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.

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.

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.

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.

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.

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+).

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'.

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.

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.

Memantine: a NMDA receptor antagonist that improves memory by restoration of homeostasis in the glutamatergic system--too little activation is bad, too much is even worse.

The neurotransmitter glutamate activates several classes of metabotropic receptor and three major types of ionotropic receptor--alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). The involvement of glutamate mediated neurotoxicity in the pathogenesis of Alzheimer's disease (AD) is finding increasing scientific acceptance. Central to this hypothesis is the assumption that glutamate receptors, in particular of the NMDA type, are overactivated in a tonic rather than a phasic manner. Such continuous, mild, chronic activation ultimately leads to neuronal damage/death. Additionally, impairment of synaptic plasticity (learning) may result not only from neuronal damage per se but may also be a direct consequence of this continuous, non-contingent NMDA receptor activation. Complete NMDA receptor blockade has also been shown to impair neuronal plasticity, thus, both hypo- and hyperactivity of the glutamatergic system leads to dysfunction. Memantine received marketing authorization from the EMEA (European Medicines Agency) for the treatment of moderate to severe AD in Europe and was subsequently also approved by the FDA (Food and Drug Administration) for use in the same indication in the USA. Memantine is a moderate affinity, uncompetitive NMDA receptor antagonist with strong voltage-dependency and fast kinetics. This review summarizes existing hypotheses on the mechanism of action (MOA) of memantine in an attempt to understand how the accepted interaction with NMDA receptors could allow memantine to provide both neuroprotection and reverse deficits in learning/memory by the same MOA.

Neuroprotective activity of selective mGlu1 and mGlu5 antagonists in vitro and in vivo.

The neuroprotective potential of allosteric mGlu5 and mGlu1 antagonists such as 6-methyl-2-(phenylethynyl)-pyridin (MPEP)/[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), was tested in vitro in organotypic hippocampal cultures and in the middle cerebral artery occlusion model of stroke in vivo. Both classes of agent have high selectivity toward mGlu sub-types and are active in animal models of various diseases indicating satisfactory CNS penetration. In organotypic hippocampal cultures MPEP showed high neuroprotective potency against sub-chronic (12 days) insult produced by 3-NP with an IC50 of c.a. 70 nM. In contrast, although the mGlu1 antagonist EMQMCM was also protective, it seems to be weaker yielding an IC50 of c.a. 1 microM. Similarly, in the transient (90 min) middle cerebral artery occlusion model of ischaemia in rats, MTEP seems to be more effective than EMQMCM. MTEP, at 2.5 mg/kg and at 5 mg/kg provided 50 and 70% neuroprotection if injected 2 h after the onset of ischaemia. At a dose of 5 mg/kg, significant (50%) neuroprotection was also seen if the treatment was delayed by 4 h. EMQMCM was not protective at 5 mg/kg (given 2 h after occlusion) but at 10 mg/kg 50% of neuroprotection was observed. The present data support stronger neuroprotective potential of mGlu5 than mGlu1 antagonists.

Memantine as an example of a fast, voltage-dependent, open channel N-methyl-D-aspartate receptor blocker.

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 N-methyl-D-aspartate (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 with 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.

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.

The antiallodynic effect of NMDA antagonists in neuropathic pain outlasts the duration of the in vivo NMDA antagonism.

Clinical reports have described a long-lasting relief in neuropathic pain patients treated with NMDA receptor antagonists; it is unclear, however, what mediates this effect. In this work, we have used two NMDA antagonists of different class to investigate if the antiallodynic effects in a rat neuropathy model can outlast their in vivo NMDA antagonism. Both the uncompetitive NMDA antagonist ketamine and the glycine(B) antagonist MRZ 2/576 inhibited neuronal responses to iontophoretic NMDA in anaesthetised rats with the time course consistent with their known pharmacokinetics (t(1/2) approximately 10-12min, similar in control and nerve-injured rats). Surprisingly, the antiallodynic effects of the same doses of the NMDA antagonists in the neuropathic pain model were long-lasting (>3h with ketamine, >24h with MRZ 2/576). The effect of ketamine was further prolonged (>24h) when combined with a short-acting opioid, fentanyl, which only produced a short effect ( approximately 40min) when given alone. The duration of centrally mediated side effects of ketamine and MRZ 2/576 was short, similar to the in vivo NMDA antagonism. We speculate that NMDA receptor blockade may down-regulate the central sensitisation triggered by nerve injury, resulting in a long-lasting antiallodynic effect. Development of short-acting NMDA antagonists could represent a strategy for improving their tolerability.

mGlu1 and mGlu5 receptor antagonists lack anticonvulsant efficacy in rodent models of difficult-to-treat partial epilepsy.

Modulation of metabotropic glutamate (mGlu) receptors represents an interesting new approach for the treatment of a range of neurological and psychiatric disorders. Several lines of evidence suggest that functional blockade of group I (mGlu1 and mGlu5) receptors may be beneficial for treatment of epileptic seizures. This study was conducted to investigate whether mGlu1 or mGlu5 receptor antagonists have the potential to block partial or secondarily generalized seizures as occurring in partial epilepsy, the most common and difficult-to-treat type of epilepsy in patients. For this purpose, we systemically administered novel highly selective and brain penetrable group I mGlu receptor antagonists, i.e., the mGlu1 receptor antagonist EMQMCM [3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate] and the mGlu5 receptor antagonist MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine), at doses appropriate for mGlu1 or mGlu5 receptor-mediated effects in rodent models of partial seizures. Two models were used: the 6-Hz electroshock model of partial seizures in mice and the amygdala-kindling model in rats. Clinically established antiepileptic drugs were included in the experiments for comparison. Antiepileptic drugs exerted significant anticonvulsant effects in both models, while EMQMCM and MTEP were ineffective in this regard, although both compounds were administered up to doses associated with essentially full receptor occupancy and with typical mGlu receptor-mediated effects in rodent models of anxiety or pain. Brain microdialysis for determining extracellular levels of MTEP following i.p. administration in rats substantiated that effective brain concentrations were reached at times of our experiments in seizure models. The present results do not support a significant anticonvulsant potential of group I mGlu receptor antagonists in rodent models of difficult-to-treat partial epilepsy.

Peripherally acting NMDA receptor/glycineB site receptor antagonists inhibit morphine tolerance.

The present study focused on the role of peripheral ionotropic N-methyl-D-aspartate (NMDA) receptors in the development of tolerance to morphine-induced antinociception. An initial experiment revealed that NMDA channel blocker memantine, and NMDA receptor/glycine(B) site antagonist MRZ 2/576 inhibited maximal electroshock-induced convulsions (MES) in female NMR mice with respective potency of 5.93 and 20.8 mg/kg, while other NMDA receptor/glycine(B) site antagonists MRZ 2/596 and MDL 105,519 were ineffective, supporting lack of CNS activity of the latter two agents. This observation was also supported by blood-brain barrier experiments in vitro. In male Swiss mice, morphine (10 mg/kg) given for 6 days twice a day (b.i.d.) produced tolerance to its antinociceptive effects in the tail-flick test. The NMDA receptor/glycine(B) site antagonists, MRZ 2/576 at 0.03, 0.1, 0.3 mg/kg and MRZ 2/596 at 0.1, 0.3, 3 and 10 mg/kg attenuated the development of morphine tolerance. Similarly, in male C57/Bl mice, morphine (10 mg/kg) given for 6 days b.i.d. produced tolerance to its antinociceptive effects in the tail-flick test. Like in Swiss mice, in C57/Bl mice morphine tolerance was attenuated by both MRZ 2/576 and MRZ 2/596. Another NMDA receptor/glycine(B) site receptor antagonist, MDL 105,519 (that very weakly penetrates to the central nervous system) also inhibited morphine tolerance at the dose of 1 but not 0.1 mg/kg. Moreover, both naloxone hydrochloride (5 and 50 mg/kg) and centrally inactive naloxone methiodide (50mg/kg) inhibited morphine tolerance suggesting the involvement of peripheral opioid receptors in this phenomenon. The present data suggest that blockade of NMDA receptor/glycine(B) sites in the periphery may attenuate tolerance to the antinociceptive effects of morphine.

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.

Mrz 2/579, a fast kinetic NMDA channel blocker, reduces the development of morphine tolerance in awake rats.

The purpose of the present study was to investigate whether uncompetitive NMDA antagonists with fast channel blocking kinetics, which show fewer side effects in man than compounds such as ketamine, affect the development of tolerance to continuous exposure to morphine. Rats were trained on the Randall--Selitto apparatus before being implanted, under halothane anaesthesia, with primed mini-osmotic pumps (240 microl/day). Six rats were implanted with a vehicle filled pump, seven with a morphine filled pump (28.8 mg/kg/day), and eight with a pair of pumps, one containing morphine and the other Mrz 2/579, a new NMDA antagonist (40 mg/kg/day). A fourth group was implanted with a morphine filled pump followed 25 h later by a Mrz 2/579 filled pump. Paw withdrawal tests were undertaken immediately before, and at 2, 4, 6, 8, 10, 12, 24, 48 and 72 h after the first pump was implanted. Before pump implantation, withdrawal thresholds were 120+/-7 g (mean+/-SEM, n=30). Vehicle infusion had no effect on withdrawal thresholds, whereas morphine infusion increased them significantly at 2 and 4 h after pump implantation (+2 h: 208+/-14 g; P<0.001 vs. control). From 6 h the antinociception elicited by morphine declined progressively; at 10 h withdrawal thresholds were significantly lower than the 2 h post-treatment value (P<0.001). In rats treated with morphine plus Mrz 2/579, thresholds remained significantly higher between 10--72 h post-implantation than with morphine alone (P<0.05). In contrast, infusion of the same level of Mrz 2/579 once tolerance had developed did not reverse tolerance. These results indicate that fast NMDA channel blockers such as Mrz 2/579 may prove to be useful in enhancing analgesia to continuous morphine administration.