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Flumazenil attenuates the pituitary response to CRH in healthy males
EUROPEANNEUROPSYCHOPHARMACOLOGY ELSEVIER
European Neuropsychopharmacology 6 (1996) 323-325
Short communication
Flumazenil attenuates the pituitary response to CRH in healthy males Andreas Strfhle*, Klaus Wiedemann Max Planck Institute of Psychiatry, Clinical Institute, Kraepelinstr. 2-I0, 80804, Munich, Germany
Received 22 January 1996; accepted 4 June 1996
Abstract Differential effects of flumazenil (an antagonist) and midazolam (an agonist at the benzodiazepine receptor) were studied in 8 healthy males. Adrenocorticotropic hormone (ACTH) and cortisol responses to a corticotropin-releasing hormone (CRH) challenge were measured. ACTH response was significantly lower after flumazenil administration than after saline or midazolam. No significant treatment effect was found for the CRH-induced cortisol secretion. Our results agree with previous reports that point to an agonistic effect of flumazenil on pituitary-adrenocortical system activity and, moreover, may further support the anxiolytic activity of flumazenil in stimulated stress.
Keywords: Flumazenil; CRH; Midazolam; Benzodiazepine; ACTH; Cortisol; Pituitary
1. Introduction: Flumazenil is a specific benzodiazepine receptor ligand that in animal and human studies has been shown to antagonize most of the pharmacological effects of benzodiazepines, which include sedative, muscle-relaxing, anxiolytic, and anticonvulsant effects (Hunkeler et al., 1981; Darragh et al., 1982; Dorow and Duka, 1986). Recently, agonistic as well as inverse agonistic effects of flumazenil on behavior, sleep EEG, and electrophysiological parameters have been reported (Sch6pf et al., 1984; Polc, 1988; Nutt et al., 1990; Steiger et al., 1994). In animals, the activating effects of corticotropin-releasing hormone (CRH) administration were attenuated by flumazenil, which indicated partial agonistic effects (Britton et al., 1988). In addition, healthy volunteers showed an anxiolytic effect of flumazenil during public speaking, which is a model of stimulated stress (Kapczinski et al., 1994). These behavioral findings support an activated pituitaryadrenocortical system impact on the modulation of the partial agonist activity of flumazenil. To characterize the effects of agonistic and antagonistic ligands on CRHstimulated pituitary-adrenocortical activation and to explore possible mutual influences, we administered *Corresponding author. Tel. +49 89 30622379; fax +49 89 30622601. 0924-977X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0924-977X(96)00040-5
flumazenil and the benzodiazepine receptor agonist, midazolam, before CRH administration.
2. Experimental procedures
2.1. Subjects Eight healthy male volunteers between 22 and 30 years of age were enrolled in the study after a thorough medical examination to rule out physical or psychiatric illness, drug intake or life styles that might have interfered with the study. The study was approved by the Ethics Comittee of the Max Planck Institute of Psychiatry and written informed consent was obtained from each subject.
2.2. Study design Subjects received in a double-blind, randomized order an infusion of either 2 mg flumazenil (1 mg at both 18.00 and 18.30 h), 0.5 mg midazolam (18.00 h) or saline on 3 occasions separated by at least 3 days. At 19.00 h, 100 Ixg CRH were administered intravenously. Plasma ACTH and cortisol concentrations were then repeatedly determined at 18.00 (baseline) and further until 21.00 h.
A. Strrhle, K. Wiedemann / European Neuropsychopharmacology 6 (1996) 323-325
324
2.3. Assays Commercial radioimmuno- and immunoradiometric assays (ICN Biomedicals, Carson, CA and Nichols Institute, San Juan Capistrano, CA, USA) were used for plasma cortisol and ACTH measurements. Intra- and interassay coefficients of variation were <7%, respectively.
Table 1 CRH-induced cortisol and ACTH secretion after placebo, flumazenil and midazolam
AUC-cortisol AUC-ACTH
Placebo
Flumazenil
Midazolam
541.0-+ 103.6 113.8_+66.8
445.9_+77.9 61.2_+28.5
584.6_+ 164.7 96.35-+31.84
AUC expressed as arbitrary units (mean values_+s.d.): 19.00 to 21.00 h, linear background subtracted.
2.4. Statistics Areas under the curve (AUC) values were calculated according to the trapezoid rule and expressed as arbitrary units. The AUC values without linear background of the different treatments were compared by multivariate analyses of variance (MANOVA). In the case of significant treatment effects, group-by-group comparisons were calculated with univariate F-tests. A nominal level of significance (alpha=0.05) was accepted.
3. Results
Fig. 1 shows the mean plasma concentrations of cortisol and ACTH secretions during CRH stimulation. Table 1 shows the respective AUC's. MANOVA yielded a nonsignificant trend in the overall treatment effect (F=5.06; DF=2,6; P=0.052) for the AUC of cortisol secretion. For ACTH secretion, the AUC (19.00 to 21.00 h) showed a
CRH-stimulated ACTH Secretion pg/ml 5O 45 -x-Placebo ~ ~. 4O •.e-Midazolam 35 3O 25 20 15 10 5 0 18:00 18:30 19:00 19:15 19:30 20:00 20:30 21:00 ~
n
h
h
h
u
CRH-stimulated Cortisol Secretion ng/ml 200 -x-Placebo 175 !-8-Midazolam 150 ~ F l u m ~ " ~ 125 100 75 50 25 0
~
I
t
n
n
I
u
18:00 18:30 19:00 19:15 19:30 20:00 20:30 21:00 Fig. 1. CRH-stimulated ACTH and cortisol secretion. Data indicate mean ACTH (top) and cortisol (bottom) secretion.
significant treatment effect (F=5.28; DF=2,16; P=0.047). Univariate F-tests (DF=I,7) indicated significant differences between flumazenil and placebo (F=7.40; P=0.030) and between flumazenil and midazolam (F=9.53; P = 0.018), but not between midazolam and placebo (F=0.90; P =0.374).
4. Discussion
The major finding of our study was that flumazenil administration in healthy males resulted in a significantly blunted ACTH response to CRH, whereas midazolam had no effect on ACTH response. We also found a tendency towards a blunted cortisol secretory response. These results support an agonistic effect of flumazenil. Most studies with benzodiazepine receptor agonists report a reduction of pituitary-adrenocortical activity (Cristensen et al., 1992), whereas inverse agonists increase the secretion of corticosteroids (De Boer et al., 1991). Our results are further supported by the observations of Steiger et al. (1994), who also demonstrated an intrinsic agonistic activity of flumazenil on the pituitary-adrenocortical system and an inverse agonistic activity on sleep EEG recordings. An explanation for the effects observed in our study may be that corticotropin itself alters the antagonistic activity of flumazenil (Britton et al., 1988). In the rat conflict test and during human public speaking, which are both models of stimulated stress, the partial agonist potency of flumazenil is emphasized presumably by an enhanced release of CRH and/or ACTH. Recently Patchev et al. (1994) reported that tetrahydroprogesterone, a potent benzodiazepine receptor modulator, counteracts CRH-induced anxiety in rats by alteration of its release. Therefore, flumazenil may exert its effects via endogenous ligands of the benzodiazepine receptor, thus leading to an attenuation of CRH-mediated ACTH release. Although direct effects of the applied benzodiazepine receptor ligands on endogenous CRH secretion cannot be ruled out with the present design, the observed decrease in ACTH secretion after flumazenil administration may not necessarily be explained by an inhibition of CRH secretion. The negative results for midazolam have to be assigned to other causes, such as its rapid pharmac~kinetics or distinct dose-response relations (Pohorecky et al., 1988). Furthermore, it should be kept in mind that the inhibiting effects of benzodiazepines on the HPA system were found to depend on the dosage
A. Str6hle, K. Wiedemann / European Neuropsychopharmacology 6 (1996) 323-325
( P o h o r e c k y et al., 1988). W i t h the present design a distinct suppression o f C R H neurons ( K a l o g e r a s et al., 1990) cannot be investigated. Our observations strengthen t h e v i e w that b e n z o d i a z e p i n e receptors are influenced in a highly c o m p l e x m a n n e r by the pituitary-adrenocortical system. W h e t h e r agonistic or inverse agonistic effects p r e d o m i n a t e appears to d e p e n d on the input o f transmitters such as C R H .
References Britton, K.T., Lee, G. and Koob, G.F. (1988) Corticotropin releasing factor and amphetamine exaggerate partial agonist properties of benzodiazepine antagonist Ro 15-1788 in the conflict test. Psychopharmacology 94, 306-311. Cristensen, E, Lolk, A., Gram, L.F. and Kragh-Sorensen, E (1992) Benzodiazepine-induced sedation and cortisol suppression. Psychopharmacology 106, 511-516. Darragh, A., Lambe, R., Kenny, M., Brick, I., Taafe, W.O. and O'Boyle, C. (1982) Ro 15-1788 antagonises the central effects of diazepam in man without altering diazepam bioavailibility. Br. J. Clin. Pharmacol. 14, 677-682. De Boer, S.F., Van der Gugten, J. and Slangen, J.L. (1991) Effects of chlordiazepoxide, flumazenil and DCMCM on plasma catecholamine and corticosterone concentrations in rats. Pharmacol. Biochem. Behav. 38, 13-19. Dorow, R. and Duka, T. (1986) Anxiety: its generation by drugs and their withdrawal. In: Biggio C. and Costa E. (Eds.), GABAergic Transmission and Anxiety. Raven Press, New York, pp. 221-225.
325
Hunkeler, W., Mohler, H., Pieri, L., Polc, E, Bonetti, E.E and Cumin R. (1981) Selective antagonists of benzodiazepines. Nature 290, 514516. Kalogeras, K.T., Calogero, A.E., Kuribayiashi, Y., Khan, I., Gallucci, W.T., Kling, M.A., Chrousos, G.E and Gold, P.W. (1990) In vitro and in vivo effects of the triazolobenzodiazepine alprazolam on hypothalamic-pituitary-adrenal function: pharmacological and clinical implications. J. Clin. Endocrinol. Metab. 70, 1462-1471. Kapczinski, F., Curran, H.V., Gray, J. and Lader, M. (1994) Flumazenil has an anxiolytic effect in stimulated stress. Psychopharmacology 114, 187-189. Nutt, D.J., Glue, E, Lawson, C. and Wilson, S. (1990) Flumazenil provocation of panic attacks. Arch. Gen. Psychiatry 47, 917-925. Patchev, V.K., Shoaib, M., Holsboer, F. and Almeida, O.F. (1994) The neurosteroid tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release and gene expression of corticotropin-releasing hormone in the rat hypothalamus. Neuroscience 62, 265-271. Pohorecky, L., Cotler, S., Carbone, J.J. and Roberts, E (1988) Factors modifying the effect of diazepam on plasma corticosterone levels in rats. Life Sci. 43, 2159-2167. Polc, E (1988) Electrophysiology of benzodiazepine receptor ligands: multiple mechanism and sites of action. Prog. Neurobiol. 3 l, 350-423. SchiSpf, J., Laurian, S. and Gaillard, J.M. (1984) Intrinsic activity of the benzodiazepine antagonist Ro 15-1788 in man: an electrophysiological investigation. Pharmacopsychiatry 17, 79-83. Steiger, A., Guldner, J., Lauer, C.J., Meschenmoser, C., Pollm~icher, T. and Holsboer, F. (1994) Flumazenil exerts intrinsic activity on sleep EEG and nocturnal hormone secretion. Psychopharmacology 113, 334-338.
European Neuropsychopharmacology 6 (1996) 323-325
Short communication
Flumazenil attenuates the pituitary response to CRH in healthy males Andreas Strfhle*, Klaus Wiedemann Max Planck Institute of Psychiatry, Clinical Institute, Kraepelinstr. 2-I0, 80804, Munich, Germany
Received 22 January 1996; accepted 4 June 1996
Abstract Differential effects of flumazenil (an antagonist) and midazolam (an agonist at the benzodiazepine receptor) were studied in 8 healthy males. Adrenocorticotropic hormone (ACTH) and cortisol responses to a corticotropin-releasing hormone (CRH) challenge were measured. ACTH response was significantly lower after flumazenil administration than after saline or midazolam. No significant treatment effect was found for the CRH-induced cortisol secretion. Our results agree with previous reports that point to an agonistic effect of flumazenil on pituitary-adrenocortical system activity and, moreover, may further support the anxiolytic activity of flumazenil in stimulated stress.
Keywords: Flumazenil; CRH; Midazolam; Benzodiazepine; ACTH; Cortisol; Pituitary
1. Introduction: Flumazenil is a specific benzodiazepine receptor ligand that in animal and human studies has been shown to antagonize most of the pharmacological effects of benzodiazepines, which include sedative, muscle-relaxing, anxiolytic, and anticonvulsant effects (Hunkeler et al., 1981; Darragh et al., 1982; Dorow and Duka, 1986). Recently, agonistic as well as inverse agonistic effects of flumazenil on behavior, sleep EEG, and electrophysiological parameters have been reported (Sch6pf et al., 1984; Polc, 1988; Nutt et al., 1990; Steiger et al., 1994). In animals, the activating effects of corticotropin-releasing hormone (CRH) administration were attenuated by flumazenil, which indicated partial agonistic effects (Britton et al., 1988). In addition, healthy volunteers showed an anxiolytic effect of flumazenil during public speaking, which is a model of stimulated stress (Kapczinski et al., 1994). These behavioral findings support an activated pituitaryadrenocortical system impact on the modulation of the partial agonist activity of flumazenil. To characterize the effects of agonistic and antagonistic ligands on CRHstimulated pituitary-adrenocortical activation and to explore possible mutual influences, we administered *Corresponding author. Tel. +49 89 30622379; fax +49 89 30622601. 0924-977X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0924-977X(96)00040-5
flumazenil and the benzodiazepine receptor agonist, midazolam, before CRH administration.
2. Experimental procedures
2.1. Subjects Eight healthy male volunteers between 22 and 30 years of age were enrolled in the study after a thorough medical examination to rule out physical or psychiatric illness, drug intake or life styles that might have interfered with the study. The study was approved by the Ethics Comittee of the Max Planck Institute of Psychiatry and written informed consent was obtained from each subject.
2.2. Study design Subjects received in a double-blind, randomized order an infusion of either 2 mg flumazenil (1 mg at both 18.00 and 18.30 h), 0.5 mg midazolam (18.00 h) or saline on 3 occasions separated by at least 3 days. At 19.00 h, 100 Ixg CRH were administered intravenously. Plasma ACTH and cortisol concentrations were then repeatedly determined at 18.00 (baseline) and further until 21.00 h.
A. Strrhle, K. Wiedemann / European Neuropsychopharmacology 6 (1996) 323-325
324
2.3. Assays Commercial radioimmuno- and immunoradiometric assays (ICN Biomedicals, Carson, CA and Nichols Institute, San Juan Capistrano, CA, USA) were used for plasma cortisol and ACTH measurements. Intra- and interassay coefficients of variation were <7%, respectively.
Table 1 CRH-induced cortisol and ACTH secretion after placebo, flumazenil and midazolam
AUC-cortisol AUC-ACTH
Placebo
Flumazenil
Midazolam
541.0-+ 103.6 113.8_+66.8
445.9_+77.9 61.2_+28.5
584.6_+ 164.7 96.35-+31.84
AUC expressed as arbitrary units (mean values_+s.d.): 19.00 to 21.00 h, linear background subtracted.
2.4. Statistics Areas under the curve (AUC) values were calculated according to the trapezoid rule and expressed as arbitrary units. The AUC values without linear background of the different treatments were compared by multivariate analyses of variance (MANOVA). In the case of significant treatment effects, group-by-group comparisons were calculated with univariate F-tests. A nominal level of significance (alpha=0.05) was accepted.
3. Results
Fig. 1 shows the mean plasma concentrations of cortisol and ACTH secretions during CRH stimulation. Table 1 shows the respective AUC's. MANOVA yielded a nonsignificant trend in the overall treatment effect (F=5.06; DF=2,6; P=0.052) for the AUC of cortisol secretion. For ACTH secretion, the AUC (19.00 to 21.00 h) showed a
CRH-stimulated ACTH Secretion pg/ml 5O 45 -x-Placebo ~ ~. 4O •.e-Midazolam 35 3O 25 20 15 10 5 0 18:00 18:30 19:00 19:15 19:30 20:00 20:30 21:00 ~
n
h
h
h
u
CRH-stimulated Cortisol Secretion ng/ml 200 -x-Placebo 175 !-8-Midazolam 150 ~ F l u m ~ " ~ 125 100 75 50 25 0
~
I
t
n
n
I
u
18:00 18:30 19:00 19:15 19:30 20:00 20:30 21:00 Fig. 1. CRH-stimulated ACTH and cortisol secretion. Data indicate mean ACTH (top) and cortisol (bottom) secretion.
significant treatment effect (F=5.28; DF=2,16; P=0.047). Univariate F-tests (DF=I,7) indicated significant differences between flumazenil and placebo (F=7.40; P=0.030) and between flumazenil and midazolam (F=9.53; P = 0.018), but not between midazolam and placebo (F=0.90; P =0.374).
4. Discussion
The major finding of our study was that flumazenil administration in healthy males resulted in a significantly blunted ACTH response to CRH, whereas midazolam had no effect on ACTH response. We also found a tendency towards a blunted cortisol secretory response. These results support an agonistic effect of flumazenil. Most studies with benzodiazepine receptor agonists report a reduction of pituitary-adrenocortical activity (Cristensen et al., 1992), whereas inverse agonists increase the secretion of corticosteroids (De Boer et al., 1991). Our results are further supported by the observations of Steiger et al. (1994), who also demonstrated an intrinsic agonistic activity of flumazenil on the pituitary-adrenocortical system and an inverse agonistic activity on sleep EEG recordings. An explanation for the effects observed in our study may be that corticotropin itself alters the antagonistic activity of flumazenil (Britton et al., 1988). In the rat conflict test and during human public speaking, which are both models of stimulated stress, the partial agonist potency of flumazenil is emphasized presumably by an enhanced release of CRH and/or ACTH. Recently Patchev et al. (1994) reported that tetrahydroprogesterone, a potent benzodiazepine receptor modulator, counteracts CRH-induced anxiety in rats by alteration of its release. Therefore, flumazenil may exert its effects via endogenous ligands of the benzodiazepine receptor, thus leading to an attenuation of CRH-mediated ACTH release. Although direct effects of the applied benzodiazepine receptor ligands on endogenous CRH secretion cannot be ruled out with the present design, the observed decrease in ACTH secretion after flumazenil administration may not necessarily be explained by an inhibition of CRH secretion. The negative results for midazolam have to be assigned to other causes, such as its rapid pharmac~kinetics or distinct dose-response relations (Pohorecky et al., 1988). Furthermore, it should be kept in mind that the inhibiting effects of benzodiazepines on the HPA system were found to depend on the dosage
A. Str6hle, K. Wiedemann / European Neuropsychopharmacology 6 (1996) 323-325
( P o h o r e c k y et al., 1988). W i t h the present design a distinct suppression o f C R H neurons ( K a l o g e r a s et al., 1990) cannot be investigated. Our observations strengthen t h e v i e w that b e n z o d i a z e p i n e receptors are influenced in a highly c o m p l e x m a n n e r by the pituitary-adrenocortical system. W h e t h e r agonistic or inverse agonistic effects p r e d o m i n a t e appears to d e p e n d on the input o f transmitters such as C R H .
References Britton, K.T., Lee, G. and Koob, G.F. (1988) Corticotropin releasing factor and amphetamine exaggerate partial agonist properties of benzodiazepine antagonist Ro 15-1788 in the conflict test. Psychopharmacology 94, 306-311. Cristensen, E, Lolk, A., Gram, L.F. and Kragh-Sorensen, E (1992) Benzodiazepine-induced sedation and cortisol suppression. Psychopharmacology 106, 511-516. Darragh, A., Lambe, R., Kenny, M., Brick, I., Taafe, W.O. and O'Boyle, C. (1982) Ro 15-1788 antagonises the central effects of diazepam in man without altering diazepam bioavailibility. Br. J. Clin. Pharmacol. 14, 677-682. De Boer, S.F., Van der Gugten, J. and Slangen, J.L. (1991) Effects of chlordiazepoxide, flumazenil and DCMCM on plasma catecholamine and corticosterone concentrations in rats. Pharmacol. Biochem. Behav. 38, 13-19. Dorow, R. and Duka, T. (1986) Anxiety: its generation by drugs and their withdrawal. In: Biggio C. and Costa E. (Eds.), GABAergic Transmission and Anxiety. Raven Press, New York, pp. 221-225.
325
Hunkeler, W., Mohler, H., Pieri, L., Polc, E, Bonetti, E.E and Cumin R. (1981) Selective antagonists of benzodiazepines. Nature 290, 514516. Kalogeras, K.T., Calogero, A.E., Kuribayiashi, Y., Khan, I., Gallucci, W.T., Kling, M.A., Chrousos, G.E and Gold, P.W. (1990) In vitro and in vivo effects of the triazolobenzodiazepine alprazolam on hypothalamic-pituitary-adrenal function: pharmacological and clinical implications. J. Clin. Endocrinol. Metab. 70, 1462-1471. Kapczinski, F., Curran, H.V., Gray, J. and Lader, M. (1994) Flumazenil has an anxiolytic effect in stimulated stress. Psychopharmacology 114, 187-189. Nutt, D.J., Glue, E, Lawson, C. and Wilson, S. (1990) Flumazenil provocation of panic attacks. Arch. Gen. Psychiatry 47, 917-925. Patchev, V.K., Shoaib, M., Holsboer, F. and Almeida, O.F. (1994) The neurosteroid tetrahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety and alters the release and gene expression of corticotropin-releasing hormone in the rat hypothalamus. Neuroscience 62, 265-271. Pohorecky, L., Cotler, S., Carbone, J.J. and Roberts, E (1988) Factors modifying the effect of diazepam on plasma corticosterone levels in rats. Life Sci. 43, 2159-2167. Polc, E (1988) Electrophysiology of benzodiazepine receptor ligands: multiple mechanism and sites of action. Prog. Neurobiol. 3 l, 350-423. SchiSpf, J., Laurian, S. and Gaillard, J.M. (1984) Intrinsic activity of the benzodiazepine antagonist Ro 15-1788 in man: an electrophysiological investigation. Pharmacopsychiatry 17, 79-83. Steiger, A., Guldner, J., Lauer, C.J., Meschenmoser, C., Pollm~icher, T. and Holsboer, F. (1994) Flumazenil exerts intrinsic activity on sleep EEG and nocturnal hormone secretion. Psychopharmacology 113, 334-338.