Glutamate receptor antagonists attenuate experimental catalepsy in rats

A long-term akinesia induced by haloperidol used as an experimental model of catalepsy helped to reveal that a dicationic derivatives adamantane (IEM-1754) and phenylcyclohexyl (IEM-1925) exerted different degrees of inhibition of the haloperidol effect: the IEM-1754 proved to be not inferior to the most effective NMDA antagonist MK-801. A relatively low potency of the IEM-1925 may be due to its obvious equal effects both on the NMDA and the AMPA receptor channels. A good correlation between the anticataleptic effects of the glutamate antagonists and the NMDA receptor blocking activity, were found. The AMPA receptor blockade might negatively affect the anticataleptic potency of the drugs under study.     

Mechanisms of blockade of glutamate receptors channels: the significance for structural and physiological investigations

The mechanism of blocking effect of phenylcyclohexyl derivative, IEM-1925, on ionotropic glutamate receptors of the NMDA and AMPA types has been studied on the rat isolated brain neurons. The whole-cell configuration of patck clanp recording technique was used equilibrium conditions and -80 mV holding potential, the IEM-1925 manifests nonselective action on open channels of both receptors. However, the prominent differences in the mechanism of the blocking effect were revealed. Although IEM-1925 can not enter the closed channels of both types, its molecule are able to leave closed channels of the AMPA but not the NMDA receptors. Hyperpolarization reduces removal of blocker from the open channels of the NMDA receptors. Contrary to that, hyperpolarization facilitates going out of the IEM-1925 to cytozol from both open and closed channels. Evidently, the bloker can pass through the AMPA receptor channels into the cell, and the gating mechanism of these channels is located above the binding site for the blocker. The blocking action of the IEM-1925 on the NMDA and AMPA receptors was compared with its potency to weaken the tremor evoked by subcutaneous injection of arecoline to mice. The observed differences in the mechanisms of action help to explain the ambiguous effects of channel blocking drugs on experimental models of pathological processes.     

Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats

The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.     

Comparison of the anticonvulsant activity of organic mono- and di-cations and their potential to inhibit NMDA and AMPA glutamate receptors

Effects of mono- and dicationic derivatives of adamantine and phenylcyclohexyl were studied on: (i) open channels of NMDA and AMPA glutamate receptors in the experiments on the isolated rat brain neurones, and (ii) convulsions induced by intraventricular injections of NMDA or kainate in mice. Monocations inhibited the NMDA receptors in vitro and prevented convulsions induced by NMDA in vivo, but failed to affect both the AMPA receptors and kainite-induced convulsions. Dications (IEM-1754 and IEM-1925) revealed both anti-NMDA and anti-AMPA potency in vitro, were highly effective against kainite-induced convulsions and excelled monocations in preventing the NMDA-induced ones. Evidently some steps connected with the AMPA receptor activity are involved in the genesis of the NMDA-induced convulsions. Anticonvulsant potency of IEM-1754 and IEM-1925 is comparable with those of known NMDA receptor inhibitors: memantine and MK-801. The IEM-1754 and IEM-1925 show no side effects. An incomplete correspondence between the activity in vitro and in vivo found studying some derivatives, may be due to peculiarities of their pharmacokinetics.     

Involvement of ionotropic glutamate receptors in the genesis of arecoline-induced tremor

The muscarinic agonist arecoline (6 mg/kg, subcutaneously in mice) induced a long-lasting tremor. The inhibitory potency of non-competitive antagonists of ionotropic glutamate receptors has been studied. These antagonists are the derivatives of adamantane and phenylcyclohexyl. A part of them: monocationic compounds, selectively block the NMDA-receptor channels, their dicationic analogues affecting both channels of the NMDA- and the AMPA-glutamate receptors. Monocationic blockers effectively reduced the arecoline-evoked tremor and their potency correlated with ability to block the NMDA-receptor channels. Dicationic blockers revealed protective effect only in low range doses (0.0001-0.01 microM/kg). Further increase of the dose reduced or completely abolished this effect. This suggests that the NMDA-receptors are involved in the genesis of arecoline-evoked tremor. The only moderate blockade of the AMPA-receptors potentiates the drug blocking action but the prevalent blockade of these receptors impedes the effect on arecoline-evoked tremor.     

Role of NMDA and AMPA glutamate receptors in the mechanism of korazol-induced convulsions in mice

The potency of mono- and dikationic derivatives of adamantane and phenylcyclohexyl to prevent seizures induced in mice by intraperitoneal administration of 80 mg/kg pentylenetetrazol (corazol), was studied. Monocationic derivatives of phenylcyclohexyl, being the selective channel blockers of NMDA glutamate receptors, as well memantine and MK-801 in micromolar concentrations, prevented both clonic and tonic components of corazol-induced convulsions. Their dicatonic derivatives which are channel blockers of NMDA and AMPA types of glutamate receptors, failed to prevent clonic seizures but at submicromolar concentrations prevented the tonic extensions provoked by corazol. Evidently, convulsive action of corazol originating from suppression of GABA-ergic inhibition is realized through activation of glutamergic synaptic transmission, and NMDA receptors are mainly involved in genesis of clonic seizures whereas activation of AMPA receptors is important for the tonic component of the corazol-induced syndrome.     

Channel blocking drugs as tools to study glutamate receptors in insect muscles and molluscan neurons

The action of three dicationic drugs, derivatives of adamantane (IEM-1460 and IEM-1754) and phencyclidine (IEM-1925), on glutamate receptors (GluRs) at the insect neuromuscular junction (Calliphora vicina larva) and on neurons of the freshwater gastropodian molluscPlanorbarius corneus has been studied using the voltage clamp technique. In the presence of concanavalin A complex glutamate-induced currents recorded from molluscan neurons reflected mainly the opening of cationic channels as a result of decreased desensitisation and inhibition of a chloride component. Under these conditions all drugs studied inhibited the stationary component of glutamate-gated cationic currents in a dose-dependent manner (IC₅₀s were 0.1 μM, 1.0 μM and 19.2 μM for the action of IEM-1925, IEM-1460 and IEM-1754 respectively). The same rank order of potency: IEM-1925 > IEM-1460 > IEM-1754 was observed in both the insect and mollusc. The results of these experiments are compared with those obtained earlier on vertebrate GluRs. Open-channel blocking drugs may help to identify and classify GluRs of invertebrates, and could be used as tools to elucidate the involvement of GluRs in the transmission at certain synapses.     

The participation of glutaminergic transmission in the mechanism of movement disorders induced by reverse rotation in white mice]

Administration of MK-801 or IEM-1754 prevented akinesia in mice induced by reversing rotation, not less effectively than scopolamine. Quaternary adamantane derivative IEM-1857 was ineffective. IEM-1925 enhanced the locomotor disturbance induced by reversing rotation due, probably, to different spectrum of its antiglutamate action. The data obtained suggest involvement of glutamate synaptic transmission in development of locomotor disturbances of a vestibular origin.     

9-Aminoacridine blocks NMDA and AMPA receptors by different mechanisms

Tricyclic mono- and dicationic compounds (derivatives of 9-aminoacridine) antagonize AMPA and NMDA glutamate receptors. The aim of the present study was to compare mechanisms of the 9-aminoacridine action on AMPA and NMDA receptors. Experiments were carried out by whole-cell patch-clamp technique on native receptors from rat brain neurons. An important peculiarity of the 9-aminoacridine action on NMDA receptors is the large slope of the concentration dependence, which suggests the binding of two molecules in the channel. AMPA receptors blockade also demonstrated interesting features. In contrast to the NMDA receptor channel block, inhibition of AMPA receptors is voltage-independent. 9-Aminoacridine and its dicationic analog demonstrated similar anti-AMPA activity. For classical AMPA-receptor channel blockers (derivatives of adamantane and phenylcyclohexyl) it was demonstrated that dicationic analogs are much more potent than monocationic analogs. We conclude that 9-aminoacridine binds to a specific site in AMPA receptors. This finding opens a possibility to develop a new family of non-competitive antagonists of AMPA receptors.     

Structural characteristics of ionotropic glutamate receptors revealed by channel blockade

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule. The second group includes NMDA receptor and both nicotinic cholinoreceptors. Contrary, here the blocking potency of monocations and dications are practically equal irrespective of molecule length. The data obtained suggest that hydrophobic and nucleophilic components of the binding site are located close to each other in the channels of the NMDA receptor type but are separated by approximately 10 A in the AMPA receptor channel.     

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

Antipunishment effects of acute and repeated administration of phencyclidine and NPC 12626 in rats.

The effects of phencyclidine (PCP) and NPC 12626 on punished responding were examined using a modified Geller-Seifter procedure in rats. Both drugs are known to antagonize N-methyl-D-aspartate (NMDA) receptor mediated neurotransmission, albeit at different sites on the NMDA receptor complex. Rats were trained to lever press for food reinforcement under a multiple schedule, with responding in one component reinforced under a fixed-interval 60-sec schedule, while each response in the other component resulted in both food and brief electric shock. Both PCP and NPC 12626 produced selective increases in punished responding, although the effects were not as large as those produced by chlordiazepoxide. Repeated daily administration of each of these drugs for 6 days resulted in increases in punished responding during different portions of the treatment. A 5 mg/kg dose of chlordiazepoxide produced increases over the last 2 days of administration. PCP (2 mg/kg) produced an increase only during the second session, whereas NPC 12626 (30 mg/kg) produced increases for all but the first and fifth days of the 6-day regimen. Both competitive and noncompetitive NMDA antagonists can have antipunishment effects in this model.     

Pharmacological antagonists of excitant amino acid action

Pharmacological receptors for excitant amino acids have been classified into three major types found within the vertebrate central nervous system (CNS). The three types of receptor are exemplified by the action of the selective agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate. Several compounds have been discovered which are selective antagonists of NMDA-evoked excitations, the most potent to date being 2-amino-5-phosphonovalerate (APV). Depression of synaptic excitation by NMDA receptor antagonists indicates a physiological role of these receptors in various regions of the CNS.Potent and selective antagonists for kainate or quisqualate receptors have yet to be developed. However, glutamate diethyl ester (GDEE) and γ-D-glutamylglycine (DGG), applied microelectrophoretically, selectively depress quisqualate and kainate-evoked responses, respectively. 2-Amino-4-phosphonobutyrate (APB) and $\text{cis}$-2, 3-piperidine dicarboxylate (PDA) are relatively non-selective antagonists of the three types of excitant receptor. Depression of APV-resistant spinal transmission by PDA and synaptically localized kainate binding in the hippocampus suggest that kainate and/or quisqualate receptors are also involved in excitatory transmission.     

Discovery of novel and orally active NR2B-selective N-methyl-D-aspartate (NMDA) antagonists, pyridinol derivatives with reduced HERG binding affinity

Novel NR2B antagonists with an amide tether were found by an approach to avoid pharmacophoric similarity to dofetilide. Structure-activity relationship investigation led to N-[cis-4-hydroxy-4-(5-hydroxypyridin-2-yl)cyclohexyl]-3-henylpropanamide as an orally active NR2B-subtype selective N-methyl-D-aspartate (NMDA) receptor antagonist with very weak HERG (human ether-a-go-go related gene) binding (IC(50)> 30 microM). This compound exhibited potent in vivo anti-allodynic activity in the mouse partial sciatic nerve ligation (PSL) model (minimum effective dose=10 mg/kg, po).     

Excitatory amino acid receptors and synaptic excitation in the mammalian central nervous system.

At least two different types of excitatory amino acid receptors have been identified in the mammalian and amphibian central nervous systems. One type ('NMDA receptors') appears to be important in amino acid-mediated synaptic excitation, NMDA being the most potent and specific exogenous agonist for this type of receptor. Many antagonists have selective blocking actions at these NMDA receptors, and such substances are also selective antagonists of synaptic excitation in the vertebrate spinal cord. It is proposed that these receptors are transmitter receptors activated by an excitatory amino acid. In addition, extrasynaptic receptors, activated by domoate, kainate, quisqualate and L-glutamate, but not by NMDA, and only weakly by L-aspartate, have been identified on dorsal root fibres of the immature rat.     

The synaptic and nonsynaptic glycine transporter type-1 inhibitors Org-24461 and NFPS alter single neuron firing rate in the rat dorsal raphe nucleus. Further evidence for a glutamatergic-serotonergic interaction and its role in antipsychotic action

Single neuron firing rate was recorded from dorsal raphe nucleus of anesthetized rats. The firing rate of raphe neurons varied from 4 to 8 discharge per second before drug administration and this neuronal activity was decreased by L-701,324 (2 mg/kg i.v. injection), a competitive antagonist of glycineB binding site of N-methyl-D-aspartate (NMDA) receptors. The glycine transporter type-1 (GlyT1) antagonists Org-24461 (10 mg/kg i.v.) and NFPS (3 mg/kg i.v.) reversed the inhibitory effect of L-701,324 on single neuron activity recorded from dorsal raphe nucleus of the rat. Org-24461 and NFPS both tended to increase the raphe neuronal firing rate also when given alone but their effect was not significant. This finding serves further evidence that glutamate released from axon terminals of the cortico-striatal projection neurons stimulates serotonergic neurons in the raphe nuclei and this effect is mediated at least in part by postsynaptic NMDA receptors. Thus, GlyT1 inhibitors are able to reverse the hypofunctional state of NMDA receptors, suggesting that these drugs may have beneficial therapeutic effects in neurological and psychiatric disorders characterized with impaired NMDA receptor-mediated transmission.     

Characterization of ionotropic glutamate receptors in insect neuro-muscular junction

Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure-activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6-7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 A. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific.     

Synthesis, radiosynthesis and in vivo evaluation of 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-[(11)C]one, as a potent NR(1A)/2B subtype selective NMDA PET radiotracer

Recently, a new series of potent and highly subtype-selective 1-(heteroarylalkynyl)-4-benzylpiperidine antagonists of the NMDA receptors has been described by Pfizer Laboratories. In this series, 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-one (1) was identified as a selective antagonist for the NR1(A)/2B subtype, displaying IC(50) values for inhibition of the NMDA responses of 5.3 nM for this subtype (compared to NR1(A)/2A: 35 microM and NR1(A)/2C>100 microM) and was active in rat at a relatively low dosage (10mg/kg po). Derivative 1 has been synthesized in four chemical steps in good overall yield and labelled with carbon-11 at its benzoimidazolone ring using [(11)C]phosgene. The pharmacological profile of [(11)C]-1 was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [(11)C]-1 was extremely low (0.07% I.D./mL on average at 30 min) and rather uniform across the different brain structures. This in vivo brain regional distribution of [(11)C]-1 did not match with autoradiographic or binding data obtained with other NR2B subtype-selective NMDA ligands. Competition studies with ifenprodil (20 mg/kg, ip, 30 min before the radiotracer injection) failed to demonstrate specific binding of the radiotracer in the brain. In view of these results, and especially considering the low brain penetration of the radiotracer, [(11)C]-1 does not have the required properties for imaging NMDA receptors using positron emission tomography.