The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hyrpercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at α5-, α2-, and α3- but low intrinsic efficacy at α1-containing GABA(A) receptors is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26μg/kg pyridostigmine bromide 30min prior to 2× LD(50) soman exposure and 1min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2× LD(50) of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg showed less efficacy than 1mg/kg probably due to non-specific actions at sites other than GABA(A) receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as hippocampus neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.

The clinical use of diazepam or midazolam to control organophosphate (OP) nerve agent-induced seizure activity is limited by their unwanted effects including sedation, amnesia, withdrawal, and anticonvulsant tolerance. Imidazenil is an imidazo-benzodiazepine derivative with high intrinsic efficacy and selectivity for α2-, α3-, and α5- but low intrinsic efficacy for α1-containing GABA(A) receptors. We have previously shown that imidazenil is more efficacious than diazepam at protecting rats and mice from diisopropyl fluorophosphate (DFP)-induced seizures and neuronal damage without producing sedation. In the present study, we compared the tolerance liability of imidazenil and diazepam to attenuate the seizure activity and neurotoxic effects of DFP. Rats received protracted (14 days) oral treatment with increasing doses of imidazenil (1-4 mg/kg), diazepam (5-20 mg/kg), or vehicle. Eighteen hours after the last dose of the protracted treatment schedule, rats were tested for anticonvulsant tolerance after a 30 min pretreatment with a single test dose of imidazenil (0.5 mg/kg) or diazepam (5 mg/kg) prior to a DFP challenge (1.5 mg/kg). The anticonvulsant (modified Racine score scale) and neuroprotective (fluoro-jade B staining) effects of diazepam were significantly reduced in protracted diazepam-treated animals whereas the effects of imidazenil were not altered in protracted imidazenil-treated animals. The present findings indicate that protracted imidazenil treatment does not produce tolerance to its protective action against the neurotoxic effects of OP exposure.

Recent evidence suggests that alpha1-containing GABA(A) receptors mediate the sedative, amnestic, and to some extent the anticonvulsant actions of non-selective benzodiazepine (BZ) receptor ligands, such as diazepam (DZ). Anxiolytic and in part, anticonvulsant actions of BZ ligands are mediated by alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. This has resulted in increasing interest in developing BZ ligands with selective actions at GABA(A) receptors, including alpha2-, alpha3-, and alpha5-subunits, but devoid of efficacy at alpha1-containing receptors. To refine their spectrum of pharmacological actions, efforts are being made to minimize unwanted effects such as sedation, amnesia, and tolerance liabilities. A prototype for such BZ ligands is imidazenil (IMD), an imidazo-benzodiazepine carboxylic acid derivative that elicits potent anticonvulsant and anxiolytic actions at doses virtually devoid of sedative, cardio-respiratory depressant and amnestic effects, and anticonvulsant tolerance liability. To define the pharmacological profile of IMD and its derivatives, we compared the anticonflict (anxiolytic), anti-proconflict (antipanic), anti-bicuculline (BIC), and maximal electroshock seizure (MES) effects, and the suppression of locomotor activity by imidazo-benzodiazepine carboxylic acid derivatives to those of DZ and bretazenil (BTZ). We report here that IMD and one of its derivatives (RO 25-2775) possess dose-dependent anticonflict, anti-proconflict, and anti-BIC actions but failed to suppress locomotor activity. Like DZ, the other IMD derivatives (enazenil, RO 25-2776, and RO 25-2847) not only elicit dose-dependent anticonflict, anti-proconflict, anti-BIC, anti-MES effects but also suppress locomotor activity. In contrast, none of the IMD derivatives studied shows any similarity to BTZ, which elicits anticonflict, anti-proconflict actions and suppresses locomotor activity but is virtually inactive against BIC-induced tonic-clonic convulsions.

Organophosphate (OP)-nerve agent poisoning may lead to prolonged epileptiform seizure activity, which can result in irreversible neuronal brain damage. A timely and effective control of seizures with pharmacological agents can minimize the secondary and long-term neuropathology that may result from this damage. Diazepam, the current anticonvulsant of choice in the management of OP poisoning, is associated with unwanted effects such as sedation, amnesia, cardio-respiratory depression, anticonvulsant tolerance, and dependence liabilities. In search for an efficacious and safer anticonvulsant benzodiazepine, we studied imidazenil, a potent anticonvulsant that is devoid of sedative action and has a low intrinsic efficacy at alpha1- but is a high efficacy positive allosteric modulator at alpha5-containing GABA(A) receptors. We compared the potency of a combination of 2mg/kg, i.p. atropine with:(a) imidazenil 0.05-0.5mg/kg i.p. or (b) equipotent anti-bicuculline doses of diazepam (0.5-5mg/kg, i.p.), against diisopropyl fluorophosphate (DFP; 1.5mg/kg, s.c.)-induced status epilepticus and its associated neuronal damage. The severity and frequency of seizure activities were determined by continuous radio telemetry recordings while the extent of neuronal damage and neuronal degeneration were assessed using the TUNEL-based cleaved DNA end-labeling technique or neuron-specific nuclear protein (NeuN)-immunolabeling and Fluoro-Jade B (FJB) staining, respectively. We report here that the combination of atropine and imidazenil is at least 10-fold more potent and longer lasting than the combination with diazepam at protecting rats from DFP-induced seizures and the associated neuronal damage or ongoing degeneration in the anterior cingulate cortex, CA1 hippocampus, and dentate gyrus. While 0.5mg/kg imidazenil effectively attenuated DFP-induced neuronal damage and the ongoing neuronal degeneration in the anterior cingulate cortex, dentate gyrus, and CA1 hippocampus, 5mg/kg or a higher dose of diazepam is required to produce similar protective effects. These finding suggests that imidazenil, a non-sedating anticonvulsant BZ ligand, is a more potent, effective, and safer drug than diazepam in protecting rats from DFP-induced seizures and the associated neuronal damage and/or ongoing neuronal degeneration.

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes:(i) pretreatment with reversible blockers of AChE,(ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 mug/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.

Flumazenil (FLU), a specific benzodiazepine (BZ) receptor antagonist has been used in the treatment of acute BZ intoxication or the alleviation of BZ-induced withdrawal syndrome on the basis of its weak partial agonist action at GABA(A) receptors. However, given to patients, FLU can worsen diazepam-induced withdrawal syndrome by lowering seizure threshold. We therefore investigated whether imidazenil, a selective positive allosteric modulator of GABA action at GABA(A) receptors containing alpha5 subunit, can antagonize diazepam-induced sedative action and suppression of locomotor activity without affecting diazepam anti-bicuculline action. We report here that while FLU (16.5 micromol/kg) showed no effect on locomotor activity and bicuculline-induced convulsion, it completely antagonized diazepam (10.5 micromol/kg) anti-bicuculline action and the suppression of locomotor activity. However, imidazenil (0.76 micromol/kg) elicited anti-bicuculline action and was dose-dependently antagonized by FLU (16.5 and 33 micromol/kg). Furthermore, imidazenil showed no effect on path length traveled but slightly decreased (40%) horizontal activity when compared to diazepam (85%), and maintained the anti-bicuculline action of diazepam to a threshold level similar to that observed with diazepam. Whereas cross-tolerance between BZs has been reported in animals and humans, we previously reported the absence of cross-tolerance between imidazenil and diazepam. Thus, we suggest that imidazenil might be more effective than FLU at alleviating the withdrawal syndrome associated with long-term BZ administration.

RATIONALE: Cortical gamma-aminobutyric acid (GABA)ergic neurons contribute to the orchestration of pyramidal neuron population firing as follows:(1) by releasing GABA on GABA(A) and GABA(B) receptors,(2) by releasing reelin in the proximity of integrin receptors located on cortical pyramidal neuron dendritic spines, and (3) through reelin contributing to the regulation of dendritic spine plasticity by modulating dendritic resident mRNA translation. In schizophrenia (SZ) and bipolar (BP) postmortem brains, the downregulation of mRNAs encoding glutamic acid decarboxylase 67 (GAD(67)) and reelin decreases the cognate proteins coexpressed in prefrontal cortex (PFC) GABAergic neurons. This finding has been replicated in several laboratories. Such downregulation suggests that the neuropil hypoplasticity found in the PFC of SZ and BP disorder patients may depend on a downregulation of GABAergic function, which is associated with a decrease in reelin secretion from GABAergic neuron axon terminals on dendrites, somata, or axon initial segments of pyramidal neurons. Indirectly, this GABAergic neuron downregulation may play a key role in the expression of positive and negative symptoms of SZ and BP disorders. OBJECTIVES: The above described GABAergic dysfunction may be addressed by pharmacological interventions to treat SZ and BP disorders using specific benzodiazepines (BZs), which are devoid of intrinsic activity at GABA(A) receptors including alpha(1) subunits but that act as full positive allosteric modulators of GABA action at GABA(A) receptors containing alpha(2), alpha(3), or alpha(5) subunits. These drugs are expected to enhance GABAergic signal transduction without eliciting sedation, amnesia, and tolerance or dependence liabilities. RESULTS AND CONCLUSIONS: BZs, such as diazepam, although they are efficient in equilibrating GABA(A) receptor signal transduction in a manner beneficial in the treatment of positive and negative symptoms of SZ, may not be ideal drugs, because by mediating a full positive allosteric modulation of GABA(A) receptors containing the alpha(1) subunit, they contribute to sedation and to the development of tolerance after even a brief period of treatment. In contrast, other BZ-binding site ligands, such as 6-(2bromophenyl)-8-fluoro-4H-imidazo [1,5-a][1,4] benzodiazepine-3-carboxamide (imidazenil), which fail to allosterically and positively modulate the action of GABA at GABA(A) receptors with alpha(1) subunits but that selectively allosterically modulate cortical GABA(A) receptors containing alpha(5) subunits, contribute to the anxiolytic, antipanic, and anticonvulsant actions of these ligands without producing sedation, amnesia, or tolerance. Strong support for the use of imidazenil in psychosis emerges from experiments with reeler mice or with methionine-treated mice, which express a pronounced reelin and GAD(67) downregulation that is also operative in SZ and BP disorders. In mice that model SZ symptoms, imidazenil increases signal transduction at GABA(A) receptors containing alpha(5) subunits and contributes to the reduction of behavioral deficits without producing sedation or tolerance liability. Hence, we suggest that imidazenil may be considered a prototype for a new generation of positive allosteric modulators of GABA(A) receptors, which, either alone or in combination with neuroleptics, should be evaluated in GABAergic dysfunction operative in the treatment of SZ and BP disorders with psychosis.

Convulsions are major and life-threatening signs of organophosphate (OP) nerve agents induced neurotoxicity. Thus, early intervention with anticonvulsant drugs to control seizure propagation and the consequent irreversible neuronal damage that may occur during OP exposure is essential. Diazepam is the standard anticonvulsant used in the therapeutic management of OP poisoning. However, its use has been associated with several unwanted effects including, sedation, amnesia, and in the large doses used for such treatment, respiratory depression. Moreover, protracted administration of diazepam has been associated with tolerance and dependence liabilities. In this study, we compared the efficacy and safety of diazepam (full allosteric modulator of GABA action) to that of imidazenil (partial, selective allosteric modulator of GABA action) as preventive treatment against diisopropyl fluorophosphate (DFP)-induced convulsions and mortality. Our results show that imidazenil is more potent and efficacious than diazepam in protecting rats against DFP-induced convulsions and death. Moreover, imidazenil was effective at doses (1 and 0.5 mg/kg) we have previously shown to be devoid of sedation, amnesia, respiratory depression, or tolerance and/or dependence. In contrast, diazepam was effective at doses (5 and 2.5 mg/kg) that produce sedation, amnesia, and ataxia. Furthermore, the combination of imidazenil with atropine was more potent and efficacious than that with diazepam.