Potentiation of the depressant effect of alcohol by flunitrazepam in rats: an electrocorticographic, respiratory and electrocardiographic study.

Alcohol, a widely commercialized psychotropic drug, and the benzodiazepine Flunitrazepam, an anxiolytic widely prescribed for patients with anxiety and insomnia problems, are well known drugs and both act on the central nervous system. The misuse and the association of these two drugs are public health concerns in several countries and could cause momentary, long-lasting and even lethal neurophysiological problems due to the potentiation of their adverse effects in synergy. The present study observed the result of the association of these drugs on electrophysiological responses in the brain, heart, and respiratory rate in Wistar rats. 8 experimental groups were determined: control, one alcohol group (20% at a dose of 1 ml/100 g VO), three Flunitrazepam groups (doses 0.1; 0.2 and 0.3 mg/kg) and three alcohol-Flunitrazepam groups (20% at a dose of 1 ml/100 g VO of alcohol, combined with 0.1; 0.2 and 0.3 mg/kg of Flunitrazepam, respectively). The results showed that there was a more pronounced reduction in alpha and theta wave power in the alcohol-Flunitrazepam groups, a decrease in the power of beta oscillations and greater sedation. There was a progressive decrease in respiratory rate linked to the increase of Flunitrazepam dose in the alcohol-Flunitrazepam associated administration. It was observed alteration in heart rate and Q-T interval in high doses of Flunitrazepam. Therefore, we conclude that the association alcohol-Flunitrazepam presented deepening of depressant synergistic effects according to the increase in the dose of the benzodiazepine, and this could cause alterations in low frequency brain oscillations, breathing, and hemodynamics of the patient.

Anxiety-like Behavior and GABAAR/BDZ Binding Site Response to Progesterone Withdrawal in a Stress-Vulnerable Strain, the Wistar Kyoto Rats.

Stress susceptibility could play a role in developing premenstrual anxiety due to abnormalities in the hypothalamus-pituitary-adrenal (HPA) axis and impairments in the GABAA receptors' benzodiazepine (BDZ) site. Hence, we studied the stress-vulnerable Wistar Kyoto rat strain (WKY) to evaluate progesterone withdrawal (PW) effects on anxiety, HPA axis response, and to explore indicators of GABAA functionality in the BDZ site. For five days, ovariectomized WKY rats were administered 2.0 mg/kg of progesterone. Twenty-four hours after the last administration, rats were tested in the anxiety-like burying behavior test (BBT) or elevated plus maze test (EPM), and corticosterone was determined. [3H]Flunitrazepam binding autoradiography served as the BDZ binding site index of the GABAA receptor in amygdala nuclei and hippocampus's dentate gyrus (DG). Finally, different doses of diazepam in PW-WKY rats were tested in the BBT. PW induced anxiety-like behaviors in both BBT and EPM compared with No-PW rats. PW increased corticosterone, but was blunted when combined with PW and BBT. PW increased [3H]Flunitrazepam binding in the DG and central amygdala compared with No-PW rats. Diazepam at a low dose induced an anxiogenic-like response in PW rats, suggesting a paradoxical response to benzodiazepines. Overall, PW induced anxiety-like behavior, a blunted HPA axis response, and higher GABAAR/BZD binding site sensitivity in a stress-vulnerable rat strain. These findings demonstrate the role of stress-susceptibility in GABAAR functionality in a preclinical approximation of PMDD.

Delayed creatine supplementation counteracts reduction of GABAergic function and protects against seizures susceptibility after traumatic brain injury in rats.

Traumatic brain injury (TBI) is a devastating disease frequently followed by behavioral disabilities including post-traumatic epilepsy (PTE). Although reasonable progress in understanding its pathophysiology has been made, treatment of PTE is still limited. Several studies have shown the neuroprotective effect of creatine in different models of brain pathology, but its effects on PTE is not elucidated. Thus, we decided to investigate the impact of delayed and chronic creatine supplementation on susceptibility to epileptic seizures evoked by pentylenetetrazol (PTZ) after TBI. Our experimental data revealed that 4 weeks of creatine supplementation (300 mg/kg, p.o.) initiated 1 week after fluid percussion injury (FPI) notably increased the latency to first myoclonic and tonic-clonic seizures, decreased the time spent in tonic-clonic seizure, seizure intensity, epileptiform discharges and spindle oscillations induced by a sub-convulsant dose of PTZ (35 mg/kg, i.p.). Interestingly, this protective effect persists for 1 week even when creatine supplementation is discontinued. The anticonvulsant effect of creatine was associated with its ability to reduce cell loss including the number of parvalbumin positive (PARV+) cells in CA3 region of the hippocampus. Furthermore, creatine supplementation also protected against the reduction of GAD67 levels, GAD activity and specific [3H]flunitrazepam binding in the hippocampus. These findings showed that chronic creatine supplementation may play a neuroprotective role on brain excitability by controlling the GABAergic function after TBI, providing a possible new strategy for the treatment of PTE.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage.

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.

The Effect of Flunitrazepam (Rohypnol(®) ) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology.

This study investigated the potential effects of flunitrazepam (known as "date rape drug") on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal-Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.

Notas de farmacología: estudios de disolución de medicamentos del sistema nervioso central / Pharmacology notes: drug dissolution studies of the central nervous system

El propósito de los estudios in vitro es comparar las características de liberación del principio activo contenido en una forma farmacéutica sólida oral mediante la cantidad, o porcentaje de la dosis, disuelta en función del tiempo en condiciones controladas y validadas. Se realizó una comparación de disolución in vitro de flunitrazepam (Rohypnol®, Somnidual® e Inervon®) y de lamotrigina (Lamictal® y Epilepax®). Para los perfiles de disolución de flunitrazepam, una de las marcas comerciales tuvo un perfil de disolución muy diferente a las otras dos marcas. Para el caso de lamotriginano existió diferencia para aquellos pH que se consideran fundamentales para la disolución del comprimido en el tracto gastrointestinal. Los resultados obtenidos de los estudios in vitro son simplemente orientadores, permiten tan solo guiar la puesta en marcha del ensayo de bioequivalencia entre el test evaluado in vitro y la referencia utilizada en el mismo ensayo. No obstante, estos resultados pueden constituirse en un elemento de apoyo a la presunción de bioinequivalencia como causa de un evento adverso (ineficacia o toxicidad) detectado en los programas de farmacovigilancia actualmente en marcha.

Notas de farmacología: estudios de disolución de medicamentos del sistema nervioso central / Pharmacology notes: drug dissolution studies of the central nervous system

El propósito de los estudios in vitro es comparar las características de liberación del principio activo contenido en una forma farmacéutica sólida oral mediante la cantidad, o porcentaje de la dosis, disuelta en función del tiempo en condiciones controladas y validadas. Se realizó una comparación de disolución in vitro de flunitrazepam (Rohypnol®, Somnidual® e Inervon®) y de lamotrigina (Lamictal® y Epilepax®). Para los perfiles de disolución de flunitrazepam, una de las marcas comerciales tuvo un perfil de disolución muy diferente a las otras dos marcas. Para el caso de lamotriginano existió diferencia para aquellos pH que se consideran fundamentales para la disolución del comprimido en el tracto gastrointestinal. Los resultados obtenidos de los estudios in vitro son simplemente orientadores, permiten tan solo guiar la puesta en marcha del ensayo de bioequivalencia entre el test evaluado in vitro y la referencia utilizada en el mismo ensayo. No obstante, estos resultados pueden constituirse en un elemento de apoyo a la presunción de bioinequivalencia como causa de un evento adverso (ineficacia o toxicidad) detectado en los programas de farmacovigilancia actualmente en marcha.

Synaptosomal membrane-based Langmuir-Blodgett films: a platform for studies on γ-aminobutyric acid type A receptor binding properties.

In this work we used Langmuir-Blodgett films (LB) as model membranes to study the effect of molecular packing on the flunitrazepam (FNZ) accessibility to the binding sites at the GABAA receptor (GABAA-R). Ligand binding data were correlated with film topography analysis by atomic force microscopy images (AFM) and SDS-PAGE. Langmuir films (LF) were prepared by the spreading of synaptosomal membranes (SM) from bovine brain cortex at the air-water interface. LBs were obtained by the transference, at 15 or 35 mN/m constant surface pressure (π), of one (LB15/1c and LB35/1c) or two (LB35/2c) LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference was performed in a serial manner, which allowed the accumulation of a great number of samples. SDS-PAGE clearly showed a 55 kDa band characteristic of GABAA-R subunits. Detrended fluctuation analysis of topographic data from AFM images exhibited a single slope value (self-similarity parameter α) in CONglass and a discontinuous slope change in the α value at an autocorrelation length of ∼100 nm in all LB samples, supporting the LF transference to the substrate. AFM images of CONglass and LB15/1c exhibited roughness and average heights that were similar between measurements and significantly lower than those of LB35/1c and LB35/2c, suggesting that the substrate coverage in the latter was more stable than in LB15/1c. While [(3)H]FNZ binding in LB15/1c did not reach saturation, in LB35/1c the binding kinetics became sigmoid with a binding affinity lower than in the SM suspension. Our results highlight the π dependence of both binding and topological data and call to mind the receptor mechanosensitivity. Thus, LB films provide a tool for bionanosensing GABAA-R ligand binding as well as GABAA-R activity modulation induced by the environmental supramolecular organization.

Effect of synthetic steroids on GABAA receptor binding in rat brain.

Neuroactive steroids, like allopregnanolone (A) and pregnanolone (P), bind to specifics sites on the GABAA receptor complex and modulate receptor function. They are capable to inhibit or stimulate the binding of GABAA receptor-specific ligands, like t-butyl-bicyclophosphorothionate, flunitrazepam and muscimol. We have previously characterized a set of oxygen-bridged synthetic steroids (SS) analogs to A or P using synaptosomes. Considering that the subunit composition of the GABAA receptor throughout the central nervous system affects the magnitude of the modulation of the GABAA receptor by NAS, we evaluated the action of two selected SS, in brain sections containing the cerebral cortex (CC) and hippocampus (HC) using quantitative receptor autoradiography. Both SS affected the binding of the three ligands in a similar way to A and P, with some differences on certain CC layers according to the ligand used. One of the SS, the 3α-hydroxy-6,19-epoxypregn-4-ene-20-one (compound 5), behaved similarly to the natural neuroactive steroids. However, significant differences with compound 5 were observed on the HC CA2 region, making it steroid suitable for a specific action. Those differences may be related to structural conformation of the SS and the subunits' composition present on the receptor complex.

Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons.

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.

Central α- and ß-thujone: similar anxiogenic-like effects and differential modulation on GABAA receptors in neonatal chicks.

The convulsant effects of α-thujone are attributed to inhibitory actions on the GABAA receptor. We investigated, for the first time, the effects of α-thujone or ß-thujone administrated centrally on the fear/anxiety behaviour of 3-day-old chicks in an Open Field and their modulation on the GABAA receptor. Higher doses were convulsant by eliciting a toxic and excitatory action, with the results showing that a dose of 78 nmol of either of the two diastereoisomers had an anxiogenic-like effect observed as an increased latency to ambulate and a reduced locomotor activity in an Open Field. Nevertheless, only the central administration of α-thujone reversed the increase induced by acute stress in the flunitrazepam-sensitive GABAA receptor recruitment. These findings demonstrated that α-thujone, when intracerebroventricularly administered, suppressed the GABAA receptor recruitment induced by acute stress, maybe due to α-thujone blocking the benzodiazepine binding site or another site of the GABAA complex. However, it should not be discarded that acute stress associated with novelty may have induced the recruitment of a subpopulation of GABAA receptors more sensitive to α-thujone than to the constitutive receptors, or that this monoterpene could have inhibited any protein or enzyme trafficking that modulated the phosphorylation of the receptor involved in the turnover of GABAA receptor. ß-Thujone showed behavioural effects similar to its diastereoisomer α-thujone. However, its action mechanism may have been mediated by other neurotransmitter systems, such as the serotonergic one or by a different biological effectiveness due to a distinct stereochemistry at the specific site of the GABAA receptor.

GABA-induced uncoupling of GABA/benzodiazepine site interactions is mediated by increased GABAA receptor internalization and associated with a change in subunit composition.

Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24-25h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5-10min (t½=3min) initiates a process that results in uncoupling hours later (t½=12h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of α3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABA-induced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism that negatively regulates GABAergic transmission under physiological conditions in which synaptic GABAA receptors are transiently activated for several minutes.

Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo

Flunitrazepam (FNZ) is a sedative benzodiazepine prescribed for the short-term treatment of insomnia. However, there are concerns regarding possible carcinogenic or genotoxic effects of this medicine. Thus, the aim of this study was to evaluate the cytotoxic, clastogenic and aneugenic effects of FNZ in hepatoma cells from Rattus norvegicus (HTC) in vitro and in bone marrow cells of Wistar rats in vivo. These effects were examined in vitro following treatment with 0.2, 1.0, 5.0 or 10 μg/mL FNZ using a micronucleus test with a cytokinesis block or in vivo using a chromosomal aberration test following treatment with 7, 15 or 30 μg/mL/kg body weight. The results showed that the benzodiazepine concentrations tested were not cytotoxic, aneugenic or clastogenic. However, considering the adverse effects of using this benzodiazepine, more studies are required.

Flunitrazepam (FNZ) é um sedativo benzodiazepínico prescrito para o tratamento da insônia em curto prazo. Entretanto, existe a preocupação com relação aos possíveis efeitos carcinogênicos ou genotóxicos causados por este fármaco. Então, o objetivo deste estudo foi avaliar os efeitos citotóxicos, clastogênicos e aneugênicos do FNZ em células de hepatoma de Rattus norvegicus (HTC) in vitro e em células de medula óssea de ratos Wistar in vivo. Foram testadas as concentrações de 0,2, 1,0 e 10 μg/mL de FNZ pelo teste do micronúcleo com bloqueio de citocinese in vitro e 7, 15 e 30 μg/mL/kg de peso corpóreo para o teste de aberração cromossômica in vivo. Os resultados mostraram que as concentrações do benzodiazepínico testadas não foram citotóxicas, aneugênicas ou clastogênicas. Entretanto, considerando os efeitos adversos do uso deste benzodiazepínico, mais estudos são necessários.

Anxiolytic-like effect of lavender essential oil inhalation in mice: participation of serotonergic but not GABAA/benzodiazepine neurotransmission.

ETHNOPHARMACOLOGICAL RELEVANCE: Lavandula angustifolia (lavender) inhalation has been used in folk medicine for the treatment of anxiety, and clinical and animal studies have corroborated its anxiolytic effect, although its mechanism of action is still not fully understood. AIMS OF THE STUDY: The objective of the present study was to determine whether the GABAA/benzodiazepine complex or serotonin neurotransmission mediates the anxiolytic-like effect of lavender essential oil. MATERIALS AND METHODS: Male Swiss mice were subjected to the marble-burying test after being exposed to the aroma of lavender essential oil (1-5%), amyl acetate (5%; used as a behaviorally neutral odor), or distilled water for 15 min via inhalation. Additionally, the effect of 5% lavender essential oil was also evaluated in mice subjected to the elevated plus maze. GABAA/benzodiazepine mediation was evaluated by pretreating the mice with the GABAA receptor antagonist picrotoxin before the marble burying test and [(3)H]flunitrazepam binding to the benzodiazepine site on the GABAA receptor. Serotonergic mediation was studied by pretreating the mice with O-methyl-[3H]-N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635), a serotonin 5-HT1A receptor antagonist before the marble burying test. We also evaluated changes in the pharmacologically induced serotonin syndrome and the effects of combined administration of subeffective doses of lavender essential oil and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). RESULTS: Lavender essential oil (1-5%) decreased the number of marbles buried compared with the control and amyl acetate groups. In the elevated plus maze, 5% lavender essential oil inhalation increased the percentage of time spent on and number of entries into the open arms compared with controls. No effect was seen in the number of closed arm entries or number of beam interruptions in the automated activity chamber. Pretreatment with the GABAA receptor antagonist picrotoxin (0.5mg/kg) did not modify the behavioral effect of 5% lavender essential oil in the marble-burying test. Lavender essential oil also did not alter [(3)H]flunitrazepam binding to the benzodiazepine site on the GABAA receptor. Pretreatment with the serotonin 5-HT1A receptor antagonist WAY100635 (3mg/kg) blocked the anxiolytic-like effect of lavender essential oil and the 5-HT1A receptor agonist 8-OH-DPAT (3mg/kg). A combination of ineffective doses of 8-OH-DPAT (0.5mg/kg) and lavender essential oil (0.1%) reduced the number of marbles buried. Finally, 5% lavender essential oil attenuated the serotonin syndrome induced by 40 mg/kg fluoxetine plus 80 mg/kg 5-hydroxytryptophan. CONCLUSIONS: These results indicate an important role for the serotonergic system in the anxiolytic-like effect of lavender essential oil.

Recruitment of GABA(A) receptors and fearfulness in chicks: modulation by systemic insulin and/or epinephrine.

One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation fearfulness.

Probing the combined effect of flunitrazepam and lidocaine on the stability and organization of bilayer lipid membranes. A differential scanning calorimetry and dynamic light scattering study.

Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔHp, ΔHm, Tp, Tm) and decreased the cooperativity of the main-transition (ΔT(1/2,m)). FNZ decreased ΔHm and, at least up to 59 µM, also decreased ΔHp. In conjunction with LDC, FNZ induced a recovery of ∆T(1/2,m) control values and increased ΔHm even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC-LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid-LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug-membrane binding on LDC-FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.

Coupling between GABA(A)-R ligand-binding activity and membrane organization in ß-cyclodextrin-treated synaptosomal membranes from bovine brain cortex: new insights from EPR experiments.

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with ß-cyclodextrin (ß-CD). The mere pre-incubation (PI) at 37°C accompanying the ß-CD treatment was an underlying source of perturbations increasing [(3)H]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4°C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the ß-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not ß-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with ß-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, ß-CD is not completely eliminated from the system through centrifugation washings. It was concluded that ß-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual ß-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

N,N'-dicyclohexylsulfamide and N,N'-diphenethylsulfamide are anticonvulsant sulfamides with affinity for the benzodiazepine binding site of the GABA(A) receptor and anxiolytic activity in mice.

A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABA(A) receptor. The most active compounds, N,N'-dicyclohexylsulfamide (7) and N,N'-diphenethylsulfamide (10), competitively inhibited the binding of [(3)H]-flunitrazepam to the benzodiazepine binding site with K(i)±SEM values of 27.7±4.5µM (n=3) and 6.0±1.2µM (n=3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound 7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3mg/kg) and the plus-maze assay (at 1 and 3mg/kg). Compound 10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1mg/kg. Locomotor activity of mice was not modified by compound 7 or 10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABA(A) receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders.

Rats with different thresholds for DMCM-induced clonic convulsions differ in the sleep-time of diazepam and [(3)H]-Ro 15-4513 binding.

The current study investigated the possible inherent relationship between convulsions and sleep involving the GABA(A)/benzodiazepine site complex. The aim of this study was to determine if rats with high (HTR) and low (LTR) thresholds for clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ in the following aspects: (1) sensitivity to the hypnotic effects of the GABA(A) positive allosteric modulators diazepam, pentobarbital and ethanol and (2) in the binding of [(3)H]-flunitrazepam, a benzodiazepine agonist, measured by autoradiography, and [(3)H]-Ro 15-4513, a benzodiazepine partial inverse agonist, to membranes from discrete brain regions. The LTR subgroup presented a shorter diazepam-induced sleeping time compared to that of the HTR subgroup. Biochemical assays revealed that the LTR subgroup did not differ in [(3)H]-flunitrazepam binding compared to the HTR subgroup. With respect to the binding of [(3)H]-Ro 15-4513, the LTR subgroup had higher binding in the brainstem and lower binding in the striatum compared to the HTR subgroup. These results suggest that differences in the benzodiazepine site on the GABA(A) receptor may underlie the susceptibility to DMCM-induced convulsions and sensitivity to the hypnotic effect of diazepam.

Participation of the GABAergic system on the glutamate release of frontal cortex synaptosomes from Wistar rats with experimental autoimmune encephalomyelitis.

We previously found that the glutamate release was decreased in synaptosomes from rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Various other reports have shown a deficit in the expression of proteins associated with GABAergic neurotransmission in the neocortex of patients with multiple sclerosis and it was also demonstrated that the activation of GABAA receptors leads to an inhibition of glutamate release. Now, in order to evaluate the events that may affect the neuronal function in EAE synaptosomes, we analyzed the participation of the GABAergic system in glutamate release and in the flunitrazepam-sensitive GABAA receptor density. This revealed alterations in the GABAergic system of the frontal cortex synaptosomes from EAE animals. GABA induced a decrease in the 4-aminopyridine-evoked glutamate release in control synaptosomes which was abolished by picrotoxin, a GABAA receptor antagonist. In contrast, synaptosomes from EAE rats showed a loss in the inhibition of glutamate release mediated by GABA. Furthermore, the flunitrazepam-sensitive GABAA receptor density was decreased during the acute stage of the disease in synaptosomes from EAE rats. We also observed a loss of inhibition in the Ca2+-dependent phosphorylation of synapsin I mediated by GABA in nerve terminals from EAE animals, which could explain the loss of GABAergic regulation on evoked glutamate release. The changes observed in the GABAA receptor density as well as the loss of GABAergic inhibition of glutamate release were partially reverted in cortical synaptosomes from recovered EAE animals. These results suggest that the decrease in the flunitrazepam-sensitive GABAA receptor density may explain the observed failure of GABAergic regulation in the glutamate release of synaptosomes from EAE rats, which might contribute to the appearance of clinical symptoms and disease progression.