Treatment of depression with the CRH-1-receptor antagonist R121919: endocrine changes and side effects

Journal of Psychiatric Research 37 (2003) 525–533 www.elsevier.com/locate/jpsychires

Treatment of depression with the CRH-1-receptor antagonist R121919: endocrine changes and side effects Heike E. Ku¨nzel*, Astrid W. Zobel, Thomas Nickel, Nibal Ackl, Manfred Uhr, Annette Sonntag, Marcus Ising, Florian Holsboer Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, D-80804 Munich, Germany Received 28 November 2002; received in revised form 22 April 2003; accepted 28 April 2003

Abstract A dysregulation of the hypothalamus-pituitary-adrenocortical (HPA) system has been hypothesized to account for a myriad of cardinal symptoms of affective disorders. Specifically, increased CRH signalling via CRH type 1 receptors is thought to be an important factor in the pathogenesis of major depression and anxiety disorders. Consequently, a number of drugs have been developed in order to target the postulated increase in CRH/CRH 1 receptor signalling. One of these compounds, R121919, binds with high affinity to CRH1 receptors antagonising the action of CRH. R121919 was recently tested in an open-label study conceptualized as a safety and tolerability study. As part of this study, a thorough endocrine evaluation and detailed clinical laboratory analysis were assessed several times during 30 days of treatment with two different dose regimens of R121919 (5–40 mg vs. 40–80 mg) in 24 patients with a major depressive episode. During treatment with the experimental drug no serious side effects were noted. In particular, there were no adverse effects or impairment of the hypothalamic–pituitary–gonadal system, the hypothalamic–pituitary– thyroid axis, the renin–angiotensin system, prolactin or vasopressin secretion. Furthermore, no changes in the serum corticotropin and cortisol concentrations and in the responsivity of corticotropin and cortisol following a CRH stimulation test were noted. No effects of R121919 on clinical laboratory parameters including liver enzymes, EEG and ECG were observed. These results encourage the development of other CRH-1-R antagonists as a novel class of antidepressive drugs. # 2003 Elsevier Ltd. All rights reserved. Keywords: Corticotropin-releasing hormone; CRH-1-receptor antagonist; Depression; Side effects

1. Introduction Dysregulation of the hypothalamic–pituitary–adrenal (HPA) system in patients with depression is reflected by an increased cortisol secretion at baseline (Halbreich et al., 1985), by an exaggerated ACTH and cortisol response in the combined dexamethasone/corticotropin releasing hormone (CRH) test (Heuser et al., 1994; Zobel et al., 2001), and by elevated levels of CRH in the cerebrospinal fluid corresponding to decreased CRH binding in the prefrontal cortex of depressed patients (Nemeroff et al., 1984; Hartline et al., 1996; Holsboer, 1999).

* Corresponding author. Tel.: +49-89-30622-209; fax: +49-8930622-605. E-mail address: [email protected] (H.E. Ku¨nzel). 0022-3956/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-3956(03)00070-0

In numerous animal studies, CRH proved to be an important modulator not only of the stress hormones ACTH and cortisol secretion but also for behavioural adaptation to stressful conditions (reviews: Owens and Nemeroff, 1999; Holsboer, 1999). Up to now, two different types of CRH-receptors have been found to be expressed in the brain of rodents and primates, the CRH 1- and CRH 2-receptor. The CRH 1-receptor (CRHR1) is found in many brain areas, e.g. the brain stem, cortex, amygdala and anterior pituitary and in the cerebellum. The CRH 2-receptor is expressed in the anterior pituitary, lateral septum and in non-neuronal structures (Sanchez et al., 1999; review: Reul and Holsboer, 2002). A dysregulation of CRH-release with an inadequately elevated secretion of CRH seems to promote the development of psychiatric diseases (reviews: Holsboer, 1999, 2000; Owens and Nemeroff, 1991, 1999).

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The hypothesis of a CRH over-expression in the brain as a basic mechanism of the pathogenesis in depression was also derived from several animal studies. Stimulation of CRH receptors, e.g. through transgenic over-expression may lead to a number of cardinal symptoms of depression, e.g. increased anxiety, loss of libido and anorexia (Stenzel-Poore et al., 1994; van Gaalen et al., 2002). In contrast, suppression of CRH 1 receptor (CRHR1) function either by antisense treatment (Liebsch et al., 1995, 1999; Skutella et al., 1994, 1998) or by genetic deletion of the receptor (Timpl et al., 1998; Smith et al., 1998) result in decreased anxiety-like behaviour. On the basis of these results a CRHR1 antagonist R121919 (formerly NBI 30775) was developed to challenge the CRH/CRHR1- hypothesis pharmacologically (Chen et al., 1996). This substance is a selective CRH receptor 1 antagonist with a Ki ( S.E.) of 2.8 ( 3.5) nM that penetrates the blood–brain barrier and occupies the CRH-1-receptors in a dose dependent manner, as shown by ex vivo autoradiography in rats (Keck et al., 2001; Heinrichs et al., 2002). The compound is rapidly absorbed after oral administration, and plasma peak concentrations are reached within 1–2 h after intake. Half-life after an administration of a single dose was about 1 h and after repeated administration about 2–4 h. Terminal elimination of the compound ranges from 30 to 50 h, (average 40 h). In vitro studies in human liver cells showed that the CYP3A4 and CYP3A5 are involved in the metabolism of R121919 resulting in a sister compound R142900. There are no data thus far suggesting that R121919 is an enzyme inducer. We previously reported that, within the limitations of an exploratory study, R121919 was found to have a beneficial effect upon depressive symptoms among patients with major depression (Zobel et al., 2000). In the current report we present our extended data from this study on the safety and tolerability of R121919. Even though R121919 may exert antidepressive effects by curtailing depressive symptoms, the compound could theoretically cause serious side effects, e.g. by suppressing pituitary responsivity to stressors or by inducing adverse effects in other endocrine systems. A detailed evaluation of endocrine effects including parameters of the HPA-system (ACTH, cortisol, UFC, DHEA, aldosterone), vasopressin as a key modulator of HPAaxis activity, the gonadotropic axis (LH, testosterone, progesterone, estrogen), thyroid hormone axis (TSH, T3, T4), the growth hormone axis (IGF-1, HGH), renin as a parameter of the cardiovascular system and prolactin are presented together with the general side effect profile. Although the clinical development of R121919 was discontinued we report a detailed safety and tolerability evaluation because there are several back up components with different chemical structures but similar pharmacologial properties.

2. Methods 2.1. Patients and design Twenty-four patients with a single or recurrent major depressive episode according to the criteria of DSM IV and ICD 10 were enrolled. Patients were required to have a score equal to 18 or above in the 21 item Hamilton Depression Rating Scale (HDRS) at screening (5 days before initiation of the treatment) and at day 0. All psychoactive medication was stopped with the beginning of the screening period for at least 5 days. Only chloralhydrate up to 2500 mg/day was allowed as a sleeping aid. Further details on inclusion and exclusion criteria, the ethical regulation, and the design of the study are reported by Zobel et al. (2000). Briefly, two panels with two different dose escalation regimens were conducted. Panel 1 (6 male, 4 female patients; age=43.8  11.4) escalated every 10 days from 5, 20 to 40 mg daily and panel 2 (7 male, 7 female patients; age=46.7  16.1) started with 40, 60 up to 80 mg daily. For a detailed description of the endocrine assessments and the safety monitoring see Table 1. 2.2. Laboratory assessments A detailed analysis of clinical laboratory parameters was performed five times during the study trial at screening, day 10, 20, 30 and 32, after the end of the trial at day 32 including the following blood chemistry parameters: haemoglobin, haematocrit, red blood cells (RCB), white blood cells (WBC), white blood cell differential count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, protein, alkaline phosphatase, AST (Aspartat-Aminotransferase, GOT), ALT (Alanine-Aminotransferase, GPT), gamma-glutamyl-transferase (GGT), lactat-dehydrogenase (LDH), total bilirubine, urea, uric acid, creatinine, sodium potassium, chloride, calcium, glucose. Urine analysis included protein, glucose and occult blood. Cardial safety and elevation of the probability to suffer from seizures during treatment with R121919, safety was proven by repeated performance of electroencephalography (EEG) and electrocardiography (ECG). Additionally we measured blood pressure, heart rate and body weight repeatedly every 10 days during the study trial. 2.3. Hormonal assessments HPA-axis secretory capacity was kept under surveillance with repeated CRH-stimulation tests (day 9, 19, 29), circadian profiles at 08:00, 11:00, 04:00, 08:00 and 08.00 of plasma ACTH and cortisol and urinary free cortisol (at 08:00, 11:00, 04:00, 08:00 and 08:00 the next day, performed at screening and on days 10, 20, 30),

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H.E. Ku¨nzel et al. / Journal of Psychiatric Research 37 (2003) 525–533 Table 1 Schedule of safety and endocrine assessments during the study Screening

Day 1

Day 4

Day 9

Clinical routine EEG ECG

Day 10

Day 14

Day 19

Clinical routine EEG ECG CRH stimulation test

Day 20

Day 24

Day 29

Clinical routine EEG ECG CRH stimulation test

Day 30

Day 32

Clinical routine EEG ECG

Clinical routine ECG

CRH stimulation test

24 h urinary free cortisol 24 h profile cortisol and ACTH

24 h urinary free cortisol 24 h profile cortisol and ACTH

24 h urinary free cortisol 24 h profile cortisol and ACTH

24 h urinary free cortisol 24 h profile cortisol and ACTH

At 08:00 h: vasopressin HGH HGH DHEA, renin TSH,T3,T4 TSH,T3,T4 aldosterone prolactin prolactin HGH, IGF-1 estrogen, progesteron, testosteron, LH TSH,T3,T4 prolactin

Vasopressin HGH DHEA, renin TSH,T3,T4 aldosterone prolactin HGH, IGF-1 estrogen, progesteron, testosteron, LH TSH,T3,T4 prolactin

Vasopressin HGH DHEA, renin TSH,T3,T4 aldosterone prolactin HGH, IGF-1 estrogen, progesteron, testosteron, LH TSH,T3,T4 prolactin

Vasopressin HGH DHEA,renin TSH,T3,T4 aldosterone prolactin HGH, IGF-1 estrogen, progesteron, testosteron, LH TSH,T3,T4 prolactin

and concentrations of morning plasma cortisol and ACTH (every fourth morning; see Table 1). The CRHtest was performed according to a previously described protocol (Holsboer et al., 1986). The endocrine evaluation included gonadal hormones (luteinizing hormone (LH) and testosterone in men and estrogen and progesterone in women), insulin like growth factor (IGF-1), human growth hormone (hGH), dehydroepiandrosterone (DHEA), aldosterone, vasopressin, plasma renin activity (PRA), thyreoidea stimulating hormone (TSH), trijodothyronine (T3), thyroxine (T4) and prolactin. The plasma ACTH and cortisol levels were determined by radio-immunoassay kits (Nichols Institute, San Juan Capistrano, CA and ICN Biomedicals, Carson, CA, respectively). GH was measured using Nichols Luma human GH and chemiluminescence immunometric assay (Nichols Institute). Prolactin concentrations were measured using a two-site immunoluminometric assay (Liaison, Prolactin, Byk-Sangtec Diagnostica, Dietzenbach, Germany). Intra- and interassay coefficients of variation were below 10% for all hormones. For each hormone, all samples were analysed in the same assay. Aldosterone and IGF-1, TSH, estrogen, progesterone, testosterone, LH and vasopressin was measured in an external laboratory using radioimmuno assays.

2.5. Data analysis Differences between the two panel groups during the treatment with R121919 were assessed by analyses of variance (ANOVA) for repeated measures with treatment panel as between-subjects factor and time of examination as within-subjects factor contrasted by the simple deviations from the first time of examination. Additionally, independent t-tests were performed to assess panel differences separately for each time point of examination. Fisher’s exact tests were applied to assess panel differences in case of dichotomous data. The main goal of this study was to assess the safety of R121919 regarding endocrine and other hematological and clinical chemistry effects. We also repeatedly measured EEG and ECG. In safety studies, false negative results (no statistical differences between the panels or no change compared to baseline) can be misleading and mask a drug’s harmful effects potentially leading to serious consequences for the patients on the compound. Therefore, we employed an anticonservative approach without a-level adjustment for multiple comparisons in order to minimize the risk for false negative results.

3. Results 3.1. General clinical side effects

2.4. Pharmacokinetics The plasma concentrations of R121919 and of the major active metabolite R142900 were measured by HPLC by Janssen Pharmaceutica.

We investigated side effects during the study and compared the prevalence of single adverse events between the low dose (panel 1) and the high dose (panel 2) panel.

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No serious side effects such as impairment of excitability or conduction in the heart or disturbance of brain activity (e.g. EEG signs for seizures) were observed at any of the time points. Most common changes in ECG were intermittent bradycardia and tachycardia as well as a first degree atrio-ventricular blocking, which means that no serious cardiac side effects were induced. Lowering of dromotropic times or negative inotropic and bathmotropic effects were not observed during treatment with R121919. EEG was not affected by the compound. Only one female patient showed a local deceleration, which was considered as elevated cerebral excitability on days 20 and 30. This observation continued after switching treatment to various common antidepressants.

when patients were treated with common antidepressants (see Fig. 1). Vascular parameters such as blood pressure and pulse stayed within normal ranges during the study trial. Weight was monitored during the study trial as well. In panel 1 weight showed no significant change under medication whereas during treatment with the higher dose in panel 2 the mean weight decreased slightly but not statistically different. Additionally all adverse events like headache, nausea, dizziness, obstipation, urogenital infection and lumbago were noted during the treatment in both panels. Although some adverse events were reported none of them seem to be related to the drug. Furthermore, no significant differences in the prevalence of adverse events between the lower and higher dose panels were not observed (see Table 2).

3.3. Clinical chemistry

3.4. Effects on the HPA-system

There was no significant effect of R121919 on any of the assessed laboratory parameters. No impairment of differential blood count was found. Liver enzymes remained within normal range. A significant elevation was found in the follow up examination,

3.4.1. CRH stimulation test: The intactness of the HPA-axis under basal conditions and following CRH stimulation was analysed three times during the study. As already reported in our previous report (Zobel et al., 2000) there were no significant

3.2. EEG and ECG

Fig. 1. Liver enzymes during treatment with R121919 and follow up. Mean scores and standard errors (S.E.M.) are presented. Circles indicate liver enzymes in panel 1, triangles represent liver enzymes in panel 2. P-values of a contrast analysis assessing change from baseline and after discontinuation of R121919 are reported. All values remained within the normal range and showed no significant elevation under treatment with R121919. After starting follow up treatment with different common antidepressants a significant increase in all liver enzymes was observed when compared to the values obtained at day 30.

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H.E. Ku¨nzel et al. / Journal of Psychiatric Research 37 (2003) 525–533 Table 2 Adverse events during treatment with R121919a

Headache Nausea Dizziness Sickness Obstipation Suicidal ideations Laboratory changes ECG EEG Abdominal pain Urogenital infection Skin affection Lumbago Others

Panel 1 (N=10)

Panel 2 (N=14)

Fisher’s exact test (one-sided P, 1< 2)

2 2 1 0 1 0 3 3 1 0 0 2 0 4

4 0 1 2 3 1 7 5 0 2 1 0 2 4

0.506 1.000 1.000 0.330 0.437 0.583 0.290 0.561 1.000 0.330 0.583 1.000 0.330 1.000

a No serious side effects were observed during administration of R121919. Comparing the frequency of side effects between both panels, there was no significant difference between the low and high dose panel.

changes in ACTH and cortisol concentrations during dose escalation or between both panels. 3.4.2. Morning and circadian profiles of plasma ACTH and cortisol secretion: Morning ACTH and cortisol concentrations showed no significant changes during the study course or between both panels. Circadian rhythms of ACTH and cortisol secretion were analysed four times (screening, days 10, 20, 30) during the study period and were found to be unchanged during treatment with R121919. Averaging the circadian concentrations of plasma ACTH and cortisol resulted in a trend towards a decrease in ACTH, when day 10 was compared with screening. However this trend was only seen in panel 1 where the dosage was lower. Between both panels there was no significant difference. In all, plasma ACTH and cortisol values remained within the normal range. 3.4.3. Urinary free cortisol: Urinary free cortisol concentrations were analysed four times (screening, days 10, 20, 30) during the study trial in the 24 h urine collections. The mean values tended to decrease with increasing dose. Although this trend became significant in panel 2, there was no significant difference between panel 1 and panel 2. 3.4.4. DHEA and aldosterone: Plasma concentrations of DHEA and of aldosterone were not affected by treatment with R121919. 3.5. Hormones of the hypothalamic–pituitary–gonadal system Luteinizing hormone (LH), testosterone in men and estrogen and progesterone in women were measured

four times during the study trial. Gonadal hormone concentrations remained within normal ranges at all time points. LH showed a trend toward an elevation at days 10 and 20 in panel 1 and a decrease at day 10 in panel 2. No significant changes in gonadal hormones were observed. A comparison of hormone concentrations between the panels 1 and 2 did not achieve statistical significance. 3.6. Growth hormones The plasma levels of human growth hormone (hGH) did not showed significant changes, neither during dose escalation nor between the panels. Insuline like growth factor (IGF-1) plasma concentrations were within normal ranges during the study trial. Furthermore, we did not find significant differences between the two panels. 3.7. Plasma renin and vasopressin Circadian plasma renin activity (PRA) was assessed at the same time points as ACTH and cortisol in addition to the morning of every fourth day. PRA did not show significant differences during the study or between both panels. The circadian rhythm remained unaffected. We observed a non-significant increase in vasopressin concentrations between screening and day 30 in panel 1 and a decrease in panel 2. However, plasma vasopressin concentration always remained within the normal range. Between both panels we found a significant difference only on day 10 (panel 2 > panel 1; P=0.003) (see Table 3). 3.8. Thyroid hormones Pituitary–thyroid functions were assessed by measuring plasma TSH, triiodothyronine T3 and thyroxine T4 concentrations. We observed lower concentrations of TSH in panel 2 at screening, which increased slightly during the treatment. During the treatment period plasma T3 and T4 hormone concentration remained within normal ranges and showed no significant differences. We found that T3 was significantly higher in panel 2 than in panel 1. T4 seemed to decrease from screening until the end of treatment, but the changes were not significant.

Table 3 Plasma vasopressin during treatment with R121919 in panel 1 and 2 Vasopressin (pg/ml) Screening Day 10 Day 20 Day 30

Panel 1 (M S.E.M.) 3.18 0.77 1.88 0.30 2.81 0.61 4.42 0.92

Panel 2 (MS.E.M.) 6.58 1.97 5.39 0.86 4.68 1.11 3.15 0.56

Panel 1 vs. panel 2 n.s. 0.003 n.s. n.s.

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Table 4 Plasma prolactin during treatment with R121919 in panel 1 and 2 Prolactin (ng/ml) Screening Day 4 Day 10 Day 14 Day 20 Day 24 Day 30 Day 32

Panel 1 (MS.E.M.) 16.6 4.2 15.7 2.5 14.3 2.6 13.6 2.3 15.4 2.7 14.7 2.6 15.1 3.4 14.6 2.6

Panel 2 (MS.E.M.) 13.8 3.0 13.3 1.7 16.7 3.8 14.8 3.6 17.0 3.2 14.2 1.9 16.2 2.9 16.8 4.1

Panel 1 vs. panel 2 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

3.9. Prolactin Plasma prolactin concentrations remained unchanged in panel 1, however there was a trend toward an increase in panel 2 (see Table 4). 3.10. Pharmacokinetics of R121919 In both dose escalation panels plasma concentrations of R121919 as well as its primary metabolite R142900 increased over time (see Fig. 2). The increases of both compounds are steeper in panel 2 using the higher dose increments compared with panel 1 which is reflected by

Fig. 2. Plasma levels of R121919 and R142900. Mean scores and standard errors (S.E.M.) of R121919 and of its active metabolite R142900 are presented. Circles show plasma levels in panel 1, solid lines with triangles indicate plasma levels in panel 2. Dashed lines with squares show plasma levels of four cases in panel 2 who did not complete the study protocol for different reasons (withdrawal of consent in three cases, worsening of symptoms in one case). Plasma concentrations were significantly higher in panel 2, and an increase of the plasma concentrations parallel to the dose escalation was observed.

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a significant interaction between dosage and panel (R121919: P=001; R142900: P=0.005). 3.11. Prediction of outcome by CRH-tests We found a relationship between the ACTH-response to CRH-stimulation in the CRH-test and HAMD scores. ACTH response (
AUC) to CRH stimulation on day 9 was negatively correlated with treatment response according to HDRS criteria (reduction 550%) on day 30 indicating that patients with an attenuated ACTH response at day 9 in the CRH-test would respond more favourably to treatment with the CRH-1receptor antagonist. No significant correlation between the cortisol response in the CRH-stimulation test and treatment response was found.

4. Discussion CRH-receptors are widely distributed across the brain and are also found in peripheral tissues, e.g. in the heart, gut and testes. Moreover the HPA-system is known to interact with several other endocrine systems, e.g. with the thyroid, somatotropic and gonadal system. Therefore by blocking CRH-1-receptors, it must be ruled out that this intervention causes effects in hormonal systems that may limit the usefulness of this class of compounds in general. Because maintenance of appropriate HPA secretory activity at baseline and under challenge conditions is pertinent for all psychotropic drugs targeting the HPA-system, it is mportant that under the treatment conditions used in this study there were no significant changes in the morning plasma levels and the 24 h rhythm of ACTH and cortisol. UFC mean values showed a significant reduction during treatment with R121919, but remained within normal ranges at every time point. The slight reduction of peripheral plasma ACTH concentrations does not seem to be a specific effect of R121919 since it has also been demonstrated following treatment with many other antidepressants (Holsboer and Barden, 1996; Holsboer, 2000). This is reflected in the studies monitoring HPA function during treatment with various antidepressants (reviews: Holsboer-Trachsler et al., 1994; Heuser et al., 1996; Zobel et al., 2001) and by studies investigating the adrenal cortex during antidepressant treatment (Rubin and Phillips, 1993; Rubin et al., 1996). The reactivity of the HPA-axis particularly of the CRH receptors at pituitary corticotrophs was surveyed by a CRH-stimulation test and repeated three times during the study. As already briefly reported by Zobel et al. 2000, we found no significant effects on the ability of the pituitary corticotrophs to react appropriately to stimulation by intravenous administration of CRH. One explanation for this finding was provided by Keck et al.

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(2001), who showed that at dosages of R121919 completely blocking central CRH1 receptors in the rat there are still sufficient receptors available at the pituitary to preserve responsivity to CRH. Additionally, CRH-2receptors are not functionally impaired by R121919 potentially allowing the response to CRH in the absence of functioning CRH1R (Radulovic et al., 1999; Sanchez et al., 1999). Vasopressin is known to have a synergizing effect on the HPA-axis in humans and rats and modulating effects on behaviour itself (von Bardeleben et al., 1985; Scott and Dinan, 1998; Wotjak et al., 1996). Additionally van Londen et al. (1997) showed elevated plasma vasopressin concentrations in patients with depression when compared with healthy controls. Indeed, we found significant changes in the peripheral vasopressin concentration, which however may not be considered as systematic, due to the increase of vasopressin concentrations during panel 1 and the decrease in panel 2. All values remained within the normal range and were not predictive for response of the patient to the drug. Measurement of peripheral vasopressin concentrations only poorly reflected the neuropeptide concentration interacting with the HPA-axis, because vasopressin secreted into the pituitary portal blood circle, synergies with CRH at the level of the corticotrophic cells to produce enhanced ACTH. These data, on plasma vasopressin concentrations are not in complete agreement with those reported by van Londen and coworkers (1997). The renin activity was monitored, because of its close relation to the HPA-system. Cortisol and aldosterone are able to stimulate the renin secretion in peripheral tissue. Therefore changes in plasma renin concentrations could be possibly affected by R121919. As we found the HPA-system to be unimpaired by R121919 at baseline the lack of effects upon renin concentrations during the study trial was expected. Gonadal hormone secretion is also known to be affected by stress through increased CRH secretion (Schweiger et al., 1999; Meller et al., 2001; Young and Korszun, 1998). Male rats normally show a decrease of testosterone secretion in response to stress and gonadal hormones are reported to be decreased among patients with depression. Following acute administration of R121919, the stress induced decrease of testosterone was prevented in rats (Keck et al., 2001). In the current study R121919 was devoid of effects on progesterone and estrogen in women or testosterone in men, allowing us to conclude that treatment with R121919 for 30 days does not cause a disturbance of gonadal hormone secretion in either of the sexes. Some studies have shown that CRH influences the activity of the hGH secretion (Barbarino et al., 1990; Wiedemann et al., 1991). For example, HPA hyperactivity has been associated with a reduction of hGH

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secretion (review: Steiger and Holsboer, 1997; Lesch et al., 1988). Growth hormone secretion showed no changes, as in most patients the concentrations measured were below the detection limit. IGF-1, the opposing hormone of hGH, showed a reduction at two time points. Since these effects did not appear to be systematic during the study, the changes are most likely not mediated by treatment with R121919. The hypothalamic-thyroid-axis which is integrated with the HPA system has also been reported to be altered among patients with depression (Jackson, 1998; Haggerty and Prange, 1995). In our sample we observed a significant increase in plasma TSH concentration from screening to day 10 and a significant difference in concentrations of plasma T3 between the panels. Since all other changes of plasma thyroid hormone concentration were not significant, an impairment of thyroid hormone secretion by medium-term administration of R121919 is unlikely. Many animal studies (e.g. Torner et al., 2001) demonstrate that prolactin reduces the HPA responsiveness to stress and anxiety-related behaviour. Similarly, Keck et al., (2003) observed that R121919 produces a dose-dependent reduction of stress-induced prolactin increase following acute administration of R121919 in rats. In our study prolactin concentrations remained within the normal range. In contrast to the animal data, which reflect acute effects, we found a trend toward an increase of prolactin in panel 2. This finding probably reflects a slightly reduced activation of the HPA-system along with reduction of anxiety related behaviour. The attenuation of prolactin secretion by a modulation of the HPA-system goes along with findings of Rupprecht et al. (1987) who reported an attenuated suppressibility of prolactin by glucocorticoids in depressed patients. As CRH-1-receptors are also found in non-endocrine peripheral tissue like skeletal muscle or heart (Aguilera et al., 1987), it was important that serious effects on the vascular system, and in a particular laboratory parameters directly and indirectly related to the cardiovascular system be excluded. We analysed changes in haematological parameters and liver enzymes, which are often affected during drug treatment with common psychopharmacological substances. We found no changes in differential blood count or a suppression of immune modulating cells. Liver tissue itself has no CRH-receptors, but often, constituents of compounds are able to cause increases in liver enzymes. None of the liver enzymes analysed showed significant elevations during treatment with R121919, however, a significant increase in GGT, ASAT, ALAT and AP was found during follow-up medication, when common antidepressants were administered. It is important to note that in this patient sample no signs of liver toxicity were observed. In a parallel study conducted elsewhere, two healthy con-

trols receiving much higher doses of R121919, exhibited reversible elevations of liver enzymes which prompted discontinuation of the clinical development of R121919. Based on the data reported here and the absence of CRH receptors in the liver it is unlikely that observed toxic effects are related to a mechanism-based CRH receptor blockade. Toxicological studies with back-up compounds having different chemical structures, but the same pharmacology are awaited in order to clarify the particular chemical property of R121919 that prompted adverse effects on liver cells, when administered in very high dosages. Additionally we registered all adverse events reported by the patients, irrespective of whether they were related to the drug or not. When comparing both panels we found no significant difference in the frequency of these reports, and none of the reported adverse events appeared to be specifically related to the experimental compound. Summarising our results we confirm and extend our previous report that blocking CRH1 receptors by R121919 at doses that presumably suffice for antidepressant activity does not precipitate endocrine and other laboratory effects that are potentially harmful. This conclusion with respect to the endocrine systems and stress-hormone signalling is of particular importance since it allows us to reject the possibility that potential therapeutics targeting the blockade of the CRRH1 receptor will not adversely interact with these systems in a way that would limit their clinical utility. Although clinical development of this particular CRH1R antagonist has been discontinued it is expected that new back up compounds will become available in the near future that will confirm and extend the conclusions drawn from this first study in patients with major depressive disorders.

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