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NEUROPHARMACOLOGY

Characterization of Two Novel N-Methyl-D-aspartate Antagonists: EAA-090 (2-[8,9-Dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic Acid) and EAB-318 (R--Amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic Acid Hydrochloride)

Discovery Neuroscience (L.S., D.C., D.K., R.S., M.S., S.L.) and Chemical Sciences (R.B.), Wyeth Research, Princeton, New Jersey; Department of Neuroscience (R.S.Z.), Albert Einstein College of Medicine, Bronx, New York; and Departments of Psychiatry and Neuropathology (J.O.), Washington University School of Medicine, St. Louis, Missouri

Received January 26, 2004; accepted April 6, 2004.

	Abstract

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Two novel N-methyl-D-aspartate (NMDA) antagonists with unique chemical structures, EAA-090 (2-[8,9-dioxo-2, 6-diazabicyclo[5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) and EAB-318 (R--amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride), were compared with CGS-19755 (Selfotel) in ligand binding, electrophysiology, and neuroprotection assays. CGS-19755, EAA-090 and EAB-318 inhibited [³H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to NMDA receptors with IC₅₀ values of 55, 28, and 7.9 nM, respectively. All three compounds decreased the duration of spontaneous synaptic currents and inhibited NMDA-activated currents in rat hippocampal neurons. IC₅₀ values for inhibition of current induced by 10 µM NMDA were 795, 477, and 69 nM for CGS-19755, EAA-090, and EAB-318, respectively. The NMDA antagonists protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate- and NMDA-induced neurotoxicity. In experiments in which different NMDA receptor splice variants and subtypes were expressed in Xenopus oocytes, all three antagonists preferentially blocked NMDA-elicited currents mediated by N-methyl-D-aspartate receptor (NR)1 splice variants containing the N-terminal insertion. They also favored NR2A-versus NR2B- or NR2C-containing NMDA receptors, with EAA-090 showing the greatest selectivity. EAA-090 was 10 times more potent at blocking NR2A-versus NR2B- or NR2C-containing NMDA receptors. In addition to being the most potent NMDA antagonist, EAB-318 inhibited -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors. The combination of NMDA and AMPA/kainate block enabled EAB-318 to protect neurons against ischemia induced cell death.

Still, clinical trials with several different NMDA antagonists for the treatment of stroke have been unsuccessful due to lack of efficacy and/or side effects (Muir and Lees, 1995). Many compounds induced psychotic behavior similar to that seen with phencyclidine (PCP) and/or hypotension and other cardiovascular side effects. In some clinical trials, there was also a higher incidence of death in stroke patients treated with NMDA antagonist compared with placebo.

NMDA receptors are assembled from a combination of NR1 and NR2 subunits. A single gene encodes the NR1 subunit; however, alternative splicing with three different exons gives rise to eight different variants (Durand et al., 1992, 1993; for review, see Zukin and Bennett, 1995). Four genes encode NR2 subunits: NR2A, NR2B, NR2C, and NR2D (Monyer et al., 1992). Different NR1 splice variants assemble with various NR2 subunits to create a number of NMDA receptor subtypes. These are expressed in different regions of the brain and at different times in development. Drugs that selectively block specific NMDA receptor subtypes may provide neuroprotection, without producing unwanted side effects.

To increase potency and selectivity, NMDA antagonists with novel structures were synthesized (Fig. 1). EAA-090 (2-[8,9-dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) is an NMDA antagonist with an unique squaric acid moiety (3,4-diamino-3-cyclobutene-1,2-dione) in place of a polar amino acid group (Kinney et al., 1998). The squaric acid moiety is structurally similar to amino acids, but it lacks the basicity, acidity, or nucleophilicity of typical amino acids. Previous results showed that EAA-090 was as potent as CGS-19755 (Selfotel), the standard competitive NMDA antagonist, in blocking [³H]CPP binding to NMDA receptors in rat brain homogenates and in preventing NMDA-induced lethality in mice (Childers et al., 2002).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Chemical structures of CGS-19755, EAA-090, and EAB-318.

EAB-318 (R--amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride) has both phosphonic acid and -amino acid functional groups. Unlike many NMDA antagonists, EAB-318 has an unusual AP-6 spacing using a heterocycle (namely, 5-chlorobenzimidazole), rather than the typical AP-3, AP-5, or AP-7. In [³H]CPP binding and NMDA-induced lethality assays; EAB-318 was more potent than CGS-19755 or AP-7 (Baudy et al., 2001).

In this study, we compared the efficacy of EAA-090 and EAB-318 to CGS-19755 at inhibiting binding of ligands to several glutamate receptors, blocking NMDA-induced currents, and protecting against glutamate- and ischemia-induced toxicity. We also evaluated the compounds' selectivity for different NR1 and NR2 subunit combinations.

	Materials and Methods

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Binding Assays
Membranes were prepared from rat whole brain for [³H]CPP, [³H]glycine and [³H]TCP binding assays (Murphy et al., 1987a) and from rat frontal cortex (London and Coyle, 1979) and forebrain (Murphy et al., 1987b) for [³H]kainate and [³H]AMPA binding assays, respectively. [³H]CPP and [³H]TCP are used as ligands for NMDA and PCP binding sites, respectively. [³H]CPP, [³H]glycine, [³H]kainate, and [³H]AMPA binding assays were performed as described previously (London and Coyle, 1979; Murphy et al., 1987a,b; Monahan et al., 1989). [³H]TCP binding assays were carried out as described previously by Ransom and Stec (1988) with modifications. Briefly, rat whole brain membranes (0.2–0.5 mg) were combined with 3 µM L-glutamate, 1 µM glycine, 2.5 nM [³H]TCP, test compound, and enough 5 mM Tris-HCl, pH 7.4, buffer to obtain a final volume of 1 ml in glass test tubes. After a 60-min incubation period at 25°C, the incubate was filtered under vacuum over Whatman GF/B filters presoaked with 0.05% polyethyleneimine and rapidly washed three times with 2-ml volumes of cold buffer. The filters were processed and counted using conventional liquid spectroscopy. Nonspecific binding was defined by 100 µM MK-801.

In each of the binding assays, total specific radioactivity in dpm was defined as the difference between "total" and "nonspecific" dpm. Specific dpm in the presence of test compound (i.e., total dpm minus the dpm in the presence of test compounds) was expressed as a percentage of the total specific binding. When test compounds were examined for a concentration-response relationship, specific counts determined for each of five to 10 concentrations were analyzed using a nonlinear regression of dpm versus the log of the test compound concentration from which an IC₅₀ value was calculated.

Rat Neuronal Cultures
Rat hippocampal cultures were prepared using methods modified from Furshpan and Potter (1989). Cells were plated on glass cover-slips treated with polylysine and laminin in 35-mm dishes for electrophysiology, or in 96-well plates (Falcon Plastics, Oxnard, CA) for neurotoxicity experiments. After a few hours, the cultures were fed with Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with the addition of 5% rat serum (Harlan, Indianapolis, IN), 5% fetal bovine serum (Invitrogen), Mito+ (Collaborative Research, Bedford, MA), and penicillin/streptomycin.

A few days later, when glial cells were confluent, 10 µM cytosine arabinofuranoside (Sigma-Aldrich, St. Louis, MO) was added to stop proliferation of non-neuronal cells. After the first week, the cultures were fed weekly with medium containing 2.5% rat serum, 2.5% fetal bovine serum, and 1 mM kynurenate (Sigma/RBI, Natick, MA) and 10 mM added MgCl₂. Kynurenate is a nonspecific blocker of glutamate receptors, and elevated levels of magnesium block calcium and sodium influx through NMDA receptor channels. The combination of both protects neurons from glutamate-induced toxicity (Furshpan and Potter, 1989). Cultures of cortical neurons in 24-well plates were similarly prepared.

Electrophysiology
Hippocampal Neurons. Whole-cell currents were recorded from hippocampal neurons after 3 to 6 weeks in culture. Patch pipettes, pulled on a Sutter P97 micropipette puller, had resistances of 3 to 5 M when filled with 125 mM potassium aspartate, 20 mM KCl, 1 mM KH₂PO₄, 5 mM HEPES, 5 mM NaCl, 1 mM MgCl₂, and 10 mM EGTA. Gigaohm seals were obtained in external bath perfusion solution containing Hanks' balanced salt solution (HBSS) with 10 mM HEPES, 10 mM glucose, 1 mM kynurenate, and 10 mM MgCl₂, pH 7.4. To measure synaptic or NMDA-induced currents, the following magnesium-free external solution was used: 150 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, along with 10 to 20 µM glycine (pH 7.4) to promote NMDA activity. During bath application of NMDA, 1 µM tetrodotoxin was added to block action potentials and synaptic currents.

Spontaneous synaptic and NMDA-induced currents were recorded with an Axopatch 200 amplifier and PClamp (Axon Instruments Inc., Union City, CA) software. Data were analyzed using PClamp and Origin (OriginLab Corp, Northampton, MA) programs. Neurons were voltage clamped at negative holding potentials, and the amplitude and duration of synaptic currents were measured. NMDA currents were induced by bath perfusion of NMDA. The amplitude of the current induced by 10 or 100 µM NMDA in the presence of a given concentration of drug was divided by the amplitude of the control current taken immediately before the drug trial to determine the percentage of inhibition. Data from several experiments on different cells at each drug concentration were collected, and the mean and standard error were calculated. From these data, concentration-response curves were generated, and an IC₅₀ for each compound was determined.

Oocytes. NR1₁₀₀ cDNA was previously isolated from a ventral midbrain cDNA library (Durand et al., 1992). NR1₁₁₁ was from R. Axel (Columbia University College of Physicians and Surgeons, New York). NR1₀₁₁ and NR2A-C cDNAs were gifts of S. Nakanishi (Kyoto University Faculty of Medicine, Kyoto, Japan). To generate templates for transcription, circular plasmid cDNA were linearized with NotI (NR1₀₁₁ and NR2A) or BamH1 (NR1₁₀₀, NR1₁₁₁, NR2B, and NR2C). Transcription reactions were performed with T7 or T3 polymerase (MEGAscript transcription kit; Ambion, Austin, TX; 4 h at 37°C) in the presence of capped analog or mMessage mMachine transcription kit, 2 h at 37°C.

Oocytes were collected from anesthetized Xenopus laevis as described previously (Kushner et al., 1988). After removing the follicular layer, stage V and VI oocytes were injected with in vitro-transcribed RNA (about 20 ng/cell) and maintained at 18°C in culture buffer (103 mM NaCl, 2.5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 5 mM HEPES, pH 7.5). Three to 7 days after injection, oocytes were clamped at –60 mV in Mg²⁺-free Ca²⁺ Ringer (116 mM NaCl, 2 mM KCl, 1 mM CaCl₂, and 10 mM HEPES, pH 7.2) with a Dagan two-electrode voltage-clamp amplifier. Responses were elicited by application of NMDA (100 µM) with glycine (10 µM) in the presence and absence of different concentration of each drug. Concentration-response curves were calculated by the following equation: I = I_max (A/A + k_d)n, where I is the measured steady-state current amplitude normalized to the maximal current I_max, and A is the concentration of agonist; K_d, the apparent affinity constant; and n, the Hill coefficient, are free parameters. Data points represent means ± S.E.M. of responses of an average of five oocytes.

Neurotoxicity Assays
Chick Embryo Retina Studies. Intact retinas from 15-day-old chick embryos were gently removed, sliced into thirds, and incubated in a balanced salt solution containing 1.2 mM CaCl₂, 5 mM KCl, 0.9 mM MgCl₂, 123 mM NaCl, 0.44 mM KH₂PO₄, 22 mM NaHCO₃, 5 mM glucose, and 0.03 mM phenol red. The buffer is oxygenated and brought to pH 7.4 by equilibrating it with 95% O₂, 5% CO₂. Glutamate, NMDA, kainic acid, or AMPA was added at a minimal concentration sufficient to produce a maximum fully developed lesion across the retinal section in 30 min. Concentrations above that cause the same degree of damage, but doses below that cause either little or no damage. All incubations are for a duration of 30 min after which the retinas are fixed by immersion in a phosphate-buffered aldehyde solution (1% paraformaldehyde plus 1.5% glutaraldehyde), and then postfixed in osmium tetroxide and embedded in araldite. Histological sections (1 µm in thickness) displaying the full radial extent of the retina from the ora serrata to the nerve head are stained with methylene blue/azure II and evaluated by light microscopy.

Test compounds were added at various concentrations to the incubation medium for the 30-min duration of the experiment. For each treatment condition, at least six retinal segments were evaluated. Typically, for agents with significant neuroprotective properties, a threshold concentration could be found at which the agent began to provide partial protection and a second threshold at which total protection was consistently obtained. A concentration-response evaluation was performed for each agent, and the lowest concentration at which the agent provided total protection (in all of six retinal segments studied at that concentration) was used as a measure of its potency.

The chick embryo retina assay was also used to determine whether a test compound could protect against simulated ischemia (oxygen/glucose deprivation). In this protocol, glucose is removed from the medium, and all procedures are performed in a 100% nitrogen atmosphere. Incubation of the retina under simulated ischemia conditions for 30 min causes a pattern of degeneration that seems very similar to that observed when the retina is incubated for 30 min in the presence of a toxic concentration of glutamate. A test compound is added to this incubation at various concentrations to determine whether it can inhibit or prevent simulated ischemic neuronal degeneration.

Cultured Rat Brain Neurons. After 4 to 6 weeks in culture, cortical and hippocampal neurons were washed three times and fed with test compound in HBSS containing 44 mM sodium bicarbonate and 10 mM glucose. Glutamate (30 µM), NMDA (10 µM), or AMPA (10 µM) was added to induce neurotoxicity. There were at least four wells of each drug concentration in each experiment. Control wells contained HBSS with 1 mM kynurenate and 10 mM MgCl₂. Initial neurotoxicity experiments used hippocampal cultures grown in 96-well plates. The number of hippocampal neurons remaining in five predetermined fields in each well was counted 24 h after the addition of 30 µM glutamate to determine the amount of neuroprotection afforded by each drug.

Due to the great variability in results and difficulty in counting large numbers of wells, neurotoxicity in later experiments was measured with a lactate dehydrogenase (LDH) assay. The amount of LDH released in culture medium is directly correlated with the number of dead neurons. For unknown reasons, results of LDH measurements taken from neurons cultures in 96-well plates were more variable than those from cultures in 24-well plates. Therefore, cultures grown in 24-well plates were used for LDH assays. To generate the large number of neurons required for 24-well plates, neurons from cortical brain regions were used. At different time points after treatment, 50 µl of medium was removed from each well and transferred to wells in flat bottom 96-well plates. The medium sample was diluted with 250 µl of phosphate buffer (33 mM KH₂PO₄, 66 mM K₂HPO₄) containing -NADH (8 mg/100 ml; Sigma-Aldrich). Plates were kept in the dark for at least 20 min. After 10 µl of 22.7 mM pyruvate (Sigma-Aldrich) was added to each well, each plate was immediately placed in a plate reader (Molecular Devices, Sunnyvale, CA), and the O.D. at 340 nm was read every 15 s for 5 min. The concentration of LDH in the medium is proportional to rate of enzyme activity, which can be determined by measuring the initial slope in the change in O.D.₃₄₀. At the end of the experiment, 0.2% Triton X-100 (Sigma-Aldrich) was added to each well to lyse all cells (both neurons and glia), and a final sample was taken to determine the total LDH activity in each well. The amount of LDH at each time point for each well was normalized to the total LDH in the well, allowing data from multiple wells with the same treatment to be pooled.

	Results

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Binding Assays. Results of rat brain membrane binding assays with radioligands to NMDA, PCP, AMPA, kainate, and glycine binding sites are shown in Table 1. CGS-19755, EAA-090, and EAB-318 inhibit CPP binding to NMDA receptors with IC₅₀ values of 55, 28, and 7.9 nM, respectively. IC₅₀ values for TCP binding to the PCP site are more than a 100-fold greater, indicating that the compounds are competitive NMDA receptor antagonists. By contrast, MK-801 has no effect on CPP binding, but it does inhibit TCP binding with an IC₅₀ value of 9 nM. At 100 µM, CGS-19755, EAA-090, and EAB-318 inhibit glycine binding by 45, 33, and 48%, respectively. EAB-318 also inhibits AMPA (43%) and kainate (22%) binding at 100 µM.

Effects on Spontaneous Synaptic Currents. Hippocampal neurons grown in culture for three or more weeks were voltage clamped at –60 mV. When perfused with magnesium-free HBSS, spontaneous synaptic currents occur. These currents are produced by glutamate released from presynaptic neurons, which activates AMPA and NMDA receptors on the recorded neuron. AMPA receptors desensitize rapidly, whereas current flowing through NMDA receptors is sustained as long as glutamate is present (Trussell et al., 1988; Tang et al., 1989). Without magnesium to block NMDA receptors, the synaptic current durations are long, lasting up to a couple of seconds. EAA-090 or CGS-19755 (at 10 µM) reduced the duration of synaptic currents without affecting the peak amplitude and fast desensitizing AMPA component (Fig. 2A). The fast desensitizing current can be inhibited by addition of the AMPA receptor-specific antagonist YM90K (data not shown). EAB-318, at 1 µM, shortened the duration of synaptic currents, and at 10 µM, blocked the fast desensitizing component (Fig. 2B).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effects of EAA-090, EAB-318, and CGS-19755 on synaptic currents in cultured hippocampal neurons. Synaptic currents were recorded from rat hippocampal neurons cultured for at least 3 weeks. Neurons were voltage clamped at –60 mV. Eliminating extracellular magnesium from the bath solution prevented magnesium block of NMDA receptor currents and increased synaptic current duration. EAA-090 and CGS-19755 each reduced synaptic current duration without affecting the peak amplitude. Similar effects were seen with 1 µM EAB-318. At 10 µM, EAB-318 blocked both the fast desensitizing AMPA and the sustained NMDA components of the synaptic current.

Inhibition of NMDA-Induced Currents in Neurons. Pure NMDA currents in cultured rat hippocampal neurons activated by bath application of NMDA (in the presence of 1 µM tetrodotoxin to block synaptic currents) were reduced by EAA-090 or EAB-318 (Fig. 3). Concentration-response curves for EAA-090, EAB-318, and CGS-19755 are shown in Fig. 4A. The IC₅₀ values for inhibition of current induced by 10 µM NMDA are 795 nM for CGS-19755, 477 nM for EAA-090, and 69 nM for EAB-318. Increasing the concentration of NMDA to 100 µM shifted the dose-response curves to the right for EAA-090 and EAB-318, consistent with their competitive antagonist mechanism of action. IC₅₀ values for the two compounds at 100 µM NMDA are 2.35 µM and 317 nM for EAA-090 and EAB-318, respectively (Fig. 4, B and C).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Block of NMDA currents by EAA-090 and EAB-318. Cultured rat hippocampal neurons were voltage clamped at –60 mV. Bath application of 100 µM NMDA produces a large inward current that is decreased by EAA-090 (A) or EAB-318 (B).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for NMDA current inhibition. CGS-19755, EAA-090, and EAB-318 inhibit NMDA currents in a concentration-dependent manner. IC50 values for CGS-19755, EAA-090, and EAB-318 with 10 µM NMDA are 795, 477, and 69 nM, respectively (A). Increasing the concentration of NMDA from 10 to 100 µM shifts the dose-response curve to the right for EAA-090 (B) and EAB-318 (C), demonstrating a competitive mechanism of action. IC50 values at 100 µM NMDA for EAA-090 and EAB-318 are 2350 and 317 nM, respectively. Each data point represents mean and standard error from at least three experiments on three different neurons.

Inhibition of Specific NR1 Splice Variants and NR/NR2 Subunit Combinations. The effect of the antagonists on different NMDA receptor subtypes was determined by recording NMDA-elicited currents from Xenopus oocytes expressing the NR1 splice variants NR1₀₁₁ (NR1-1a), NR1₁₁₁ (NR1-1b), and NR1₁₀₀ (NR1-4b) in the presence and absence of CGS-19755, EAA-090, or EAB-318 at varying concentrations (Table 2; for review of nomenclature, see Zukin and Bennett, 1995). As with neurons, EAB-318 is much more potent than EAA-090 and CGS-19755 at inhibiting NMDA-elicited currents mediated by each of the NR1 splice variants expressed individually. EAA-090 is more potent than CGS-19755 at blocking NR1₀₁₁, but the two antagonists inhibit NR1₁₁₁ and NR1₁₀₀ receptors with comparable IC₅₀ values. All three compounds are 4 to 5 times less potent at blocking NR1₀₁₁ than NR1₁₁₁ and NR1₁₀₀ receptors. None of the compounds are selective for NR1₁₁₁ versus NR1₁₀₀ receptors.

Because NR1 receptors are coexpressed with NR2 subunits in neurons, it is more relevant to know the effects of the blockers on different NR1/NR2 combinations. IC₅₀ values for the three NR1 splice variants coexpressed with NR2A, NR2B, or NR2C in oocytes are shown in Table 3. With the addition of NR2A or NR2B subunits, all three drugs still preferentially block NR1₁₁₁ and NR1₁₀₀ over NR1₀₁₁.Incontrast, adding NR2C decreases the potency of all three blockers and eliminates their selectivity for different NR1 splice variants. IC₅₀ values for CGS-19755 block of NR1 splice variants are not affected by coexpression with NR2A, but they are increased 2- to 3-fold with NR2B and 3- to 12-fold with NR2C. IC₅₀ values for EAA-090 are also higher with the addition of NR2B and NR2C subunits. However, they are 2 to 6 times lower with NR2A subunits. EAB-318's potency is not affected by NR2A or NR2B, but its IC₅₀ values are more than 10-fold higher with NR2C. EAB-318 is still much more potent than EAA-090 and CGS-19755 at blocking NR1/NR2A and NR1/NR2B current, but at NR1/NR2C receptors, it is equipotent to EAA-090. EAA-090 is more potent than CGS-19755 at NR1/NR2A and NR1/NR2C, and equipotent at NR1/NR2B receptors.

Protection against NMDA and Glutamate-Induced Neurotoxicity. EAA-090 and EAB-318 were evaluated for their ability to protect against NMDA and glutamate-induced toxicity in chick retinas and cultured rat brain neurons (Table 4; Fig. 5). With 40 µM NMDA, EAA-090 and CGS-19755 were each able to provide complete neuroprotection in chick retinas at 3 to 5 µM. EAB-318 was much more potent, with 100 to 300 nM sufficient to provide total protection. Increasing the concentration of NMDA reduced the neuroprotective activity of each compound, but increasing the concentration of compound restored this. The potency of EAB-318 is similar to MK-801, a noncompetitive NMDA antagonist. At 250 µM, EAB-318 also completely blocked neurotoxicity induced by 15 µM AMPA, and at 500 µM, it prevented neurotoxicity induced by 25 µM kainate, 600 µM glutamate or ischemia. Neither EAA-090 nor CGS-19755, at concentrations up to 1 mM, was effective against these insults (Table 4).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Neuroprotective actions of CGS-19755, EAA-090, and EAB-318. A, visual counts of surviving neurons in hippocampal cultures grown in 96-well plates after exposure to 30 µM glutamate (n = 3–7 experiments/point, 3 wells/experiment). CGS-19755 and EAA-090 have similar IC50 values (1.66 and 1.63 µM, respectively), whereas EAB-318 is much more potent (IC50 of 0.48 µM). Due to the greater variability and laboriousness associated with counting neurons by eye, neurotoxicity was subsequently measured using an LDH assay with cultured cortical neurons. B, neuroprotection against 30 µM glutamate. The amount of LDH was normalized to the total amount of LDH produced by neurons in control wells without drugs. IC50 values for CGS-19755 and EAA-090 are similar to those with hippocampal cultures, 1.7 and 1.48 µM, respectively. Again, EAB-318 is much more potent with an IC50 value of 0.08 µM. C, neuroprotection against 10 µM NMDA (4 wells/condition). IC50 values for CGS-19755, EAA-090, and EAB-318 are 3.31, 2.48, and 0.33 µM, respectively.

Full concentration-response curves for the neuroprotective actions of EAA-090 and EAB-318 against NMDA and glutamate induced toxicity in cultured rat brain neurons were generated. IC₅₀ values for neuroprotection against 30 µM glutamate are 1.66 µM for CGS-19755, 1.63 µM for EAA-090, and 0.48 µM for EAB-318 (Fig. 5A). These results were acquired by counting the number of hippocampal neurons remaining in 96-well plates after 24-h exposure to glutamate. Similar concentration-response curves were obtained when neuroprotection was assessed by measuring LDH levels in culture medium of cortical neurons grown in 24-well plates. IC₅₀ values for those experiments are 1.71 µM for CGS-19755, 1.48 µM for EAA-090, and 80 nM for EAB-318 (Fig. 5B). With 10 µM NMDA, IC₅₀ values for neuroprotection are 3.31, 2.48, and 0.33 µM for CGS-19755, EAA-090, and EAB-318, respectively (Fig. 5C).

EAB-318 is also able to protect cortical neurons against neurotoxicity induced by 10 µM AMPA at concentrations from 100 to 1000 µM (Fig. 6). Inhibition of AMPA-induced neurotoxicity by 1 mM EAB-318 is similar to that of 10 µM YM90K with 100 µM CGS-19755, EAA-090, or EAB-318. The combination of both AMPA and NMDA antagonism prolongs the time cortical neurons are able to survive ischemic insults (Mosinger et al., 1991; Goldberg and Choi, 1993). EAB-318 protects cortical neurons for longer periods of ischemia than CGS-19755 or EAA-090 (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6. AMPA-induced neurotoxicity. Cortical neurons cultured for 5 months are very vulnerable to excitotoxicity induced by 10 µM AMPA. LDH activity was measured 24 h after addition of AMPA. Results are from n = 4 wells/condition. CGS-19755 and EAA-090 alone offer only partial protection against neurotoxicity induced by AMPA. EAB-318 at 1 mM provides maximal protection against AMPA toxicity, as did the addition of 10 µM YM90K, a specific AMPA receptor antagonist, to 100 µM of each of the three compounds.

	Discussion

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In this study, two novel competitive NMDA receptor blockers, EAA-090 and EAB-318, were compared with CGS-19755 in several in vitro assays. EAA-090 showed similar efficacy to CGS-19755 in ligand binding, electrophysiology, and neurotoxicity assays, whereas EAB-318 was 10 times more potent. All three compounds selectively blocked current produced by NR1 receptors with the N-terminal exon (NR1₁₀₀ and NR1₁₁₁) better than receptors without (NR1₀₁₁). The observation that the N-terminal insert enhances the compounds' affinities is of interest because having the N-terminal insert reduces agonist affinity at NR1 receptors by a factor of 4 to 5 (Durand et al., 1993). This suggests that the molecular determinants for agonist and antagonist binding to NMDA receptors may be different. Within the hippocampus, expression of NR1₁₀₀ and NR1₁₁₁ mRNA is more prominent in CA3 than CA1 or dentate gyrus, suggesting the drugs might preferentially target CA3 in the mature brain.

The three blockers are also more potent at inhibiting current produced by NMDA receptors with NR2A than receptors with NR2B or NR2C subunits. EAA-090 is the most selective, blocking NR1/NR2A receptors with a 10-fold higher potency than NR1/NR2B and NR1/NR2C receptors. EAB-318 is 3 times more potent in blocking NR1/NR2A compared with NR1/NR2B receptors and is much less potent at blocking NR1/NR2C receptors. In adult rat brain, NR2A mRNA is expressed prominently in neocortex and hippocampus, selected thalamic nuclei, and in the molecular and granule cell layers of the cerebellum. NR2B mRNA expression is largely confined to the forebrain with highest intensity in the olfactory cortex and caudate putamen. In contrast, NR2C is largely restricted to the cerebellum, with some labeling in the thalamus (Laurie et al., 1997).

Previous publications have suggested that NMDA receptor antagonists that selectively target NR2B subunit-containing receptors may be more effective in treating stroke than nonselective antagonists because they produce fewer central nervous system side effects (Fischer et al., 1997; Gill et al., 2002). Some of these compounds include ifenprodil, Ro 25-6981, and Ro 63-1908, which respectively are 400, 5000, and >20,000 times more potent at blocking NMDA receptors containing NR2B versus NR2A subunits (Williams, 1993; Fischer et al., 1997; Gill et al., 2002). In addition to being very selective for NR2B receptors, these compounds preferentially block active versus inactive NMDA receptor channels, which may explain their greater tolerability in animals and humans than traditional NMDA receptor antagonists (Gill et al., 2002). Nevertheless, many of these compounds failed in clinical trials for stroke due to lack of efficacy.

Despite its greater selectivity for the NR1/NR2A subtype, EAA-090 has a better separation between efficacious and toxic doses than CGS-19755. At doses where EAA-090 showed neuroprotection in the rat permanent middle cerebral artery occlusion model (3 mg/kg i.v.), it produced no vacuolization in the brain (Zaleska et al., 1998). Even at a higher dose range (30–50 mg/kg i.v.) that caused vacuolization in some animals, the vacuole reaction was very mild with EAA-090. By contrast, CGS-19755 provided neuroprotection at 10 mg/kg i.v. and induced vacuoles at 20 mg/kg i.v., suggesting a smaller margin of safety. In drug discrimination studies, EAA-090 generalized to PCP in some animals at 35 mg/kg i.v., whereas CGS-19755 generalized in all animals at 11 mg/kg i.v. (Childers et al., 2002). These results suggest that, unlike CGS-19755, EAA-090 may have a wider separation between a therapeutically effective dose and a dose that produces side effects. For a more definitive safety evaluation, it will be necessary to determine whether a wide margin of safety can be demonstrated for the irreversible type of neurotoxic reaction that noncompetitive NMDA antagonists, such as MK-801 and PCP, are known to cause (Fix et al., 1993). This issue was not addressed in the present study. However, EAA-090 was found to be safe in standard animal toxicity assays and in phase I human clinical trials. It is not clear why EAA-090 has a greater margin of safety than CGS-19755. One possible explanation is its unique squaric acid structure prevents it from interacting with other receptors or proteins. Aside from the NMDA receptor, EAA-090 had no affinity for any ligand binding or substrate sites at over 60 receptors, ion channels, or enzymes screened.

EAB-318 also has a better separation than CGS-19755 between its efficacious dose in the middle cerebral artery occlusion model and the doses that induced vacuolization or generalization to PCP. In addition to being one of the most potent NMDA receptor antagonists, EAB-318 is a weak AMPA/kainate receptor blocker. At concentrations of 10 µM or more, it inhibits the fast transient component of synaptic currents mediated by AMPA receptors, and reduces AMPA-induced neurotoxicity in chick retina and rat cortical neurons. The combination of both NMDA and AMPA antagonism makes EAB-318 an effective neuroprotective agent in models of ischemia-induced toxicity and may be an advantage in preventing neuronal damage after stroke (Goldberg and Choi, 1993). In summary, EAA-090 and EAB-318 are novel competitive NMDA receptor antagonists that are more efficacious and safer than CGS-19755 and thus may be better drugs for the treatment of stroke and other neurodegenerative diseases.

	Acknowledgements

 
We thank Madelon Price for superb technical assistance. We also thank John Moyer and Margaret Zaleska for coordinating the EAA-090 studies.

	Footnotes

 
DOI: 10.1124/jpet.104.066092.

ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; NR, N-methyl-D-aspartate receptor; EAA-090, 2-[8,9-dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic acid; EAB-318, R--amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride; CPP, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid; TCP, [1-(2-thienyl)cyclohexy-l]piperidine; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; O.D., optical density; YM90K, 6-(1H-imidazol-11-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride; Ro 25-6981, R-(R*,S*)--(4-hydroxyphenyl)--methyl-4-(phenylmethyl)-1-peperidine propanol; Ro 63-1908, 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol.

¹ Current address: Discovery Toxicology, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., Princeton, NJ 08543.

² Current address: St. Jude Children's Research Hospital, Developmental Neurobiology, Memphis, TN 38105.

Address correspondence to: Dr. Stephen Lin, Discovery Neuroscience, Wyeth Research, CN8000, Princeton, NJ 08543. E-mail: lins{at}wyeth.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Baudy RB, Fletcher H, Yardley JP, Zaleska MM, Bramlett DR, Tasse RP, Kowal DM, Katz AH, Moyer JA, and Abou-Gharbia MA (2001) Design, synthesis, SAR and biological evaluation of highly potent benzimidazole-spaced phosphono--amino acid competitive NMDA antagonists of the AP-6 type. J Med Chem 44: 1516–1529.[CrossRef][Medline]

Childers WE, Abou-Gharbia MA, Moyer JA, and Zaleska MM (2002) EAA-090. Drugs Future 2002 27: 633–638.[CrossRef]

Choi D (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: 623–634.[CrossRef][Medline]

Clark GD and Rothman SM (1987) Blockade of excitatory amino acid receptors protects anoxic hippocampal slices. Neuroscience 21: 665–671.[CrossRef][Medline]

Collingridge GL and Bliss TV (1995) Memories of NMDA receptors and LTP. Trends Neurosci 18: 54–56.[CrossRef][Medline]

Collingridge GL and Bliss TVP (1987) NMDA receptors-their role in long-term potentiation. Trends Neurosci 10: 288–293.[CrossRef]

Durand GM, Bennett MVL, and Zukin RS (1993) Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C. Proc Natl Acad Sci USA 90: 6731–6735.[Abstract/Free Full Text]

Durand GM, Gregor P, Zheng X, Bennett MVL, Uhl GR, and Zukin RS (1992) Cloning of an apparent splice variant of the rat N-methyl-D-aspartate receptor NMDAR1 with altered sensitivity to polyamines and activators of protein kinase C. Proc Natl Acad Sci USA 89: 9359–9363.[Abstract/Free Full Text]

Fischer G, Mutel V, Trube G, Malherbe P, Kew JNC, Mohacsi E, Heitz MP, and Kemp JA (1997) Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro. J Pharmacol Exp Ther 283: 1285–1292.[Abstract/Free Full Text]

Fix AS, Horn JW, Wightman KA, Johnson CA, Long GG, Storts RW, Farber N, Wozniak DF, and Olney JW (1993) Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK(+)801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex. Exp Neurol 123: 204–215.[CrossRef][Medline]

Furshpan EJ and Potter DD (1989) Seizure-like activity and cellular damage in rat hippocampal neurons in cell culture. Neuron 3: 199–207.[CrossRef][Medline]

Gill R, Alanine A, Bourson A, Buttelmann B, Fischer G, Heitz M-P, Kew JNC, Levet-Trafit B, Lorez H-P, Malherbe P, et al. (2002) Pharmacological characterization of Ro 63-1908 (1-[2-(4-hydroxy-phnoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist. J Pharmacol Exp Ther 302: 940–948.[Abstract/Free Full Text]

Goldberg M and Choi D (1993) Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. J Neurosci 13: 3510–3524.[Abstract]

Kinney WA, Abou-Gharbia M, Garrison DT, Schmid J, Kowal DM, Bramlett DR, Miller TL, Tasse RP, Zaleska MM, and Moyer JA (1998) Design and synthesis of [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)-ethyl]phosphonic acid (EAA-090), a potent N-methyl-D-aspartate antagonist, via the use of 3-cyclobutene-1,2-dione as an achiral -amino acid bioisostere. J Med Chem 41: 236–246.[CrossRef][Medline]

Kushner L, Lerma J, Zukin RS, and Bennett MVL (1988) Coexpression of N-methyl-D-aspartate and phencyclidine receptors in Xenopus oocytes injected with rat brain mRNA. Proc Natl Acad Sci USA 85: 3250–3254.[Abstract/Free Full Text]

Laurie DJ, Bartke I, Schoepfer R, Naujoks K, and Seeburg PH (1997) Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies. Mol Brain Res 51: 23–32.[Medline]

London ED and Coyle JT (1979) Specific binding of [³H]kainic acid receptor sites in rat brain. Mol Pharmacol 15: 492–505.[Abstract/Free Full Text]

Monahan JB, Corpus VM, Hood WF, Thomas JW, and Compton RP (1989) Characterization of a [³H]glycine recognition site as a modulatory site of the N-methyl-D-aspartate receptor complex. J Neurochem 53: 370–375.[CrossRef][Medline]

Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, and Seeburg PH (1992) Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science (Wash DC) 256: 1217–1221.[Abstract/Free Full Text]

Morris RGM, Anderson E, Lynch GS, and Baudry M (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature (Lond) 319: 774–776.[CrossRef][Medline]

Mosinger J, Price M, Bai H, Xiao H, Wozniak D, and Olney J (1991) Blockade of both NMDA and non-NMDA receptors is required for optimal protection against ischemic neuronal degeneration in the in vivo adult mammalian retina. Exp Neurol 113: 10–17.[CrossRef][Medline]

Muir KW and Lees KR (1995) Clinical experience with excitatory amino acid antagonist drugs. Stroke 26: 503–513.[Abstract/Free Full Text]

Murphy DE, Schneider J, Boehm C, Lehmann J, and Williams J (1987a) Binding of [³H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid to rat membranes: a selective, high-affinity ligand for N-methyl-D-aspartate receptors. J Pharmacol Exp Ther 240: 778–783.[Abstract/Free Full Text]

Murphy DE, Snowhill EW, and Williams M (1987b) Characterization of quisqualate recognition sites in rat brain tissue using DL-[³H]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay. Neurochem Res 12: 775–782.[CrossRef][Medline]

Nakanishi S (1992) Molecular diversity of glutamate receptors and implications for brain function. Science (Wash DC) 258: 597–603.[Abstract/Free Full Text]

Olney JW, Labruyere J, Wang G, Sesma M, Wozniak D, and Price MT (1991) NMDA antagonist neurotoxicity: mechanism and protection. Science (Wash DC) 254: 1515–1518.[Abstract/Free Full Text]

Ransom RW and Stec NL (1988) Cooperative modulation of [³H]MK-801 binding to the N-methyl-D-aspartate receptor-ion channel complex by L-glutamate, glycine and polyamines. J Neurochem 51: 830–836.[CrossRef][Medline]

Rothman SM and Olney JS (1986) Glutamate and the pathophysiology of hypoxicischemic brain damage. Ann Neurol 19: 105–111.[CrossRef][Medline]

Seeburg PH (1993) The molecular biology of mammalian glutamate receptor channels. Trends Neurosci 16: 359–364.[CrossRef][Medline]

Tang C, Dichter M, and Morad M (1989) Quisqualate activates a rapidly inactivating high conductance ionic channel in hippocampal neurons. Science (Wash DC) 243: 1474–1477.[Abstract/Free Full Text]

Trussell L, Thio L, Zorumski C, and Fischbach G (1988) Rapid desensitization of glutamate receptors in vertebrate central neurons. Proc Natl Acad Sci USA 85: 2834–2838.[Abstract/Free Full Text]

Williams K (1993) Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol Pharmacol 44: 851–859.[Abstract]

Zaleska MM, Boast CA, Marquis KL, Tasse RP, Lin SS, Hartupee DA, Price MT, Olney JW, Childers WE, Abou-Gharbia MA, et al. (1998) The preclinical neuropharmacological profile of EAA-090, a novel competitive NMDA receptor antagonist, in Proceedings from 7th International Symposium on Pharmacology of Cerebral Ischemia, Abstract 69.

Zukin RS and Bennett MVL (1995) Alternatively spliced isoforms of the NMDARI receptor subunit, Trends Neurosci 18: 306–313.[CrossRef][Medline]

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