Hypothalamic-pituitary-adrenal axis activity in panic disorder: Effects of alprazolam on 24 h secretion of adrenocorticotropin and cortisol

.I ,n,‘rhm

Res.. Vol 30. No. 2, pp. 79-93. 1996 CopyrIght cj 1996 Elsewer Science Ltd Prmted m Great Bntain. All rights reserved 0022-3956!96 $15.00+ 00

Pergamon 0022-3956(95)000354

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVITY IN PANIC DISORDER: EFFECTS OF ALPRAZOLAM ON 24 h SECRETION OF ADRENOCORTICOTROPIN AND CORTISOL JAMES

L. ABELSON,* University

of Michigan

GEORGE

C. CURTIS

and OLIVER

Department of Psychiatry, Anxiety Ann Arbor, Michigan. U.S.A.

Disorders

G. CAMERON Program,

(Received 16 February 1995; raised 28 Ju!,~ 1995; accepted 22 August 1995) Summary-Pre-clinical and some clinical evidence suggests that central overdrive within the hypothalamic-pituitary-adrenal (HPA) axis may play a role in panic disorder, and that the anti-panic efficacy of alprazolam may involve its ability to inhibit this drive. Detailed examination of 24 h secretion of adrenocorticotropin (ACTH) and cortisol in 20 panic patients had revealed subtle HPA axis abnormalities prior to treatment. In order to determine whether these abnormalities resolve with alprazolam therapy, these patients were re-studied over a full circadian cycle after 12 weeks on alprazolam. Alprazolam produced substantial improvement in clinical status which was accompanied by nearly full resolution of pre-treatment hypercortisolemia. The impact of treatment on ACTH was more complex and influenced by symptom severity. The results are consistent with the hypotheses that HPA axis regulation is subtly disturbed in panic disorder and that impact on the HPA axis may play a role in alprazolam’s mechanism of efficacy. Copyright LF, 1996 Elsevier Science Ltd.

Introduction It has been suggested that excessive activity in the hypothalamic-pituitary adrenal (HPA) axis, driven by corticotropin-releasing hormone (CRH) neurons, may play an etiologic role in panic disorder as well as in depression (Gold et al., 1990). The ability of benzodiazepines (BZs), and particularly alprazolam, to inhibit HPA axis activity has been cited as supporting a role for CRH and the HPA axis in panic (Gold et al., 1990; Kalogeras et al., 1990). Extensive animal research (see De Souza (1990) for review) reveals that benzodiazepine (BZ) receptor agonists consistently attenuate both stress- and drug-induced HPA axis activation and that BZ receptor inverse agonists can elicit anxiety-like behavior accompanied by HPA axis activation. In vitro evidence suggests that BZs may alter HPA axis activity via direct inhibition of hypothalamic CRH neurons (Calogero et al., 1988). Alprazolam’s IO-fold greater potency than diazepam in these models may explain its increased potency in the treatment of panic (Gold et al., 1990). Confirmation that alprazolam blocks panic by inhibiting the CRH system (Kalogeras et al., 1990) would provide strong support for the hypothesis that CRH plays an etiologic role in the disorder. *Correspondence Drive, Ann Arbor.

to: James L. Abelson, M.D.. Ph.D., Rm C435. Med Inn Bldg/0840. MI 481094840, U.S.A. (Tel: 313-764-5348; Fax: 313-936-7868). 79

1500 E Medical

Center

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Although pre-clinical data suggest a role for CRH in panic disorder (Pihoker & Nemeroff, 1993) and a role for inhibition of HPA axis activity in alprazolam’s anti-panic efficacy, clinical evidence is inconsistent. Patients with panic disorder may or may not have blunted adrenocorticotropin (ACTH) responses to CRH (Brambilla et al., 1992; Holsboer et al., 1987; Rapaport et al., 1989; Roy-Byrne et al., 1986); they sometimes show elevated rates of cortisol non-suppression following dexamethasone (DST), but not always (Coryell & Noyes, 1988; Curtis et al., 1982); and they may or may not have elevated urinary free cortisol (UFC) (Kathol et al., 1988; Uhde et al., 1988). There is clear evidence that BZs do acutely reduce HPA axis activity in normal humans (Breier et al., 1992; Schuckit et al., 1992; Schtirmeyer et al., 1988) and in patients with depression (Gram & Christensen, 1986) and panic disorder (Roy-Byrne et al., 1991). However, there is little information on longerterm effects. Treatment studies in panic suggest that alprazolam reduces baseline cortisol but does not affect ACTH response to CRH (Brambilla et al., 1992); reduces UFC, but only in “complicated” patients (Lopez et al., 1990); and does not affect DST results (Coryell et al., 1989). In order to further define the status of the HPA axis in patients with panic disorder, we studied it intensively via 24 h monitoring of ACTH and cortisol (Abelson & Curtis, in press). Panic patients had elevated overnight cortisol secretion. Patients who entered the study through routine referral channels had more extensive hypercortisolemia than those who were recruited by advertisements, despite similar illness severity. Patients with less frequent panic attacks (I 2/week) had elevations in daytime ACTH not seen in the more frequent panickers, but the patients with more frequent attacks had reduced ACTH/cortisol ratios and a shifted ACTH circadian cycle, suggesting more chronic, central overdrive. If the efficacy of alprazolam in the treatment of panic disorder is linked to its ability to inhibit the CRH system, then successful treatment with alprazolam should correct these pretreatment abnormalities. To test this hypothesis we entered all patients studied in our pretreatment evaluation of the axis in an open-label, structured treatment trial using alprazolam as the sole therapeutic intervention. We then repeated the intensive evaluation of HPA axis activity after at least 10 weeks of treatment, while patients were still on medication. Subjects and methods Patients and controls

Twenty patients with panic disorder and 12 normal control subjects were studied. All subjects were evaluated using a Structured Clinical Interview (SCID-UPrev) (Spitzer & Williams, 1986). All were between 18 and 42 years of age; were medically healthy as confirmed by history, physical examination and screening laboratory tests; reported no exposure to psychoactive medication for at least 2 weeks prior to study and passed a urine drug screen and plasma benzodiazepine screen at time of entry; and were on no medication that might effect HPA axis activity. All females with child-bearing potential were using contraceptive precautions other than birth control pills, had regular menstrual cycles, had a negative urine pregnancy screen at entry and were studied within 7 days of onset of menses. Patients were recruited from routine referrals to the University of Michigan Anxiety

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Disorders Program and from respondents to newspaper advertisements. All patients met DSM-III-R criteria for panic disorder, with or without agoraphobia and had experienced at least one panic attack per week during the prior 3 weeks. The majority (n = 11) had no psychotropic medication exposure. Only five had exposure within the past month (all on low or as-needed doses of alprazolam totaling less than 1 mg/day). Additional description of the patients is available elsewhere (Abelson & Curtis, in press). Control subjects were recruited by advertisements in community newspapers. They had no current or past history of any Axis I disorder by SCID and reported no Axis 1 disorder in any first degree relatives. Patient subjects received all clinical care during the 10 week treatment protocol at no charge. One patient withdrew from the study due to dissatisfaction with treatment and her data were discarded. Control subjects were studied once and were paid $200. Design and procedures

Patients completed an initial detailed evaluation of HPA axis functioning, a structured IO-week treatment protocol utilizing alprazolam pharmacotherapy (open-label, flexible dose), and repeat detailed evaluation of the HPA axis. The HPA axis assessments included both 24 h monitoring of ACTH and cortisol levels and a CRH stimulation test. This report will present the effects of treatment with alprazolam on 24 h HPA axis measures. Subjects were scheduled for admission to a Clinical Research Center (CRC) after screening, evaluation and provision of informed consent following full explanation of procedures. On admission, and at least 1 h before blood sampling began, an intravenous (IV) catheter was placed in a forearm vein and kept open with a heparinized normal saline drip. Beginning at 1800 h and continuing for 24 h, blood samples were drawn every 15 min into chilled tubes, placed on ice, spun in a refrigerated centrifuge within 2 h, and frozen for later analysis for ACTH and cortisol. An extension tube permitted sampling from outside of the subjects’ bed curtains overnight (230&0700 h). Subjects remained at bed rest except for bathroom use. They were served regular hospital meals, receiving dinner prior to initiation of blood sampling on day 1, and breakfast at approximately 0800 h and lunch at about noon on day 2. The last sample for the 24 h monitoring was obtained at 1745 h on day 2. A CRH stimulation test was performed between 1750 h and 2000 h on day 2, after which subjects were fed dinner and discharged. Patients returned to the clinic 1 week after the CRC stay. Clinical assessment scales were completed and alprazolam treatment initiated. The initial dose was 0.5 mg three times per day. This was adjusted if prior experience with BZs or alcohol suggested low or high tolerance for sedative side effects. Patients returned to the clinic on a weekly basis for 5 weeks, and on alternating weeks for 558 additional weeks, for clinical assessments and review of alprazolam side effects. Alprazolam dosages were advanced until panic attacks were abolished. a maximum of 10 mg/day was reached, or side effects precluded further advancement. If side effects blocked dose increases before panic attacks were fully controlled, dosage was held constant or reduced to the last tolerated level. If panic attacks continued at the next visit, further attempts to increase the dose were undertaken. No other psychoactive medications were permitted during the study and no non-pharmacological

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interventions were offered. After a minimum of 9 weeks of treatment (to insure at least 10 weeks between the two blood sampling procedures), patients returned to the CRC for repeat evaluation of HPA axis function. The procedure for the second HPA axis evaluation was identical to the first. When it was completed, patients were referred back to the Anxiety Disorders Clinic where they were treated in accordance with the clinic’s standard clinical care. Measures

Assays were conducted at a commercial laboratory (Hazelton Washington, Inc.) using commercially available kits. The ACTH was measured using the immunoradiometric (IRMA) assay kit from Nichols Institute (San Juan Capistrano, CA, U.S.A.). Cortisol was measured using the Kallestead QuanticoatTM radioimmunassay from Sanofi Diagnostics (Chaska, MN, U.S.A.). Sensitivities were 0.5 pg/dl for the cortisol assay and 1.5 pg/ml for the ACTH assay. All coefficients of variation were less than 17%. At the time of each blood sample the CRC nurse recorded whether the subject appeared to be asleep or awake. We calculated a rough “sleep quality” measure by counting for each subject their longest consecutive streak of “asleep” observation points and multiplying this number by 15 min. The result reflects the ability of subjects to maintain the appearance of sleep, uninterrupted by activity interpreted by nurses as suggestive of wakefulness. Assessment of patients’ clinical status included the Panic Attack and Anticipatory Anxiety Scale (PAAAS), the Marks/Sheehan Phobia Scale, the Sheehan Disability Scale, and the Hamilton Anxiety Scale. All instruments were adopted from the Cross-National Collaborative Study (Ballenger et al., 1988). Panic attack frequency was determined from the PAAAS and recorded as the average number of full-symptom attacks per week over the month prior to evaluation for the study. Agoraphobic avoidance was recorded as the sum of the avoidance scores on the agoraphobic items of the Phobia Scale. Total disability was recorded as the sum of the scores on the four subscales of the Disability Scale and reflects subjective evaluation of impairment in arenas of work, social life/leisure and family/home responsibilities. Analyses

All hormonal data were log-transformed prior to analysis to improve their fit to a normal distribution curve. Main analyses compared patients’ pre-treatment data to their “on alprazolam” data, using paired t tests which examined 24 h, overnight and daytime means for ACTH and cortisol. Follow-up comparisons broke the 24 h period down into six 4 h epochs. These will be reported only when they add information not available from the main analyses. We did not re-study control subjects a second time, so in order to test deviation from “normal“ in patients on alprazolam, we compared them to the same control data used in the pre-treatment analyses. Circadian rhythm characteristics were examined using a commercially available, multioscillator modeling program (CosifitTM) (Teicher & Barber, 1990). This program applies a best-fit 24 hr cosine curve to each subject’s data and then adds additional oscillators and determines the best-fit model. Our data were best fit by a three oscillator model in which

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the first oscillator was fixed at one cycle/day (circadian), the second oscillator was fixed at two cycles/day (hemicircadian), and the third oscillator was allowed to vary in frequency (the program determines the “ultradian” frequency that produces the best-fit curve). The hemicircadian oscillator appeared to partially adjust the circadian rhythm to its natural, non-sinusoidal shape. The third (ultradian) oscillator appeared to capture activity in transient bursts that were independent of the circadian rhythm. The program automatically provides a mesor. amplitudes and times of acrophase of the circadian, hemicircadian and ultradian rhythms, and goodness-of-fit correlation coefficients (measuring how well the data are fit by the model) for each subject. Cosinor data are reported only when they add information not available from the main analyses. Pulse-wave characteristics of cortisol secretion were quantified using the Kushler-Brown pulse detection algorithm (Kushler & Brown, 1991) which uses an exponential decay model based on the known half-life of a hormone and locates pulses with appropriate decay curves. It provides estimates of baseline levels, the number of pulses, and average pulse amplitude. Because it requires from three to five samples per half-life, we were not able to apply it to the ACTH data. In the body of the results section data will be presented as means k SD.We will present raw data means rather than the log transformed values used in analyses since the raw values are more meaningful to most readers. All statistical tests were considered significant at a p value = 0.05. Results As reported elsewhere (Abelson & Curtis, in press), patients and controls did not differ significantly in age (30.6 f 6.1 and 28.1 + 4.4 years, respectively) or sex distribution (8/20 and 3/12 male, respectively). Patients were re-assessed an average of 12 weeks after initiation of alprazolam therapy. They were taking an average of 4.1 mg/day of alprazolam (range: 1.5-8 mg), the majority on a three-times-per-day schedule. Significant improvements were seen on all severity of illness variables (panic attack frequency, agoraphobic avoidance, Hamilton Anxiety scores and total disability). At the time of the second assessment, the patients had a mean Hamilton Anxiety score of 6.4 + 5.7, reduced from 15.3 _t 7.8 at entry. They were experiencing an average of 0.8 f 1.5 panic attacks per week, reduced from 3.5 k 2.7 at entry. The clinical response to alprazolam in these patients has been previously reported (Abelson & Curtis, 1993). Cortisol Treatment reduced cortisol secretion throughout the circadian cycle, with significant reductions in 24 h, overnight and daytime means for the total patient group (see Table 1). As can be seen in Figure 1, the 24 h cortisol secretion curve for patients on alprazolam was nearly identical to that of controls. Prior to treatment, patients had elevated overnight cortisol secretion relative to controls. Patients derived from regular clinic referral sources (“clinic patients”) were significantly more elevated than patients who responded to advertisements (“ad patients”); and the clinic patients had elevated cortisol relative to controls throughout the 24 h cycle (Table 1).

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Table 1 Mean (k SD) Cortisol Levels (pggldl)for Untreated Control Subjects and,for Patients and Patienr Subgroups Before and During Treatment with Alprazolam a Pre-treatment 24 h Patients, all (n = 20) Clinic patients (6) Ad patients (14) High frequency PA Pts (10) Low frequency PA Pts (JO) Control subjects (n = 12)

Overnight

7.3 + 1.9 8.6 k.2.3” 6.7 k 1.4

5.3 k 2.9’ 6.8 k 4.7’ 4.7 i 1.6

7.1 + 2.3

5.6 i

3.8

7.4 * 1.5’ 6.1 If: 1.2

cortisol Daytime

24 h

on alprazolam

Overnight

Daytime

5.7 & 1.3++ 5.7 * 1.2 5.7 f 1.4

3.1 f 1.0++ 2.7 + 0.9 3.2 + 1.0

8.0 * 1.9++ 8.2 + 1.7 7.8 + 2.0

9.0 rf: 1.9

5.8 f

1.4

3.4 * 1.0

8.0 k 2.2

5.0 * 1.8

9.8 + 2.1

5.6 f

1.3

2.8 + 0.9

7.9 k 1.6

3.7 f

8.3 + 1.7

1.4

9.4 * 2.0 10.4 * 2.0 8.9 + 1.8

Cortisol

Statistical tests compared: (1) patients (both pre-treatment and on alprazolam) to controls using unpaired t tests; significant differences are marked with an asterisk (*); and (2) all patients on alprazolam to all patients pretreatment using paired I tests; significant differences are marked with a plus (‘). ’ or + p < 0.05. ‘* or ++ p < 0.01.

After 12 weeks of alprazolam therapy, all of these differences had disappeared. Neither the total patient group nor any patient subgroups differed from control subjects on any cortisol measure when patients were re-tested on alprazolam (Table 1). Treated clinic patients and ad patients did not differ from each other on any cortisol measure. Though symptoms improved and cortisol declined over the 12 weeks of alprazolam therapy, we found no significant correlations between change in any of the symptom severity measures and change in any cortisol measure. Alprazolam substantially improved our ability to model the patients’ cortisol data with cosinor curves. The “goodness-of-fit” correlation coefficient in the best-fit three oscillator model was significantly higher on alprazolam (Y = 0.85 f 0.09) than it was prior to treatment (r = 0.77 f 0.11; t = 3.6, p = 0.002). Prior to treatment, the total patient group and the clinic patients had significantly lower goodness-of-fit correlations than the control group but, on alprazolam, both groups no longer differed from the controls. Treatment significantly reduced the amplitude of the ultradian rhythm in patients (from 2.3 + 0.9 pg/dl to 1.9 f 0.6 pg/dl; t = 2.2, p = 0.04) with no change in its frequency (average of 5 cycles/24 h before and after treatment). The elevated ultradian amplitude relative to controls seen in clinic patients prior to treatment was corrected by alprazolam (reduced from 2.6 f pg/dl to 1.8 f 0.3 pg/dl, which no longer differed from the control value of 1.6 + 0.5 pg/dl; p = 0.41). Alprazolam had no significant impact on the amplitude or timing of the circadian rhythm. Confirmation that alprazolam reduced activity within the higher frequency, shorterduration components of cortisol’s secretory pattern was provided by the pulse-wave analyses. The frequency of pulses detectable by our algorithm did not change with treatment (7.9 + 1.8 pulses/24 h before treatment vs 7.5 f 1.5 after treatment, t = 0.6, p = 0.57).

HPA Axis in Alprazolam-treated

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Panic

25.00

20.00 A

E 15.00 3I S E

10.00

Y 5.00

0.00

12.00 10.00 B 3# S Z .E 5 0

6.00 6.00 4.00 2.00 0.00

4

I

Figure 1. The ACTH (upper panel) and cortisol (lower panel) levels measured every 15 min and averaged over 2 h epochs for 24 h in panic patients prior to treatment and after an average of I2 weeks on alprazolam. and in normal control subjects. Graphical presentation is meant to provide a visual depiction of the results, but to avoid figures that are too complex to visually process comfortably, error bars are not included. Statistically meaningful differences are described in the text and tables.

Pulse amplitude was significantly reduced (6.69 f 1.34 pg/dl vs 4.48 k 1.22 pg/dl; t = 2.9, p = 0.01) but baseline cortisol showed no change (2.74 k 0.8 vs 2.89 + 0.5 pg/dl). ACTH Treatment with alprazolam had little effect on ACTH secretion. There were no significant changes (p > .7 in all cases) in 24 h, overnight, or daytime means. The total patient group did not differ from controls in mean ACTH levels either before or after treatment. These

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Table 2

Mean(+ SD) ACTH Levels @g/ml) for Untreated Control Subjects and,for Patients and Patient Subgroups Before and During Treatment with Alprazolam” Pre-treatment 24 h Patients, all (n = 20) Clinic patients (6) Ad patients (14) High frequency PA Pts (10) Low frequency PA Pts (10) Control subjects (n = 12)

ACTH (pg/ml)

Overnight

ACTH

(pg/ml) on alprozolam

Daytime

24 h

Overnight

Daytime

14.0 * 5.9 14.2 k 6.5 13.9 + 5.9

10.3 + 5.0 10.3 + 4.6 10.4 + 5.4

18.4 f 7.8 18.8 f 8.6 18.3 & 7.7

13.5 * 4.8 13.8 i 3.3 13.3 & 5.4

8.7 * 4.5 8.1 k 3.2 9.0 f 5.1

17.8 k 6.5 18.8 * 4.1 17.3 * 7.4

11.2 & 4.6

8.7 k 3.4

14.8 + 6.4

12.5 + 5.6

9.7 & 5.9

15.9 i

16.7 k 6.0

12.0 k 6.0

22.1 + 7.5*

14.5 + 3.9

7.7 k 2.6

19.7 f 5.4

12.0 f 3.9

8.3 f 3.5

15.5 * 4.9

7.2

* Statistical tests compared: (1) patients (both pre-treatment and on alprazolam) to controls using unpaired t tests; significant differences @ < 0.05) are marked with an asterisk (*); and (2) all patients on alprazolam to all patients pre-treatment using paired I tests; no significant differences were found.

results are presented in Table 2. The ACTH circadian secretion curves for patients before and during treatment and for controls are presented in Figure 1 (top panel). Though patients on alprazolam generally had lower levels than they did pre-treatment, differences were not significant. However, the apparent elevation of ACTH by alprazolam during the initial epoch (1800-2200 h) was significant (7.8 _+ 4.0 pre-treatment vs 10.2 f 4.5 pg/ml t = 2.4, p = 0.02) on alprazolam; The ACTHjcortisol

ratio

The sensitivity of the adrenal cortex to stimulation by ACTH was estimated by calculating the ACTH/cortisol ratio. Group means were calculated for the entire 24 h cycle, overnight and 4 h epochs. No differences between patients and controls were seen prior to treatment. Alprazolam significantly increased the ratio over the entire 24 h (t = 2.3, p = 0.04) overnight (t = 2.4, p = 0.02) and during the 4 h epochs between 1800 and 2200 h (t = 2.4, p = 0.03) and between 0200 and 0600 h (t = 2.6, p = 0.02). There were non-significant increases in the other epochs. Patients on alprazolam did not differ from the control group in ACTH/cortisol ratios. l@kts

of illness severity

Prior to treatment (see Table 2) we had found significant elevations in daytime ACTH in patients with low panic attack frequency (I 2 attacks/week, “low PA patients”) relative to both controls and patients with higher frequency attacks (> 2 attacks/week, “high PA patients”). No significant changes in ACTH levels were seen in response to treatment, neither in the total patient group nor in any subgroups, but the group differences seen prior to treatment were attenuated. As can be seen in Table 2, ACTH levels rose (non-significantly) with treatment in the high frequency PA patients and fell (non-significantly) in the low PA patients, resulting in convergence of the two groups. Despite the convergence,

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however, the low PA patients were still elevated relative to the high PA patients during two of three epochs that had revealed significant elevations pre-treatment (18OC~2200 h, t = 2.6,~ = 0.02; and 1000-1400 h, t = 2.2,~ = 0.04). Although ACTH levels were higher in males than females, and the low PA patient group had a non-significantly higher proportion of males (50% vs 30%) the elevated ACTH seen in the low PA groups was not a confound of sex. Amongst female subjects analysed separately, low PA patients (n = 5) still had higher ACTH levels than high PA patients (n = 7) during the daytime (100&1800 h; t = 2.3,~ = 0.04). Prior to treatment, we had found a reduced ACTH/cortisol ratio in the high PA patients. suggestive of increased adrenal reactivity to ACTH. The analysis compared the two patient groups and controls in an ANOVA, using female patients only because of the effect of sex on ACTH/cortisol ratios (Roelfsema et al., 1993), and excluded the period of intrinsic, circadian activation (0200-1000 h) when high ambient ACTH levels could obscure subtle differences in adrenal sensitivity. The significant group difference in this ANOVA disappeared with patients on alprazolam (F = 0.02, p = 0.82) due to a nearly significant rise in the ACTH/cortisol ratio in the high PA patients (from 1.3 + 0.6 to 2.3 f 1.5; t = 2.2, p = 0.07). The ratio remained unchanged in the low PA patients (2.8 + 0.9). The ratio for the control group was 2.5 &- 1.2. The pre-treatment abnormality in ACTH circadian profile seen in high PA patients, reflected in a highly significant group-by-time interaction (p = 0.0003) in a repeated measures ANOVA comparing them to controls (ACTH averaged into 2 h epochs over the 24 h) and in a significantly earlier time of circadian acrophase @ = 0.01) in the cosinor analyses, was attenuated by treatment. The group-by-time ANOVA interaction no longer reached significance (p = 0.07); and the groups now had nearly identical times of circadian acrophase Gr, = 0.82). At entry, high and low frequency PA patients did not differ in age or duration of illness, but high PA patients were significantly more ill on all severity measures (Abelson & Curtis, 1993). They remained significantly more disabled after 12 weeks on alprazolam (12 + 7.1 vs 4.2 + 4.6, t = 2.9, p = 0.009); but PA frequency was now nearly identical (0.8 k 1.9 vs 0.7 + 0.8, t = 0.2, p = 0.88) and differences on the other severity measures were no longer significant (agoraphobic avoidance: 3.9 f 4.1 vs 2.0 + 2.6, t = 1.2, p = 0.23; and Hamilton Anxiety score: 8.0 &- 5.4 vs 4.7 + 5.3, t = 1.4, p = 0.19). The high PA patients were on significantly higher doses of alprazolam (adjusted for weight) after 12 weeks of treatment (5.2 + 1.5 mg vs 3.0 f 1.6 mg, t = 3.1, p = 0.007). Because the high and low PA groups differed in disability and alprazolam dose and both of these factors could affect ACTH levels, we re-analysed ACTH levels during the two epochs where significant differences had been found (1800-2200 and 1000-1400 h), using disability and alprazolam dose as covariates in an analysis of covariance (ANCOVA). The group differences remained nearly significant ((I < 0.06) for both epochs even when differences in disability were taken into account. When covaried for differences in alprazolam dose, the group difference remained significant for the 1800-2200 h epoch (p < 0.01) but no longer reached significance for the 1000-1400 h epoch @I = 0.33). Because we had seen reduced ACTH/cortisol ratios in our female, high PA patients prior to treatment and because high PA patients required higher doses of alprazolam to

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adequately control attacks, we wondered whether the degree of adrenal sensitivity to ACTH prior to treatment might predict these patients’ final dose of alprazolam. Because illness severity could also play a role, the relationships between illness severity measures and dose were also examined. We limited these analyses to the female patients (n = 12). Both ACTH/cortisol ratio and agoraphobic avoidance prior to treatment significantly predicted alprazolam dose (Y = -0.7, p = 0.01 and r = +0.6, p = 0.05, respectively). Other measures of illness severity (PA frequency, disability, Hamilton Anxiety score) did not predict dose. When ACTH/cortisol ratio and agoraphobic avoidance were analysed in a multiple, stepwise regression, only the ACTH/cortisol ratio was entered (the partial correlation for agoraphobic avoidance was 0.1). The only clinical measure which yielded a wide enough range of outcomes at follow-up to permit meaningful statistical analysis was total disability. When we divided the female patients into two groups on the basis of their remaining disability after 12 weeks of treatment and compared them on pre-treatment measures of HPA axis activity, we found that those patients with greater residual impairment (total disability rating 2 10) had significantly lower ACTH/cortisol ratios prior to treatment (1.3 ) 0.6 vs 2.6 + 1.O, t = 2.8, p = 0.02) and lower “waking” ACTH secretion (1800-2200 h, t = 3.0, p = 0.01; 1000~1800 h, t = 2.4, p = 0.04). They also had been more severely ill prior to treatment on all clinical measures (PA frequency, agoraphobic avoidance, disability, Hamilton A scores; p < 0.01 for all). “Sleep” and cortisol

Prior to treatment, patients who “slept” better in the CRC (i.e. were better able to maintain the appearance of uninterrupted sleep) had lower 24 h cortisol secretion (r = - 0.5,~ < 0.05). Sleep quality prior to treatment did not predict sleep quality on alprazolam. On alprazolam there was no longer a relationship between “sleep” and 24 h cortisol (r = -0.1, p = 0.83). However, sleep quality prior to treatment was able to significantly predict 24 h cortisol secretion 12 weeks later on alprazolam (r = -0.5, p = 0.03). Those who had more trouble maintaining the appearance of sleep at time of initial study still had greater cortisol secretion when restudied, even though they appeared to sleep better. Discussion As reported elsewhere (Abelson & Curtis, in press), panic patients had elevated overnight cortisol levels prior to treatment. The ACTH levels were not similarly elevated, but ACTH secretion nevertheless appeared dysregulated. Patients with less frequent panic attacks (I 2/week) had elevated daytime ACTH, while high frequency panic attack patients had normal levels but a decreased ACTH/cortisol ratio and a shifted circadian rhythm. Twelve weeks of treatment with alprazolam produced substantial symptom resolution (Abelson & Curtis, 1993). The present data demonstrate that alprazolam also significantly reduced cortisol levels, “correcting” the cortisol abnormalities that had been seen prior to treatment, but had little overall effect on ACTH levels, with a resultant increase in the ACTH/cortisol ratio. Interpretation of ACTH data was again complicated by the impact of panic attack

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frequency, but pre-treatment dysregulation in ACTH secretion also appeared to normalize with therapy. Although we cannot definitively rule out the possibility that changes were a simple consequence of re-testing, evidence of test-re-test stability in cortisol activity (Schreiber et al., 1988) and the known inhibitory effects of BZs on the HPA axis argue against this possibility. Our data suggest that the well-established acute cortisol suppressive effects of BZs (Adam et al., 1984; Breier et al., 1992; Gram & Christensen, 1986; Schuckit et al., 1992; Schtirmeyer et al., 1988) are sustained during chronic administration, at least when alprazolam is therapeutically administered to panic patients. The data also indicate that BZs reduce cortisol by reducing activity in higher frequency cortisol pulses or oscillations (reflected in reduced ultradian and pulse amplitude in the absence of changes in circadian amplitude or “baseline“ levels) and by producing smoother, more tightly controlled rhythms (reflected in improved goodness-of-fit correlations in cosinor modeling). Whether the changes are pharmacological effects that would occur in the absence of a psychiatric disorder and symptom change cannot be determined from our data. since we do not have repeat assessments on a non-ill control group or on an ill, but unimproved, group of patients. However, our support of the a priori hypothesis, based on pre-clinical and prior clinical literature, that effective alprazolam therapy would be associated with reduced HPA axis activity, supports the possibility that the anxiolytic efficacy of alprazolam may be related to its ability to inhibit the HPA axis. We did not find significant direct relationships between symptom improvement and change in 24 h or overnight cortisol, but a variety of factors made detection of such relationships difficult. These include initial value effects, the consistency of change in both cortisol levels and symptoms across subjects, and the differential effects of alprazolam on ACTH secretion. depending on illness severity (see below). The fact that all pre-treatment elevations in cortisol levels disappeared on alprazolam is consistent with treatment studies in depression (Linkowski et al., 1987). It is also consistent with two out of the three longitudinal studies of HPA axis function in panic disorder patients treated with BZs (Brambilla et al., 1992; Coryell et al., 1989; Lopez et al., 1990). which showed that alprazolam therapy was associated with correction of pre-treatment HPA axis abnormalities. It suggests that hypercortisolemia may be a state marker in panic disorder, as it appears to be in depression, rather than a trait marker of vulnerability. Though the strength of this impression is tempered by a number of important factors (see below), the data do support the hypothesis that HPA axis over activity in panic patients is linked to the active presence of symptomatic disorder. Though alprazolam had little overall impact on ACTH levels, interpretation of the ACTH data is complicated by the effects of PA frequency. Prior to treatment. low frequency PA patients had increased daytime ACTH levels, perhaps reflecting the increased acute reactivity to environmental perturbations proposed by others (Roy-Byrne et al., 1986). The ACTH levels were not elevated in the high frequency P.4 patients, perhaps due to adrenal sensitization from chronic exposure to ACTH, reflected in reduced ACTH/cortisol ratios. Alprazolam had differing effects on ACTH secretion in the two groups, appearing to raise secretion in the high frequency group and lower it in the low frequency group. The result was to produce some convergence towards “normal” in both groups. The decreased ACTH,icortisol ratio seen prior to treatment in the high PA group largely disappeared and

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the shift seen in their circadian rhythm was ameliorated. The daytime elevations in ACTH secretion seen in the low PA patients were attenuated. The changes seen in ACTH are less striking than those seen for cortisol, perhaps because of the following factors: (a) more rapid fluctuations in ACTH levels, producing increased variability over the circadian cycle; (b) greater variability in the ACTH assay; and (c) smaller sample sizes produced by the differential impact of panic attack frequency on ACTH levels. Replication is needed, but our ACTH data, like the cortisol data, are consistent with the hypotheses that HPA axis dysregulation in panic patients resolves with treatment and that alprazolam’s anti-panic efficacy may be linked to its ability to inhibit HPA axis activity. The impression that HPA axis hyperactivity is a state marker in panic that resolves with treatment is tempered by a number of observations. One is the presence of a pharmacological agent known to inhibit HPA axis activity at the time of re-testing. Until we are able to demonstrate that hypercortisolemia resolves in conjunction with illness remission, even in the absence of interventions which inhibit HPA function in their own right, we cannot be certain that HPA axis dysregulation is state-dependent. Inhibition of the axis may be an epiphenomenon of effective drug treatments and not a necessary concomitant of efficacy. Inability to detect continuing HPA axis dysregulation in drug-treated patients may then result from the masking of a trait abnormality by the drug-related epiphenomenon. Because drug effects can be quite enduring (Schittecatte et al., 1992) clearer resolution of this issue may require treatment studies in which symptom remission is achieved by nonpharmacological means. Although interpretation of post-hoc correlational analyses must be considered preliminary, one intriguing relationship was noted which also complicates a simple state-marker interpretation of HPA axis dysregulation. Cortisol elevations prior to treatment correlated with an observational measure of sleep quality; and even though a direct relationship between sleep and 24 h cortisol no longer existed after 12 weeks on alprazolam, pretreatment sleep quality significantly predicted 24 h cortisol levels 12 weeks later while on treatment. This linkage across a 3 month time span between behavior and the endocrine measure suggests that, despite the reduction in both symptoms and cortisol levels induced by alprazolam, a significant portion of the variance in cortisol secretion at time 2 is related to an enduring characteristic of individuals that manifests itself at time 1 in their inability to sustain sleep in the experimental setting. Perhaps hypercortisolemia is linked to an enduring trait but is most robustly detectable only when individuals with the trait are examined under certain conditions (Cloninger, 1986). Though speculative, this kind of model may be of heuristic value in shaping further study of so-called “state/trait” issues. A simple state-marker interpretation of HPA axis abnormalities is also complicated in our data by some persistence of ACTH differences between high and low PA patients despite equally full resolution of panic attacks in the two groups. The elevated ACTH levels seen during “waking” hours in low PA patients relative to high PA patients did not entirely resolve with 12 weeks of treatment. The remaining difference may be partially confounded by alprazolam dose differences, since the high PA patients required higher doses of alprazolam to control their panic attacks; but at least the evening differences (1800-2200 h) between the two groups appear independent of dose effects. The difference also is not explained by persisting symptom severity differences since at follow-up the groups had

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nearly identical panic attack frequencies and did not differ in agoraphobic avoidance or Hamilton Anxiety score. They did differ in residual disability, but the ACTH differences remained when covaried for disability. The persistent ACTH differences suggest that the pre-treatment relationship between illness severity and ACTH secretion reflects a robust phenomenon with some degree of resistance to change. We cannot determine whether this resistance is due to persistent HPA axis dysregulation in either the high or low frequency of panic groups, or in both. It is entirely possible that adrenal sensitization has occurred in the more severely ill patients and requires more than 12 weeks to resolve. Perhaps with a more prolonged protocol remaining group differences would have disappeared. Further study of this question is needed. If high PA patients had greater adrenal sensitization than low PA patients and this persisted to some degree over 12 weeks of treatment, it may be of interest to explore whether adrenal sensitization is linked to treatment resistance. Greater residual disability after 12 weeks oftreatment in our female patients was in fact associated with reduced ACTH/cortisol ratios prior to treatment; also, lower pre-treatment ACTH/cortisol ratios predicted greater alprazolam dose requirements to achieve remission of panic attacks. Future research should address potential linkages between pre-treatment illness severity, pre-treatment adrenal sensitization and treatment resistance as manifested by a need for higher doses of alprazolam to control attacks and more residual functional impairment. The ability of pre-treatment HPA axis dysregulation to predict long-term functional outcomes in panic (Coryell et al., 1991) is consistent with such linkages and complicates a simple state-marker interpretation of HPA axis abnormalities. Despite relative resolution with treatment, pre-treatment HPA axis dysregulation may mark something that endures and is related to less effective functioning. Although our data cannot by itself shed definitive light on the mechanisms through which alprazolam inhibits activity within the HPA axis, the mechanism involved must be compatible with an overall reduction in cortisol levels, with little change in ACTH, and the appearance of differential effects on ACTH levels depending on initial panic attack frequency. It is difficult to describe a single mechanism that can fully account for these results. Reduced cortisol levels in the absence of reduced ACTH suggests some direct inhibition of cortisol release at the adrenal level. However, the differential behavior of ACTH in the two groups, despite reduction of cortisol in both, does not appear explicable on the basis of purely peripheral effects. The pre-clinical evidence, indicating that BZ inhibition of the HPA axis involves GABAA receptor-mediated inhibition of CRH release from hypothalamic neurons (Calogero et al., 1988; De Souza, 1990), also suggests a central mechanism. A primarily central mechanism is compatible with the differential behavior of ACTH. In the high PA patients, reduction in CRH drive would allow their adrenals to recover from previously enhanced sensitivity to ACTH, raising the ACTH/cortisol ratio, and reducing the amount of feedback inhibition by cortisol per unit ACTH released. With reduced feedback inhibition, lower cortisol levels could now be maintained despite little change or a slight rise in ACTH levels. If the higher ACTH/cortisol ratio seen in the low PA patients prior to treatment reflected less adrenal sensitization, then these patients might not show similar corrective effects in response to alprazolam, and reductions in both cortisol and ACTH may be seen. Further study will clearly be needed to more fully define

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mechanisms. Our data are compatible with a primarily central effect; but they also suggest the possibility of some direct effects at the adrenal level. Acknowledgemenrs-Michael Bronzo, M.D., and the CRC nurses provided critical assistance in management and execution of the study. Sandy Ostrowski (UM-CRC), Brenda Benson (NIMH) and Sam Listwak (NIMH) managed the blood samples and facilitated completion of assays. Hedieh Haghighatgou, M.S.W., assisted in data management and David Mauger, Ph.D. (UM-Biostatistics), helped with statistical analyses. Philip W. Gold, M.D. (NIMH), helped in the development of the protocol and oversaw completion of ACTH assays. Thomas Uhde, M.D. (Wayne State University), supervised completion of the cortisol assays. Elizabeth Young, M.D., provided helpful review of earlier drafts of this manuscript. This research was supported in part by Clinical Research Center Grant MO1 RR00042 and by a grant from The Upjohn Company.

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