Synthesis and anticonvulsant activity of novel and potent 2,3-benzodiazepine AMPA/kainate receptor antagonists.

We have previously shown that 1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) in analogy to the structurally related 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (4a-h) and 1-aryl-3, 5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-thiones (5a-c). The activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and compared to that of 1 and 3b,c. Compounds 4a,b and 5a-c antagonize seizures induced by AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared with that of the parent compounds 1 and 1-(4-aminophenyl)-3, 4-dihydro-4-methyl-3-methylcarbamoyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine (2, GYKI 53655).     

Novel potent AMPA/kainate receptor antagonists: synthesis and anticonvulsant activity of a series of 2-[(4-alkylsemicarbazono)-(4-amino-phenyl)methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters

In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemicarbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1 (compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylenetetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of AMPA or kainate (KA). Using the patch-clamp technique in primary cultures of granule neurons we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives reduced KA and ATPA currents to a larger extent than that shown by reference compound 1. Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application of KA (100 microM).     

Role of AMPA and GluR5 kainate receptors in the development and expression of amygdala kindling in the mouse

The role of AMPA and GluR5-containing kainate receptors in the development and expression of amygdala kindling was examined using the selective 2,3-benzodiazepine AMPA receptor antagonist GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine] and the decahydroisoquinoline mixed AMPA receptor and GluR5 kainate receptor antagonist LY293558 {(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline- 3-carboxy lic acid)}. Administration of GYKI 52466 (5-40 mg/kg, intraperitoneally) and LY293558 (10-40 mg/kg, intraperitoneally) prior to daily kindling stimulation in mice produced a dose-dependent suppression of the rate of development of behavioral kindled seizure activity and reduced the duration of the stimulation-induced electrographic afterdischarge. In drug-free stimulation sessions after the initial drug-treatment sessions, there was an acceleration in the rate of kindling development compared with the rate during the preceding drug-administration period; the "rebound" rate was also greater than the kindling rate in saline-treated control animals. In fully kindled animals, both GYKI 52466 and LY293558 produced a dose-dependent suppression of evoked seizures (ED(50), 19.3 and 16.7 mg/kg, respectively). Although AMPA receptors appear to be critical to the expression of kindled seizures, since kindling development progressed despite the suppression of behavioral seizure activity, AMPA receptors are less important to the kindling process. LY293558 was modestly less effective at suppressing behavioral seizures during kindling and was not superior to GYKI 52466 in retarding the overall extent of kindling development, indicating that GluR5 kainate receptors do not contribute to epileptogenesis in this model.     

beta-Adrenoceptor blockade enhances the anticonvulsant effect of glutamate receptor antagonists against maximal electroshock.

In this study, we evaluated whether beta-adrenoceptor antagonists may modify the protective efficacy of dizocilpine (MK-801), a NMDA receptor antagonist, and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), a non-NMDA (AMPA/kainate) receptor antagonist, against maximal electroshock-induced seizures in mice. Propranolol, acebutolol, metoprolol and atenolol were used in doses that did not alter the electroconvulsive threshold. Propranolol potentiated the anticonvulsant activity of MK-801 and GYKI 52466, significantly lowering their ED(50) values from 0.38 and 15.0 to 0.15 (P<0.001) and 8.4 mg/kg (P<0.001), respectively. Similarly, metoprolol lowered the ED(50) of MK-801 and GYKI 52466 from 0.38 and 15.0 to 0.17 (P<0.05) and 11.2 mg/kg (P<0.05). Acebutolol enhanced the protective action of GYKI 52466, lowering its ED(50) value from 15.0 to 12.2 mg/kg (P<0.05), but not that of MK-801. Atenolol, not penetrating the blood-brain barrier, did not affect the anticonvulsive efficacy of MK-801 and GYKI 52466. In conclusion, beta-adrenoceptor antagonists may act synergistically with excitatory amino acid receptor antagonists to inhibit generalised tonic-clonic seizures.     

Synthesis and anticonvulsant activity of N-3 substituted 2,3-benzodiazepines

A series of new 3-alkylcarbamoyl-1-aryl-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-ones was synthesized starting from the corresponding 3-N-unsubstituted derivatives, previously described as noncompetitive AMPA-type glutamate receptor antagonists. The new compounds proved to protect against seizures induced by means of auditory stimulation in DBA/2 mice and some of them showed anticonvulsant properties comparable or better than those of GYKI 52466, the prototype of 2,3-benzodiazepine noncompetitive AMPA receptor antagonists.     

Synthesis and anticonvulsant activity of new 2,3-benzodiazepines as AMPA receptor antagonists.

Novel 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-ones (12a-j) were prepared and their anticonvulsant effects were evaluated by using various models of experimental epilepsy. The seizures were evoked both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. Some of these compounds possess marked anticonvulsant properties in all tests employed. Compounds 12 antagonise seizures induced by AMPA in analogy to the structurally-related 1-(4'-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3- benzodiazepine (1) (GYKI 52466), a well-known non-competitive AMPA-receptor antagonist. On the other hand, these novel 2,3-benzodiazepines exhibit anticonvulsant properties that are not affected by flumazenil, but are reversed by aniracetam. In addition, when compared to model compound 1, compounds 12 show a longer-lasting anticonvulsant activity and a lower toxicity. A structure-activity relationship study carried out on compounds 12 as well as analogous 7,8-dimethoxy derivatives 2 offers an approach for designing more potent agents.     

Effects of non-competitive AMPA receptor antagonists injected into some brain areas of WAG/Rij rats, an animal model of generalized absence epilepsy

CFM-2 [1-(4-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one] and THIQ-10c [N-acetyl-1-(4-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline], are two non-competitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) receptor antagonists, which demonstrated to antagonize generalized tonic-clonic seizures in different animal models. We have evaluated the effects of such compounds in a genetic animal model of absence epilepsy, the WAG/Rij rat. Animals were focally microinjected into specific brain areas of the cortico-thalamic circuit in order to evaluate the effects of these compounds on the number and duration of epileptic spike-wave discharges (SWDs) and better characterize the role of AMPA neurotransmission in this animal model. The focal microinjection of the two AMPA antagonists into some thalamic nuclei (ventralis posteromedialis (VPM), reticularis (NRT), ventralis posterolateralis (VPL) and the primary somatosensory forelimb region (S1FL)) was, generally, not able to significantly modify the occurrence of SWDs. Whereas, both compounds were able to reduce the number and duration of SWDs dose-dependently when microinjected into the peri-oral region of the primary somatosensory cortex (S1po). These findings suggest that AMPA receptor antagonists might play a role in absence epilepsies and that it might depend on the involvement of specific neuronal areas.     

Synthesis and evaluation of pharmacological and pharmacokinetic properties of 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones

A series of 2,3-benzodiazepine derivatives has been previously described as noncompetitive AMPA-type glutamate receptor antagonists potentially useful for treatment of epilepsy. To further explore the structure-activity relationships of AMPA antagonists, a series of 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones (6) was synthesized starting from the corresponding bicyclic 1-aryl-3, 5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-ones (2, CFM). The new compounds were found to possess anticonvulsant effects against seizures induced both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. In addition, they antagonize the AMPA-induced seizures, and their anticonvulsant activity is reversed by pretreatment with aniracetam, thus suggesting the involvement of AMPA receptors. The pharmacological studies revealed that the 11H-[1,2,4]triazolo[4, 5-c][2,3]benzodiazepin-3(2H)-ones (6) herein reported show anticonvulsant activity comparable to that of their bicyclic precursors. Furthermore, an HPLC study put in evidence that these tricyclic derivatives 6 were converted in vivo into the corresponding 2, the agents likely to be mainly responsible for the anticonvulsant properties observed.     

Negative allosteric modulation of AMPA-preferring receptors by the selective isomer GYKI 53784 (LY303070), a specific non-competitive AMPA antagonist

GYKI 53784 or LY303070 [(-)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine] belongs to a new family of 2,3-benzodiazepine compounds (also called homophtalazines) selective and non-competitive antagonists at alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. These compounds include the original GYKI-52466, its more potent derivative GYKI 53655 and the active isomer of the latter, GYKI 53784. This review summarizes current knowledge of this novel AMPA antagonist: GYKI 53784. GYKI 53784 is the most potent of the compounds in the 2,3-benzodiazepine class, blocking AMPA receptor-mediated responses. In contrast to the compounds of the quinoxalinedione family, that block AMPA as well as kainate receptors, GYKI 53784 does not block the activation of kainate receptors. Furthermore, GYKI 53784 does not act at the same receptor site as positive AMPA modulators (i.e., cyclothiazide, BDP-12, 1-BCP or aniracetam). GYKI 53784 is a powerful neuroprotective agent in both in vitro and in vivo models of AMPA receptor-mediated excitotoxicity. In contrast to NMDA receptor antagonists, whose favorable clinical actions are compromised by important side effects such as the impairment of memory functions, the selective AMPA antagonist, GYKI 53784, may be of potential clinical value, both in acute (stroke and trauma) and chronic (Alzheimer's disease, epilepsy) neurological disorders.     

AMPA/kainate-related mechanisms contribute to convulsant and proconvulsant effects of 3-nitropropionic acid

The role of the glutamatergic system in the convulsant and proconvulsant action of a mitochondrial toxin, 3-nitropropionic acid, was studied in mice. The occurrence of 3-nitropropionic acid-induced seizures was inhibited by the -amino-2,3-dihydro-5-methyl-3-oxo-isoxazole-propionate (AMPA)/kainate receptor antagonists, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione disodium (NBQX) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HCl (GYKI 52466), with ED₅₀ of 14.1 (7.9–25.2) and 7.2 (5.3–9.6) mg/kg, respectively. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and 3-(2-carboxypiperazine-4-yl)propenyl-1-phosphonic acid (CPPene), were ineffective. Moreover, 3-nitropropionic acid given in a subthreshold dose potently enhanced seizures generated by intracerebroventricular administration of AMPA and kainate, lowering their CD₅₀ from 0.98 (0.83–1.17) and 0.73 (0.64–0.83) to 0.55 (0.45–0.66) (P<0.001) and 0.58 (0.51–0.65) (P<0.05) nmol, respectively. In contrast, NMDA action was not changed by 3-nitropropionic acid application. We conclude that AMPA/kainate-mediated events are involved in proconvulsive and convulsive effects of 3-nitropropionic acid.     

Effects of GYKI 52466 and some 2,3-benzodiazepine derivatives on hippocampal in vitro basal neuronal excitability and 4-aminopyridine epileptic activity

In order to determine whether the anticonvulsant effect of 2, 3-benzodiazepines is also displayed in a model of in vitro epilepsy, such as the "epileptiform" hippocampal slice, we studied the effects of 2,3-benzodiazepine 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxe-5H 2,3-benzodiazepine hydrochloride (GYKI 52466) and some new 2,3-benzodiazepine derivatives on CA1 basal neuronal excitability and on CA1 epileptiform burst activity produced by 4-aminopyridine in rat hippocampal slices. The results showed that GYKI 52466 affected basal neuronal excitability as evidenced by its influence on the magnitude of the CA1 orthodromic-evoked field potentials. 2,3-Benzodiazepines showed their antiepileptic effect also in an in vitro model of experimental epilepsy. The effects of the new 2,3-benzodiazepine derivatives suggest that the methylenedioxidation in positions 7 and 8 of the 2,3-benzodiazepine ring is the main structural modification for the antiepileptic effect of 2,3-benzodiazepines to take place.     

Receptor site specificity for the acute effects of beta-N-methylamino-alanine in mice

DL-beta-N-methylamino-alanine (DL-BMAA; 1-10 mumol i.c.v.) in mice induced a syndrome of: ataxia, ptosis, scratching, jumping, myoclonic jerks, clonic muscle spasms and tonic seizure, which was unaffected by pretreatment with D(-)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylate (D(-)-CPPene; i.p.), or by co-administration of gamma-D-glutamylamino-methylsulphonate (gamma-D-GAMS with DL-BMAA; i.c.v.). Pretreatment with 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466; i.v.) decreased the incidence of clonic seizures for DL-BMAA, kainic acid and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (RS-AMPA; i.c.v.). These results suggest an involvement of the AMPA/quisqualate subtype of excitatory amino acid receptors in acute BMAA toxicity.     

The non-N-methyl-D-aspartate receptor antagonists, GYKI 52466 and NBQX are anticonvulsant in two animal models of reflex epilepsy.

The effect of i.p. or i.v. administration of the non-N-methyl-D-aspartate antagonists, GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepin e.HCl, molecular weight 330) and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline, molecular weight 342) on sound-induced seizures in rats and photically induced myoclonus in baboons was studied. In both species an anticonvulsant effect occurred 15-60 min after administration of GYKI 52466 or NBQX. The ED50 value for clonic seizure suppression for GYKI 52466 at 30 min was 39 (rats, i.p.) and at 15 min was 13 (Papio papio, i.v.) mumol kg-1 and for NBQX at 30 min was 40 (rats, i.p.) and at 15 min approximately 10 (Papio papio, i.v.) mumol kg-1. Side effects were not observed in rats; apparent side effects in baboons probably arose from drug formulation. The anticonvulsant actions of GYKI 52466 and NBQX suggest a possible role for non-NMDA antagonists in the therapy of epilepsy.     

Comparative anticonvulsant activity of some 2,3-benzodiazepine derivatives in rodents

The anticonvulsant activities of some 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)/kainate receptor antagonists, noncompetitive (2,3-benzodiazepines) and a competitive 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), were compared in different experimental seizure models. In particular, compounds were evaluated against audiogenic seizure in DBA/2 mice, maximal electroshock seizure (MES) test and various chemoconvulsant models; both groups showed a protective action against audiogenic seizure, MES- and pentylenetetrazole (PTZ)-induced seizures. All 2,3-benzodiazepines were also protective against clonic and tonic seizures and lethality induced by 4-aminopyridine, kainate, AMPA and 3-mercaptopropionic acid but were ineffective against NMDA-induced seizures. NBQX was unable to affect 4-aminopyridine-, mercaptopropionic acid- and NMDA-induced seizures. The duration of anticonvulsant action of 33 micromol/kg of some 2,3-benzodiazepine in DBA/2 mice, genetically susceptible to audiogenic seizures, was also investigated. The derivatives possessing a thiocarbonyl group at the C-4 position of heptatomic ring showed higher anticonvulsant activities and longer lasting protective effects. We conclude that all 2,3-benzodiazepines studied are effective against various models of experimental epilepsy and the presence of thiocarbonyl groups at the C-4 position of heptatomic ring is able to increase the anticonvulsant effect of these compounds.     

Group III mGlu receptor agonists potentiate the anticonvulsant effect of AMPA and NMDA receptor block

We report the anticonvulsant action in DBA/2 mice of two mGlu Group III receptor agonists: (R,S)-4-phosphonophenylglycine, (R,S)-PPG, a compound with moderate mGlu8 selectivity, and of (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid, ACPT-1, a selective agonist for mGlu4alpha receptors. Both compounds, given intracerebroventricularly at doses which did not show marked anticonvulsant activity, produced a consistent shift to the left of the dose-response curves (i.e. enhanced the anticonvulsant properties) of 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one hydrochloride, CFM-2, a noncompetitive AMPA receptor antagonist, and 3-((+/-)-2-carboxypiperazin-4-yl)-1-phosphonic acid, CPPene, a competitive NMDA receptor antagonist, in DBA/2 mice. In addition, (R,S)-PPG and ACPT-1 administered intracerebroventricularly prolonged the time course of the anticonvulsant properties of CFM-2 (33 micromol/kg, i.p.) and CPPene (3.3 micromol/kg, i.p.) administered intraperitoneally. We conclude that modest reduction of synaptic glutamate release by activation of Group III metabotropic receptors potentiates the anticonvulsant effect of AMPA and NMDA receptor blockade.     

Anticonvulsant activity and plasma level of 2,3-benzodiazepin-4-ones (CFMs) in genetically epilepsy-prone rats.

Anticonvulsant properties of some 2,3-benzodiazepine derivatives acting as alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) antagonists have been examined in vivo in the genetically epilepsy-prone rats using an audiogenic seizures assay. 2,3-Benzodiazepin-4-ones (CFMs) are nonselective AMPA antagonists that have been found to be potent anticonvulsant compound is in acute models of epilepsy. Because very little is known about their actions in a chronic model of epilepsy, and no correlations exist between anticonvulsant potency and plasma levels of these derivatives, we planned to investigate such a relationship. Maximal anticonvulsant protection occurred 15-60 min after the IP administration of GYKI 52466, 30-90 min after CFM-2, and 45-120 min after CFM-3. In addition, maximal anticonvulsant effect was observed 60-120 min after the IP administration of CFM-4 and at 90 min after CFM-5. The therapeutic index revealed that GYKI 52466 was slightly more toxic than CFM-2 and CFM-3. The time course of plasma levels of rats treated showed that peak plasma concentration was observed 45 min after IP administration of CFM-2 and CFM-3 and 75 min after CFM-4 and CFM-5. Following IP administration of CFM-3 two curves were detected, one is referred to the injected compound, and the other to its demethylated metabolite, which corresponds to CFM-2. Also. for the nitroderivative CFM-4 two curves were detected: one of an injected compound and the second due to its reduced metabolite (CFM-2). Finally, three different metabolites were detected in rat plasma after IP administration of CFM-5. The present study demonstrated that CFMs showed a significant protection against auditory stimulation during the period of peak plasma concentrations, suggesting a marked inhibition of those brain structures involved in the initiation and/or spreading of the audiogenic seizures.     

Interaction of GYKI 52466, a selective non-competitive antagonist of AMPA/kainate receptors, with conventional antiepileptic drugs in amygdala-kindled seizures in rats.

GYKI 52466 [1,4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive AMPA/kainate receptor antagonist, administered i.p. at the dose of 5 mg/kg, exerted a significant anticonvulsant effect, as it decreased seizure and afterdischarge durations, being ineffective at 2 mg/kg. Subsequently, GYKI 52466 (2 mg/kg) was combined with antiepileptic drugs at doses ineffective in fully kindled rats. Co-administration of GYKI 52466 with clonazepam (0.003 mg/kg i.p.) resulted in a significant reduction of seizure severity (by 20%), seizure duration (by 31%) and afterdischarge duration (by 24%). Co-injection of GYKI 52466 with valproate (75 mg/kg i.p.) also resulted in the respective 8%, 16%, and 17% reductions of the three studied seizure parameters. No protection was observed when GYKI 52466 was co-administered with carbamazepine (20 mg/kg i.p.), phenobarbital (20 mg/kg i.p.), or diphenylhydantoin (40 mg/kg i.p.). Combinations of GYKI 524662 with antiepileptic drugs did not cause any significant motor (rotarod test) or long-term memory deficits (passive avoidance task). Only GYKI 52466 administered alone at 5 mg/kg, caused a significant impairment of retention in amygdala-kindled rats. The interaction at a pharmacokinetic level, at least in case of the combination of GYKI 52466 with valproate, can be excluded because GYKI 52466 did not interfere with the free plasma level of valproate. These results give further support to the idea of a potential clinical benefits of the combined treatment of AMPA/kainate receptor antagonists with some antiepileptic drugs.     

Effects of some AMPA receptor antagonists on the development of tolerance in epilepsy-prone rats and in pentylenetetrazole kindled rats

The non-selective alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor antagonists, 2,3-benzodiazepine derivatives CFM-1 (3,5-dihydro-7,8-dimethoxy-1-phenyl-4H-2,3-benzodiazepin-4-one) and CFM-2 (1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin -4-one), following intraperitoneal (i.p.) administration, were studied against audiogenic seizures in genetically epilepsy-prone rats (GEPRs) or pentylenetetrazole induced kindling in rats. After acute i.p. administration the ED50 values of CFM-1 against the clonic and tonic phases of the audiogenic seizures 30 min after pretreatment were 40 (16-100) and 13 (8-25) micromol kg(-1), respectively. The animals used for chronic study were treated i.p. daily (at 10 h) for 4 weeks with CFM-1 (20 or 50 micromol kg(-1)). Chronic treatment for 2 weeks with CFM-1 gave ED50 values against clonic and tonic seizures of 39 (22-69) and 16 (8-25) micromol kg(-1), respectively, whereas chronic treatment for 4 weeks gave ED50 values against clonic and tonic seizures of 42 (18-98) and 17 (7-41.3) micromol kg(-1), respectively. The duration of anticonvulsant activity observed between 0.5 and 4 h following administration of CFM-1 was similar for acute and chronic treatment. Two groups of Sprague-Dawley rats received CMF (20 or 50 micromol kg(-1)) 30 min before a subconvulsant dose of pentylentetrazole (25 mg kg(-1) i.p.) which is able to increase seizure severity in control animals (i.e., chemical kindling). Pretreatment with CFM-2 delayed the progression of seizure rank during repeated administration of pentylentetrazole. At the end of the period of repeated pentylentetrazole treatment (6 weeks) the mean seizure score was 0 in vehicle treated controls, 4.3 in animals treated with vehicle + pentylentetrazole, 2.2 in rats treated chronically with CFM-2 (20 micromol kg(-1) i.p.) + pentylentetrazole and 1.0 in rats treated repeatedly with CFM-2 (50 micromol kg(-1) i.p.) + pentylenetetrazole. CFM-2 was also able to antagonize the long-term increase in sensitivity of the convulsant effects of GABA function inhibitors in pentylentetrazole-kindled animals. Thus, the administration of a challenge dose of pentylentetrazole (15 mg kg(-1) i.p.) or picrotoxin (1.5 mg kg(-1) i.p.) 15 or 30 days after the end of the repeated treatment showed that animals treated with CFM-2 were significantly protected against seizures induced by pentylentetrazole or picrotoxin. The data suggest that, following repeated treatment, tolerance to the novel AMPA receptor antagonists does not develop (CFM-1 in genetically epilepsy-prone rats and CFM-2 in the pentylentetrazole kindling model of epilepsy). Thirteen minutes after drug injection on days 1, 14 and 28 of chronic treatment the motor impairment induced by these compounds was studied with a rotarod apparatus. The TD50 values for CFM-1 or CFM-2-induced impairment of locomotor performance were similar following acute and repeated treatment. The data also suggest that some novel 2,3-benzodiazepines may have clinical potential for some types of epilepsy.     

Riluzole, a glutamate release inhibitor, and motor behavior

Riluzole (2-amino-6-trigluoromethoxy benzothiazole) has neuroprotective, anticonvulsant, anxiolytic and anesthetic qualities. These effects are mediated by blockade of glutamate transmission, stabilizing of sodium channels and blockade of γ-aminobutyric acid (GABA) reuptake. The action profile of riluzole is dominated by its effects on glutamate transmission which are predominately mediated by N-methyl-D-aspartate (NMDA) receptor-linked processes in vitro. In vivo studies show that blockade and stimulation of the different NMDA receptor complex binding sites or AMPA receptors modulate motor behavior in a characteristic manner. It was therefore interesting to examine if blockade of glutamatergic transmission by riluzole induced similar behavioral effects as direct NMDA/AMPA receptor antagonists and if these effects are mediated by a specific receptor. The effects of riluzole alone and in combination with several other neuroactive compounds on the central nervous system was assessed by behavioral paradigms to evaluate sniffing behavior, locomotion, ataxia and rigidity. Accompanying compounds included the NMDA receptor agonist NMDA, the partial glycine site agonist D-cycloserine (DCS), and the α-amino-3-hydroxy-5-phenyl-4-isoxazolepropionic acid (AMPA) receptor antagonist GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo-diazepine HCl]. Riluzole influenced neither stereotyped sniffing behavior nor locomotion but impaired motor coordination and attenuated rigidity induced by blockade of dopamine D1 and D2 receptor antagonists when given alone. At higher doses spontaneous behavioral activity decreased and motor coordination was more impaired. Augmentation of the riluzole effects were observed when NMDA, but not GYKI 52466, was coadministered. The glycine site agonist DCS increased the anticataleptic properties of riluzole. The results indicate that when given alone, riluzole has a behavioral profile resembling that of competitive NMDA receptor antagonists. However, coadministration of riluzole with NMDA/AMPA receptor ligands suggests that this assumption is incorrect, and that riluzole affects glutamatergic transmission by a more indirect mechanism. Nevertheless, the profile of riluzole together with its pre- and postsynaptic blockade of glutamatergic transmission implies beneficial properties in diseases where an overactive glutamate system induces chronic neurotoxicity and/or acute behavioral effects. Received: 15 December 1997 / Accepted: 12 May 1998     

Comparison of neuroprotective efficacy of competitive and non-competitive AMPA antagonists in vitro

The neuroprotective efficacy of the most potent competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and three recently developed 2,3-benzodiazepine non-competitive AMPA antagonists (GYKI 52466, 53405 and 53655) was investigated in primary cultures of rat telencephalic neurons. NBQX protected cultured neurons against AMPA (20 μM for 21-23 h) induced toxicity with EC(50) of 0.5 μM. In the same test GYKI 52466, 53405 and 53655 had EC(50) values of 10.6, 9.3 and 5.1 μM, respectively. Thus we found the competitive antagonist NBQX to be ten times more effective neuroprotectant in vitro than the most potent non-competitive GYKI compound (GYKI 53655).