low concentrations of NBQX (50-250 nM) actually potentiated plateau AMPA responses--an effect likely to be due to a reduction in the degree of AMPA-induced desensitization. Similar effects on response kinetics, were seen with kainate such that the IC50s for NBQX in antagonizing initial and plateau components of current responses to kainate 400 microM were 18.1 +/- 2.9 nM and 298 +/- 27 nM respectively whereas the IC50s for GYKI 52466 against kainate 50 microM were 17.3 +/- 1.8 microM and 15.5 +/- 3.3 microM respectively. These differences are likely to be due to the different modes of action of the two antagonists--NBQX shifted kainate concentration responses curves to the right in a parallel fashion indicative of competitive antagonism whereas the effects of GYKI 52466 were largely noncompetitive. There was, however, some indication for a small allosteric influence of GYKI 52466 on the affinity of the glutamate recognition site of the AMPA/kainate receptor. Estimation of Kbs using the Cheng-Prussoff relationship revealed little difference in the affinity of NBQX in antagonizing plateau responses to AMPA (Kb 23.2 nM) and kainate (Kb 57.1 nM) and indicate that the effects of these two agonists are mediated at a common receptor under the experimental conditions used. Moreover, the differential effects of NBQX on peak and plateau components of AMPA (50 microM) responses was associated with a desensitization-induced, paradoxical increase in the agonist affinity and was probably not due to any change in the affinity of NBQX.(ABSTRACT TRUNCATED AT 400 WORDS)

Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist--a review of preclinical data.

N-methyl-D-aspartate (NMDA) receptor antagonists have therapeutic potential in numerous CNS disorders ranging from acute neurodegeneration (e.g. stroke and trauma), chronic neurodegeneration (e.g. Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS) to symptomatic treatment (e.g. epilepsy, Parkinson's disease, drug dependence, depression, anxiety and chronic pain). However, many NMDA receptor antagonists also produce highly undesirable side effects at doses within their putative therapeutic range. This has unfortunately led to the conclusion that NMDA receptor antagonism is not a valid therapeutic approach. However, memantine is clearly an uncompetitive NMDA receptor antagonist at therapeutic concentrations achieved in the treatment of dementia and is essentially devoid of such side effects at doses within the therapeutic range. This has been attributed to memantine's moderate potency and associated rapid, strongly voltage-dependent blocking kinetics. The aim of this review is to summarise preclinical data on memantine supporting its mechanism of action and promising profile in animal models of chronic neurodegenerative diseases. The ultimate purpose is to provide evidence that it is indeed possible to develop clinically well tolerated NMDA receptor antagonists, a fact reflected in the recent interest of several pharmaceutical companies in developing compounds with similar properties to memantine.

Budipine is a low affinity, N-methyl-D-aspartate receptor antagonist: patch clamp studies in cultured striatal, hippocampal, cortical and superior colliculus neurones.

The NMDA receptor antagonistic effects of budipine were assessed using concentration- and patch-clamp techniques on cultured striatal, hippocampal, cortical and superior colliculus neurones. Inward current responses of striatal neurones to NMDA (200 microM) at -70 mV were antagonized by budipine in a concentration-dependent manner (50% inhibitory concentration (IC50) 59.4 +/- 10.7 microM, n = 17) with 24 times lower potency than memantine but similar potency to amantadine. In striatal neurones, budipine blocked outward currents at +70 mV with an IC50 of 827 microM, suggesting that the binding site is less deep in the channel (delta = 0.45) than for memantine. However, more detailed analysis of the fractional block by budipine 300 microM in hippocampal neurones gave a delta-value of 0.90, but revealed that 28% block is mediated at a voltage-independent site. This voltage-insensitive site was accessible in the absence of agonist. Budipine exhibited concentration-dependent open channel blocking kinetics (kappa(on) = 0.71 x 10(4) M(-1) s(-1)) whereas the fast offset rate was concentration-independent (kappa(off) = 0.63 s(-1)). Calculation of the ratio kappa(off)/kappa(on) revealed an apparent Kd value of 88.7 microM. Budipine, memantine and amantadine had similar effects against NMDA-induced currents in cultured hippocampal, cortical and superior colliculus neurones, although amantadine was somewhat more potent in cultured striatal neurones. The relevance of NMDA receptor antagonism to the anti-Parkinsonian effects of budipine remains to be established.

Patch clamp studies on the kinetics and selectivity of N-methyl-D-aspartate receptor antagonism by memantine (1-amino-3,5-dimethyladamantan).

Memantine (1-amino-3,5-dimethyladamantan) was tested as an antagonist of N-methyl-D-aspartate (NMDA) receptors on cultured superior collicular and hippocampal neurones using the patch clamp technique and its actions were compared to those of Mg2+ ions, ketamine, dextrorphan, dextromethorphan, phencyclidine and dizocilpine (MK-801). Memantine (2-33 microM) concentration-dependently antagonized responses to NMDA 100 microM with an IC50 of 2.92 +/- 0.05 microM. In contrast, current responses to (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (L-AMPA 50-100 microM) and gamma-amino butyric acid (GABA 10 microM) were unaffected by Memantine 8 microM. Memantine 8 microM caused a non-parallel shift of the NMDA concentration-response curve to the right in a manner indicative of uncompetitive open channel block. The effects of memantine were similar to ketamine in that both antagonists were weakly use- and strongly voltage-dependent. In contrast, MK-801, phencyclidine and dextrorphan showed much slower kinetics that was reflected in their marked use- and weaker voltage-dependency. The antagonistic effects of memantine were not reversed by increasing concentrations of glycine (0.1-100 microM) ruling out the possibility of an interaction of memantine with the strychnine-insensitive glycine modulatory site associated with the NMDA receptor-channel complex. Memantine (1-100 microM) also selectively antagonized responses to NMDA (40 microM) in the cortical wedge preparation with IC50 of 12.9 +/- 1.5 microM.

Different binding affinities of NMDA receptor channel blockers in various brain regions--indication of NMDA receptor heterogeneity.

The N-methyl-D-aspartate (NMDA) receptor-channel complex exists in multiple forms which probably have different physiological and pharmacological properties. To further evaluate this concept of different NMDA receptor subtypes, receptor binding and autoradiographic techniques were used to study the phencyclidine (PCP) binding site of the NMDA receptor ion-channel complex. [3H]MK-801 was employed to characterize binding properties of (+)-MK-801, (-)-MK-801, phencyclidine (PCP), (+/-)-ketamine, amantadine (1-amino-adamantane) and memantine (3,5-dimethyl-1-amino-adamantane) in different brain regions. Saturation experiments on homogenized membranes revealed the existence of single classes of binding sites in cortex and cerebellum but with significant different affinities between these regions (KD/Cortex = 4.59 nM, Bmax/Cortex = 0.836 pmol/mg protein; KD/Cereb. = 25.99 nM, Bmax/Cereb. = 0.573 pmol/mg protein) suggesting that the lower affinity in cerebellum indicates another population of NMDA receptor channels. In contrast, in striatum there was clear evidence for two binding sites (KD/high = 1.43 nM, Bmax/high = 0.272 pmol/mg protein; KD/low = 12.15 nM, Bmax/low = 1.76 pmol/mg protein). Displacement studies (autoradiography and binding) revealed a lower affinity for unlabeled (+)-MK-801 in striatum which was clearly not the case for memantine. In cerebellar membranes there was a significant decrease in the affinity for both MK-801 enantiomers and PCP but not for the 1-amino-adamantanes. In contrast, all compounds showed lowered affinity in the dentate gyrus. These findings support NMDA receptor heterogeneity which may be of particular relevance for the development of subtype-selective drugs.

Zinc changes AMPA receptor properties: results of binding studies and patch clamp recordings.

The influence of zinc ions on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors was investigated using binding studies with [3H]AMPA to rat cortical membranes and patch clamp recordings from cultured superior colliculus neurones. In Tris-HCl buffer, zinc (1-10 mM) significantly increased the specific binding of [3H]AMPA whereas this increase was negligible in the presence of CaCl2 (2.5 mM) and KSCN (100 mM). This effect was associated with a dramatic increase in Bmax but a decrease in both agonist and antagonist affinity. Association and dissociation experiments showed that equilibrium [3H]AMPA binding is reached with faster kinetics in the presence of zinc. At low concentrations (0.3 mM) zinc also concentration-dependently potentiated both peak and plateau components of whole cell current responses to AMPA (100 microM). This effect was accompanied by a reduction of the degree, and slowing of the rate, of AMPA receptor desensitisation. In contrast, higher concentrations of zinc (1-3.0 mM) inhibited AMPA responses to some degree, but slowed desensitisation further. This ability of zinc to change AMPA receptor properties may be relevant to neurotoxicity associated with AMPA receptor activation.

Uncompetitive NMDA receptor antagonists attenuate NMDA-induced impairment of passive avoidance learning and LTP.

In general, N-methyl-D-aspartate (NMDA) receptor antagonists inhibit learning and long term potentiation (LTP). However, it has been suggested that direct tonic, i.e. non-temporal, activation of NMDA receptors, in contrast to learning, may lead to an increase in synaptic "noise" and, in turn, to a loss of association detection. In the present study, a two-choice passive avoidance task and LTP in vitro (CA1 hippocampal region) were used to address this issue. Dark avoidance learning was impaired by systemic NMDA administration (starting at 25 mg/kg) that was not related to either toxic effects or state-dependent learning. NMDA-induced amnesia was antagonized by ((+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801) and 1-amino-3,5-dimethyladamantane (memantine), starting at low doses of 0.05 and 2.5 mg/kg, respectively, in a bell-shaped dose-response relationship. A competitive NMDA receptor antagonist CGP-39551 failed to reverse NMDA-induced amnesia. In hippocampal slices, NMDA (10 microM) depressed (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolproprionic acid (AMPA) receptor-mediated field potentials in CA1 and also caused a moderate reduction of LTP induction/expression. It was this latter effect that was antagonized by memantine (1 microM). Thus, under conditions of tonic activation of NMDA receptors, uncompetitive NMDA receptor antagonists can paradoxically reverse deficits in learning and synaptic plasticity.

Interactions of GYKI 52466 and NBQX with cyclothiazide at AMPA receptors: experiments with outside-out patches and EPSCs in hippocampal neurones.

In outside-out patches from cultured hippocampal neurones, glutamate (1 mM) applied for 1 ms evoked currents which rose rapidly (tau(on) 451 +/- 31 micros) to a peak and then deactivated with slower kinetics (1.95 +/- 0.13 ms). Offset time constants were significantly slower with longer application durations (tau(off) 3.10 +/- 0.19, 3.82 +/- 0.25, 4.80 +/- 0.65 and 7.56 +/- 0.65 ms with 10, 20, 100 and 500 ms applications respectively). Desensitization was complete within 100 ms with a similar rate for all application durations (4.74 +/- 0.34 ms with 100 ms applications). GYKI 52466 reduced inward peak currents with an IC50 of 11.7 +/- 0.6 microM and had similar potency on steady-state currents to longer glutamate applications. GYKI 52466 had no significant effect on desensitization or deactivation time constants but caused a modest and significant prolongation of onset kinetics at higher concentrations. Cyclothiazide (100 microM) potentiated steady-state currents 25-fold at 100 ms and caused a modest but significant slowing in onset kinetics (601 +/- 49 micros with 1 ms applications) but a more pronounced prolongation of deactivation time constants (5.55 +/- 0.66 ms with 1 ms applications). In 50% of neuronal patches cyclothiazide completely eliminated desensitization. In those patches with residual desensitization, the rate was not significantly different to control (5.36 +/- 0.43 ms with 100 ms applications). Following 100 ms applications of glutamate, GYKI 52466 had IC50s of 11.7 +/- 1.1 microM and 75.1 +/- 7.0 microM in the absence and presence of cyclothiazide (100 microM) respectively. Onset kinetics were slowed from 400 +/- 20 micros to 490 +/- 30 micros by cyclothiazide (100 microM) and then further prolonged by GYKI 52466 (100 microM) to a double exponential function (tau(on1) 1.12 +/- 0.13 ms and tau(on2) 171.5 +/- 36.5 ms). GYKI 52466 did not re-introduce desensitization but concentration-dependently weakened cyclothiazide's prolongation of deactivation time constants (1 ms applications: 5.01 +/- 0.71, 4.47 +/- 0.80 and 2.28 +/- 0.64 ms with GYKI 52466 30, 100 and 300 microM respectively). NBQX reduced peak current responses with an IC50 of 28.2 +/- 1.3 nM. Paradoxically, steady-state currents with 500 ms applications of glutamate were potentiated from 3.3 +/- 1.2 pA to 29.4 +/- 6.4 pA by NBQX (1 nM). Higher concentrations of NBQX then antagonized this potentiated response. The potency of NBQX in antagonizing steady-state currents to 500 ms applications of glutamate (IC50 120.9 +/- 30.2 nM) was 2-fold less than following 100 ms applications (IC50 67.7 +/- 2.6 nM). NBQX had no effect on rapid onset, desensitization or deactivation time constants. However, a slow relaxation of inhibition was seen with longer applications. NBQX was 2-5-fold less potent against inward currents in the presence of cyclothiazide (100 microM) depending on the application duration but had no effect on the rapid onset, desensitization or deactivation time constants. The same relaxation of inhibition was seen as with NBQX alone. NBQX (1 microM) reduced AMPA receptor-mediated EPSC amplitude to 7 +/- 1% of control with no effect on kinetics. Cyclothiazide (330 microM) caused a 2.8-fold prolongation of the decay time constant (control 26.6 +/- 2.2 ms, cyclothiazide 74.2 +/- 7.6 ms, n = 9). Additional application of NBQX (1 microM) partly reversed this prolongation to 1.9 fold (47.7 +/- 2.5 ms, n = 5). These results support previous findings that cyclothiazide also allosterically influences AMPA receptor agonist/antagonist recognition sites. There were no interactions between NBQX and cyclothiazide on desensitization or deactivation time constants of glutamate-induced currents but clear interactions on EPSC deactivation kinetics. This raises the possibility that the interactions of NBQX, GYKI 52466 and cyclothiazide on AMPA-receptor-mediated EPSC kinetics observed are due to modulation of glutamate-release at presynaptic AMPA receptors.

The in vivo relevance of the varied channel-blocking properties of uncompetitive NMDA antagonists: tests on spinal neurones.

The voltage dependence and channel-blocking kinetics of uncompetitive NMDA receptor antagonists have been well-described using in vitro techniques, but there is little evidence concerning the functional significance of these properties in vivo. We have now compared the effects of NMDA antagonists that display varied profiles of voltage-dependent block in vitro, on responses of spinal neurones in anaesthetised rats. The compounds examined were the uncompetitive channel blockers memantine, ketamine and MK-801 and, for comparison, an antagonist that acts at the strychnine-insensitive glycine binding site (MRZ 2/502). Using frequency of spike discharge as an indicator of somatic depolarisation, we have compared the effects of these antagonists on responses evoked by iontophoretic NMDA application and on synaptic responses evoked by pinch or electrical stimulation (the latter eliciting "wind-up"). The effectiveness of the antagonists was directly but variably related to the discharge frequency of the test response. The rank order of dependence on firing rate matched the rank order of voltage dependence reported in vitro, namely: memantine > ketamine > MK-801> or = MRZ 2/502. Doses that reduced responses to iontophoretic application of NMDA were less effective at reducing responses to pinch, perhaps due to the major non-NMDA component of the synaptic response. Memantine preferentially reduced "wind-up" relative to responses to pinch, whereas ketamine and MK-801 reduced both types of synaptic responses in parallel. This "filtering" by low affinity, voltage-dependent NMDA antagonists such as memantine, of non-physiological activity whilst leaving normal synaptic events relatively untouched, may contribute to their more favourable clinical profile.

Anxiolytic activity of glycine-B antagonists and partial agonists--no relation to intrinsic activity in the patch clamp.

On the basis of animal models, anxiety was one of the first suggested clinical applications of partial agonists of the glycineB site coupled to the NMDA receptor. It is not certain, however, whether these findings can be extended to full glycineB antagonists and what is the relation between intrinsic activity (degree of NMDA receptor antagonism) and anxiolytic effect. In the present study several NMDA receptor antagonists, including several glycineB antagonists/partial agonists, were tested for anxiolytic activity in the Vogel conflict test and the elevated plus-maze. Additionally, the intrinsic activities of the glycineB partial agonists used [ACPC, (R,+)-HA-966 and D-cycloserine] were compared in patch-clamp experiments in cultured neurones. In the plus-maze the most striking increase in the time spent in open arms (index of anxiolytic effect) was seen after diazepam and D-cycloserine (at doses that did not change locomotion). Also reliable (dose-dependent), although weaker, anxiolytic activity was produced by the uncompetitive NMDA receptor antagonist (+)MK-801 and the competitive antagonist CGP 39551. Modest anxiolytic-like effect in the plus-maze was also observed after the glycineB antagonist L-701,324 and the partial agonist (+,R)-HA-966. Uncompetitive antagonists memantine and amantadine, the glycineB partial agonist ACPC (up to 600 mg/kg) or the full antagonists MRZ 2/570, MRZ 2/571 and MRZ 2/576 had no effect. In the Vogel conflict test neither memantine, nor any of the full glycineB antagonists tested (L-701,324 and MRZ 2/576), showed anxiolytic activity. Patch-clamp studies revealed that the intrinsic activity of (+,R)-HA-966, D-cycloserine and ACPC was 13, 57 and 92%, respectively, as compared to that of glycine itself (100%). In conclusion, for the agents tested there is no clear relation between the levels of intrinsic activity, i.e. degree of NMDA receptor inhibition, and anxiolytic activity. Moreover, L-701,324 and MRZ-type glycineB full antagonists do not exchibit anxiolytic activity in the elevated plus-maze and Vogel conflict test.

Do kappa opioids mimic sigma agonists as amino acid antagonists?

The kappa opioids tifluadom and U-50,488 H, when tested on spinal neurones of rats in vivo and frogs in vitro, had no selective effect on responses to microelectrophoretically or bath applied N-methyl-D-aspartate, quisqualate or kainate. In the same preparations ketamine selectively reduces responses to N-methyl-D-aspartate. Amino acid antagonism by dissociative anaesthetics and sigma opioids is thus not mediated by that binding site which kappa and sigma opioids have been reported to have in common.

Comparison of the potency, kinetics and voltage-dependency of a series of uncompetitive NMDA receptor antagonists in vitro with anticonvulsive and motor impairment activity in vivo.

The amino-adamantane derivatives memantine (1-amino-3,5-dimethyladamantane) and amantadine (1-amino-adamantane) are relatively low affinity, uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists which have been used clinically in the treatment of dementia and Parkinson's disease respectively for several years without serious side effects. The aim of this study was to test whether memantine, amantadine and other low affinity uncompetitive NMDA receptor antagonists also have better therapeutic indices than high affinity antagonists in preclinical models of epilepsy by assessing the potency, kinetics and voltage-dependency of open channel blockade for a series antagonists in vitro and comparing these effects to anticonvulsive and motor impairment activity in vivo. The compounds tested were memantine, amantadine, 14 other amino-adamantanes, (+)-MK-801, ketamine, dextrorphan, dextromethorphan and phencyclidine. The offset kinetics of open-channel blockade assessed with whole cell patch clamp recordings from cultured superior colliculus neurones were highly correlated to potency i.e. the less potent antagonists showed faster unblocking kinetics (Koff, r = 0.904). Although, onset kinetics as assessed by Kon were not correlated to potency (r = 0.023), tau on estimated at IC50 is perhaps a more meaningful measure of onset kinetics at equieffective concentrations and was also well correlated to potency (r = -0.863). All amino-adamantanes tested were strongly voltage-dependent. There was also a good correlation between the in vitro potencies of uncompetitive NMDA receptor antagonists assessed with patch clamp recordings and displacement of equilibrium [3H](+)-MK-801 binding and their in vivo activity against maximal electroshock (MES) and pentylenetetrazol (PTZ) induced tonic convulsions and NMDA-induced lethality in mice. Memantine and four other amino-adamantanes with somewhat lower potency and faster blocking kinetics had better therapeutic indices (ED50 rotarod and traction reflex over ED50 in MES-induced convulsions; TI = 2-4) than substances with higher affinity such as ketamine, dextrorphan and (+)-MK-801 (TI < 2). However, amantadine and several other amino-adamantanes with lower potency than memantine actually had poorer therapeutic indices (TI < or = 0.5) which may have been due to additional actions at other ion channels or receptors at the doses necessary to protect against seizures. In fact, ED50 in the MES test was negatively-correlated to therapeutic indices (traction r = -0.790, rotarod r = -0.797) i.e. the less potent uncompetitive antagonists had worse therapeutic indices. The data from the present study do not lend support to the idea that low affinity, open channel NMDA receptor blockers are also effective in models of epilepsy at doses having little effect on physiological processes. It should be stressed that these data do not contradict the known therapeutic safety of memantine and amantadine in dementia and Parkinson's disease respectively. Thus the good clinical profile of memantine in dementia has been attributed not only to its fast blocking/unblocking kinetics but also to its strong voltage-dependency. These biophysical properties may allow therapeutically-relevant concentrations to block chronic, low level pathological activation of NMDA receptors whilst leaving their synaptic activation intact. Precisely these properties may also underlie the poor therapeutic indices seen in the present study on antiepileptic activity due to the synaptic nature of both seizures and normal glutamatergic transmission.

Amino-alkyl-cyclohexanes are novel uncompetitive NMDA receptor antagonists with strong voltage-dependency and fast blocking kinetics: in vitro and in vivo characterization.

The present study characterized the in vitro NMDA receptor antagonistic properties of novel amino-alkyl-cyclohexane derivatives and compared these effects with their ability to block excitotoxicity in vitro and MES-induced convulsions in vivo. The 36 amino-alkyl-cyclohexanes tested displaced [3H]-(+)-MK-801 binding to rat cortical membranes with K(i)s between 1.5 and 143 microM. Current responses of cultured hippocampal neurones to NMDA were antagonized by the same compounds with a wide range of potencies (IC50s of 1.3-245 microM, at -70 mV) in a use- and strongly voltage-dependent manner (delta 0.55-0.87). The offset kinetics of NMDA receptor blockade was correlated with equilibrium affinity (Corr Coeff. 0.87 P < 0.0001). As an example, MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) had similar blocking kinetics to those previously reported for memantine (K(on) 10.67 +/- 0.09 x 10(4) M(-1) s(-1), K(off) 0.199 +/- 0.02 s(-1), K(d) = K(off)/K(on) = 1.87 microM c.f. IC50 of 1.29 microM). Most amino-alkyl-cyclohexanes were protective against glutamate toxicity in cultured cortical neurones (e.g. MRZ 2/579 IC50 2.16 +/- 0.03 microM). Potencies in the three in vitro assays showed a relatively strong cross correlation (all corr. coeffs. > 0.72, P < 0.0001). MRZ 2/579 was also effective in protecting hippocampal slices against 7 min. hypoxia/hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC50 of 7.01 +/- 0.24 microM. MRZ 2/579 showed no selectivity between NMDA receptor subtypes expressed in Xenopus oocytes but was somewhat more potent than in patch clamp experiments-IC50s of 0.49 +/- 0.11, 0.56 +/- 0.01 microM, 0.42 +/- 0.04 and 0.49 +/- 0.06 microM on NR1a/2A /2B, /2C and 2/D, respectively. In contrast, memantine and amantadine were both 3-fold more potent at NR1a/2C and NR1a/2D than NR1a/2A receptors. All Merz amino-alkyl-cyclohexane derivatives inhibited MES-induced convulsions in mice with ED50s ranging from 3.6 to 130 mg/kg i.p. The in vivo and in vitro potencies correlated indicating similar access of most compounds to the CNS. MRZ 2/579 administered at 10 mg/kg resulted in peak plasma concentrations of 5.3 and 1.4 microM following i.v. and p.o. administration respectively, which then declined with a half life of around 170-210 min. Analysis of A.U.C. concentrations indicates a p.o./i.v. bioavailability ratio for MRZ 2/579 of 60%. MRZ 2/579 injected i.p. at a dose of 5 mg/kg resulted in peak brain extracellular fluid (ECF) concentrations of 0.78 microM (brain microdialysates). Of the compounds tested MRZ 2/579, 2/615, 2/632, 2/633, 2/639 and 2/640 had affinities, kinetics and voltage-dependency most similar to those of memantine and had good therapeutic indices against MES-induced convulsions. We predict that these amino-alkyl-cyclohexanes, which all had methyl substitutions at R1, R2, and R5, at least one methyl or ethyl at R3 or R4 and a charged amino-containing substitution at R6, could be useful therapeutics in a wide range of CNS disorders proposed to involve disturbances of glutamatergic transmission.