E.H.F. Wong

Non-competitive antagonists of excitatory amino acid receptors

Abstract Non-competitive antagonists have become important tools for investigating the basic mechanisms of NMDA receptor function. Such compounds (e.g. MK-801, PCP) are thought to act at the level of the NMDA receptor-associated ion channel and many show a marked use-dependence in their antagonist properties. Little information is available concerning selective non-competitive antagonists of quisqualate or kainate receptors. A number of structurally diverse compounds act as non-competitive antagonists of glutamate receptors at invertebrate neuromuscular junctions, although it remains to be determined whether these compounds have effects at excitatory amino acid receptors in the mammalian CNS. The ability of the non-competitive NMDA receptor antagonists to penetrate the brain following systemic administration, and the use-dependency of their antagonism may confer distinct therapeutic advantages in the treatment of neurological disorders where an overstimulation of NMDA receptors is thought to occur (such as in epilepsy or cerebral ischaemia).

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

Abstract: The potent noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist [3H]MK‐801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]‐MK‐801 was considerably higher in 5 mM Tris‐HCl (pH 7.4) than in previous studies using Krebs‐Henseleit buffer. [3H]MK‐801 labels a homogenous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus > cortex > olfactory bulb = striatum > medulla‐pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]HIMK‐801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N‐allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high‐affinity sites labelled by the σ receptor ligand (+)‐[3H]SKF 10,047. [3H]MK‐801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK‐801 labels a high‐affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK‐801 and other noncompetitive NMDA receptor antagonists.

The novel anticonvulsant MK‐801 binds to the activated state of the N‐methyl‐d‐aspartate receptor in rat brain

1 The influence of endogenous and exogenous acidic amino acids on the binding of [3H]‐MK‐801, a selective, non‐competitive antagonist of N‐methyl‐d‐aspartate (NMDA) receptors, has been investigated in rat cerebral cortex crude synaptic membranes (CSM). 2 Removal of endogenous glutamate and aspartate from CSM by repeated washing reduced the affinity of [3H]‐MK‐801 for its binding site (with no change in the total number of binding sites) and increased NMDA‐sensitive l‐[3H]‐glutamate binding. 3 In washed CSM, competitive NMDA antagonists of the dl‐α‐amino‐ω‐phosphonocarboxylate series reduced [3H]‐MK‐801 binding and NMDA‐sensitive L‐[3H]‐glutamate binding, the most active compounds being 2‐amino‐5‐phosphonovalerate (AP5) and 2‐amino‐7‐phosphono‐heptanoate (AP7). 4 Exogenous excitatory amino acid agonists enhanced the binding of [3H]‐MK‐801 to washed CSM by up to 700%. A selective involvement of NMDA receptors in these effects was indicated by the excellent correlation between EC50s for stimulation of [3H]‐MK‐801 binding and IC50s for inhibition of NMDA‐sensitive l‐[3H]‐glutamate binding in the same membranes. 5 The selective, competitive NMDA receptor antagonist D‐AP5 blocked the l‐glutamate‐induced increase in [3H]‐MK‐801 binding in a competitive manner with a pA2 value of 6.0. 6 These results seem to reflect a molecular interaction between two distinct components of the NMDA receptor complex: the transmitter recognition site and the site through which MK‐801 exerts its antagonist effects, possibly the ion channel.

The discovery of rofecoxib, [MK 966, VIOXX®, 4-(4′-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

DFU (5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone) was identified as a novel orally active and highly selective cyclo‐oxygenase‐2 (COX‐2) inhibitor. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid‐dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX‐2 (IC50=41±14 nM) over COX‐1 (IC50>50 μM). Indomethacin was a potent inhibitor of both COX‐1 (IC50=18±3 nM) and COX‐2 (IC50=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX‐1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore‐challenged human platelets (IC50>50 μM and 4.1±1.7 nM, respectively). DFU caused a time‐dependent inhibition of purified recombinant human COX‐2 with a Ki value of 140±68 μM for the initial reversible binding to enzyme and a k2 value of 0.11±0.06 s−1 for the first order rate constant for formation of a tightly bound enzyme‐inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s−1, respectively, were obtained for indomethacin. The enzyme‐inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time‐dependent inhibition by DFU was decreased by co‐incubation with arachidonic acid under non‐turnover conditions, consistent with reversible competitive inhibition at the COX active site. Inhibition of purified recombinant human COX‐1 by DFU was very weak and observed only at low concentrations of substrate (IC50=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX‐2, inhibition was time‐independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX‐1. DFU inhibited lipopolysaccharide (LPS)‐induced PGE2 production (COX‐2) in a human whole blood assay with a potency (IC50=0.28±0.04 μM) similar to indomethacin (IC50=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC50>97 μM) than indomethacin at inhibiting coagulation‐induced TXB2 production (COX‐1) (IC50=0.19±0.02 μM). In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX‐1 with an IC50 value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC‐58125 were about 10 times more potent inhibitors of COX‐1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS‐398∼SC‐57666>SC‐58125>CGP 28238∼etodolac>L‐745,337>DFU. DFU inhibited dose‐dependently both the carrageenan‐induced rat paw oedema (ED50 of 1.1 mg kg−1 vs 2.0 mg kg−1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg−1 vs 1.5 mg kg−1 for indomethacin). The compound was also effective at reversing LPS‐induced pyrexia in rats (ED50=0.76 mg kg−1 vs 1.1 mg kg−1 for indomethacin). In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg−1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg−1), meloxicam (3 mg kg−1) or etodolac (10–30 mg kg−1). A 5 day administration of DFU in squirrel monkeys (100 mg kg−1) did not affect chromium leakage in contrast to diclofenac (1 mg kg−1) or naproxen (5 mg kg−1). The results indicate that COX‐1 inhibitory effects can be detected for all selective COX‐2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX‐1, a consistent high selectivity of inhibition of COX‐2 over COX‐1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX‐1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti‐inflammatory effect to gastropathy can be achieved with a selective COX‐2 inhibitor.

Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.

1 An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK‐801, in rat and gerbil brain sections. 2 Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nm [3H]‐MK‐801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 μm unlabelled MK‐801 amounted to 55–60% of total. 3 Phencyclidine, (±)‐SKF 10047, ketamine and 2‐aminophosphonovaleric acid (APV) (all 100 μm) prevented the specfic binding of [3H]‐MK‐801. l‐Glutamate and N‐methyl d‐aspartate (NMDA) (100 μm) had no effect. However, l‐glutamate prevented the inhibition by APV. 4 The highest concentrations of [3H]‐MK‐801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5 The distribution of [3H]‐MK‐801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]‐TCP) but not with high‐affinity σ sites labelled with [3H]‐3‐PPP. 6 The density of [3H]‐MK‐801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7 These results indicate that binding sites for [3H]‐MK‐801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.

The interaction between MK-801 and receptors for N-methyl-D-aspartate: functional consequences.

Electrophysiological studies using a cortical slice preparation revealed that MK-801 is a potent, non-competitive, antagonist of N-methyl-D-aspartate (NMDA)-induced depolarisations. Also, MK-801 was a highly selective antagonist, exhibiting a high degree of use-dependency. It completely blocked responses to NMDA and quinolinic acid but had no effect on responses produced by kainic acid or quisqualic acid. Using [3H]MK-801, high affinity binding sites for MK-801 were detected in membranes of the rat brain. The pharmacological specificity and regional distribution of binding sites for MK-801 were consistent with an action of the compound at the level of the NMDA receptor-associated ion channel. Administered parenterally, MK-801 had a remarkable neuroprotective role. It caused essentially complete protection against loss of neurones produced by injection of neurotoxic doses of NMDA or quinolinic acid into the striatum or hippocampus. Furthermore, MK-801 was highly effective in preventing loss of hippocampal neurones following bilateral occlusion of the common carotid arteries in the gerbil.

A role for receptors of N-methyl-D-aspartic acid in the discriminative stimulus properties of phencyclidine

Ketamine and (+)-N-allylnormetazocine ((+)-NANM) were found to generalize in a rat operant drug discrimination paradigm to the interoceptive stimulus induced by phencyclidine (PCP). Intraperitoneal administration of the non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, MK-801, and intracerebroventricular injection of the competitive antagonist, 2-DL-amino-7-phosphonoheptanoic acid (2-APH), also resulted in a dose-dependent generalisation to the PCP discriminative stimulus. The results suggest that NMDA receptor antagonism may play an important role in the mediation of the discriminative stimulus properties of PCP. The low potency of MK-801 and 2-APH to displace [3H](+)-NANM binding in vitro argues against an involvement of the haloperidol-sensitive sigma recognition site in the behaviour.

Solubilization of the N-Methyl-d-Aspartate Receptor Channel Complex from Rat and Porcine Brain

Abstract: The N‐methyl‐D‐aspartate (NMDA) receptor complex as defined by the binding of [3H]MK‐801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK‐801, could be stored for up to 1 week at 4°C with <25% loss in activity. Receptor preparations from both species exhibited [3H]MK‐801 binding properties in solution very similar to those observed in membranes (Bmax= 485 ± 67 fmol/mg of protein, KD= 11.5 ± 2.9 nM in rat; Bmax= 728 ± 108 fmol/mg of protein, KD= 7.1 ± 1.6 nM in pig, n = 3). The pharmacological profile of the solubilized [3H]MK‐801 binding site was virtually identical to that observed in membranes. The rank order of potency of: MK‐801 > (‐)‐MK‐801 = thienylcyclohexylpiperidine > dexoxadrol > SKF 10,047 > ketamine, for inhibition of [3H]MK‐801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes. After removal of small‐molecular‐weight endogenous substances by gel filtration, the following effects were observed: (1) NMDA receptor agonists such as L‐glutamate enhanced binding of [3H]MK‐801 in a concentration‐dependent manner; (2) glycine stimulated [3H]MK‐801 binding in the presence of submaximal concentrations of glutamate; (3) in the absence of glutamate and glycine, the divalent cations Mg2+, Ca2+, and Mn2+ stimulated [3H]MK‐801 binding (EC50: 10–100 μM), whereas in the presence of glutamate and glycine all divalent cations tested were inhibitory with a rank order of potency of Zn2+ > Cd2+ Mn2+ > Mg2+ > Ca2+. In summary, the soluble NMDA receptor, labelled at the cation channel site with [3H]MK‐801, maintains the ability to bind NMDA agonists, glycine, and divalent cations in a manner akin to that observed in membranes.

[3H]MK-801 binding to N-methyl-D-aspartate receptors solubilized from rat brain: effects of glycine site ligands, polyamines, ifenprodil, and desipramine.

Abstract: The N‐methyl‐d‐aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK‐801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small‐molecular‐weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in μM): glycinc, 0.31; d‐serine, 0.20; d‐cycloserine, 1.46; (+)‐HA‐966, 4.06; and 7‐chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK‐801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 μM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK‐801 binding approached equilibrium, their ability to enhance [3H]MK‐801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK‐801 binding under both equilibrium and nonequilibrium conditions, although the high‐affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specifc [3H]MK‐801 binding under nonequilibrium conditions with an IC50 of 4 μM, and this value was unaltered when [3H] MK‐801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines, and desipramine are integral components of the NMDA receptor protein.