ALPRAZOLAM (ALP), a benzodiazepine that activates

0021-972X/99/$03.00/0 Vol. 84, No. 8 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society The Inhibitory Effect of Alprazolam, a Benzodiazepine, Overrides the Stimulatory Effect of Metyrapone-Induced Lack of Negative Cortisol Feedback on Corticotroph Secretion in Humans* EMANUELA ARVAT, BARBARA MACCAGNO, JOSEFINA RAMUNNI, LIDIA DI VITO, ROBERTA GIORDANO, LAURA GIANOTTI, FABIO BROGLIO, FRANCO CAMANNI, AND EZIO GHIGO Division of Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy ABSTRACT Alprazolam (ALP), a benzodiazepine that activates -aminobutyric acid-ergic receptors, inhibits the activity of hypothalamo-pituitaryadrenal (HPA) axis, probably via inhibition of hypothalamic CRH and/or arginine vasopressin release. To further clarify the effects of ALP on the HPA axis in humans, in six normal young women (26 34 yr old) we studied the effects of 0.02 mg/kg ALP (administered orally at 0700 h) or placebo on ACTH, cortisol (F), and 11-deoxycortisol (S) levels assayed after placebo or metyrapone (MET; 0.04 g/kg administered orally at 2300 h the night before). After placebo administration, ACTH, F, and S levels showed a progressive decrease from 0700 1200 h (P 0.03). At 0700 h, ACTH, F, and S levels before ALP overlapped with those after placebo. At 1200 h, ACTH, F, and S levels after ALP were lower than those after placebo (P 0.03). MET pretreatment strongly increased ACTH (P 0.03) and S (P 0.02) while clearly inhibiting F (P 0.03) levels at 0700 h. After MET, Received April 14, 1999. Accepted May 10, 1999. Address all correspondence and requests for reprints to: E. Ghigo, M.D., Divisione di Endocrinologia, Ospedale Molinette, C. so Dogliotti 14, 10126 Torino, Italy. E-mail: camanni@pianeta.net. * This work was supported by MURST (Grant 9706151106, Rome, Italy) and FSMEM. ACTH levels did not show any decrease up to 1200 h; similarly, S levels persisted similar up to 1200 h, whereas F levels at 1200 h were significantly increased (P 0.03). At 0700 h, MET-induced ACTH and F levels before ALP overlapped with those after MET alone. The MET-induced ACTH levels at 1200 h were markedly inhibited by ALP (P 0.05). At 1200 h after MET and ALP, a clear reduction of S levels (P 0.02) and an insignificant F reduction were also found. In conclusion, our present data show that ALP inhibits basal and, much more, metyrapone-induced corticotroph secretion. These findings indicate that the inhibitory effect of central -aminobutyric acid-ergic activation by ALP overrides the stimulatory effect of the MET-induced lack of negative F feedback on corticotroph secretion. These results also point toward potential contraindication of ALP administration in patients with suspected hypoadrenalism. (J Clin Endocrinol Metab 84: 2611 2615, 1999) ALPRAZOLAM (ALP), a benzodiazepine that activates central -aminobutyric acid (GABA)-ergic receptors (1), possesses an inhibitory effect on the activity of hypothalamo-pituitary-adrenal (HPA) axis (2 10). In humans, ALP decreases urinary free cortisol (F) (11), the ACTH and F responses to stress (5, 6, 9, 10), arginine vasopressin (AVP) (8), naloxone (7), and hexarelin (12). Thus, ALP has a peculiar inhibitory activity on the HPA axis that has also been demonstrated in patients with panic disorders, in whom this benzodiazepine is widely used (13 15). In both animals and humans, ALP does not modify the ACTH response to CRH (3, 10, 16), suggesting that it acts at the central nervous system (CNS) level via inhibition of hypothalamic CRH release. In fact, ALP does not modify either spontaneous or CRH-stimulated ACTH release from isolated rat pituitary cells (3, 17), whereas it inhibits CRH release in both the locus coeruleus and the paraventricular nucleus (3, 18 20). These findings agree with the inhibitory influence of GABA on CRH-secreting neurons in animals (3, 21, 22). On the other hand, benzodiazepines could also act via inhibition of hypothalamic AVP release (16, 23) and/or though other central mechanisms, such as 2 -adrenergic agonism, which inhibits central noradrenergic activity (5, 24 26). One of the most important factors controlling the activity of HPA is the negative feedback action of glucocorticoids (27, 28). In fact, metyrapone (MET), which inhibits 11 -hydroxylase and thus the conversion of 11-deoxycortisol (S) to F, is one of the most potent stimuli of corticotroph secretion (29 31). The lack of negative F feedback on corticotroph is probably coupled with enhanced hypothalamic CRH and AVP release (28, 32). We aimed to verify the inhibitory effect, if any, of GABAergic activation by ALP on corticotroph secretion, particularly when stimulated by the MET-induced lack of negative F feedback in humans. Thus, we studied the effect of ALP on ACTH, F, and S secretion both in basal conditions and after MET pretreatment in normal young volunteers. Drugs Subjects and Methods Tablets containing 0.5 mg ALP (Xanax) were purchased from Pharmacia & Upjohn, Inc. (Milan, Italy). Capsules containing 250 mg MET (Metopirone) were purchased from Ciba Laboratories (Horsham, UK). 2611

2612 ARVAT ET AL. JCE&M 1999 Vol 84 No 8 Study design Six normal young women (age, 26 34 yr; weight, 50 58 kg) were studied in their early follicular phase. The study was approved by the local ethical committee, and informed consent to participate in it was obtained from all subjects. All subjects underwent various treatments in random order and at least 3 days apart. The tests started at 0700 h after an overnight fast and 30 min after venous cannulation, kept patent by slow infusion of isotonic saline. All subjects received the following treatments in different sessions: 1) placebo and placebo (at 2300 h and 0700 h, respectively), 2) placebo (at 2300 h) and ALP (1.0 mg, orally, at 0700 h; 0.02 mg/kg in subjects 54.5 1.8 kg); 3) MET (2.0 g, orally, at 2300 h; 0.04 g/kg in subjects 54.5 1.8 kg) and placebo (at 0700 h), and 3) MET and ALP (at 2300 and 0700 h, respectively). Blood samples were taken at 2300, 0700, 0730, 0800, 0830, 0900, 0930, 1000, and 1200 h. All samples from an individual subject were analyzed together for ACTH, F, and S secretion. Plasma ACTH levels (picograms per ml) were measured in duplicate by immunoradiometric assay (Allegro HS-ACTH, Nichols Institute Diagnostic, San Juan Capistrano, CA). The sensitivity of the assay was 1.0 pg/ml. The inter- and intraassay coefficients of variation ranged from 6.9 8.9% and from 1.1 3.0%, respectively. Serum F levels (micrograms per L) were measured in duplicate by RIA (CORT-CTK 125, immunoradiometric assay, Sorin, Saluggia, Italy). The sensitivity of the assay was 4.0 g/l. The inter- and intraassay coefficients of variation ranged from 6.6 7.5% and from 3.8 6.6%, respectively. Serum S levels (micrograms per L) were measured in duplicate by RIA (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The inter- and intraassay coefficients of variation ranged from 2.1 5.9% and from 13.7 11.6%, respectively. The hormone levels (mean sem) are expressed either as absolute values or as areas under curves. The statistical analysis was carried out using Wilcoxon test. Results ACTH, F, and S levels at 2300 h in various sessions were similar. After placebo placebo administration, ACTH, F, and S levels showed a progressive decrease from 0700 1200 h [53.2 4.8 vs. 19.8 5.4 pg/ml (P 0.03), 209.3 22.0 vs. 100.8 8.6 g/l (P 0.03), and 1.9 0.2 vs. 1.2 0.1 g/l (P 0.02), respectively; Figs. 1 and 2]. At 0700 h, ACTH, F, and S levels before ALP overlapped with those after placebo. At 1200 h, ACTH, F, and S levels after ALP were lower than those after placebo [6.8 2.5 pg/ml (P 0.03), 78.4 24.2 g/l (P 0.03), and 0.6 0.1 g/l (P 0.02), respectively; Fig. 1]. MET pretreatment strongly increased ACTH (263.5 45.0 pg/ml; P 0.03) and S (134.5 12.5 g/l; P 0.02), but clearly inhibited F (14.5 5.6 g/l; P 0.03) at 0700 h. After MET, ACTH levels did not show any decrease up to 1200 h (242.5 94.0 pg/ml); similarly, S levels persisted up to 1200 h (145.7 5.6 g/l,) whereas F levels significantly increased at 1200 h (75.6 25.9 g/l; P 0.03; Fig. 2). At 0700 h, MET-induced ACTH and F levels before ALP overlapped with those after MET alone. The MET-induced ACTH levels at 1200 h were markedly inhibited by ALP (99.5 28.6 pg/ml; P 0.05). At 1200 h after ALP, a clear reduction in S levels (75.8 8.1 g/l; P 0.02) and a slight reduction in F levels (51.9 11.8 g/l) were also found (Fig. 3). FIG. 1. Mean ( SEM) ACTH, F, and S levels after ALP (0.02 mg/kg, orally, at 0700 h) or placebo in normal subjects. Side-effects Mild sleepiness was recorded after ALP administration in all subjects, whereas MET induced heartburn in four of them. No medication was required, and no test had to be stopped.

EFFECTS OF ALPRAZOLAM ON HPA AXIS IN HUMANS 2613 FIG. 2. Mean ( SEM) ACTH, F, and S levels after MET (0.04 g/kg, orally, at 2300 h the night before) or placebo in normal subjects. Discussion The present study in humans shows that ALP enhances the spontaneous decline of HPA activity during the morning hours and, particularly, markedly inhibits the corticotroph response to the MET-induced lack of negative F feedback mechanism. ALP also inhibits the MET-induced increase in S levels. The inhibitory effect of benzodiazepines, particularly of FIG. 3. Mean ( SEM) ACTH, F, and S levels after MET (0.04 g/kg, orally, at 2300 h the night before) alone and combined with ALP (0.02 mg/kg, orally, at 0700 h) or placebo in normal subjects. ALP, on the activity of HPA axis has been clearly shown in both animals and humans (2 10). The evidence that, according to data in animals (3, 17 20), ALP inhibits the ACTH and F responses to CNS-mediated stimuli as well as to AVP, but not to CRH, in humans (5 10, 12, 16, 27) suggested that it acts via inhibition of endogenous

2614 ARVAT ET AL. JCE&M 1999 Vol 84 No 8 CRH release, although other researchers proposed an AVPmediated mechanism (16, 23). Our findings show that the inhibitory effect of ALP on HPA activity is operative even in basal conditions. In fact, the physiological decrease in ACTH and F levels in the morning hours was significantly enhanced by GABAergic activation, according to data showing that ALP is able to reduce 24-h integrated urinary F concentrations in humans (11). The most impressive finding of the present study is the strong inhibitory effect of ALP on the corticotroph hypersecretion as well as the marked S increase that follows the MET-induced lack of negative F feedback. The ACTH hypersecretion after the MET-induced lack of negative F feedback probably reflects enhanced hypothalamic CRH and AVP release (28, 32), although a pituitary mechanism should also be taken into account (26). In fact, exogenous CRH infusion further enhances the MET-induced corticotroph hypersecretion in humans (33 36). Thus, the inhibitory effect of ALP on the corticotroph responsiveness to MET could be explained by an inhibitory effect of GABAergic activation overriding the stimulatory effect of MET on the activity of CRH- and/or AVP-secreting neurons. In fact, ALP does not act at the pituitary level (2, 3, 10). The CNS level whereby GABA could inhibit the HPA activity is within the hypothalamus in the paraventricular nucleus (37) or at a suprahypothalamic level within the hippocampus (38). In fact, at both levels, high GABA and CRH receptor density has been demonstrated, and both of these CNS areas have been shown to play a critical role in the negative glucocorticoid feedback mechanisms (39, 40). In conclusion, our study shows that ALP inhibits the basal and, much more, the MET-induced corticotroph secretion. 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