Medical Hypotheses 81 (2013) 1075–1077
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Endogenous ligands of benzodiazepine binding site have inverse agonistic properties Jiri Sliva a,b,⇑, Ladislav Hess c, Martin Votava a, Jiri Malek d a
Department of Pharmacology, 2nd Faculty of Medicine, Charles University, V Úvalu 84, Prague 5 150 06, Czech Republic Department of Pharmacology, 3rd Faculty of Medicine, Charles University, Ruská 87, Prague 10 10034, Czech Republic c Institute for Clinical and Experimental Medicine, Laboratory of Experimental Anaesthesiology, Prague, Czech Republic d Department of Anaesthesiology and Critical Care Medicine, 3rd Faculty of Medicine, Charles University, Ruská 87, Prague 10 10034, Czech Republic b
a r t i c l e
i n f o
Article history: Received 12 June 2013 Accepted 8 October 2013
a b s t r a c t Benzodiazepines have been widely used in clinical praxis for many decades. They act as GABAA receptor agonists and possess muscle-relaxant, hypnotic-sedative, anticonvulsant, and anxiolytic properties. Flumazenil acts as a benzodiazepine receptor antagonist (subunits a1, a2, a3, and a5) or partial agonist (subunits a4 and a6). It competitively inhibits the activity at the benzodiazepine recognition site on the GABA/ benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. In our experiments, administration of flumazenil in rabbits was surprisingly associated with anxiolytic effects similar to those of midazolam. Additionally, flumazenil significantly and dose-dependently decreased the total number of vocalizations in rats, i.e. it was anxiolytic. These observations seem to be in contrast to the effect of flumazenil in humans, where it is believed to produce mainly anxiogenic effects. It seems that in individuals, who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent, while in individuals without any signs of anxiety, flumazenil can also act as anxiogenic agent. Thus, we hypothesize that flumazenil is associated with decreased intensity of anticipatory anxiety due to occupancy of benzodiazepine binding sites by an endogenous ligand with inverse agonistic properties. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Benzodiazepines (BZDs) are widely used around the world, mainly, but not limited, for their anxiolytic and hypnotic properties. BZDs act selectively on GABAA receptors, which mediate inhibitory synaptic transmission throughout the central nervous system. They bind specifically to a regulatory site on these receptors and act allosterically to increase the affinity of GABA for the receptor. Thus, they enhance the response to GABA by facilitating the opening of GABA-activated chloride channels. Although the BZD binding site is relatively well described, no endogenous ligand has been discovered so far and we can only indirectly predict its potential activity. Flumazenil acts as a benzodiazepine receptor antagonist (subunits a1, a2, a3, and a5) or partial agonist (subunits a4 and a6). Therefore it competitively inhibits the activity at the benzodiazepine recognition site on the GABA/benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. The main clinical indication of flumazenil is the management of benzodiazepine overdose and/or to reverse the effects of benzodiazepines
⇑ Corresponding author at: Department of Pharmacology, 2nd Faculty of Medicine, Charles University, V Úvalu 84, Prague 5 150 06, Czech Republic. Tel.: +420 257296451; fax: +420 257296452. E-mail address: [email protected] (J. Sliva). 0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.10.006
after general anesthesia; one potential adverse drug reaction is an anxiogenic effect. However, some studies have suggested that flumazenil itself also causes sedation and to some extent even produces anxiolytic effects, especially in anticipation anxiety [1]. Surprisingly, there is very little evidence regarding direct flumazenil effects on behavior, despite several interesting findings in the scientific literature showing anxiolytic effects of this antagonist. Chronic administration of flumazenil in rats has been associated with significantly longer times in open arms and with fewer fecal boluses in plusmaze tests compared to placebo. Additionally, flumazenil treated animals showed increased exploratory activity during the holeboard test and no change in drinking following a ‘shock experience’ in the drinking-punishment test [2]. Anxiolytic activity of flumazenil has also been observed in humans, where a double-blind study showed significantly reduced anxiety induced by simulation of public speaking [3]. In this paper we hypothesize that this ‘‘paradoxical’’ effect of flumazenil on behavior is caused by characteristics of endogenous ligands of BZD binding sites. Hypothesis Based on available knowledge [3–5] and our preliminary results [6], we predict that the putative endogenous ligand of the BZD
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J. Sliva et al. / Medical Hypotheses 81 (2013) 1075–1077
binding site on GABAA receptors has inverse agonistic properties. On the one hand, agonists of BZD binding sites are antagonized by flumazenil; while on the other hand, flumazenil is able to antagonize symptoms caused by benzodiazepine withdrawal [1,7–9]. It has been suggested that flumazenil ‘‘resets’’ GABAA receptors for normal function [10]. There is a lot of preclinical and clinical data showing the reversal of benzodiazepine-induced sedation by flumazenil [11]. This effect can be simply explained by its competitively antagonistic activity at the above mentioned sites of GABAA receptors. Notwithstanding, there is little data showing the effect of flumazenil or other antagonists. One study that does address this issue shows that flumazenil produced an anxiolytic-like effects in both timid and aggressive mice [12]. Such observations seem to be contrary to the observed effects of flumazenil in humans, where it is believed to produce mainly anxiogenic effects (SmPC Anexate, Roche, UK). However, this can be explained by the baseline status of the individual. It seems that in individuals who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent. It was proposed in the early 90’s that the anxiety level of the subject determines flumazenil’s effect on anxiety: when anxiety is high, such as during benzodiazepine withdrawal, flumazenil would become anxiolytic: however, when anxiety is low, flumazenil increases anxiety [1]. Similar results were found in our experiments (see Fig. 1) and also in the above mentioned study [12]. In individuals without any anxiogenic signs, flumazenil actually seems to act as anxiogenic agent, which is also supported by observations in humans (SmPC Anexate, Roche, UK) and in observations with sociable mice. In sociable mice study, flumazenil was found to produced anxiogenic-like effects [12]. This biphasic effect of flumazenil can be explained by the presence of an endogenous ligand for BZD binding site. If the hypothesized ligand triggers anxiogenic effects after binding to the BZD receptor site, then flumazenil would be able to alleviate this effect. In a situation, where no such ligand is bound to this site, flumazenil blocks the activity of the GABAA receptor and produces mild anxiolytic effects. Therefore, in concordance with our observation, we suggest that flumazenil doesn’t possess antagonistic activity that is only characterized by increased anxiety. Due to the hypothetical character of endogenous BZD binding site ligands, flumazenil can reverse its action and act as an anxiolytic agent, providing effects similar to those of a full BZD agonist. Data in support of and against hypothesis In our preliminary experiments with flumazenil, we observed that administration of flumazenil (0.1 mg/kg), to rabbits, was asso-
ciated with sedative and anxiolytic effects, which was, in some parameters, similar to that of midazolam (0.5 mg/kg). In treated rabbits, the loss of the righting reflex was achieved after approximately 3 min, irrespective of drug (midazolam or flumazenil). On the other hand, midazolam caused a more profound decrease in blood pressure and pulse rate. Such results were confirmed using the ‘ultrasonic vocalization test of anxiety’ in rats. The rats were administered various doses of flumazenil (0.1, 1 and 10 mg/kg) and the anxiolytic effect of flumazenil was observed. The study was placebo controlled and three different doses of flumazenil, based on literature data, were used. While saline had no influence on the duration and length of recorded amplitudes, flumazenil significantly and dose-dependently decreased the total number of vocalizations (22 kHz; see Fig. 1). Furthermore, although the majority of experiments showed that flumazenil can precipitate withdrawal symptoms, there was also evidence that flumazenil was capable of preventing and reversing increased anxiety associated with benzodiazepine withdrawal in both animals and humans [1,7,8]. In a study using the ‘elevated plus maze’ in rats, an acute challenge with flumazenil (10 mg/kg) induced anxiolytic-like effects similar to, although not identical, to those seen following acute administration of diazepam [13]. This finding was very similar to our findings, where the effect of flumazenil was, to some extent, similar to that of midazolam. Our hypothesis is further supported by several studies where subjects with anxiety or panic disorders displayed decreased binding of radiolabelled flumazenil, suggesting occupancy of BZD binding sites or a decrease in GABAA neurons [14,15]. We believe that if BZD binding sites are occupied by an endogenous BZD binding site ligand, then an anxiety reaction is triggered. This is in concordance with previously proposed explanations of the anxiolytic effects of flumazenil observed in humans undergoing a simulation of public speaking [3]. The direct effect of flumazenil, on behavior, can be seen in very thoroughly planned studies, where subjects were divided based on their initial level of anxiety or aggressive behavior [12]. If a preselection of subjects, based on their level of anxiety, is not performed, the behavioral effects of flumazenil are often hidden due to the heterogenicity of the cohort [9]. One double edged fact that favors our hypothesis is that so far no endogenous benzodiazepine receptor ligand has been found that contradicts our explanation, however, the other edge suggests the possibility that no such ligand exists and that the site may simply be a peculiar protein conformation capable of fine tuning GABA function, possibly by altering maximal efficacy, or rate of desensitization [16]. Consequences of the hypothesis and discussion Our hypothesis might also elucidate some effects of flumazenil in clinical practice. It explains administration of flumazenil is associated with decreased intensity of anticipatory anxiety and why flumazenil can antagonize some symptoms caused by BZD withdrawal. A substance with antagonistic properties, such as flumazenil, can decrease the level of anxiety due to occupancy of BZD binding sites by an endogenous ligand with inverse agonistic properties. This hypothesis is supported by laboratory as well as sparse clinical data. Its clinical implication can be used in alleviating anxiety symptoms (e.g. in children, ethanol or BZD withdrawal) without causing sedation, hypnosis or memory impairment often characterized by use of benzodiazepines for such purposes.
Fig. 1. Duration of ultrasonic (US) vocalization in rats 30 min after flumazenil (0.1, 1, 10 mg/kg i.p.) administration. The US vocalization was measured for 10 min and was triggered by shocks, on the previous day, from an electric grid in the floor.
Conflict of interest Authors declare no conflict of interests.
J. Sliva et al. / Medical Hypotheses 81 (2013) 1075–1077
Acknowledgement This work was supported by grant IGA MZ NS/10503-3 from the Czech Ministry of Health.
References [1] File SE, Hitchcott PK. A theory of benzodiazepine dependence that can explain whether flumazenil will enhance or reverse the phenomena. Psychopharmacology (Berl) 1990;4(101):525–32. [2] Urbancic M, Gadek MA, Marczynski TJ. Chronic exposure to flumazenil: anxiolytic effect and increased exploratory behavior. Pharmacol Biochem Behav 1990;3(35):503–9. [3] Kapczinski F, Curran HV, Gray J, et al. Flumazenil has an anxiolytic effect in simulated stress. Psychopharmacology (Berl) 1994;1(114):187–9. [4] Baldwin HA, File SE. Reversal of increased anxiety during benzodiazepine withdrawal: evidence for an anxiogenic endogenous ligand for the benzodiazepine receptor. Brain Res Bull 1988;5(20):603–6. [5] Urbancic M, Gadek M, Marczynski TJ. Chronic flumazenil (Ro 15-1788) facilitates acquisition and retention of a swim-escape response in rats. Neuropsychobiology 1990;1(24):17–24. [6] Hess L, Votava M, Slíva J, et al. Sedative and anxiolytic properties of flumazenil in rats and rabbits. Eur J Anaesthesiol 2013;30:150.
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[7] Licata SC, Rowlett JK. Abuse and dependence liability of benzodiazepine-type drugs: GABA(A) receptor modulation and beyond. Pharmacol Biochem Behav 2008;1(90):74–89. [8] Hood S, O’Neil G, Hulse G. The role of flumazenil in the treatment of benzodiazepine dependence: physiological and psychological profiles. J Psychopharmacol 2009;4(23):401–9. [9] Saxon L, Borg S, Hiltunen AJ. Reduction of aggression during benzodiazepine withdrawal: effects of flumazenil. Pharmacol Biochem Behav 2010;2(96):148–51. [10] Whitwam JG. Flumazenil: a benzodiazepine antagonist. BMJ 1988;6655(297):999–1000. [11] Sanders LD, Piggott SE, Isaac PA, et al. Reversal of benzodiazepine sedation with the antagonist flumazenil. Br J Anaesth 1991;4(66):445–53. [12] Uhlirova L, Sustkova-Fiserova M, Krsiak M. Behavioral effects of flumazenil in the social conflict test in mice. Psychopharmacology (Berl) 2004;3(171):259–69. [13] Divljakovic J, Milic M, Namjoshi OA, et al. BetaCCT, an antagonist selective for alpha(1)GABA(A) receptors, reverses diazepam withdrawal-induced anxiety in rats. Brain Res Bull 2013;91:1–7. [14] Malizia AL, Cunningham VJ, Bell CJ, et al. Decreased brain GABA(A)benzodiazepine receptor binding in panic disorder: preliminary results from a quantitative PET study. Arch Gen Psychiatry 1998;8(55):715–20. [15] Cameron OG, Huang GC, Nichols T, et al. Reduced gamma-aminobutyric acid(A)-benzodiazepine binding sites in insular cortex of individuals with panic disorder. Arch Gen Psychiatry 2007;7(64):793–800. [16] Nutt DJ, Malizia AL. New insights into the role of the GABA(A)-benzodiazepine receptor in psychiatric disorder. Br J Psychiatry 2001;179:390–6.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Endogenous ligands of benzodiazepine binding site have inverse agonistic properties Jiri Sliva a,b,⇑, Ladislav Hess c, Martin Votava a, Jiri Malek d a
Department of Pharmacology, 2nd Faculty of Medicine, Charles University, V Úvalu 84, Prague 5 150 06, Czech Republic Department of Pharmacology, 3rd Faculty of Medicine, Charles University, Ruská 87, Prague 10 10034, Czech Republic c Institute for Clinical and Experimental Medicine, Laboratory of Experimental Anaesthesiology, Prague, Czech Republic d Department of Anaesthesiology and Critical Care Medicine, 3rd Faculty of Medicine, Charles University, Ruská 87, Prague 10 10034, Czech Republic b
a r t i c l e
i n f o
Article history: Received 12 June 2013 Accepted 8 October 2013
a b s t r a c t Benzodiazepines have been widely used in clinical praxis for many decades. They act as GABAA receptor agonists and possess muscle-relaxant, hypnotic-sedative, anticonvulsant, and anxiolytic properties. Flumazenil acts as a benzodiazepine receptor antagonist (subunits a1, a2, a3, and a5) or partial agonist (subunits a4 and a6). It competitively inhibits the activity at the benzodiazepine recognition site on the GABA/ benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. In our experiments, administration of flumazenil in rabbits was surprisingly associated with anxiolytic effects similar to those of midazolam. Additionally, flumazenil significantly and dose-dependently decreased the total number of vocalizations in rats, i.e. it was anxiolytic. These observations seem to be in contrast to the effect of flumazenil in humans, where it is believed to produce mainly anxiogenic effects. It seems that in individuals, who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent, while in individuals without any signs of anxiety, flumazenil can also act as anxiogenic agent. Thus, we hypothesize that flumazenil is associated with decreased intensity of anticipatory anxiety due to occupancy of benzodiazepine binding sites by an endogenous ligand with inverse agonistic properties. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Benzodiazepines (BZDs) are widely used around the world, mainly, but not limited, for their anxiolytic and hypnotic properties. BZDs act selectively on GABAA receptors, which mediate inhibitory synaptic transmission throughout the central nervous system. They bind specifically to a regulatory site on these receptors and act allosterically to increase the affinity of GABA for the receptor. Thus, they enhance the response to GABA by facilitating the opening of GABA-activated chloride channels. Although the BZD binding site is relatively well described, no endogenous ligand has been discovered so far and we can only indirectly predict its potential activity. Flumazenil acts as a benzodiazepine receptor antagonist (subunits a1, a2, a3, and a5) or partial agonist (subunits a4 and a6). Therefore it competitively inhibits the activity at the benzodiazepine recognition site on the GABA/benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. The main clinical indication of flumazenil is the management of benzodiazepine overdose and/or to reverse the effects of benzodiazepines
⇑ Corresponding author at: Department of Pharmacology, 2nd Faculty of Medicine, Charles University, V Úvalu 84, Prague 5 150 06, Czech Republic. Tel.: +420 257296451; fax: +420 257296452. E-mail address: [email protected] (J. Sliva). 0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.10.006
after general anesthesia; one potential adverse drug reaction is an anxiogenic effect. However, some studies have suggested that flumazenil itself also causes sedation and to some extent even produces anxiolytic effects, especially in anticipation anxiety [1]. Surprisingly, there is very little evidence regarding direct flumazenil effects on behavior, despite several interesting findings in the scientific literature showing anxiolytic effects of this antagonist. Chronic administration of flumazenil in rats has been associated with significantly longer times in open arms and with fewer fecal boluses in plusmaze tests compared to placebo. Additionally, flumazenil treated animals showed increased exploratory activity during the holeboard test and no change in drinking following a ‘shock experience’ in the drinking-punishment test [2]. Anxiolytic activity of flumazenil has also been observed in humans, where a double-blind study showed significantly reduced anxiety induced by simulation of public speaking [3]. In this paper we hypothesize that this ‘‘paradoxical’’ effect of flumazenil on behavior is caused by characteristics of endogenous ligands of BZD binding sites. Hypothesis Based on available knowledge [3–5] and our preliminary results [6], we predict that the putative endogenous ligand of the BZD
1076
J. Sliva et al. / Medical Hypotheses 81 (2013) 1075–1077
binding site on GABAA receptors has inverse agonistic properties. On the one hand, agonists of BZD binding sites are antagonized by flumazenil; while on the other hand, flumazenil is able to antagonize symptoms caused by benzodiazepine withdrawal [1,7–9]. It has been suggested that flumazenil ‘‘resets’’ GABAA receptors for normal function [10]. There is a lot of preclinical and clinical data showing the reversal of benzodiazepine-induced sedation by flumazenil [11]. This effect can be simply explained by its competitively antagonistic activity at the above mentioned sites of GABAA receptors. Notwithstanding, there is little data showing the effect of flumazenil or other antagonists. One study that does address this issue shows that flumazenil produced an anxiolytic-like effects in both timid and aggressive mice [12]. Such observations seem to be contrary to the observed effects of flumazenil in humans, where it is believed to produce mainly anxiogenic effects (SmPC Anexate, Roche, UK). However, this can be explained by the baseline status of the individual. It seems that in individuals who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent. It was proposed in the early 90’s that the anxiety level of the subject determines flumazenil’s effect on anxiety: when anxiety is high, such as during benzodiazepine withdrawal, flumazenil would become anxiolytic: however, when anxiety is low, flumazenil increases anxiety [1]. Similar results were found in our experiments (see Fig. 1) and also in the above mentioned study [12]. In individuals without any anxiogenic signs, flumazenil actually seems to act as anxiogenic agent, which is also supported by observations in humans (SmPC Anexate, Roche, UK) and in observations with sociable mice. In sociable mice study, flumazenil was found to produced anxiogenic-like effects [12]. This biphasic effect of flumazenil can be explained by the presence of an endogenous ligand for BZD binding site. If the hypothesized ligand triggers anxiogenic effects after binding to the BZD receptor site, then flumazenil would be able to alleviate this effect. In a situation, where no such ligand is bound to this site, flumazenil blocks the activity of the GABAA receptor and produces mild anxiolytic effects. Therefore, in concordance with our observation, we suggest that flumazenil doesn’t possess antagonistic activity that is only characterized by increased anxiety. Due to the hypothetical character of endogenous BZD binding site ligands, flumazenil can reverse its action and act as an anxiolytic agent, providing effects similar to those of a full BZD agonist. Data in support of and against hypothesis In our preliminary experiments with flumazenil, we observed that administration of flumazenil (0.1 mg/kg), to rabbits, was asso-
ciated with sedative and anxiolytic effects, which was, in some parameters, similar to that of midazolam (0.5 mg/kg). In treated rabbits, the loss of the righting reflex was achieved after approximately 3 min, irrespective of drug (midazolam or flumazenil). On the other hand, midazolam caused a more profound decrease in blood pressure and pulse rate. Such results were confirmed using the ‘ultrasonic vocalization test of anxiety’ in rats. The rats were administered various doses of flumazenil (0.1, 1 and 10 mg/kg) and the anxiolytic effect of flumazenil was observed. The study was placebo controlled and three different doses of flumazenil, based on literature data, were used. While saline had no influence on the duration and length of recorded amplitudes, flumazenil significantly and dose-dependently decreased the total number of vocalizations (22 kHz; see Fig. 1). Furthermore, although the majority of experiments showed that flumazenil can precipitate withdrawal symptoms, there was also evidence that flumazenil was capable of preventing and reversing increased anxiety associated with benzodiazepine withdrawal in both animals and humans [1,7,8]. In a study using the ‘elevated plus maze’ in rats, an acute challenge with flumazenil (10 mg/kg) induced anxiolytic-like effects similar to, although not identical, to those seen following acute administration of diazepam [13]. This finding was very similar to our findings, where the effect of flumazenil was, to some extent, similar to that of midazolam. Our hypothesis is further supported by several studies where subjects with anxiety or panic disorders displayed decreased binding of radiolabelled flumazenil, suggesting occupancy of BZD binding sites or a decrease in GABAA neurons [14,15]. We believe that if BZD binding sites are occupied by an endogenous BZD binding site ligand, then an anxiety reaction is triggered. This is in concordance with previously proposed explanations of the anxiolytic effects of flumazenil observed in humans undergoing a simulation of public speaking [3]. The direct effect of flumazenil, on behavior, can be seen in very thoroughly planned studies, where subjects were divided based on their initial level of anxiety or aggressive behavior [12]. If a preselection of subjects, based on their level of anxiety, is not performed, the behavioral effects of flumazenil are often hidden due to the heterogenicity of the cohort [9]. One double edged fact that favors our hypothesis is that so far no endogenous benzodiazepine receptor ligand has been found that contradicts our explanation, however, the other edge suggests the possibility that no such ligand exists and that the site may simply be a peculiar protein conformation capable of fine tuning GABA function, possibly by altering maximal efficacy, or rate of desensitization [16]. Consequences of the hypothesis and discussion Our hypothesis might also elucidate some effects of flumazenil in clinical practice. It explains administration of flumazenil is associated with decreased intensity of anticipatory anxiety and why flumazenil can antagonize some symptoms caused by BZD withdrawal. A substance with antagonistic properties, such as flumazenil, can decrease the level of anxiety due to occupancy of BZD binding sites by an endogenous ligand with inverse agonistic properties. This hypothesis is supported by laboratory as well as sparse clinical data. Its clinical implication can be used in alleviating anxiety symptoms (e.g. in children, ethanol or BZD withdrawal) without causing sedation, hypnosis or memory impairment often characterized by use of benzodiazepines for such purposes.
Fig. 1. Duration of ultrasonic (US) vocalization in rats 30 min after flumazenil (0.1, 1, 10 mg/kg i.p.) administration. The US vocalization was measured for 10 min and was triggered by shocks, on the previous day, from an electric grid in the floor.
Conflict of interest Authors declare no conflict of interests.
J. Sliva et al. / Medical Hypotheses 81 (2013) 1075–1077
Acknowledgement This work was supported by grant IGA MZ NS/10503-3 from the Czech Ministry of Health.
References [1] File SE, Hitchcott PK. A theory of benzodiazepine dependence that can explain whether flumazenil will enhance or reverse the phenomena. Psychopharmacology (Berl) 1990;4(101):525–32. [2] Urbancic M, Gadek MA, Marczynski TJ. Chronic exposure to flumazenil: anxiolytic effect and increased exploratory behavior. Pharmacol Biochem Behav 1990;3(35):503–9. [3] Kapczinski F, Curran HV, Gray J, et al. Flumazenil has an anxiolytic effect in simulated stress. Psychopharmacology (Berl) 1994;1(114):187–9. [4] Baldwin HA, File SE. Reversal of increased anxiety during benzodiazepine withdrawal: evidence for an anxiogenic endogenous ligand for the benzodiazepine receptor. Brain Res Bull 1988;5(20):603–6. [5] Urbancic M, Gadek M, Marczynski TJ. Chronic flumazenil (Ro 15-1788) facilitates acquisition and retention of a swim-escape response in rats. Neuropsychobiology 1990;1(24):17–24. [6] Hess L, Votava M, Slíva J, et al. Sedative and anxiolytic properties of flumazenil in rats and rabbits. Eur J Anaesthesiol 2013;30:150.
1077
[7] Licata SC, Rowlett JK. Abuse and dependence liability of benzodiazepine-type drugs: GABA(A) receptor modulation and beyond. Pharmacol Biochem Behav 2008;1(90):74–89. [8] Hood S, O’Neil G, Hulse G. The role of flumazenil in the treatment of benzodiazepine dependence: physiological and psychological profiles. J Psychopharmacol 2009;4(23):401–9. [9] Saxon L, Borg S, Hiltunen AJ. Reduction of aggression during benzodiazepine withdrawal: effects of flumazenil. Pharmacol Biochem Behav 2010;2(96):148–51. [10] Whitwam JG. Flumazenil: a benzodiazepine antagonist. BMJ 1988;6655(297):999–1000. [11] Sanders LD, Piggott SE, Isaac PA, et al. Reversal of benzodiazepine sedation with the antagonist flumazenil. Br J Anaesth 1991;4(66):445–53. [12] Uhlirova L, Sustkova-Fiserova M, Krsiak M. Behavioral effects of flumazenil in the social conflict test in mice. Psychopharmacology (Berl) 2004;3(171):259–69. [13] Divljakovic J, Milic M, Namjoshi OA, et al. BetaCCT, an antagonist selective for alpha(1)GABA(A) receptors, reverses diazepam withdrawal-induced anxiety in rats. Brain Res Bull 2013;91:1–7. [14] Malizia AL, Cunningham VJ, Bell CJ, et al. Decreased brain GABA(A)benzodiazepine receptor binding in panic disorder: preliminary results from a quantitative PET study. Arch Gen Psychiatry 1998;8(55):715–20. [15] Cameron OG, Huang GC, Nichols T, et al. Reduced gamma-aminobutyric acid(A)-benzodiazepine binding sites in insular cortex of individuals with panic disorder. Arch Gen Psychiatry 2007;7(64):793–800. [16] Nutt DJ, Malizia AL. New insights into the role of the GABA(A)-benzodiazepine receptor in psychiatric disorder. Br J Psychiatry 2001;179:390–6.
