Antagonism of AMPA receptors produces anxiolytic-like behavior in rodents: effects of GYKI 52466 and its novel analogues.

RATIONALE: Although emerging number of data supports the role of glutamate receptors and the potential of their antagonists in anxiety disorders, the involvement of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in anxiety is less well characterized. OBJECTIVE: To evaluate the anxiolytic potential of 2,3-benzodiazepine (2,3BDZ) type AMPA receptor antagonists in various models of anxiety. MATERIALS AND METHODS: Whole-cell currents, hippocampal field potentials, elevated plus maze (EPM), meta-chlorophenylpiperazine (mCPP)-induced anxiety model, Vogel test in rats and light-dark test (LD) in mice were used to determine AMPA/kainite receptor properties and anxiolytic-like activity of a series of 2,3BDZ-type compounds. RESULTS: The reference compound GYKI 52466 was proved active in two anxiety models in non-sedative doses: minimal effective dose (MED) was especially low in EPM (0.01 mg/kg) GYKI 53405 and GYKI 53655 showed anxiolytic-like activity in two tests (EPM and mCPP). EGIS-8332 was active in EPM and LD while EGIS-9637 showed anxiolytic-like potency in EPM, mCPP and Vogel model. EGIS-10608 was the most effective compound among 2,3BDZs tested in EPM and Vogel models (MEDs are 0.01 and 2.5 mg/kg, respectively). 2,3BDZs were active in anxiety models at doses lower than those produced sedative effects. NBQX showed anxiolytic-like activity in EPM only (3 mg/kg). CONCLUSIONS: The results show that non-competitive AMPA receptor antagonists can profoundly block anxiety-like behavior in rodents independently from their motor depressant activity. However, the sedative properties at higher doses might limit their therapeutic utility as new anxiolytic drugs.

2,3-benzodiazepine-type AMPA receptor antagonists and their neuroprotective effects.

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1-4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure-activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.

The new 2,3-benzodiazepine derivative EGIS-8332 inhibits AMPA/kainate ion channels and cell death.

We observed in vitro neuroprotective and AMPA/kainate receptor antagonist effects of the new 2,3-benzodiazepine derivative EGIS-8332 (R,S-1-(4-aminophenyl)-7,8-methylenedioxy-4-cyano-4-methyl-3-N-acetyl-5H-3,4-dihydro-2,3-benzodiazepine) using the lactate dehydrogenase (LDH) release assay and patch clamp recordings on primary cultures of rat embryonic telencephalon neurons exposed to AMPA/kainate receptor agonists. EGIS-8332 potently decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate induced LDH release (IC(50)=5.2+/-0.4 and 7.4+/-1.3 microM, respectively) from the cells. Whole-cell patch clamp studies carried out on the ionotropic glutamate receptors N-methyl D-aspartate (NMDA), as well as AMPA (and kainate) in cultured telencephalon neurons verified that EGIS-8332 blocked steady state responses to AMPA and kainate (IC(50)=1.7+/-0.4 and 6.2+/-1.6 microM, respectively), but hardly influenced currents evoked by NMDA. EGIS-8332 also inhibited kainate-evoked response in CHO cells expressing the flop variant of GluR1 receptor and, in cerebellar Purkinje cells at similar efficiency. The stereoselectivity of the inhibitory site is established by the clearly dissimilar inhibitory potency of the enantiomer components of EGIS-8332 differing in the configuration of methyl and cyano substituents on carbon C(4): the R(-) enantiomer was found to be the efficient species. This finding suggests that the inhibitory interaction between the channel protein and drug is promoted by presence of the C(4) methyl group. The inhibition of the AMPA/kainate ion channels by EGIS-8332 is non-competitive, not use dependent, and depends neither on the closed/open state of the channel, nor the membrane potential. These findings suggest an allosteric mechanism for the inhibition. These in vitro observations suggest that the compound might be useful in the treatments of certain acute and chronic neurological syndromes initiated by derangements of ionotropic glutamate receptor function.

Comparison of the AMPA antagonist action of new 2,3-benzodiazepines in vitro and their neuroprotective effects in vivo.

PURPOSE: AMPA receptor-mediated excitotoxicity is thought to be a critical process in diseases accompanied by neuronal cell loss following a hypoxic/anoxic state of the central nervous system. It has been suggested that blockade of AMPA receptors might result in significant protection of neurons against cellular damage. For testing the hypothesis, in vitro efficacy and in vivo neuroprotective action of new 2,3-benzodiazepine (2,3BDZ) AMPA antagonists have been compared. METHODS: 2.3BDZs were tested on kainate-evoked whole-cell currents in cultured neurons as well as on population spikes (PS) in rat hippocampal slices. Data were correlated with those obtained from the spreading depression (SD) experiments in chicken retina. Compounds were also examined in the gerbil bilateral carotid occlusion model (BCO), where percentage decrease of ischemia-related hypermotility (HM), impaired spatial memory (SA), and hypoxia-induced hippocampal CA1 neuronal cell death (CA1) were evaluated. RESULTS: Certain structural modifications of classical 2,3BDZs resulted in increased in vitro activity and improved in vivo efficacy. In particular, the halogen-substituted compounds EGIS-9879 and EGIS-9883 showed the highest neuroprotective efficacy (84% and 47% protection in CA1, 71% and 82% decrease in HM, respectively; 4 x 5 mg/kg i.p.) in BCO. PS and SD were correlated to the decrease of neuronal loss in the CA1 area. Lack of significant correlation was found between PS and CA1 (r = 0.437, p = 0.079) or SD and CA1 (r = 0.380, p = 0.146). CONCLUSIONS: Several new 2.3BDZ AMPA receptor antagonists have been synthesized at EGIS Pharmaceuticals characterized by remarkable in vitro and corresponding in vivo neuroprotective properties.