Clinical Study of Flumazenil Antagonizing Remimazolam on Nausea and Vomiting After Gynecologic Day Surgery.

Purpose: To evaluate the effect of flumazenil antagonizing remimazolam on postoperative nausea and vomiting (PONV) after gynecologic day surgery. Patients and Methods: 141 cases of gynaecological daycase surgery patients in Weifang People's Hospital were selected, randomized into group F (flumazenil group, 71 cases) and group C (control group, 70 cases). Dexamethasone 5 mg, flurbiprofen axetil 50 mg, and droperidol 1 mg were given intravenously before induction of anesthesia in both groups. Anesthesia induction: Remimazolam 0.25mg / kg was injected within 1 minute. After the patient fell asleep, mivacurium chloride 0.2mg / kg was injected for 30 seconds and alfentanil 20ug / kg was injected for 30 seconds. Anesthesia maintenance: Remimazolam 1mg/kg/h and alfentanil 40ug/kg/h were continuously pumped by micro pump. Stopping the injection of remimazolam and alfentanil at the end of the operation. Flumazenil 0.2 mg was given to antagonize remimazolam in group F after 1 minute. Group C was given an equal volume of saline. The incidence of PONV in the postoperative PACU and over a 24-hour period, patient awakening time, and general patient information were recorded. Results: The incidence of PONV in both groups within 24 hours was 50.70% in group F was significantly higher than 32.86% in group C. The difference was statistically significant (P < 0.05). The incidence of PONV in the PACU was 5.6% in group F and 8.6% in group C. The difference was not statistically significant (p > 0.05). Conclusion: Flumazenil antagonism of remimazolam increases the incidence of PONV within 24 hours in gynecologic day surgery patients and has no significant effect on the incidence of PONV in the PACU.

Anxiolytic effects of N-(4,5-dihydro-5-oxo-1,2-dithiolo-[4,3,b]-pyrrole-6-yl)-N-methylformamide, a pyrroloformamide isolated from a marine Streptomyces sp., in adult zebrafish by the 5-HT system.

General anxiety disorders are among the most prevalent mental health problems worldwide. The emergence and development of anxiety disorders can be due to genetic (30-50%) or non-genetic (50-70%) factors. Despite medical progress, available pharmacotherapies are sometimes ineffective or can cause undesirable side effects. Thus, it becomes necessary to discover new safe and effective drugs against anxiety. This study evaluated the anxiolytic effect in adult zebrafish (Danio rerio) of a natural pyrroloformamide (PFD), N-(4,5-dihydro-5-oxo-1,2-dithiolo-[4,3,b]-pyrrole-6-yl)-N-methylformamide, isolated from a Streptomyces sp. bacterium strain recovered from the ascidian Eudistoma vannamei. The complete structure of PFD was determined by a detailed NMR analysis, including 1H-13C and 1H-15N-HBMC data. In addition, conformational and DFT computational studies also were performed. A group of fishes (n = 6) was treated orally with PFD (0.1, 0.5 and 1.0 mg/mL; 20 µL) and subjected to locomotor activity and light/dark tests, as well as, acute toxicity 96 h. The involvement of the GABAergic and serotonergic (5-HT) systems was investigated using flumazenil (a silent modulator of GABA receptor) and 5-HT1, 5-HT2A/2C and 5-HTR3A/3B receptors antagonists, known as pizotifen, granisetron and cyproheptadine, respectively. PFD was nontoxic, reduced locomotor activity and promoted the anxiolytic effect in zebrafish. Flumazenil did not inhibit the anxiolytic effect of the PFD via the GABAergic system. This effect was reduced by a pretreatment with pizotifen and granisetron, and was not reversed after treatment with cyproheptadine. Molecular docking and dynamics studies confirmed the interaction of PFD with the 5-HT receptor.Communicated by Ramaswamy H. Sarma.

Pyrroloformamide (PFD), isolated from the marine Streptomyces sp. associated ascidian Eudistoma vannamei, showed no toxicity in adult zebrafish but reduced its locomotor activity.The structural elucidation of PFD was determined by the analysis of 1D and 2D NMR and HRESIMS data.The density functional theory (DFT) study confirmed the existence of two conformers as determined by NMR spectra.The serotonergic system modulated the anxiolytic effect of PFD via the 5-HT receptor in adult zebrafish.Molecular docking and dynamics studies confirmed the interaction of PFD with the 5-HT receptor.

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

Antiseizure Effects of Peganum harmala L. and Lavandula angustifolia.

Peganum harmala L. and Lavandula angustifolia are two traditional herbs with probable antiseizure effects. This study evaluated the effects of these two herbal extracts on pentylenetetrazol- (PTZ-) induced seizures in mice. We prepared hydroalcoholic extracts using P. harmala seeds and the aerial parts of L. angustifolia and then randomly divided 190 mice into 19 groups. Normal saline (10 mg/kg), diazepam (2 mg/kg), P. harmala (2.5, 5, 10, 15, 30, 45, and 60 mg/kg), and L. angustifolia (200, 400, 600, and 800 mg/kg) were intraperitoneally (IP) administrated 30 min before an IP administration of PTZ (90 mg/kg). Animals were observed for behavioral changes for one hour. In addition, the effects of flumazenil and naloxone on the antiseizure activity of P. harmala and L. angustifolia were assessed. P. harmala showed antiseizure activity at the dose of 10 mg/kg; it prolonged the seizure latency and decreased the seizure duration. The mortality protection rate was 90% for this herbal extract. L. angustifolia (600 mg/kg) prolonged the seizure latency and decreased both seizure duration and mortality. Neither flumazenil nor naloxone significantly reversed the antiseizure activities of P. harmala and L. angustifolia. In mice, the hydroalcoholic extracts of P. harmala and L. angustifolia showed antiseizure activity against PTZ-induced seizures. We could not delineate the exact antiseizure mechanisms of these extracts in the current study.

Search for Novel Therapies for Essential Tremor Based on Positive Modulation of α6-Containing GABAA Receptors.

Background: Prior work using GABAA receptor subunit knockouts and the harmaline model has indicated that low-dose alcohol, gaboxadol, and ganaxolone suppress tremor via α6ßδ GABAA receptors. This suggests that drugs specifically enhancing the action of α6ßδ or α6ßγ2 GABAA receptors, both predominantly expressed on cerebellar granule cells, would be effective against tremor. We thus examined three drugs described by in vitro studies as selective α6ßδ (ketamine) or α6ßγ2 (Compound 6, flumazenil) receptor modulators. Methods: In the first step of evaluation, the maximal dose was sought at which 6/6 mice pass straight wire testing, a sensitive test for psychomotor impairment. Only non-impairing doses were used to evaluate for anti-tremor efficacy in the harmaline model, which was assessed in wildtype and α6 subunit knockout littermates. Results: Ketamine, in maximally tolerated doses of 2.0 and 3.5 mg/kg had minimal effect on harmaline tremor in both genotypes. Compound 6, at well-tolerated doses of 1-10 mg/kg, effectively suppressed tremor in both genotypes. Flumazenil suppressed tremor in wildtype mice at doses (0.015-0.05 mg/kg) far lower than those causing straight wire impairment, and did not suppress tremor in α6 knockout mice. Discussion: Modulators of α6ßδ and α6ßγ2 GABAA receptors warrant attention for novel therapies as they are anticipated to be effective and well-tolerated. Ketamine likely failed to attain α6ßδ-active levels. Compound 6 is an attractive candidate, but further study is needed to clarify its mechanism of action. The flumazenil results provide proof of principle that targeting α6ßγ2 receptors represents a worthy strategy for developing essential tremor therapies. Highlights: We tested for harmaline tremor suppression drugs previously described as in vitro α6ßδ or α6ßγ2 GABAA receptor-selective modulators. Well-tolerated flumazenil doses suppressed tremor in α6-wildtype but not α6-knockout mice. Compound 6 and ketamine failed to display this profile, likely from off-target effects. Selective α6 modulators hold promise as tremor therapy.

In Vivo Neuropharmacological Effects of Neophytadiene.

Neophytadiene (NPT) is a diterpene found in the methanolic extracts of Crataeva nurvala and Blumea lacera, plants reported with anxiolytic-like activity, sedative properties, and antidepressant-like actions; however, the contribution of neophytadiene to these effects is unknown. This study determined the neuropharmacological (anxiolytic-like, antidepressant-like, anticonvulsant, and sedative) effects of neophytadiene (0.1-10 mg/kg p.o.) and determined the mechanisms of action involved in the neuropharmacological actions using inhibitors such as flumazenil and analyzing the possible interaction of neophytadiene with GABA receptors using a molecular docking study. The behavioral tests were evaluated using the light-dark box, elevated plus-maze, open field, hole-board, convulsion, tail suspension, pentobarbital-induced sleeping, and rotarod. The results showed that neophytadiene exhibited anxiolytic-like activity only to the high dose (10 mg/kg) in the elevated plus-maze and hole-board tests, and anticonvulsant actions in the 4-aminopyridine and pentylenetetrazole-induced seizures test. The anxiolytic-like and anticonvulsant effects of neophytadiene were abolished with the pre-treatment with 2 mg/kg flumazenil. In addition, neophytadiene showed low antidepressant effects (about 3-fold lower) compared to fluoxetine. On other hand, neophytadiene had no sedative or locomotor effects. In conclusion, neophytadiene exerts anxiolytic-like and anticonvulsant activities with the probable participation of the GABAergic system.

Commonly Used Therapeutics Associated with Changes in Arousal Inhibit GABAAR Activation.

GABAA receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABAA receptor negative allosteric modulators (GABAARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABAARNAMs can restore the Excitation/Inhibition (E/I) balance in vitro and arousal in vivo, we chose to screen 11 compounds that have been reported to modulate arousal, to see if they shared a GABA-related mechanism. We determined modulation with both conventional and microfluidic patch clamp methods. We found that receptor activation was variably modulated by all 11 compounds: Rifampicin (RIF), Metronidazole (MET), Minocycline (MIN), Erythromycin (ERY), Ofloxacin (OFX), Chloroquine (CQ), Hydroxychloroquine sulfate (HCQ), Flumazenil (FLZ), Pentylenetetrazol (PTZ), (-)-Epigallocatechin Gallate (EGCG), and clarithromycin (CLR). The computational modeling of modulator-receptor interactions predicted drug action at canonical binding sites and novel orphan sites on the receptor. Our findings suggest that multiple avenues of investigation are now open to investigate large and brain-penetrant molecules for the treatment of patients with diminished CNS E/I balance.

Anxiolytic-like effects of citral in the mouse elevated plus maze: involvement of GABAergic and serotonergic transmissions.

Citral, a monoterpene which is a part of the essential oil of several medicinal plants, is generally regarded as safe for human and animal consumption. Studies have introduced citral as a functional component of some essential oils in anxiolytic and antidepressant therapies; however, the neuropharmacological characteristics of citral have not yet been reported. In the present study, we evaluated the anxiolytic activities of citral in comparison to two standard anxiolytics, diazepam and buspirone, in Swiss albino mice by intraperitoneal administration of 1, 2, 5, 10, and 20 mg/kg using elevated plus maze (EPM) and open-field test (OFT). Moreover, we also examined whether the GABAA-benzodiazepine and 5-HT1A receptor are involved in the anxiolytic-like effects of citral by pretreatment with flumazenil and WAY-100635, respectively. Citral dose-dependently decreased the number of border crossings and time spent in borders, and also the number of grooming and rearing in OFT without altering the exploratory behavior of mice. In the EPM, this monoterpene led to a significant increase in number of entries in open arms and time spent in open arms, as well as a decrease in time spent in closed arms. Pretreatment with flumazenil and WAY-100635 both could reverse the anxiolytic effects of the citral in the EPM. These results suggest that anxiolytic activity of citral occurs via the GABAA and 5-HT1A receptor modulation.

Anxiolytic-like Effect of Quercetin Possibly through GABA Receptor Interaction Pathway: In Vivo and In Silico Studies.

Scientific evidence suggests that quercetin (QUR) has anxiolytic-like effects in experimental animals. However, the mechanism of action responsible for its anxiolytic-like effects is yet to be discovered. The goal of this research is to assess QUR's anxiolytic effects in mouse models to explicate the possible mechanism of action. After acute intraperitoneal (i.p.) treatment with QUR at a dose of 50 mg/kg (i.p.), behavioral models of open-field, hole board, swing box, and light-dark tests were performed. QUR was combined with a GABAergic agonist (diazepam) and/or antagonist (flumazenil) group. Furthermore, in silico analysis was also conducted to observe the interaction of QUR and GABA (α5), GABA (ß1), and GABA (ß2) receptors. In the experimental animal model, QUR had an anxiolytic-like effect. QUR, when combined with diazepam (2 mg/kg, i.p.), drastically potentiated an anxiolytic effect of diazepam. QUR is a more highly competitive ligand for the benzodiazepine recognition site that can displace flumazenil (2.5 mg/kg, i.p.). In all the test models, QUR acted similar to diazepam, with enhanced effects of the standard anxiolytic drug, which were reversed by pre-treatment with flumazenil. QUR showed the best interaction with the GABA (α5) receptor compared to the GABA (ß1) and GABA (ß2) receptors. In conclusion, QUR may exert an anxiolytic-like effect on mice, probably through the GABA-receptor-interacting pathway.

Effects of Hyssopus Officinalis Hydroalcoholic Extract on Pentylenetetrazol-Induced Convulsive Seizures in Rat.

Hyssopus officinalis L. is one of the most important medicinal plants in traditional medicine used to treat seizures. In this study, we assessed the effects of H. officinalis hydroalcoholic extract against pentylenetetrazol (PTZ)-induced seizures in rat. The anti-seizure activity of the extract was assessed in three doses of 25, 50, and 100 mg/kg. Kindling was induced by intraperitoneal injection of PTZ (35 mg/kg) every 48 h, and H. officinalis extract was administered daily and behavioral tests performed. The possible involvement of GABA receptors in the extract activity was investigated using flumazenil. Tonic seizure threshold and mortality rate were measured following intraperitoneal injection of 60 mg/kg PTZ on the 14th day, following 14 days administration of H. officinalis hydroalcoholic extract. Blood and hippocampus samples were prepared to measure brain and serum antioxidant capacity, malondialdehyde (MDA), and nitric oxide (NO). Finally, the expression of GABA receptor gene in brain tissue was investigated. H. officinalis extract increased tonic seizure threshold and decreased mortality due to PTZ. Flumazenil, as a GABA receptor antagonist, reduced the tonic seizure threshold. Extract treatment significantly improved memory and learning, increased brain antioxidant capacity, decreased brain MDA and NO in kindled rats. It also increased GABA receptor gene expression in pre-treated groups compared to the negative control group. H. officinalis extract probably exerts potential antiepileptic effects through the GABAergic system. Also, H. officinalis extract has a supportive effect against hippocampal neuronal damage and improves memory and learning in kindled rats.

Altered Behavioral Responses Show GABA Sensitivity in Muscleblind-Like 2-Deficient Mice: Implications for CNS Symptoms in Myotonic Dystrophy.

Considerable evidence from mouse models and human postmortem brain suggests loss of Muscleblind-like protein 2 (MBNL2) function in brain is a major driver of CNS symptoms in Myotonic dystrophy type 1 (DM1). Increased hypersomnia, fatigue, and surgical complications associated with general anesthesia suggest possible sensitivity to GABAergic inhibition in DM1. To test the hypothesis that MBNL2 depletion leads to behavioral sensitivity to GABAA receptor (GABAA-R) modulation, Mbnl2 knock-out (KO) and wild-type (WT) littermates were treated with the anesthetic sevoflurane, the benzodiazepine diazepam, the imidazopyridine zolpidem, and the benzodiazepine rescue agent, flumazenil (Ro 15-1788), and assessed for various behavioral metrics. Mbnl2 KO mice exhibited delayed recovery following sevoflurane, delayed emergence and recovery from zolpidem, and enhanced sleep time at baseline that was modulated by flumazenil. A significantly higher proportion of Mbnl2 KO mice also loss their righting reflex [loss of righting reflex (LORR)] from a standard diazepam dose. We further examined whether MBNL2 depletion affects total GABAA-R mRNA subunit levels and validated RNA-sequencing data of mis-spliced Gabrg2, whose isoform ratios are known to regulate GABA sensitivity and associated behaviors. While no other GABAA-R subunit mRNA levels tested were altered in Mbnl2 KO mouse prefrontal cortex, Gabrg2S/L mRNA ratio levels were significantly altered. Taken together, our findings indicate that loss of MBNL2 function affects GABAergic function in a mouse model of myotonic dystrophy (DM1).

Inhibition of GABAA receptors in intestinal stem cells prevents chemoradiotherapy-induced intestinal toxicity.

Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce P53-dependent apoptosis in LGR5+ intestinal stem cells (ISCs). Gamma-aminobutyric acid (GABA) and its A receptors (GABAAR) are present in the gastrointestinal tract. However, the functioning of the GABAergic system in ISCs is poorly defined. We found that GABAAR α1 (GABRA1) levels increased in the murine intestine after chemoradiotherapy. GABRA1 depletion in LGR5+ ISCs protected the intestine from chemoradiotherapy-induced P53-dependent apoptosis and prolonged animal survival. The administration of bicuculline, a GABAAR antagonist, prevented chemoradiotherapy-induced ISC loss and intestinal damage without reducing the chemoradiosensitivity of tumors. Mechanistically, it was associated with the reduction of reactive oxygen species-induced DNA damage via the L-type voltage-dependent Ca2+ channels. Notably, flumazenil, a GABAAR antagonist approved by the U.S. Food and Drug Administration, rescued human colonic organoids from chemoradiotherapy-induced toxicity. Therefore, flumazenil may be a promising drug for reducing the gastrointestinal side effects of chemoradiotherapy.

First evaluation of the anxiolytic-like effects of a bromazepam­palladium complex in mice.

A significant fraction of patients are affected by persistent fear and anxiety. Currently, there are several anxiolytic drug options, however their clinical outcomes do not fully manage the symptoms. Here, we evaluated the effects of a bromazepam­palladium derivative [2-{(7-bromo-2-oxo-1,3-dihydro-2H-1,4-benzodiazepin-5-il)pyridinyl-κ2-N,N}chloropalladium(II)], [(BMZ)PdCl2], on fear/anxiety and memory-related behavior in mice. For this, female Swiss mice were treated intraperitoneally (i.p.) with saline (NaCl 0.9%) or [(BMZ)PdCl2] (0.5, 5.0, or 50 µg/kg). After 30 min, different tests were performed to evaluate anxiety, locomotion, and memory. We also evaluated the acute toxicity of [(BMZ)PdCl2] using a cell viability assay (neutral red uptake assay), and whether the drugs mechanism of action involves the γ-aminobutyric acid type A (GABAA) receptor complex by pre-treating animals with flumazenil (1.0 mg/kg, i.p., a competitive antagonist of GABAA-binding site). Our results demonstrate that [(BMZ)PdCl2] induces an anxiolytic-like phenotype in the elevated plus-maze test and that this effect can be blocked by flumazenil. Furthermore, there were no behavioral alterations induced by [(BMZ)PdCl2], as evaluated in the light-dark box, open field, and step-down passive avoidance tests. In the acute toxicity assay, [(BMZ)PdCl2] presented IC50 and LD50 values of 218 ± 60 µg/mL and 780 ± 80 mg/kg, respectively, and GSH category 4. Taken together, our results show that the anxiolytic-like effect of acute treatment with [(BMZ)PdCl2] occurs through the modulation of the benzodiazepine site in the GABAA receptor complex. Moreover, we show indications that [(BMZ)PdCl2] does not promote sedation and amnesia and presents the same toxicity as the bromazepam prototype.

A theory of the anxiolytic action of flumazenil in anxiety disorders.

BACKGROUND: Anxiety disorders are highly prevalent affecting up to 33.7% of people over a lifetime. Although many treatment options are available, they are often associated with unacceptable side-effect profiles and approximately one in three patients are treatment resistant. Allopregnanolone, a neuroactive steroid acting as a positive allosteric modulator at the GABAA receptor, is synthesised in response to stress and acts to negatively modulate the hypothalamic-pituitary-adrenal axis. FINDINGS: After chronic exposure to and withdrawal from allopregnanolone, an increase in α4ß2δ GABAA receptors results in a reduced inhibitory effect of allopregnanolone, resulting in decreased inhibition and, therefore, increased neuronal excitability. The relationship between allopregnanolone and increased α4ß2δ GABAA receptors has been demonstrated in animal models during methamphetamine withdrawal and puberty, events both associated with stress. The effect of allopregnanolone during these events is anxiogenic, a paradoxical action to its usual anxiolytic effects. Flumazenil, the GABAA receptor antagonist, has been shown to cause receptor internalisation of α4ß2δ GABAA receptors, which may results in anxiolysis. CONCLUSION: We propose that chronic stress and chronic exposure to and withdrawal from allopregnanolone in anxiety disorders result in alterations in GABAA receptor function, which can be corrected by flumazenil. As such, flumazenil may exhibit anxiolytic properties in patients with increased α4ß2δ GABAA receptor expression.

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Reversal effects of atipamezole, flumazenil, and 4-aminopyridine on bradycardia and increases in blood pressures induced by medetomidine, midazolam, and ketamine in isoflurane-anesthetized cats.

OBJECTIVE: To investigate the effects of a fixed dose of atipamezole (AT), flumazenil (FL), and 4-aminopyridine (AP), both alone and in combination, on changes in arterial blood pressure and heart rate induced by medetomidine (ME), midazolam (MI), and ketamine (KE) under isoflurane anesthesia with controlled ventilation in healthy cats. DESIGN: Prospective experimental study. SETTING: University animal research facility. ANIMALS: Healthy adult mixed-breed cats were used for 8 investigation groups (6 cats per group), with ≥2 weeks between interventions. INTERVENTIONS: Cats were anesthetized with an end-tidal isoflurane concentration of 2% under controlled ventilation. A catheter was inserted into the right or left femoral artery for arterial pressure monitoring and blood gas sampling, and ECG electrodes were placed. Upon completed preparations, cats were administered a mixture of ME (0.05 mg/kg) and MI (0.5 mg/kg), followed 10 minutes later by intramuscular KE (10 mg/kg). Twenty minutes after KE injection, the cats received IV injection with either a physiological saline solution at 0.1 mL/kg (control), or 1 of 7 variations of experimental drugs, alone or in combination: AT (0.2 mg/kg), FL (0.1 mg/kg), AP (0.5 mg/kg), AT+FL, FL+AP, AT+AP, and AT+FL+AP. Arterial blood pressure and heart rate were continuously measured over 120 minutes after administration of potential antagonists. MEASUREMENTS AND MAIN RESULTS: ME+MI+KE induced an increase in blood pressure and bradycardia. Potential antagonists alone or in combination did not significantly alter the bradycardia. FL, AP alone, and FL+AP did not significantly alter the changes in blood pressures induced by ME+MI+KE. Meanwhile, administration of AT alone or in combination reversed the increase in blood pressure induced by ME+MI+KE but transiently caused excessive hypotension. CONCLUSION: These results revealed that AT alone or in combination is effective for antagonizing hypertension induced by ME+MI+KE; however, attention should be paid to temporary hypotension in cats anesthetized with isoflurane.

Anxiolytic-like effect of chrysophanol from Senna cana stem in adult zebrafish (Danio rerio).

The aim of this study was to evaluate the anxiolytic-like effect of chrysophanol (CHRY), isolated from hexane extract of Senna cana stem and its possible mechanism of action. CHRY was obtained through chromatographic treatments and its identity was confirmed by uni and bidimensional RMN1H and RMN13C. Adult zebrafish (n = 6/group) were treated (with CHRY (4.0 or 12.0 or 40.0 mg/Kg; 20 µL; intraperitoneally) and submitted to acute toxicity and open field tests. Subsequently, other groups (n = 6/each) received CHRY for the analysis of its effect on the Light & Dark Test. The participation of the GABAergic system was also assessed using the diazepam (GABAA receptor agonist) and flumazenil (GABAA receptor antagonist). CHRY was considered non-toxic, it did not reduce the locomotor activity, and showed an anxiolytic-like effect. This effect was reduced by pre-treatment with flumazenil. The results suggest that CHRY is an anxiolytic-like agent mediated via the GABAergic system.

Sedative-hypnotic, anxiolytic and possible side effects of Salvia limbata C. A. Mey. Extracts and the effects of phenological stage and altitude on the rosmarinic acid content.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia limbata C. A. Mey. (Persian name: Maryam Goli-e-labeh dar) has been used for treating central nervous disorders such as insomnia, anxiety and depression in Persian traditional medicine. S. limbata is known for its pharmacological activities which could be at least in a part, upon the presence of rosmarinic acid (RA). However, the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extract has not yet been examined. AIM OF THE STUDY: In the current study the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extracts were evaluated. Besides, the effects of altitude and phenological stage on the RA content of S. limbata were investigated. MATERIALS AND METHODS: Sedative-hypnotic and anxiolytic effects were evaluated through the pentobarbital induced loss of righting reflex test and open field test, respectively. Flumazenil was used to reveal the mechanism of action. Possible side effects were investigated in the passive avoidance and grip strength tests. Besides, the effects of altitude and phenological stage (vegetative, flowering, and seed setting) on the RA content of S. limbata were evaluated using reversed-phase high-performance liquid chromatography (RP-HPLC). RESULTS: Following behavioral tests, sedative-hypnotic and anxiolytic effects were observed. Since the observed effects were reversed by flumazenil and no side effect on the memory and muscle strength was reported, modulation of the α1-containing GABA-A receptors could be proposed as one of the involved mechanisms. According to the RP-HPLC analysis, harvesting S. limbata in the vegetative stage at the altitude of 2500 m led to the highest content of RA (8.67 ± 0.13 mg/g dry matter). Among different extract of the plant samples collected in the vegetative stage at the altitude of 2500 m, the hydroalcoholic extract showed the highest rosmarinic acid content. CONCLUSION: The obtained results help to find the optimum situation to gain the highest content of RA as well as the pharmacological activity that could be economically important for the pharmaceutical industries.

Neuropharmacological Activity of the New Piperazine Derivative 2-(4-((1- Phenyl-1H-Pyrazol-4-yl)Methyl)Piperazin-1-yl)Ethyl Acetate is Modulated by Serotonergic and GABAergic Pathways.

BACKGROUND: Pharmacological treatments for mental disorders, such as anxiety and depression, present several limitations and adverse effects. Therefore, new pharmacotherapy with anxiolytic and antidepressant potential is necessary, and the study of compounds capable of interacting with more than one pharmacological target may provide new therapeutic options. OBJECTIVES: In this study, we proposed the design, synthesis of a new compound, 2-(4-((1- phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethyl acetate (LQFM192), pharmacological evaluation of its anxiolytic-like and antidepressant-like activities, as well as the possible mechanisms of action involved. METHODS: Administration of LQFM192 was carried out prior to the exposure of male Swiss mice to behavioral tests, such as the elevated plus-maze and forced swimming test. The involvement of the serotonergic system was studied by pretreatment with WAY-100635 or p-chlorophenylalanine (PCPA) and the involvement of the benzodiazepine site of the GABAA receptor by pretreatment with flumazenil. RESULTS: The treatment with LQFM192 at doses of 54 and 162 µmol/kg demonstrated anxiolyticlike activity that was blocked by WAY-100635, PCPA, and flumazenil pretreatments. The potential antidepressant-like activity was visualized at the same doses and blocked by WAY-100635 and PCPA. CONCLUSION: In summary, the anxiolytic-like activity of LQFM192 is mediated by the serotonergic system and the benzodiazepine site of the GABAA receptor, and the antidepressant-like activity through the serotonergic system.