Concentration and time dependant behavior of chlorpromazine interaction with supported bilayer lipid membrane

The interaction of chlorpromazine (CPZ) with supported bilaver lipid (dipalmitoyphosphatidylcholine) membrane (s-BLM) on the glassy carbon electrode (GCE) was investigated using cyclic voltammetry and ac impedance spectroscopy. The experimental data, based on the voltammetric response of Ru(NH3)(6)(3+) associated with the oxidation of CPZ on the electrode, indicated that the interaction of CPZ with s-BLM was concentration and time dependant. The interaction between them could be divided into three stages by the concentration of CPZ: low, middle and high concentration. At the first stage, s-BLM was not affected by CPZ and the interaction was only a penetration of a small quantity of CPZ molecule into s-BLM. At the second stage, the defects formed in s-BLM due to the penetration of more CPZ molecule into s-BLM. At the last stage, a high CPZ:lipid ratio reached in s-BLM, resulting in the solubilization of s-BLM. The interaction time had different effect at three stages.

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Molecular modeling and UV-vis spectroscopic studies on the mechanism of action of reversed chloroquine (RCQ)

Reversed chloroquine (RCQ) is a multiple ligand compound active against chloroquine-sensitive and resistant falciparum malaria. It is composed by a 4-aminoquinoline moiety (like that present in chloroquine (CQ)) joined to imipramine (IMP), a modulating agent that also showed intrinsic antiplasmodial activity against Brazilian Plasmodium falciparum isolates resistant to CQ. Molecular modeling and ultraviolet-visible spectroscopy (UV-vis) studies strongly suggest that the interaction between RCQ and heme is predominant through the quinoline moiety in a mechanism of action similar to that observed for CQ. (C) 2010 Elsevier Ltd. All rights reserved.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C 18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 mu m of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 103 less sample than using conventional methods). (C) 2008 Elsevier B.V. All rights reserved.

Revisão sistemática e meta-análise do uso de antidepressivos no transtorno de ansiedade generalizada

Revisão da Literatura: O Transtorno de Ansiedade Generalizada (TAG) é caracterizado por preocupação excessiva, persistente e incontrolável sobre diversos aspectos da vida do paciente. Tem prevalência entre 1,6% e 5,1% e índice de comorbidades de até 90,4%. As principais comorbidades são depressão maior (64%) e distimia (37%). Os antidepressivos podem ser eficazes no tratamento do TAG. A Medicina Baseada em Evidências (MBE) busca reunir a melhor evidência disponível com experiência clínica e conhecimentos de fisiopatologia. A melhor maneira disponível de síntese das evidências é a revisão sistemática e a meta-análise. Objetivos: Investigar a eficácia e tolerabilidade dos antidepressivos no tratamento do TAG através de uma revisão sistemática da literatura e meta-análise. Sumário do artigo científico: A revisão sistemática incluiu ensaios clínicos randomizados e controlados e excluiu estudos não-randomizados, estudos com pacientes com TAG e outro transtorno de eixo I. Os dados foram extraídos por dois revisores independentes e risco relativo, diferença da média ponderada e número necessário para tratamento (NNT) foram calculados. Antidepressivos (imipramina, paroxetina e venlafaxina) foram superiores ao placebo. O NNT calculado foi de 5,5. A evidência disponível sugere que os antidepressivos são superiores ao placebo no tratamento do TAG e bem tolerados pelos pacientes.

Social separation and diazepam withdrawal increase anxiety in the elevated plus-maze and serotonin turnover in the median raphe and hippocampus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antipanic procedures reduce the strychnine-facilitated wild running of rats

Wild running (WR) behavior of rats seen in response to intense acoustic stimulation of audiogenic seizure-paradigm is very similar to the panic flight and can be facilitated by subconvulsive doses of strychnine. The present work aimed to test whether antipanic procedures, such as dorsal periaqueductal gray (dPAG) lesion and imipramine treatments, affect the strychnine-facilitated WR. In study 1, six Wistar male adult rats with electrolytic lesion of dPAG had their WR completely blocked, whereas it was facilitated in 50% of sham-lesioned control rats by a dose of 0.5 mg/kg of strychnine administered intraperitoneal. This effect was not reproduced with a higher strychnine dose (1.0 mg/kg). In study 2, the effects of imipramine were investigated by testing 36 rats under a dose of strychnine that induces WR in 50% of subjects. They were assigned into three experimental groups: imipramine treatments of 5.0 and 10.0 mg/kg, and infusions of saline. All these treatments were subchronical with three intraperitoneal injections within 24h. Imipramine (10.0mg/kg) reduced the incidence of WR in comparison to the saline results. It is concluded that strychnine-facilitated WR is reduced by antipanic procedures and, therefore, can be viewed as a manifestation closely related to panic. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Use of the elevated T-maze to study anxiety in mice

We have recently suggested that the elevated T-maze (ETM) is not a useful test to study different types of anxiety in mice if a procedure similar to that originally validated for rats is employed. The present study investigated whether procedural (five exposures in the enclosed arm instead of three as originally described for rats) and structural (transparent walls instead of opaque walls) changes to the ETM leads to consistent inhibitory avoidance acquisition (IAA) and low escape latencies in mice. Results showed that five exposures to the ETM provoked consistent IAA, an effect that was independent of the ETM used. However, the ETM with transparent walls (ETMt) seemed to be more suitable for the study of conditioned anxiety (i.e. IAA) and unconditioned fear (escape) in mice, since IAA (low baseline latency with a gradual increase over subsequent exposures) and escape (low latency) profiles rendered it sensitive to the effects of anxiolytic and anxiogenic drugs. In addition to evaluation of drug effects on IAA and escape, the number of line crossings in the apparatus were used to control for locomotor changes. Results showed that whereas diazepam (1.0-2.0 mg/kg) and flumazenil (10-30 mg/kg) impaired IAA, FG 7142 (10-30 mg/kg) did not provoke any behavioral change. Significantly, none of these benzodiazepine (BDZ) receptor ligands modified escape latencies. The 5-HT1A partial receptor agonist buspirone (1.0-2.0 mg/kg) and the 5-HT releaser fenfluramine (0.15-0.30 mg/kg) impaired IAA and facilitated escape, while the full 5-HT1A receptor agonist, 8-OH-DPAT (0.05-0.1 mg/kg) and the 5-HT2B/2C receptor antagonist, SER 082 (0.5-2.0 mg/kg) failed to modify either response. mCPP (0.5-2.0 mg/kg), a 5-HT2B/2C receptor agonist, facilitated IAA but did not alter escape latency. Neither antidepressant utilized in the current study, imipramine (1.0-5.0 mg/kg) and moclobemide (3.0-10 mg/kg) affected IAA or escape performance in mice. The well-known anxiogenic drugs yohimbine (2.0-8.0 mg/kg) and caffeine (10-30 mg/kg) did not selectively affect IAA, although caffeine did impair escape latencies. Present results suggest the ETMt is useful for the study of conditioned anxiety in mice. However, upon proximal threats (e.g. open arm exposure), mice do not exhibit escape behavior as an immediate defensive strategy, suggesting that latency to leave open arm is not a useful parameter to evaluate unconditioned fear in this species. (C) 2003 Elsevier B.V. All rights reserved.

Ethopharmacological evaluation of the rat exposure test: A prey-predator interaction test

The rat exposure test (RET) is a prey (mouse)-predator (rat) situation that activates brain defensive areas and elicits hormonal and defensive behavior in the mouse. Here, we investigated possible correlations between the spatiotemporal [time spent in protected (home chamber and tunnel) and unprotected (surface) compartments and frequency of entries into the three compartments] and ethological [e.g., duration of protected and unprotected stretched-attend postures (SAP), duration of contact with the rat's compartment] measures (Experiment 1). Secondly, we investigated the effects of systemic treatment with pro- or anti-aversive drugs on the behavior that emerged from the factor analysis (Experiment 2). The effects of chronic (21 days) imipramine and fluoxetine on defensive behavior were also investigated (Experiment 3). Exp. 1 revealed that the time in the protected compartment, protected SAP and rat contacts loaded on factor 1 (defensive behavior), while the total entries and unprotected SAP loaded on factor 2 (locomotor activity). Exp. 2 showed that alprazolam (but not diazepam) selectively changed the defensive factor. Caffeine produced a mild proaversive-like effect, whereas yohimbine only decreased locomotor activity (total entries). Fluoxetine (but not imipramine) produced a weak proaversive-like effect. 5-HT1A/5-HT2 receptor ligands did not change any behavioral measure. In Exp. 3, chronic fluoxetine (but not imipramine) attenuated the defensive behavior factor without changing locomotion. Given that the defensive factor was sensitive to drugs known to attenuate (alprazolam and chronic fluoxetine) and induce (caffeine) panic attack, we suggest the RET as a useful test to assess the effects of panicolytic and panicogenic drugs. © 2012 Elsevier B.V.

Ethopharmacological analysis of the open elevated plus-maze in mice

Exposure of rodents to an open elevated plus-maze (oEPM) elicits antinociception and increases plasma corticosterone levels. However, no studies have yet assessed the defensive behaviour repertoire of animals in this modified test. In Experiment 1, factor analysis was employed to characterise the behavioural profile of mice exposed to the oEPM. Experiments 2 and 3 assessed the effects of acute alprazolam (0.5-1.5. mg/kg; diazepam 0.5-1.5. mg/kg), pentylenetetrazole (10.0-30.0. mg/kg), yohimbine (2.0-6.0. mg/kg), mCPP (0.3-3.0. mg/kg), and acute and chronic fluoxetine (10.0-30.0. mg/kg) and imipramine (1.0-15.0. mg/kg) on behaviours identified in Experiment 1. The factor analyses revealed that behaviour in the oEPM can largely (77% total variance) be accounted for in terms of 3 factors: factor 1 ('. depth exploration'; e.g. head-dipping on the arms), factor 2 ('. cautious exploration of arms'; e.g. flatback approach), and factor 3 ('. risk assessment'; stretched attend postures - SAP). Experiments 2 and 3 showed that, over the dose range used, alprazolam selectively attenuated all measures of defensiveness. Similar, though more modest, effects were seen with diazepam. Confirming the intensity of the emotional response to the oEPM (nociceptive, endocrine and behavioural), relatively few significant behavioural changes were seen in response to the anxiogenic compounds tested. Although acute fluoxetine or imipramine treatment failed to modify behaviour in the oEPM, chronic fluoxetine (but not chronic imipramine) attenuated total flat back approach and increased head dipping outside the central square. Together, the results indicate that the oEPM induces behavioural defensive responses that are sensitive to alprazolam and chronic fluoxetine. © 2013 Elsevier B.V.

Validação farmacológica da preferência claro-escuro em Danio rerio

A ansiedade é uma desordem complexa e com grande relevância clínica, cujo estudo com modelos animais é importante para pesquisar sobre seus mecanismos e drogas para o seu tratamento. O zebrafish figura como um potencial modelo animal para pesquisas farmacológicas da ansiedade. Um modelo de ansiedade é a preferência claro-escuro, que já foi validado comportamentalmente em zebrafish, contudo necessita de uma validação farmacológica. Objetiva-se descrever a sensibilidade da preferência claro-escuro em zebrafish adultos para as drogas mais utilizadas na clínica da ansiedade, foram administradas pela imersão do animal na solução: Benzodiazepínicos (Clonazepam); Agonistas parciais 5-HT1A (Buspirona); Antidepressivo tricíclico (Imipramina); Antidepressivo ISRS (Fluoxetina e Paroxetina); Antipsicóticos (Haloperidol e Risperidona); Psicostimulante (Dietilpropiona); Beta bloqueadores (Propranolol) e Depressores do SNC (Etanol). Os parâmetros analisados foram o tempo despendido pelo animal no ambiente escuro, o tempo da primeira latência e número de alternâncias. O clonazepam administrado por 300s aumentou o tempo no escuro na menor concentração e reduziu a atividade locomotora, a administração durante 600s da concentração intermediária diminuiu o tempo no escuro e da primeira latência, assim como aumentou a atividade locomotora, indicando efeito ansiolítico. A buspirona aumentou o tempo de permanência no escuro provavelmente devido a redução da atividade motora. A imipramina e a fluoxetina aumentaram o tempo no escuro e da primeira latência e diminuíram o número de alternâncias, indicando ação ansiogênica. A paroxetina não alterou o tempo no escuro, entretanto aumentou o tempo da primeira latência e diminuiu a atividade locomotora. O haloperidol diminuiu a ansiedade na menor concentração, curiosamente aumentou a atividade motora na maior concentração, ao contrário da risperidona que diminuiu a atividade na maior concentração. A dietilpropriona não modificou o tempo no escuro, mas aumentou o tempo da primeira latência e diminuiu a atividade motora apenas na menor concentração. O propranolol reduziu somente o tempo no escuro. O etanol foi efetivo na redução da ansiedade com a concentração intermediária e diminuiu a atividade locomotora em uma concentração menor Os dados corroboram com relatos da literatura em Danio rerio tanto neste modelo em administração intraperitoneal como em outros modelos por administração hídrica e em roedores, quando foi possível a comparação.

Administração subcrônica de nicotina modifica as respostas neuroendócrina e neuroquímica induzidas pelo teste de natação forçada

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Investigação do envolvimento de subtipos de adrenoceptores α1 no efeito anti-imobilidade da imipramina no teste de suspensão pela cauda

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

DIFFERENTIAL INVOLVEMENT OF DORSAL RAPHE SUBNUCLEI IN THE REGULATION OF ANXIETY- AND PANIC-RELATED DEFENSIVE BEHAVIORS

A wealth of evidence indicates that the dorsal raphe nucleus (DR) is not a homogenous structure, but an aggregate of distinctive populations of neurons that may differ anatomically, neurochemically and functionally. Other findings suggest that serotonergic neurons within the mid-caudal and caudal part of the DR are involved in anxiety processing while those within the lateral wings (IwDR) and ventrolateral periaqueductal gray (vIPAG) are responsive to panic-evoking stimuli/situations. However, no study to date has directly compared the activity of 5-HT and non-5HT neurons within different subnuclei of the DR following the expression of anxiety- and panic-related defensive responses. In the present investigation, the number of doubly immunostained cells for Fos protein and tryptophan hydroxylase, a marker of serotonergic neurons, was assessed within the rat DR, median raphe nucleus (MRN) and PAG following inhibitory avoidance and escape performance in the elevated T-maze, behaviors associated with anxiety and panic, respectively. Inhibitory avoidance, but not escape, significantly increased the number of Fos-expressing serotonergic neurons within the mid-caudal part of the dorsal subnucleus, caudal and interfascicular subnuclei of the DR and in the MRN. Escape, on the other hand, caused a marked increase in the activity of non-5HT cells within the IwDR, vIPAG, dorsolateral and dorsomedial columns of the PAG. These results strongly corroborate the view that different subsets of neurons in the DR are activated by anxiety- and panic-relevant stimuli/situations, with important implications for the understanding of the pathophysiology of generalized anxiety and panic disorders. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Changes in hippocampal gene expression by 7-nitroindazole in rats submitted to forced swimming stress

Nitric oxide (NO) is an atypical neurotransmitter that has been related to the pathophysiology of major depression disorder. Increased plasma NO levels have been reported in depressed and suicidal patients. Inhibition of neuronial nitric oxide synthase (nNOS), on the other hand, induces antidepressant effects in clinical and pre-clinical trials. The mechanisms responsible for the antidepressant-like effects of nNOS inhibitors, however, are not completely understood. In this study, genomic and proteomic analyses were used to investigate the effects of the preferential nNOS inhibitor 7-nitroindazole (7-NI) on changes in global gene and protein expression in the hippocampus of rats submitted to forced swimming test (FST). Chronic treatment (14 days, i.p.) with imipramine (15 mg/kg daily) or 7-NI (60 mg/kg daily) significantly reduced immobility in the FST. Saturation curves for Serial analysis of gene expression libraries showed that the hippocampus of animals submitted to FST presented a lower number of expressed genes compared to non-FST stressed groups. Imipramine, but not 7-NI, reverted this effect. GeneGo analyses revealed that genes related to oxidative phosphorylation, apoptosis and survival controlled by HTR1A signaling and cytoskeleton remodeling controlled by Rho GTPases were significantly changed by FST. 7-NI prevented this effect. In addition, 7-NI treatment changed the expression of genes related to transcription in the cAMP response element-binding pathway. Therefore, this study suggests that changes in oxidative stress and neuroplastic processes could be involved in the antidepressant-like effects induced by nNOS inhibition.