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Racemic (dl)-methadone has antagonist activity at the N-methyl-D-aspartate (NMDA) receptor. We evaluated dl-methadone, the opioid active (l-) and the opioid inactive (d-) isomers in competition binding assays. dl-Methadone and its d- and l- isomers exhibited low micromolar affinities for the [3H]MK-801-labeled non-competitive site of the NMDA receptor in both rat forebrain and spinal cord synaptic membranes, with Ki values and displacement curves similar to those of dextromethorphan, an established NMDA receptor antagonist. They lacked affinity at the [3H]CGS-19755-labeled competitive site of the NMDA receptor. Therefore, both methadone and its the d- and l- isomers differ from morphine, hydromorphone, and naltrexone in that they have non-competitive antagonist activity at the NMDA receptor. A non-opioid NMDA receptor antagonist, such as d-methadone, may improve the efficacy of morphine by attenuating the development of tolerance.

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.

The interaction of structurally constrained competitive NMDA receptor antagonists, (+/-)-cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), (2-amino-4,5-(1,2-cyclohexyl))-7-phosphonoheptanoic acid (NPC 12626), (+/-)-6-phosphonomethyl-de-cahydroisoquinoline-3-carboxylic acid (LY 274614), (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (SDZ EAB-515) and (S)-alpha-amino-5-phosphonomethyl[1,1':4',1"-terphenyl]-3-propa noi c acid (SDZ 215-439), with their receptor was assessed using radioligand binding, protection against neurotoxicity in cortical neuronal cultures and computerised molecular modelling. All compounds inhibited the specific binding of [3H]CGS 19755 and/or [3H]CGP 39653 (inhibition constants 40-2000 nM), and protected neuronal cultures from NMDA-mediated injury (IC50 values 1.3-5.6 microM). Quantitative conformational analyses indicated that the molecules fitted well to a NMDA receptor model. Our results draw attention to a deep hydrophobic pocket, defined by the bi- and terphenyl containing antagonists (SDZ EAB-515, SDZ 215-439), which may influence potency and selectivity.

A series of 3- and 4-(sulfo- and sulfonamidoalkyl)pyridine and piperidine-2-carboxylic acid derivatives as analogs of NMDA receptor antagonists was prepared. Affinity for the NMDA receptor was determined by binding assays using the specific radioligand [3H] (2SR,4RS)-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS-19755). The 3-alkylsulfonyl moiety was introduced by selective reduction of a carboxylic acid function followed by bromination, substitution by Na2SO3 and catalytic reduction. For the 4-alkylsulfonic derivatives the crucial step was the introduction of the 2-cyano function and its further conversion to 2-carboxylic acid. The most potent compound of the series was the pyridine (11a) [4-(sulfomethyl)pyridine-2-carboxylic acid] with a modest IC50 of 40 microM. A molecular modeling study has been undertaken to understand the pharmacological results. In a first step, a comparative modeling study of the active pyridine and the poorly active piperidine sulfonic acid derivatives 11a and 10a [4-(sulfomethyl)piperidine-2-carboxylic acid] and of the phosphonic homologues was performed. We propose that the binding geometry of the sulfonic moiety within the NMDA receptor is different from that of the phosphonic containing antagonists. In order to test this assumption, we have made, in a second step, a complete conformational analysis of the sulfonic acid derivatives, as well as some analogs taken from the literature, either active or inactive as NMDA antagonists. A preferred conformation of the sulfonic acids is proposed.

Several studies have indicated important functional interactions between volatile anesthetics and the N-methyl-D-aspartate (NMDA) class of glutamate receptors. In the present study, we examined the effects of diethyl ether, chloroform, methoxyflurane, halothane, enflurane, and isoflurane on (1) glutamate activation of the NMDA receptor complex, including glycine reversal of anesthetic action, as revealed by [3H]-(5R, 10S)-(+)methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, dizocilpine (MK-801) binding to the cation channel, and (2) [3H]cis-4-( phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755) binding to the glutamate recognition site of the NMDA receptor In agreement with previous studies, glutamate increased the binding of 1 nM [3H]MK-801, measured after a 1-hr incubation at 37 degrees, by up to several hundred fold. This stimulation was blocked by glutamate antagonists and potentiated by glycine with an EC50 of approximately 0.03 muM. Glycine also had a direct stimulatory effect on [3H]MK-801 binding at much higher concentrations ( > or = 10 muM). All of the anesthetics examined depressed glutamate stimulation of [3H]MK-801 binding in a concentration-dependent manner with the following order of potency: halothane > or = enflurane > methoxyflurane > chloroform > diethyl ether. This inhibition of [3H]MK-801 binding was observed at concentrations that are routinely attained in the cerebrospinal fluid during surgical anesthesia. Moreover, the inhibition was reversed rapidly following removal of the anesthetics from the assay medium. Inclusion of glycine in the incubation medium markedly attenuated anesthetic-induced inhibition of glutamate-sensitive [3H]MK-801 binding with an EC50 of between 0.1 and 1 muM. Thus, this reversal by glycine correlated with its potentiating as opposed to its direct stimulatory, effect on NMDA receptors. Anesthetic inhibition of [3H]MK-801 binding could not be overcome by raising the glutamate concentration (i.e. the interaction did not appear to be competitive with respect to glutamate) unless glycine was present. Binding of [3H]CGS 19755 to the glutamate recognition site was also inhibited by each of the anesthetics examined. However, with the exception of chloroform, all of the anesthetics were more potent inhibitors of glutamate-stimulated [3H]MK-801 binding than they were of [3H]CGS 19755 binding. [3H]CGS 19755 binding saturation curves in the presence of halothane and enflurane indicated a decrease in the density of [3H]-CGS 19755 binding sites with no change in binding affinity (i.e. the inhibition did not appear to be competitive). These findings support the idea that anesthetic drugs disrupt NMDA receptor transmission through multiple allosteric effects on the receptor-channel activation mechanisms and the glutamate binding site.

Binding of [3H]glutamate, [3H]glycine, and the glutamate antagonist [3H]CGS-19755 to NMDA-type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [3H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [3H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [3H]glycine bound to a second site with a 10-fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [3H]-Glutamate binding was not affected by glycine agonists or antagonists, nor was [3H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS-19755 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, glutamate antagonists, reduced [3H]-glycine binding in cerebellum, whereas only CGS-19755 was effective in forebrain. Glycine agonists and antagonists modulated [3H]CGS-19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.

[3H]Cis-4-phosphonomethyl-2-piperidine carboxylic acid ([3H]CGS 19755) was used to investigate the pharmacology and characteristics of the N-methyl-D-aspartate (NMDA) receptor recognition site from Triton X-100-treated membranes of rat spinal cord and cerebral cortex. The association of [3H]CGS 19755 was biphasic in both spinal cord and cerebral cortical membranes reaching a maximum after 5 min of incubation then decreasing to a steady level after an additional 10 min, suggesting that a proportion of the binding is unstable. The dissociation of the stable binding component was biphasic with rate constants at 4 degrees C of 1.55 and 0.020 min-1 for the spinal cord and 1.48 and 0.051 min-1 for the cerebral cortex. These multiple sites could not be captured in the saturation studies which were best fitted to a one-site model using non-linear regression analysis. Depending on the time of incubation with [3H]CGS 19755, KD and Bmax values differed; 9.9-26.1 nM and 25-96 fmol/mg protein vs 14.0-26.5 nM and 449-900 fmol/mg protein for spinal cord and cerebral cortex, respectively. The rank order of potency of inhibiting [3H]CGS 19755 binding was similar in both tissues: L-glutamate > CGS 19755 = CPP > NMDA. The specific NMDA agonist cis-2,4-methanoglutamate potently inhibited [3H]CGS 19755 binding as did MDL 100,925, although the latter was one order of magnitude less potent in the spinal cord than in the brain. The Hill coefficients were significantly lower than unity. In both tissues, AMPA, kainate and glycine competed poorly with [3H]CGS 19755.(ABSTRACT TRUNCATED AT 250 WORDS)

The N-methyl-D-aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P2 membranes we have investigated the effect of glutamate recognition site ligands on [3H]glycine (agonist) and (+-)-4-trans-2-carboxy-5,7-dichloro-4- [3H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([3H]L-689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on L-[3H]glutamate (agonist), DL-3-(2-carboxypiperazin-4-yl)-[3H]propyl-1-phosphonate ([3H]CPP; "C-7" antagonist), and cis-4-phosphonomethyl-2-[3H]piperidine carboxylate ([3H]CGS-19755; "C-5" antagonist) binding to the glutamate site. "C-7" glutamate site antagonists partially inhibited [3H]L-689,560 binding but had no effect on [3H]glycine binding, whereas "C-5" antagonists partially inhibited the binding of both radioligands. Glycine, D-serine, and D-cycloserine partially inhibited [3H]CGS-19755 binding but had little effect on L-[3H]glutamate or [3H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (L-687,414), and 1-amino-1-carboxycyclobutane all enhanced [3H]CPP binding but had no effect on [3H]CGS-19755 binding, and (+)-HA-966 and L-687,414 inhibited L-[3H]glutamate binding. The association and dissociation rates of [3H]L-689,560 binding were decreased by CPP and D-2-amino-5-phosphonopentanoic acid ("C-5"). Saturation analysis of [3H]L-689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [3H]L-689,560 is discussed.

Human immunodeficiency virus (HIV-1) infection often results in central nervous system (CNS) dysfunction, yet the mechanism(s) of action for HIV-1 in the CNS are not fully understood. In the present study gp120, the HIV-1 envelope glycoprotein, was shown to selectively inhibit N-methyl-D-aspartate (NMDA) receptor function. In addition to inhibiting radioligand binding to rat NMDA receptors, gp120 inhibited NMDA-induced currents in Xenopus oocytes, attenuated NMDA-stimulated calcium flux and cytotoxicity in cultured cerebellar granule cells, and provided partial protection against NMDA-induced lethality in vivo. These findings suggest that NMDA receptor complex is a possible site of action of HIV-1 within the CNS.

ES-242-1 approximately 5 are novel microbial bioxanthracenes which do not contain nitrogen. The ES-242s inhibited the binding of [3H]TCP and [3H]CGS19755 to the N-methyl-D-aspartate (NMDA) receptor complex. They had no effect on the binding of the specific ligands for the non-NMDA receptor. The biochemical and pharmacological properties of ES-242-1 were fully examined since it is the most potent of the five compounds. ES-242-1 is highly specific for the NMDA receptor; it has no effect on other receptors. Kinetic analyses indicated that ES-242-1 inhibited the binding of [3H]TCP and [3H]CGS19755 in a competitive manner, respectively, suggesting that ES-242-1 interacts with both the transmitter recognition site and the channel domain. ES-242-1 selectively inhibited NMDA-induced Ca2+ influx in primary cultures of mouse hippocampal neurons. ES-242-1 also specifically blocked the increase in cyclic GMP accumulation induced by NMDA or L-glutamate in rat cerebellar slices. In a concentration range of 0.1-1.0 microM, ES-242-1 was as potent as MK-801 in preventing glutamate-induced neurotoxicity in primary cultures of mouse hippocampal neurons. These results show that ES-242-1 is a potent and specific antagonist for the NMDA receptor. The antagonistic properties of the ES-242s appear to be due to a novel mechanism of action at the NMDA receptor.

This paper describes the pharmacological activity of D,L-(tetrazol-5-yl)glycine, a structurally novel and highly potent agonist at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor. D,L-(Tetrazol-5-yl)glycine potently displaced NMDA receptor binding to rat brain membranes as measured using [3H]CGS19755 (IC50 = 98 +/- 7 nM) and [3H]glutamate (IC50 = 36 +/- 18 nM) as ligands. D,L-(Tetrazol-5-yl)glycine did not appreciably inhibit the binding of D,L-alpha-[5-methyl-3H] amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, or [3H]glycine (IC50s greater than 30,000 nM). D,L-(Tetrazol-5-yl)glycine was more potent than NMDA or cis-methanoglutamate as a depolarizing agent in the rat cortical slice, and unlike these other agents induced rapid receptor-mediated neurotoxicity. Depolarization by D,L-(tetrazol-5-yl)glycine was antagonized by LY233053, a selective NMDA receptor antagonist. D,L-(Tetrazol-5-yl)glycine was a highly potent convulsant when given to neonatal rats (ED50 = 0.071 mg/kg i.p.). Convulsions in neonatal rats or lethality in mice induced by D,L-(tetrazol-5-yl)glycine were selectively antagonized by competitive and non-competitive NMDA receptor antagonists. D,L-(Tetrazol-5-yl)glycine is a structurally novel (tetrazole-substituted) compound that is a highly potent and selective NMDA receptor agonist. D,L-(Tetrazol-5-yl)glycine could be used to probe further NMDA receptor function in vitro and in vivo.

We have prepared a series of cis-4-(tetrazolylakyl)piperidine-2-carboxylic acids as potent and selective N-methyl-D-aspartic acid (NMDA) receptor antagonists. NMDA antagonists may prove to be useful therapeutic agents, for instance, as anticonvulsants, in the treatment of neurodegenerative disorders such as Alzheimer's disease and in the prevention of neuronal damage that occurs during cerebral ischemia. The compounds prepared were evaluated in vitro in both receptor binding assays [( 3H]CGS-19755, [3H]AMPA, and [3H]kainic acid) and in a cortical-wedge preparation (versus NMDA, quisqualic acid, and kainic acid) to determine affinity, potency, and selectivity. The new amino acids were also evaluated in vivo for their ability to block NMDA-induced convulsions in neonatal rats and NMDA-induced lethality in mice. The most potent compound of this series, 15 (LY233053), selectively displaced [3H]CGS-19755 binding with an IC50 of 107 +/- 7 nM and selectively antagonized responses due to NMDA in a cortical-wedge preparation with an IC50 of 4.2 +/- 0.4 microM. Compound 15 blocked both NMDA-induced convulsions in neonatal rats (minimum effective dose (MED) = 20 mg/kg ip) and NMDA-induced lethality in mice (MED = 5 mg/kg ip). This is the first example of an NMDA receptor antagonist that incorporates a tetrazole moiety as an omega-acid bioisostere. These amino acid antagonists are also unique from their phosphonic acid counterparts in that they have a shorter duration of action in vivo. For the treatment of acute disorders such as stroke, where an NMDA antagonist would be administered parenterally, the shorter duration of action may be beneficial, e.g., allowing for better dosage control. The combination of potent NMDA receptor antagonism and a short duration of action may make these compounds useful therapeutic agents in the treatment of a variety of neurological disorders.