Linda M. Pullan

Glycine Site Antagonist Attenuates Infarct Size in Experimental Focal Ischemia Postmortem and Diffusion Mapping Studies

BACKGROUND AND PURPOSE The glycine site on the N-methyl-D-aspartate (NMDA) receptor complex offers a therapeutic target for acute focal ischemia, potentially devoid of most side effects associated with competitive and noncompetitive NMDA antagonists. METHODS A novel glycine receptor antagonist, ZD9379, was studied in 70 Sprague-Dawley rats using the suture occlusion model of permanent middle cerebral artery occlusion (MCAO). In the first experiment, 20 rats received an initial bolus of vehicle or 10 mg/kg ZD9379 (n = 10 in each group) 30 minutes after MCAO, followed by a continuous infusion of the same dose per hour for 4 hours. Diffusion-weighted MRI with echo-planar acquisition was used to generate maps of the apparent diffusion coefficient (ADC) of water. In a second experiment, 50 rats were assigned to five groups: vehicle and 10, 5, 2.5, and 1 mg/kg ZD9379 (n = 10 in each group) with the same dosing protocol but no imaging. In both experiments, infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining. RESULTS In the first experiment, before therapy was begun, there was no significant difference in ADC-derived ischemic lesion volume between the two groups. Over time, the 10-mg/kg ZD9379-treated rats had a significant delayed regional recovery of reduced ADC values in the peripheral parietal cortex (P = .0156). Postmortem corrected infarct volume at 24 hours after MCAO was significantly smaller in the group treated with 10 mg/kg ZD9379 than in the vehicle group (119.2 +/- 52.2 versus 211.2 +/- 50.0 mm3 [mean +/- SD]; P = .0008; a reduction of 43.6%). In the second experiment, postmortem corrected infarct volumes in rats receiving 10, 5, and 2.5 mg/kg ZD9379 were significantly smaller than in those receiving vehicle, a reduction of 42.6%, 51.4%, and 42.9%, respectively (P = .0001). CONCLUSIONS This study demonstrates that 2.5- to 10-mg/kg doses of ZD9379 initiated 30 minutes after MCAO significantly reduced infarct size. Diffusion mapping disclosed a delayed treatment effect of this glycine antagonist in focal ischemia, confirmed by the postmortem study.

Neomycin Is an Agonist at a Polyamine Site on the N‐Methyl‐D‐Aspartate Receptor

Abstract: Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1‐[1‐(2‐thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N‐methyl‐D‐aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2 ‐ (2 ‐ carboxypiperazin ‐ 4 ‐ yl)[3H]propyl ‐ 1 ‐ phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and an antagonist to the second.

Comparison of binding at strychnine-sensitive (inhibitory glycine receptor) and strychnine-insensitive (N-methyl-d-aspartate receptor) glycine binding sites

We compared, for a number of ligands to the two receptors, the displacement of [3H]strychnine binding to the glycine-gated chloride channel of spinal cord and brainstem synaptic membranes to the displacement of [3H]glycine binding to the NMDA receptor complex of hippocampal and cortex synaptic membranes. Glycine and beta-alanine are recognized by both receptors. In the NMDA receptor glycine antagonists, the kynurenic acids, most of the quinoxalinediones, and the (R)-enantiomer of HA-966 had little affinity at the strychnine-sensitive site. Surprisingly, the quinoxalinedione widely used as an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor antagonist, NBQX (2,3-dihydro-6-nitro-sulfamoylbenzo[f]quinoxaline-2,3-dione) displaced [3H]strychnine binding (IC50 = 11 microM) and to a lesser extent [3H]glycine binding (IC50 = 119 microM). Of the compounds tested, only strychnine, brucine, taurine and (S)-HA-966 were more potent displacers of [3H]strychnine than of glycine binding. Generally, the two glycine recognition sites appear to have remarkably different structural requirements.

Spermine reciprocally changes the affinity of NMDA receptor agonists and antagonists

We report that the enhancement of [ 3 H]CPP binding is correlated with an inhibition of the binding of the agonist L-[ 3 H]glutamate. Spermine decreases the K d of CPP and increases the K d of L-glutamate

Stereoselectivity for the (R)‐Enantiomer of HA‐966 (l‐Hydroxy‐3‐Aminopyrrolidone‐2) at the Glycine Site of the N‐Methyl‐d‐Aspartate Receptor Complex

Abstract: HA‐966 (1‐hydroxy‐3‐aminopyrrolidone‐2) is an antagonist at the glycine allosteric site of the N‐methyl‐d‐aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA‐966 is chiral. We report stereoselectivity for the (R)‐enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)‐enantiomer has an IC50 of 4.1 ± 0.6 μM. The racemic mixture has an IC50 of 11.2 ± 0.5 μM, whereas (S)‐HA‐966 has an IC50 greater than 900 μM. In glycine‐stimulated [3H]l‐[1‐(2‐thienyl)cyclohexyl] piperidine binding, the (R)‐enantiomer inhibits with an IC50 of 121 ± 61μM, whereas the racemic mixture has an IC50 of 216 ± 113 μM and (S)‐HA‐966 is inactive. The inhibition by (R)‐HA‐966 can be prevented by the addition of glycine. (R)‐HA‐966 and racemic HA‐966, but not (S) HA‐966, also prevent N‐methyl‐d‐aspartate cytotoxicity in cortical cultures. The (R)‐enantiomer and, less potently, the (S)‐enantiomer inhibit N‐methyl‐d‐aspartate‐evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)‐HA‐966 is reversed by added glycine. In glutamate‐evoked contractions of the guinea pig ileum, (R)‐HA‐966 causes a glycine‐reversible inhibition (IC50 of about 150 μM), whereas (S)‐HA‐966 is much less potent (IC50 of greater than 1mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N‐methyl‐d‐aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N‐methyl‐d‐aspartate receptors in glutamate‐evoked contractions of the guinea pig ileum, and supports their similarity to central N‐methyl‐d‐aspartate receptors.

Synthesis and activity of spiroisoindolines as novel noncompetitive NMDA antagonists

A series of novel spiroisoindolines was designed and synthesized as potential noncompetitive NMDA antagonists. Affinities of these compounds for the noncompetitive NMDA binding site were determined using [3H]TCP and found to possess IC50s ranging from 0.065 to 17μM. In vivo testing of 2′-methylspiro-[4,5,6,7-tetrahydrobenzothiophene-4,1′-(1,3-dihydroisoindole)] (43) showed it to antagonize NMDA-induced convulsions, to be neuroprotective in a gerbil model of ischemia, and not to generalize to MK-801 in a drug discrimination paradigm.

The inhibition of [125I]ω-conotoxin GVIA binding to neuronal membranes by neomycin may be mediated by a GTP-binding protein

ω-Conotoxin GVIA (ω-CT) has been reported to block calcium currents at the L- and N-type calcium channels. In neuronal membranes ω-CT, and the aminoglycoside antibiotic neomycin, have been shown to inhibit [125I]ω-CT binding, presumably acting at the N-type calcium channel. We demonstrate here that the concentration curve for neomycin sulfate inhibition of [125I]ω-CT binding is shifted to the right by GTP analogues or fluoride, increasing the IC90 for neomycin. [125I]ω-CT binding is unaffected by these agents and in competition studies the potency of ω-CT. Ca2− or La3+ is not modulated by GTP analogues or fluoride. These results indicate that the inhibition of [125I]ω-CT binding by neomycin may be mediated by a GTP binding protein.

Lack of involvement of nitric oxide in NMDA-induced neuronal cell death in cortical culture.

In primary cultures of rat cerebral cortex, N-methyl-D-aspartate causes widespread neurotoxicity. Inhibitors of the nitric oxide generating the enzyme nitric oxide synthase has been shown to attenuate the effects of N-methyl-D-aspartate in a number of neuronal systems both in vivo and in vitro. In our experiments, the nitric oxide synthase inhibitor N-nitroarginine was ineffective at blocking neurotoxicity induced by N-methyl-D-aspartate. Cyclic guanine monophosphate, known to be synthesized in response to nitric oxide was demonstrably inhibited by identical treatments with N-nitroarginine in sister cultures. We conclude that although nitric oxide is produced in response to N-methyl-D-aspartate, it is neither necessary nor sufficient for neurotoxicity.

Synthesis and biological activity of a series of 4-aryl substituted benz[b]azepines: Antagonists at the strychnine-insensitive glycine site

Abstract A series of 4-arylbenz[b]azepine analogs were prepared and shown to act as antagonists at the strychnine-insensitive glycine receptor. The heteroaryl substituted benz[b]azepine derivative 11 also showed excellent in vivo activity.

Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex.

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.