Treatment of early and late kainic acid-induced status epilepticus with the noncompetitive AMPA receptor antagonist GYKI 52466

Purpose:  Benzodiazepines such as diazepam may fail to effectively treat status epilepticus because benzodiazepine-sensitive GABA_A receptors are progressively internalized with continued seizure activity. Ionotropic glutamate receptors, including AMPA receptors, are externalized, so that AMPA receptor antagonists, which are broad-spectrum anticonvulsants, could be more effective treatments for status epilepticus. We assessed the ability of the noncompetitive AMPA receptor antagonist GYKI 52466 to protect against kainic acid–induced status epilepticus in mice.
Methods:  Groups of animals treated with kainic acid received GYKI 52466 (50 mg/kg followed in 15 min by 50 mg/kg) or diazepam (25 mg/kg followed in 20 min by 12.5 mg/kg) beginning at 5 min of continuous seizure activity or 25 min later. The duration of seizure activity was determined by EEG recording from epidural cortical electrodes.
Results:  Both GYKI 52466 and diazepam rapidly terminated electrographic and behavioral seizures when administered early. However, diazepam-treated animals exhibited more seizure recurrences. With late administration, GYKI 52466 also rapidly terminated seizures and they seldom recurred, whereas diazepam was slow to produce seizure control and recurrences were common. Although both treatments caused sedation, GYKI 52466-treated animals retained neurological responsiveness whereas diazepam-treated animals did not. GYKI 52466 did not affect blood pressure whereas diazepam caused a sustained drop in mean arterial pressure.
Discussion:  Noncompetitive AMPA receptor antagonists represent a promising approach for early treatment of status epilepticus; they may also be effective at later times when there is refractoriness to benzodiazepines.     

Chronic low-dose maternal exposure to methylmercury enhances epileptogenicity in developing rats

Effects of continuous low-dose maternal methylmercury intoxication on the induction and propagation of ictal epileptiform activity induced by 3-aminopyridine, were investigated on the neocortex of 4-weeks-old offspring rats. Epileptogenicity was significantly increased in offspring of mercury-treated animals compared to those of controls, characterized by more frequent occurrence of periodic ictal activity, a facilitated propagation of epileptiform discharges and a strong tendency to generalization. The latency of first ictal event was slightly shorter and the average duration of individual ictal periods slightly longer in treated animals. However, the amplitude of seizure discharges was significantly smaller in treated animals than in controls. We conclude, that the synaptic and membrane mechanisms responsible for initiation and propagation of paroxysmal activity were probably facilitated, while the efficacy of cortical inhibition, in preventing initiation and spread of epileptiform discharges was reduced by mercury treatment in the developing nervous system. The smaller amplitude of paroxysmal discharges could be a sign of a remarkable loss of cortical neurons.     

Effect of a glutamate receptor antagonist (GYKI 52466) on 4-aminopyridine-induced seizure activity developed in rat cortical slices

In the present experiments we have tested the effect of the noncompetitive AMPA antagonist GYKI 52466 (20-80 microM) on spontaneous epileptic discharges developed as the consequence of 4-aminopyridine application in neocortex slices of adult rats. Parallel to the changes of spontaneous activity, the field potentials, evoked by electrical stimulation of the corpus callosum, were also analyzed. Glass microcapillary extracellular recording electrode was positioned in the third layer of the somatosensory cortex slice, while the stimulating electrode was placed at the border of the white and gray matter. 4-aminopyridine and GYKI 52466 were bath-applied. The application of 40 microM GYKI 52466 caused about 40% decrease in the frequency and the amplitude of spontaneous seizures as well as the duration of each discharges developed in 4-amino-pyridine. Pre-incubation with the AMPA antagonist effectively inhibited both the development of seizure activity and the maintenance of the discharges. GYKI 52466 also decreased the duration and amplitude of field responses evoked by stimulation of the corpus callosum. This inhibitory effect was dose-dependent. Our data in the in vitro cortex slice epilepsy model suggest that the non-competitive AMPA antagonist GYKI 52466 is a potent anticonvulsant and neuroprotective compound because it reduced the fully developed epileptic discharges or prevented their development.     

Behavioural and neurochemical interactions of the AMPA antagonist GYKI 52466 and the non-competitive NMDA antagonist dizocilpine in rats

Summary The behavioural and neurochemical effects of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine and the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) antagonist GYKI 52466, given alone or in combination, were investigated in rats. Locomotor activity was increased by dizocilpine (0.2 mg/kg), but not by GYKI 52466 (2.4 mg/kg). Dizocilpine-induced hyperlocomotion was reduced by co-administration of GYKI 52466. In dizocilpine-treated rats dopamine (DA) metabolism (measured as DOPAC [dihydroxyphenylacetic acid] or DOPAC/DA in post mortem brain tissue) was increased in the prefrontal cortex and nucleus accumbens. In GYKI 52466-treated rats serotonin was reduced in the prefrontal cortex and nucleus accumbens while DA metabolism was not affected. In rats treated with dizocilpine plus GYKI 52466, DA metabolism was increased only in the prefrontal cortex, but not in the nucleus accumbens, when compared with vehicle-treated animals. These data confirm that AMPA and NMDA antagonists do not have synergistic effects on locomotor activity. A differential role of NMDA and AMPA antagonists in the control of mesolimbic DA neurons will be discussed here.     

AMPA receptor antagonists, GYKI 52466 and NBQX, do not block the induction of long-term potentiation at therapeutically relevant concentrations

The involvement of alpha-amino-3-hydroxy-5-methylizoxazole-4-propionic acid (AMPA) receptors in induction of long-term potentiation (LTP) was examined in rat hippocampal slice preparation. Using conventional extracellular recording, excitatory postsynaptic potentials (EPSPs) and population action potentials (PSs), evoked by low-frequency stimulation of the Schaffer collateral-commissural fibres, were recorded in the CA1 region. The effects of a competitive AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2, 3-dione (NBQX), and that of a non-competitive blocker, 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) have been examined. 0.25-0.5 microM of NBQX and 20-40 microM of GYKI 52466 did not suppress the induction of LTP. LTP was attenuated only at the highest concentrations tested (1 microM NBQX or 80 microM GYKI 52466). These in vitro concentrations, however, exceed the brain levels needed for in vivo anticonvulsant action. Furthermore, even at the highest concentrations both compounds suppressed only the expression but not the induction of LTP. Namely after their washout LTP reappeared. Thus, at pharmacologically relevant concentrations these AMPA receptor antagonists apparently do not suppress LTP, a cellular mechanism underlying memory formation. These experiments suggest that in clinical practice AMPA receptor blockade may have some advantage over N-methyl-D-aspartate receptor antagonism, which is accompanied by severe memory impairment.     

In vivo, the direct and seizure-induced neuronal cytotoxicity of kainate and AMPA is modified by the non-competitive antagonist, GYKI 52466

The 2,3-benzodiazepine GYKI 52466, administered intracerebrally or systemically, was assessed for its ability to protect against the neuronal death in the brain caused by intra-hippocampal injections of the non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). In contrast to a previous report, a low intra-hippocampal dose of GYKI 52466 (25 nmol) did not protect against kainate toxicity. In order to achieve higher doses of GYKI 52466, solubilization in 2-hydroxypropyl-beta-cyclodextrin was used, and limited protection against AMPA, but not kainate toxicity was found. There was a commensurate reduction in seizure-related neuronal loss in the limbic regions of the brain. When diazepam was used to prevent seizures, GYKI 52466 had no effect on hippocampal neuronal loss caused by the direct toxicity of AMPA and kainate on hippocampal neurons. Systemic administration of GYKI 52466 had only a minimal effect on preventing neuronal death caused by AMPA. In vivo, GYKI 52466 is only weakly effective as a neuroprotective agent.     

Interactions of allosteric modulators of AMPA/kainate receptors on spreading depression in the chicken retina

The functional role of AMPA and kainate receptors in spreading depression (SD) was investigated in the isolated chicken retina. Competitive (NBQX) and non-competitive (GYKI 52466, GYKI 53405 and GYKI 53655) antagonists of the AMPA receptor inhibited AMPA-induced SD in a concentration-dependent manner. Concentrations of drugs caused 50% inhibition (IC(50) values) are 0.2, 16.6, 7.0 and 1.4 microM, respectively. AMPA receptor positive modulator cyclothiazide was more effective in the potentiation of SD evoked by AMPA than by kainate. Slight potentiation of either AMPA- or kainate-induced SD was observed only at high concentration (1 mg/ml) by the kainate receptor modulator concanavalin A. Compounds that positively modulate AMPA receptor function (cyclothiazide, IDRA-21, S 18986, 1-BCP and aniracetam) caused a concentration-dependent potentiation in SD. Concentrations of drugs that caused 50% potentiation (estimated EC(50) values) are 9, 135, 142, 450 and 1383 microM, respectively. Interaction between cyclothiazide, aniracetam or S 18986 administered with each other, or with GYKI 52466, respectively, was also investigated. When cyclothiazide and S 18986 were co-applied, their effects seemed to be additive. However, lack of additivity was obtained when S 18986 was added together with aniracetam. Positive modulators applied at equiactive concentrations reduced the inhibitory action of GYKI 52466 and differently shifted its concentration-response curve. In this respect, S 18986 was the most effective (IC(50) of GYKI 52466 changed from 16.6 to 51.9 microM). Our findings indicate the contribution of AMPA rather than kainate receptors in the mediation of retinal spreading depression. Our data further support the idea that multiple positive modulatory sites are present on the AMPA receptor complex in addition to a negative modulatory site.     

GYKI 52466 has positive modulatory effects on AMPA receptors

The 2,3-benzodiazepine derivative GYKI 52466 has been well characterized as a negative modulator of AMPA-type glutamate receptors. The present study re-examined the effects of GYKI 52466 on AMPA receptor-mediated currents in patches excised from pyramidal neurons in the hippocampal CA1 field and found that this drug has positive modulatory effects in addition to its receptor blocking action. A low concentration of GYKI 52466 (10 microM) reliably increased the steady-state current by about three-fold, while the peak current was reduced by 30% only. Higher drug concentrations produced parallel reductions in both the steady-state and peak currents. The increase in the steady-state current was not accompanied by a change in the deactivation time constant and thus, is more likely to result from a change in desensitization than a slowing of channel closing. The results indicate that GYKI 52466 modulates AMPA receptor-mediated currents in a complex manner, perhaps by acting through more than one binding site.     

Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening: the Role of 4-Methyl versus 4-Carbonyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives

2,3-Benzodiazepine derivatives are synthesized as drug candidates for a potential treatment of various neurodegenerative diseases involving the excessive activity of AMPA receptors. Here, we describe a rapid kinetic investigation of the mechanism of inhibition of the GluA2Q(flip) AMPA receptor channel opening by two 2,3-benzodiazepine derivatives, i.e. the prototypic 2,3-benzodiazepine compound GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine)] and 1-(4-aminophenyl)-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (BDZ-2). GYKI 52466 and BDZ-2 are structurally similar in that the 4-methyl group in the diazepine ring of GYKI 52466 is replaced by a carbonyl group, yielding BDZ-2. Using a laser-pulse photolysis technique with ～60 μs time resolution, we characterize the effect of the two compounds individually on the channel-opening process of the GluA2Q(flip) receptor expressed in HEK-293 cells. We find that BDZ-2 preferentially inhibits the open-channel state, whereas GYKI 52466 is more selective for the closed-channel state of the GluA2Q(flip) receptors. Each inhibitor binds independently to its own noncompetitive site, yet the two sites do not interact allosterically. The significance of these results in the context of both the structure-activity relationship and the properties of the GluA2Q(flip) receptor channels is presented.     

Influence of aminophylline and strychnine on the protective activity of excitatory amino acid antagonists against maximal electroshock-induced convulsions in mice

Summary Aminophylline reversed the protective action of both, D-3-(2-carboxypiperazine-4-yl)-1-propenyl-1-phosphonic acid (D-CPP-ene — a competitive NMDA antagonist) and valproate (used as a conventional antiepileptic drug for comparative purposes) against maximal electroshock-induced seizures. The respective ED₅₀ values of aminophylline were 55.7 and 98.4mg/kg i.p. However, aminophylline (up to 100mg/kg i.p.) did not influence the protective efficacy of 1-(4-aminophenyl)-4-methyl-7,8-methyl-enedioxy-5H-2, 3-benzodiazepine (GYKI 52466 — a non-NMDA antagonist). Strychnine affected the protection provided by D-CPP-ene, GYKI 52466, and valproate against maximal electroshock — the ED₅₀ values of strychnine for the reversal of the anticonvulsive effects of D-CPP-ene, GYKI 52466 or valproate were 0.082, 0.35 and 0.28mg/kg s.c., respectively.An involvement of strychnine sensitive glycinergic receptor-mediated events in the mechanism of the anticonvulsive activity of excitatory amino acid antagonists and valproate may be postulated. The ineffectiveness of aminophylline to reduce the anticonvulsive effects of GYKI 52466 may distinguish a new class of antiepileptic drugs offering an advantage over conventional antiepileptics in patients with epilepsy, requiring aminophylline for pulmonary reasons.