NMDA Receptor Glycine Site Antagonist Research Articles

A Role for Accumbal Glycine Receptors in Modulation of Dopamine Release by the Glycine Transporter-1 Inhibitor Org25935

Accumbal glycine modulates basal and ethanol-induced dopamine levels in the nucleus accumbens (nAc) as well as voluntary ethanol consumption. Also, systemic administration of the glycine transporter-1 inhibitor Org25935 elevates dopamine levels in nAc, prevents a further ethanol-induced dopamine elevation and robustly and dose-dependently decreases ethanol consumption in rats. Here we investigated whether Org25935 applied locally in nAc modulates dopamine release, and whether accumbal glycine receptors or NMDA receptors are involved in this tentative effect. We also addressed whether Org25935 and ethanol applied locally in nAc interact with dopamine levels, as seen after systemic administration. We used in vivo microdialysis coupled to HPLC-ED in freely moving male Wistar rats to monitor dopamine output in nAc after local perfusion of Org25935 alone, with ethanol, or Org25935-perfusion after pre-treatment with the glycine receptor antagonist strychnine or the NMDA receptor glycine site antagonist L-701.324. Local Org25935 increased extracellular dopamine levels in a subpopulation of rats. Local strychnine, but not systemic L-701.324, antagonized the dopamine-activating effect of Org25935. Ethanol failed to induce a dopamine overflow in the subpopulation responding to Org25935 with a dopamine elevation. The study supports a role for accumbal glycine receptors rather than NMDA receptor signaling in the dopamine-activating effect of Org25935. The results further indicate that the previously reported systemic Org25935–ethanol interaction with regard to accumbal dopamine is localized to the nAc. This adds to the growing evidence for the glycine receptor as an important player in the dopamine reward circuitry and in ethanol's effects within this system.

ChemInform Abstract: Synthesis of Kynurenic Analogues as Potential NMDA Receptor Glycine Site Antagonists.

ChemInform Abstract: Synthesis of Kynurenic Analogues as Potential NMDA Receptor Glycine Site Antagonists.

Ammonia at pathophysiologically relevant concentrations activates kynurenic acid synthesis in cultured astrocytes and neurons

The synthesis of the NMDA receptor glycine site antagonist kynurenic acid (KYNA) from exogenously added kynurenine was measured in cultured cerebral cortical neurons, cortical astrocytes and an oligodendroglial cell line OLN-93, incubated for 2 h in the presence or absence of ammonium acetate (“ammonia”) at 0.1–10 mM concentration. In neurons ammonia stimulated KYNA synthesis to ∼160–170% of control at 0.1–1.0 mM, did not produce any effect at 2.5 and 5 mM, and inhibited the synthesis to ∼80% of control at 10 mM concentration. In astrocytes, a stimulation to ∼200% of control occurred in the whole ammonia concentration range. No effect of ammonia was noted in OLN-93 cells. Increased KYNA synthesis may represent a cytoprotective response of astrocytes and neurons against the NMDA receptor-mediated cytotoxic activity of ammonia.

Interaction between intrathecal morphine and glutamate receptor antagonists in formalin test

The analgesic interaction between intrathecally administered morphine and the NMDA receptor antagonist, ((±)-2-amino-5-phosphonopentanoic acid; AP-5), the NMDA receptor glycine site antagonist, (5-nitro-6,7-dichloro-2,3-quinoxaline dion; ACEA 1021), or the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor antagonist (ACEA 2752) in the formalin test was investigated with a rat model of chronic lumbar intrathecal catheterization. After obtaining dose–response curves for each agent, combinations of morphine and AP-5, ACEA 1021 or ACEA 2752 were tested for their effect on the number of flinches in the formalin test and for associated side-effects, such as motor disturbances, flaccidity, and agitation/allodynia. Using isobolographic analyses, a potent analgesic synergy was observed with decreased side-effects between morphine and ACEA 2752 or AP-5. ACEA 1021 increased the analgesic effect of low-dose morphine. Spinal μ-opioid receptor activation and NMDA or AMPA receptor antagonism showed a synergistic antinociception against tonic pain. These results suggest an important direction in the management of inflammatory pain.

Synthesis of 4,6-dichloro-3-[(1-N-arylaminocarbonyl)-hydrazono]-1,3-dihydro-indole-2 -one as a potential NMDA receptor glycine site antagonist.

A synthetic procedure for the preparation of indole-2,3-dione derivatives 6 as a potential NMDA receptor glycine site antagonist with improved pharmacological profile compared with 2-carboxyindole derivative 5, starting from readily available 3,5-dichloroaniline (7), is described.

Receptor binding characteristics of the novel NMDA receptor glycine site antagonist [ 3H]GV150526A in rat cerebral cortical membranes

Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt ([ 3H]GV150526A) was characterised in rat cerebral cortical membranes. Saturation experiments indicated the existence of a high affinity binding site, with a pK d value of 9.08 ( K d=0.8 nM) and a B max of 3.4 pmol/mg of protein. A strong linear correlation was observed between the displacement potencies for [ 3H]GV150526A and [ 3H]glycine of 13 glycine site ligands ( r=0.991). The association kinetics of [ 3H]GV150526A binding was monophasic, with a k on value of 0.047 (nM) −1 min −1. Dissociation was induced by the addition of an excess of glycine, GV150526A, or 5,7-dichlorokynurenic acid (DCKA), another glycine antagonist. With GV150526A and DCKA, the dissociation curves presented similar k off values (0.068 and 0.069 min −1, respectively), as expected from ligands binding to the same site. Conversely, a significantly lower k off value (0.027 min −1) was found with glycine. Although these data may suggest that glycine agonists and antagonists bind to discrete sites with an allosteric linkage (rather than interacting competitively), the reason for this difference remains to be elucidated. It is concluded that [ 3H]GV150526A can be considered a new valuable tool to further investigate the properties of the glycine site of the NMDA receptor.

Synthesis of 7,8-dichloro-6-Nitro-1H-1,5-benzodiazepine-2,4-(3H, 5H)-dione as a potential NMDA receptor glycine site antagonist

An efficient procedure for the preparation of 7,8-dichloro-6-nitro-1H-1,5-benzodiazepine-2,4-(3H, 5H)-dione(7) as a potential lead compound for the NMDA receptor glycine binding site antagonist, starting from readily available 4,5-dichloro-2-nitroaniline(8), is described. The key step in the synthesis involves the cyclization of malonic ester amide 10 to compound 11.

P–439 - Neuroprotective effect of GV150526, an NMDA receptor glycine site antagonist, on acute subdural hematoma induced brain damage in the rat.

P–439 - Neuroprotective effect of GV150526, an NMDA receptor glycine site antagonist, on acute subdural hematoma induced brain damage in the rat.

Complex polyamine effects on [ 3H]MDL 105,519 binding to the NMDA receptor glycine site

Several studies have suggested that polyamines modulate the interaction of glycine with the NMDA receptor. We have further investigated the effects of polyamines using the NMDA receptor glycine site antagonist [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carbox ylic acid] ([3H]MDL 105,519). [3H]MDL 105,519 binding assays were performed using well washed membranes prepared from frozen rat brains. The polyamines spermine and spermidine increased the fraction of non-specific binding (determined by the addition of 1 mM glycine) in the [3H]MDL 105,519 binding assay from 40-60% when spermine or spermidine concentration was increased from 1 to 100 microM. Polyamine agonists spermine, spermidine and 1,5-(diethylamino)piperidine (30 microM) did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. Similarly, the polyamine antagonist arcaine did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. However, spermidine significantly depressed the potency of MDL 105,519 in displacing [3H]dizocilpine binding. These data suggest that [3H]MDL 105,519 may preferentially bind to a polyamine insensitive form of the NMDA receptor.

NMDA receptor glycine site antagonist (indole-2-carboxylic acid) enhances the anticonvulsive activity of antiepileptic drugs

Indole-2-carboxylic acid, an antagonist of the glycine site within NMDA receptor complex, significantly raised the threshold for electroconvulsions in mice, when given at doses 150-200 mg/kg. At a subprotective dose (100 mg/kg) it enhanced the protective activity of carbamazepine, phenobarbital and valproate, diphenylhydantoin being an exception. The activity of carbamazepine and valproate was also potentiated by indole-2-carboxylic acid administered at 50 mg/kg. moreover at the dose of 25 mg/kg it sill significantly lowered the ED50 of valproate. However, the combined treatment of this glycine site antagonist with all antiepileptic drugs studied impaired the motor performance of mice in the rotorod test. Indole-2-carboxylic acid alone at the dose range from 150 to 200 mg/kg induced motor impairment in mice. When co-administered with valproate, it significantly worsened passive avoidance behaviour. The plasma levels of antiepileptic drugs remained unchanged in the presence of indole-2-carboxylic acid. It may be concluded that although indole-2-carboxylic acid enhanced the protective activity of certain antiepileptics, its therapeutic use seems questionable due to pronounced adverse effects, as revealed by the behavioural tests.

NMDA receptor glycine site antagonist (indole‐2‐carboxylic acid) enhances the anticonvulsive activity of antiepileptic drugs

Indole-2-carboxylic acid, an antagonist of the glycine site within NMDA receptor complex, significantly raised the threshold for electroconvulsions in mice, when given at doses 150-200 mg/kg. At a subprotective dose (100 mg/kg) it enhanced the protective activity of carbamazepine, phenobarbital and valproate, diphenylhydantoin being an exception. The activity of carbamazepine and valproate was also potentiated by indole-2-carboxylic acid administered at 50 mg/kg. moreover at the dose of 25 mg/kg it sill significantly lowered the ED50 of valproate. However, the combined treatment of this glycine site antagonist with all antiepileptic drugs studied impaired the motor performance of mice in the rotorod test. Indole-2-carboxylic acid alone at the dose range from 150 to 200 mg/kg induced motor impairment in mice. When co-administered with valproate, it significantly worsened passive avoidance behaviour. The plasma levels of antiepileptic drugs remained unchanged in the presence of indole-2-carboxylic acid. It may be concluded that although indole-2-carboxylic acid enhanced the protective activity of certain antiepileptics, its therapeutic use seems questionable due to pronounced adverse effects, as revealed by the behavioural tests.

4-Substituted-kynurenic acid derivatives: A novel class of NMDA receptor glycine site antagonists

A series of 4-substituted-kynurenic acid derivatives possessing several different substituents at C4-position which are consisted of both a flexible propyloxy chain and an adjunct several type of carbonyl groups has been synthesized and evaluated for theirin vitro antagonist activity at the glycine site on the NMDA receptor. Of them, N-benzoylthiourea15c and N-phenylthiourea15a were found to have the bestin vitro binding affinity with IC(50) of 3.95 and 6.04 muM, respectively. On the other hand, in compounds12a approximately c and13 the displacement of a thiourea group to an amide or a carbamate caused a significant decrease of thein vitro binding affinity. In the SAR study of the 4-substituted kynurenic acid derivatives, it was realized that the terminal substitution pattern on a flexible C4-propyloxy chain of kynurenic acid nucleus significantly influences on the binding affinity for glycine site; the binding affinity to the NMDA receptor might be increased by the introduction of a suitable electron rich substituent at C4 of kynurenic acid nucleus.

Pharmacology of ACEA-1416: a potent systemically active NMDA receptor glycine site antagonist

Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1,4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites ( K b = 7.9 nM in oocytes, K b = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower ( K b = 3.5 μM in oocytes, K b = 1.6 μM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites ( K b > 5.9 μM) and metabotropic glutamate receptors ( K b > 57 μM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at ∼ 2 min and the biological half-time of protection was ∼ 60 min. The ED 50 measured at peak efficacy was ∼ 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.

Structure-activity relationships of 4-hydroxy-3-nitroquinolin-2(1H)-ones as novel antagonists at the glycine site of N-methyl-D-aspartate receptors.

A series of 4-hydroxy-3-nitroquinolin-2(1H)-ones (HNQs) was synthesized by nitration of the corresponding 2,4-quinolinediols. The HNQs were evaluated as antagonists at the glycine site of NMDA receptors by inhibition of [3H]DCKA binding to rat brain membranes. Selected HNQs were also tested for functional antagonism by electrophysiological assays in Xenopus oocytes expressing either 1a/2C subunits of NMDA receptors or rat brain AMPA receptors. The structure-activity relationships (SAR) of HNQs showed that substitutions in the 5-, 6-, and 7-positions in general increase potency while substitutions in the 8-position cause a sharp reduction in potency. Among the HNQs tested, 5,6,7-trichloro HNQ (8i) was the most potent antagonist with an IC50 of 220 nM in [3H]DCKA binding assay and a Kb of 79 nM from electrophysiological assays. Measured under steady-state conditions HNQ 8i is 240-fold selective for NMDA over AMPA receptors. The SAR of HNQs was compared with those of 1,4-dihydroquinoxaline-2,3-diones (QXs) and 1,2,3,4-tetrahydroquinoline-2,3,4-trione 3-oximes (QTOs). In general, HNQs have similar potencies to QXs with the same benzene ring substitution pattern but are about 10 times less active than the corresponding QTOs. HNQs are more selective for NMDA receptors than the corresponding QXs and QTOs. The similarity of the SAR of HNQs, QXs, and QTOs suggested that these three classes of antagonists might bind to the glycine site in a similar manner. With appropriate substitutions, HNQs represent a new class of potent and highly selective NMDA receptor glycine site antagonists.