Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) receptors but with the opposite functionality.

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA(A) receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. 2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response. 3. These functional characteristics were monitored with Cl- currents measurements in cloned rat alphaxbeta2gamma2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA(A) receptors with binding of [35S]-TBPS, which is a high affinity ligand specific for GABA(A) receptors with exquisite sensitivity to allosteric modulations. 4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

Azulenyl nitrones: colorimetric detection of oxyradical end products and neuroprotection in the gerbil transient forebrain ischemia/reperfusion model.

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.

Pharmacological approaches to counter the toxicity of Dopa.

Dopa and related catecholamines and their degradation products have been demonstrated to have neurotoxic potential in a number of cellular and in vivo experiments. Several mechanisms have been hypothesized to be involved including generation of prooxidant products that subsequently oxidize membrane lipids and exposed macromolecules. We have utilized a neuronal culture of cerebellar granule cells to study the toxicity of Dopa and the ability of various neuroprotective and antiparkinsonian compounds to offer protection therefrom. This model is apparently based on the ability of Dopa to non-enzymatically induce an oxidative injury to the neuronal cultures. Evidence for this arises from the equal neurotoxic potency of L- and D-Dopa in these cells and the ability of catalase, superoxide dismutase and glutathione to protect the neurons from this toxicity. Further, we found that the neuroprotective antioxidant, PNU-101033 is more effective and potent than vitamin E and deprenyl in this regard. Similarly the D2/D3 agonist, pramipexole is also capable of blocking Dopa toxicity in this model and this effect is independent of dopamine receptor affinity as both enantiomers are equally potent in this assay but disparate in receptor affinity. Also the protection by pramipexole is accompanied by the preservation of reduced glutathione. Thus, this activity seems to be a function of the oxidation potential of pramipexole and it's consequent antioxidant property. Potent antioxidants are effective blockers of Dopa toxicity. If the mechanisms involved in this toxicity have relevance to the progression of Parkinson's pathology in Dopa treated (or untreated) patients, these compounds have the potential to alter the course of the illness.

Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models.

A novel group of antioxidant compounds, the pyrrolopyrimidines, has been discovered recently. Many of these possess significantly improved oral bioavailability (56-70% in rats), increased efficacy and potency in protecting cultured neurons against iron-induced lipid peroxidative injury and as much as a 5-fold increase in brain uptake compared with the 21-aminosteroid antioxidant compound, tirilazad mesylate (U-74006F), described earlier. They appear to quench lipid peroxidation reactions by electron-donating and/or radical-trapping mechanisms. Several compounds in the series, such as U-101033E and U-104067F, demonstrate greater ability than tirilazad to protect the hippocampal CA1 region in the gerbil transient (5-min) forebrain ischemia model. Delaying treatment until 4 hr after the ischemic insult still results in significant CA1 neuronal protection. U-101033E is still effective in salvaging a portion of the CA1 neuronal population when the ischemic duration is extended to 10 min. In addition, U-101033E has been found to be protective in the context of focal cerebral ischemia, reducing infarct size in the mouse permanent middle cerebral artery occlusion model, in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage than those which are localized in the cerebral microvasculature. However, the activity of U-101033E in improving early post-traumatic recovery in mice subjected to severe concussive head injury is similar to that of tirilazad. Last, the oral bioavailability of many pyrrolopyrimidines suggests that they may be useful for certain chronic neurodegenerative disorders in which lipid peroxidation plays a role.

Effects of lazaroids and a peroxynitrite scavenger in a cell model of peroxynitrite toxicity.

We developed a cerebellar granule cell model of peroxynitrite toxicity and showed that certain sulfhydryl-containing compounds (e.g., penicillamine) present as concurrent treatments could inhibit this toxicity. In the present study, 21-aminosteroid and pyrrolopyrimidine lazaroids were tested for cytoprotection in this peroxynitrite toxicity model. In addition, we tested for added protection using a peroxynitrite scavenger concurrent treatment combined with a lazaroid post-treatment. The toxicity assay utilized cells that were previously exposed to 100 microM L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of gamma-glutamyl-cysteine synthetase, for 24 h. This sublethal concentration of BSO shifted the peroxynitrite (1-1000 microM) toxicity curve to the left by more than one-half of a log unit. The half-maximal toxicity concentration (TC50) of peroxynitrite in cells treated with BSO was 50 microM. The 21-aminosteroids, U-74006F and U-74500A, and the pyrrolopyrimidines, U-91736B and U-101033E, were tested as post-treatments. U-74006F and U-74500A had EC50 values of approximately 100 microM (concentrations which blocked 50% of the toxicity). U-91736B and U-101033E had EC50 values of 1 microM and showed 100% protection at 3-10 microM. Treatment with either 100 microM U-74006F or 1 microM U-101033E resulted in a right-hand shift (protection) in the peroxynitrite toxicity curve. Further, combination treatment of lazaroids with 1 mM penicillamine resulted in additive protection compared to either treatment alone.

D1 dopamine receptor activity of anti-parkinsonian drugs.

Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.013, 0.053, 0.25, 1.04, 2.18, 50.9 and 54.4, respectively. SKF 38393, apomorphine, dopamine and pergolide had similar intrinsic activity (100%), while the intrinsic activities of 7-OH-DPAT, bromocriptine and lisuride were 28.0%, 20.7% and 17.2%, respectively. SCH 23390, a selective D1 dopamine receptor antagonist, blocked an increase in cAMP formation produced by EC50 concentrations of all of the dopamine agonists investigated in this study. Clozapine concentration-dependently blocked pergolide-induced increases in cAMP and was approximately 1700-fold less potent than SCH 23390 (IC50: 0.97 microM and 0.56 nM, respectively). U-95666A (1-1000 microM), selective for the D2 receptors, showed no significant effect on cAMP, while pramipexole (0.1-100 microM), a D3 preferring agonist, did not elevate cAMP. These data suggest that primary cerebellar granule cell cultures are an excellent model for measuring D1 dopamine receptor-mediated changes in cellular cAMP. The results are discussed with reference to the relationship between the D1 receptor-stimulated increase in cAMP formation and the induction of dyskinesia in humans by these anti-parkinsonian drugs.

PNU-107484A with alpha isoform-dependent functional changes in alpha(x)beta2gamma2 subtypes of rat recombinant GABA(A) receptors.

1. We discovered a novel gamma-aminobutyric acidA (GABA(A)) receptor ligand displaying seemingly opposite functionalities, depending on the alpha isoform of the alpha(x)beta2gamma2 subtypes. PNU-107484A enhanced GABA-induced Cl- currents in the alpha1beta2gamma2 subtype, but inhibited the currents in the alpha3beta2gamma2 and alpha6beta2gamma2 subtypes, and its half-maximal concentrations in the subtypes were 3.1 +/- 0.5, 4.2 +/- 1, and 3.5 +/- 0.2 microM, respectively, without showing much dependency on alpha isoforms. 2. In the alpha1beta2 subtype, the drug at concentrations up to 40 microM showed no effect on GABA-induced Cl- currents, suggesting the requirement of the gamma subunit for its action. 3. PNU-107484A behaved like a positive allosteric modulator of the alpha1beta2gamma2 subtype with its binding site distinct from those for benzodiazepines, barbiturates and neurosteroids. With the alpha3beta2gamma2 subtype, the drug behaved like a non-competitive inhibitor of GABA, thus blocking Cl- currents by GABA alone or in the presence of pentobarbitone and neurosteroids. 4. It appears that PNU-107484A is a unique GABA(A) receptor ligand with alpha isoform-dependent functionalities, which may provide a basis for development of alpha isoform-selective ligands, and it could be useful as a probe to investigate the physiological roles of the various alpha isoform subtypes.

Pharmacological characterization of U-101387, a dopamine D4 receptor selective antagonist.

Dopamine D2-like receptors play an important role in the pharmacotherapy of psychotic disorders. Molecular and cellular techniques have identified distinct gene products (D2-long, D2-short, D3 and D4) displaying the D2 receptor pharmacology. However, the contribution of each subtype in antipsychotic effects of or their physiological role remain unclear. Here we describe the pharmacological effects of a selective D4 antagonist, U-101387. U-101387 displayed moderately high affinity (Ki = 10 nM) and selectivity for the dopamine D4.2 receptor expressed in clonal cell lines. It lacked measurable affinity for not only other dopamine receptors but also noradrenalin, serotonin and histamine receptor families (Ki > 2000 nM). It fully and dose-dependently antagonized quinpirole-induced cAMP inhibition (without producing any effect by itself) in stably transfected cells. U-101387 also displayed excellent oral bioavailability, brain penetration and other pharmacokinetic characteristics. Unlike classical neuroleptics (e.g., haloperidol), U-101387 neither blocked acute behavioral effects of amphetamine or apomorphine nor did it alter spontaneous locomotion by itself. Additionally, U-101387 was without effect in behavioral and biochemical tests predictive of extrapyramidal and neuroendocrine side effects. Consistent with the lack of autoreceptor function of D4, acute administration of U-101387 failed to alter dopamine neuronal firing by itself or reverse the inhibition produced by dopamine agonists and to affect monoamine turnover in areas innervated by the mesencephalic or hypothalamic dopamine neurons. However, U-101387 potently induced c-fos mRNA expression in the infralimbic/ventral prelimbic cortex to a level similar to that produced by the atypical antipsychotic, clozapine. This is consistent with the predominantly cortical distribution of the D4 receptor. Taken together, these results demonstrate that the D4-selective antagonist, U-101387, produces effects that are distinct from those of the nonselective D2 antagonists as well as D3-preferring agents. U-101387 offers a unique tool to understand the role of dopamine D4 receptors in diseases involving central dopamine systems.

Neuroprotective effects of the dopamine D2/D3 agonist pramipexole against postischemic or methamphetamine-induced degeneration of nigrostriatal neurons.

We have examined the neuroprotective efficacy of the selective dopamine (DA) D2/D3 receptor agonist pramipexole in two models of nigrostriatal (NS) degeneration. The first involves the delayed (28-day) postischemic retrograde NS degeneration that takes place in gerbils following a 10-min episode of bilateral carotid arterial occlusion-induced forebrain ischemia. In vehicle (40% hydroxypropyl cyclodextrin)-treated male gerbils, there was a 40-45% loss of NS cell bodies in the pars compacta and pars reticulata (TH immunohistochemistry and Cresyl violet histochemistry) by 28 days after ischemia/reperfusion. Daily postischemic oral dosing (1 mg/kg p.o., b.i.d., beginning at 1 h after insult) decreased the 28-day postischemic loss of NS DA neurons by 36% (P < 0.01 vs. vehicle-treated). The effect was specific for dopamine neurons since no significant salvage of hippocampal CA1 neurons was observed. In a second model, pramipexole's effects were examined on methamphetamine-induced (10 mg/kg, i.p. X 4, each 2 h apart) NS degeneration in male Swiss-Webster mice. In vehicle-treated mice, there was a 40% loss of NS neurons by day 5. In contrast, pramipexole dosing (1 mg/kg, p.o., 1 h after the last methamphetamine dose, plus daily) attenuated the NS degeneration from 40% to only 8% (P < 0.00001 vs. vehicle). We postulated that pramipexole acts in both of these models to reduce the elevated DA turnover and the associated elevation in hydroxyl radical production secondary to increased MAO activity that could be responsible for oxidative damage to the NS neurons. Indeed, in the gerbil ischemia model, we documented by HPLC-ECD a 135% postreperfusion increase in DA turnover (DOPAC + HVA/DA) at 5 min after reperfusion. Pramipexole at the 1 mg/kg, p.o., dose level was able to significantly reduce the increased DA turnover, but by only 16%. Thus, it is conceivable that other mechanisms may also contribute to pramipexole's dopaminergic neuroprotection. Based on a preliminary examination of pramipexole's oxidation potential, it appears that the compound may possess significant intrinsic antioxidant properties that might contribute to its neuroprotective effects.

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

Thyroid hormonal modulation of the binding and activity of the GABAA receptor complex of brain.

Thyroid hormones, which are known to act by genomic mechanisms in peripheral tissues, were found to influence the binding and function of the GABAA receptor complex in brain membranes. Submicromolar concentrations of triiodothyronine and thyroxine stereospecifically stimulated the binding of [35S]t-butylbicyclophosphorothionate (a convulsant ligand for the GABAA receptor complex) to highly washed rat brain membranes, while higher concentrations of the hormones inhibited radioligand binding. GABA-stimulated 36Cl-flux in isolated brain membrane sacs was inhibited by L-triiodothyronine with a half-maximally inhibitory concentration (IC50) of 10(-7) M. Patch-clamp analysis of recombinant GABAA receptor subunits expressed in human embryonic kidney-293 cells showed an inhibition of chloride currents by thyroid hormones. This effect required only the alpha 1 beta 2 subunits, and was not blocked by the benzodiazepine antagonist flumazenil. Since thyroid hormones are known to be concentrated in nerve terminal preparations and subsequently released, the hormones may have non-genomic mechanisms of action as putative neurotransmitters or neuromodulators in brain and act through GABAA receptors.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

U-90042, a sedative/hypnotic compound that interacts differentially with the GABAA receptor subtypes.

U-90042 is a structurally novel compound that has comparable affinities for binding to three recombinant subtypes of the gamma-aminobutyric acidA receptor: alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2. The relatively high affinity for the alpha 6 beta 2 gamma 2 subtype is similar to the benzodiazepine (BZ) partial inverse agonist Ro 15-4513 and different from BZ sedative/hypnotics such as diazepam and zolpidem. In the present study, U-90042 (3 mg/kg i.p.) suppressed locomotor activity and impaired rotarod performance in mice. These effects were not antagonized by flumazenil. The sedative effect was further confirmed in rats (10 mg/kg i.p.) and monkeys (1 mg/kg p.o.) by an increase of behavioral sleep and a corresponding electroencephalographic frequency spectral shift. Unlike the BZ hypnotics, U-90042 (10 mg/kg i.p.) produced no amnesia in the one-trial passive avoidance response in mice but antagonized diazepam-induced amnesia. In rats trained to discriminate an injection of diazepam from saline, U-90042 produced predominantly vehicle-appropriate responses, even at depressant doses. The in vivo diazepam-antagonist effect of U-90042 is consistent with its low intrinsic activity and diazepam-antagonism at the gamma-aminobutyric acidA alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 receptor subtypes. The receptor mechanism for the sedative/hypnotic effect is not clear at this time.

Antibody transformation by peroxynitrite as determined using capillary electrophoresis: a feasibility study.

Peroxynitrite may be a physiologically relevant endogenous neurotoxin that forms following CNS trauma when excessive levels of NO and .O2 accumulate. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP. A feasibility study was performed to evaluate the use of capillary electrophoresis as an effective tool regarding the structural transformation of antibody following exposure to peroxynitrite with or without co-incubation with a peroxynitrite scavenger. A polyclonal antibody to cAMP and a monoclonal antibody to plasminogen activation inhibitor-1 were exposed to peroxynitrite with or without penicillamine coincubation. Samples were analyzed by an Applied Biosystems analytical capillary electrophoresis system, model 270A. Initial examination of the peroxynitrite scavenger penicillamine and its reaction with peroxynitrite showed a penicillamine migration peak at about 9.1 min and a presumed s-nitro adduct of penicillamine that migrated at 10.9 min. Exposure of either antibody to peroxynitrite resulted in structural transformation of protein based on changes in migration patterns. In addition, co-incubation with penicillamine prevented this transformation and preserved the pre-peroxynitrite migration patterns of antibodies. In cases of antibody reaction, s-nitro adduct formation could be simultaneously monitored. We found capillary electrophoresis to be ideally suited to this type of analysis. With capillary electrophoresis, we were able to simultaneously monitor the effects of peroxynitrite on large proteins and a small scavenger molecule. As a result, a complete record of the reaction was obtained within a single 15-min analysis period.

Structure activity relationships of peroxynitrite scavengers an approach to nitric oxide neurotoxicity.

Nitric oxide (NO) is made by NO synthase during the conversion of arginine to citrulline. Researchers have found that they can block the actions of excitotoxins by inhibiting NO synthase. Released from excitable cells during trauma, NO may react with superoxide to form peroxynitrite. Once formed, peroxynitrite and its products can then react with proteins, lipids and nucleic acids resulting in cell injury and death. The present study was undertaken to investigate analogs of cysteine as scavengers of peroxynitrite. Peroxynitrite scavengers were assayed by Attoflo, an automated radioimmunoassay. Briefly, peroxynitrite, in a dose-dependent manner (0.1 to 10 mM), inhibited the binding of I125 cAMP to a polyclonal antibody used in the assay of cAMP. Drugs were tested for blockade of the inhibition (90%) caused by peroxynitrite at 10 mM. Cysteine blocked the inhibition of ligand/antibody binding in a dose-dependent manner (EC50 = 3 mM). Cysteine, cysteine esters, penicillamine, penicillamine esters and cysteamine were the most effective peroxynitrite scavengers. Analogs of cysteine may thereby protect cells from nitric oxide toxicity.

Pharmacology of a mixed 5-hydroxytryptamine1A/dopamine agonist.

U-67413B (4-hydroxydipropylaminodihydrophenalene) bound with high affinity to both 5-hydroxytryptamine (HT)1A and D2-dopamine (DA) receptor sites. U-67413B depressed 5-HT and DA cell firing rates and depressed synthesis of both neurotransmitters. The drug depressed mouse body temperatures by an amount similar to that for buspirone, gepirone and ipsapirone, but less than that for 8-hydroxy-N,N-dipropyl-2-aminotetralin. In rats, it produced the 5-HT1A behavioral syndrome. In contrast to 5-HT1A agonists having DA antagonist effects, U-67413B mildly depressed rather than stimulated firing rates of noradrenaline (NA) neurons in the locus ceruleus by a non-alpha-2 receptor mechanism. In behavioral tests designed to measure anxiolytic activities, U-67413B was a slightly more effective anxiolytic than standard 5-HT1A anxiolytics (buspirone, gepirone and ipsapirone). The data are consistent with the hypothesis that effects of 5-HT1A agonists on NA neuron activity are mediated through effects on dopaminergic mechanisms, and that effects on NA neurons could modulate anxiolytic activities of 5-HT1A agonists.

Anticonvulsant and related effects of U-54494A in various seizure tests.

The anticonvulsant activity of (+-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benz am ide monohydrochloride (U-54494A), a benzamide derivative chemically related to kappa opioid receptor agonists, was investigated in three selected seizure models of experimental epilepsy. In the maximal electroshock seizure test in mice, U-54494A (ED50 28 mg/kg i.p.) was effective, with a potency somewhat less than phenobarbital. In combination with clinically used antiepileptics, especially phenobarbital and carbamazepine, the anticonvulsant activity of the latter was significantly increased. More detailed studies with phenobarbital showed additive anticonvulsant effects. The anticonvulsant activity of U-54494A was partially antagonized by naloxone. On the other hand, this compound did not elevate the pentylenetetrazol seizure threshold (at high doses a tendency of proconvulsant action was seen). Furthermore, in unrestrained rats with chronically implanted electrodes, U-54494A (> or = 10 mg/kg) significantly reduced the duration of electrically evoked hippocampal afterdischarges. However, the focal stimulation threshold was not markedly increased. With respect to the possible mode of action, whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes showed that U-54494A depressed the fast sodium inward current in a concentration- and frequency-dependent manner. In summary, our results agree with earlier reports that demonstrated marked anticonvulsant effects of U-54494A in grand mal-analogous seizure tests. Moreover, in combination with some standard antiepileptics, additive effects can be found. It is suggested that, in addition to kappa opioid and excitatory amino acid receptor related effects, modulations of Na+ membrane currents may contribute to the mechanisms of action.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes of cloned GABAA receptors.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1',2',4'- oxadiazol-3'-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5'-cyclopropyl-1',2',4'-oxadiazole-3'-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart. In particular, the 5-benzoyl analogue containing a 6-fluoro group showed no interaction with the alpha 6 beta 2 gamma 2 subtype even at the concentration of 10 microM, probably due to stabilization of the benzoyl group in the out-of-plane region by the steric and electrostatic effects of the 6-fluoro group.4. We propose that the benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype shares overlapping regions with that of the alpha 1 beta 2 gamma 2 subtype, but has a sterically restricted out-of-plane region, which may be also incompatible with the 5-phenyl group of classical benzodiazepines.

Potentiation of gamma-aminobutyric acid-induced chloride currents by various benzodiazepine site agonists with the alpha 1 gamma 2, beta 2 gamma 2 and alpha 1 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acid type A receptors.

Previous studies with cloned gamma-aminobutyric acid type A receptors expressed in human embryonic kidney cells have indicated that the alpha 1 beta 2 gamma 2 and alpha 1 gamma 2 (but not alpha 1 beta 2) subtypes have benzodiazepine sites. We found in this study that even the beta 2 gamma 2 subtype displays gamma-aminobutyric acid-induced Cl- currents that are potentiated by triazolam (a triazolobenzodiazepine). The maximal efficacy of the drug among the subtypes was highest with the alpha 1 beta 2 gamma 2 subtype, followed by the alpha 1 gamma 2 and beta 2 gamma 2 subtypes. These observations led us to compare the ability of several benzodiazepine site agonists of diverse chemical structures to potentiate Cl- currents with these subtypes. With the alpha 1 gamma 2 subtype, diazepam, alpidem, zolpidem, Cl-218872, zopiclone, U-79098 (an imidazoquinoxaline derivative), and U-90167 (a diimidazoquinazoline derivative) at 5 microM potentiated Cl- currents to essentially similar levels (slightly lower for a few ligands), compared with those with the alpha 1 beta 2 gamma 2 subtype. With the beta 2 gamma 2 subtype, the type 1 ligands zolpidem, alpidem, and Cl-218872 showed no or very low levels of potentiation, whereas less selective ligands such as diazepam, zopiclone, U-78098, and U-90167 displayed levels of Cl- current potentiation comparable to those observed with the subtypes containing the alpha 1 and gamma 2 subunits. These data indicate that, in the presence of gamma 2, beta 2 may substitute for alpha 1 in forming the benzodiazepine site of limited sensitivity to the type 1 ligands. It appears that individual ligands for benzodiazepine sites have their own sets of interacting domains, which are distributed in alpha 1 and gamma 2, and the agonistic activity of type 1 ligands may be more dependent on the alpha 1-specific domains than is that of less selective ligands.