Hyperactivity of CRH neuronal circuits as a target for therapeutic interventions in affective disorders

Peptides 22 (2001) 835– 844

Hyperactivity of CRH neuronal circuits as a target for therapeutic interventions in affective disorders Martin E. Keck,* Florian Holsboer Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, D-80804 Munich, Germany Received 20 April 2000; accepted 30 August 2000

Abstract Increasing evidence suggests that the neuroendocrine changes seen in psychiatric patients, especially in those suffering from affective disorders, may be causally related to the psychopathology and course of these clinical conditions. The most robustly confirmed neuroendocrine finding among psychiatric patients with affective disorders is hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system, resulting from hyperactive hypothalamic corticotropin-releasing hormone (CRH) neurons. A large body of preclinical and clinical evidence suggests that both genetic and environmental factors contribute to the development of these HPA system abnormalities. Further, normalization of HPA system regulation was shown to be a prerequisite for favorable treatment response and stable remission among depressives. Preclinical data based on animal models including selectively bred rat lines and mouse mutants support the notion that CRH neurons are hyperactive also in neuroanatomical regions that are involved in behavioral regulation but are located outside the neuroendocrine system. This raises the question of whether more direct interventions such as CRH receptor antagonists would open a new lead in the treatment of stress-related disorders such as depression, anxiety and sleep disorders. Recent clinical observations support this possibility. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Stress; Depression; Anxiety; Corticotropin-releasing hormone; Corticotropin-releasing factor; Corticotropin-releasing hormone receptor antagonist; Hypothalamic-pituitary-adrenocortical (HPA) system; Antidepressant

1. Introduction Neuroendocrine studies strongly suggest that hyperactivity of central corticotropin-releasing hormone (CRH) circuits, resulting in a characteristic dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) system, plays a causal role in the development and course of affective and anxiety disorders. Many studies have shown that CRH, independently from its neuroendocrine effects on the HPA system acts at diverse locations within the mammalian brain as a neurotransmitter/neuromodulator to coordinate endocrine, immune, autonomic and behavioral responses to stress (review: [28,65]). At the pituitary level, the effects of CRH are amplified by vasopressin (AVP), which, after prolonged stress, is increasingly coexpressed and cosecreted from hypothalamic CRH neurons [1,33,95]. The excessive release of CRH and AVP increases the secretion of corticotropin (ACTH) from pituitary corticotropic cells, which

* Corresponding author. Tel.: ⫹89-30622-314; fax: ⫹89-30622-569. E-mail address: [email protected] (M.E. Keck).

in turn increases the release of corticosteroids from the adrenocortical gland. In psychiatric disorders such as major depression, a variety of changes in HPA system regulation have been demonstrated, among them basal hypercortisolemia, inappropriate HPA suppression by the synthetic corticosteroid dexamethasone and a paradoxically increased ACTH and cortisol secretion after CRH in dexamethasone pretreated patients (review: [25,26,83]). In depression, the use of the combined dexamethasone-CRH test proved to be the most sensitive tool for the detection of altered HPA regulation. Depending on age and gender up to 90% of patients show this neuroendocrine symptom [20]. Moreover, studies using the dexamethasone-CRH test not only agree that normalization of an initial aberrancy predicts favorable treatment response but also corroborate that persistent HPA abnormality correlates with chronicity or relapse [21,30,101]. Since activation of corticosteroid receptors suppresses the synthesis and release of CRH and AVP from the hypothalamic paraventricular nucleus (PVN) [12], these findings are consistent with the existence of functionally impaired corticosteroid receptor signaling in both depressed patients [57]

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Fig. 1. Plasma cortisol concentrations (means ⫾ SEM) in dexamethasonepretreated subjects before and after intravenous (i.v.) CRH injection. Highrisk probands (open circles; squares) from the Munich Vulnerability Study had (1) at least one first-degree relative with an affective disorder or schizophrenia, and (2) at least one first-degree relative with no current or life-time psychiatric diagnosis. The dexamethasone/CRH test was performed at index assessment (open circles; n ⫽ 47) and at follow-up investigation after 4 years (squares; n ⫽ 14). Patients suffering from major depression (diamonds; n ⫽ 18) had been drug-free for at least 2 weeks. The control group (black circles; n ⫽ 20) comprised normal volunteers without any personal or family history of psychiatric disorders. All subjects received an oral dose of dexamethasone at 23:00 h. The following day blood samples were drawn at the time points given before and after i.v. CRH injection. At both investigations high-risk probands showed abnormal test results with a cortisol release that was between that of the healthy control group and the depressed patients [56].

and in healthy subjects who have a family history of depression (Fig. 1; [56]). The latter would result in enhanced CRH release that produces not only HPA hyperactivity but also several signs and symptoms of depression, such as increased anxiety and decreased appetite, libido and sleep. The hypothesis that increased CRH release is the driving force behind these clinical findings is supported by studies showing that patients suffering from major depression had elevated CRH concentrations in the cerebrospinal fluid [61]. This finding is further supported by the observation that clinically effective antidepressants lower the level of CRH in the cerebrospinal fluid of depressed patients [11]. These observations are interpreted as a reflection of increased synaptic concentrations of CRH due to central hypersecretion, which is consistent with evidence of decreased density of CRH receptors in the frontal cortex of depressed suicide victims, reflecting an adaptive down-regulation [60]. Further evidence in support of this hypothesis comes from the recent finding of increased numbers of both CRH-secreting neurons and CRH neurons that coexpress AVP mRNA in the hypothalami of depressed patients [76,77]. However, CRH is not just expressed at the level of the hypothalamic PVN to trigger the release of ACTH but plays a role as a neurotransmitter in several brain areas (review: [91]) and in at least some of these sites, corticosteroids may even activate CRH expression. Such stimulation of CRH gene ex-

pression by corticosteroids has been described in the central nucleus of the amygdala, in the bed nucleus of the stria terminalis, and in the dorsal part of the PVN from where spinal projections of CRH neurons emerge [55,94]. Thus elevations of CRH in the CSF and the central amygdala may be viewed as a result of hypercortisolemia in these patients. Although discussed controversially (e.g. [5]), this stimulating mechanism of corticosteroid action [84], together with impaired corticosteroid receptor functioning [25,57], may be one explanation for the paradoxical finding that central CRH activity is increased in depression despite the fact that at the same time elevated plasma cortisol levels are observed. Accordingly, subgroups of eucortisolemic depressives which did not show CRH elevation in the cerebrospinal fluid have been described; these patients may have special clinical characteristics such as chronicity [14,15]. Taken together, the findings described point towards a close relationship between the dysregulation of HPA system activity which occurs in the vast majority of individuals with depression, the progression into depression, the action of antidepressants, and the development of new drugs targeting HPA axis regulation more directly than current drugs. However, the limitations of research in humans necessitate preclinical studies in suitable animal models and basic studies at the cellular and molecular level to better understand how CRH is regulated and which regulatory elements might serve as potential drug targets of the HPA system. In this article, we will summarize our efforts (1) to characterize the potential role of CRH as a causal factor in the development of depression and (2) to document how we arrived at the conclusion that CRH type 1 receptors (CRHR1) would be appropriate pharmacological targets by integrating bidirectionally studies from the bench and the bedside.

2. Antisense targeting of HPA system function Clinical and preclinical studies have underscored the notion that antidepressants most likely act through rectifying excessive HPA system activity induced by overproduction of CRH (review: [26]). As CRH is considered to be the prime mediator of psychopathological processes and neuroendocrine symptoms of affective disorders, strategies targeted against the biosyntheses of CRH and its receptors have a strong potential to provide insight into the underlying mechanisms. Indeed, rats treated with antisense oligodeoxynucleotides directed against CRH mRNA showed a reduced hypothalamic CRH content which was accompanied by decreased anxiety-related behavior [86,87]. In rat and in human brain, CRH acts via at least two different receptor subtypes (CRHR1 and CRHR2), which differ in their pharmacological profiles, ligand affinities and anatomical distribution [54,71,72]. The CRHR1 is distributed throughout the rat brain, including cortex, brain stem, amygdala and anterior pituitary [7,75]. In contrast, the two CRHR2 splice

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variants (␣ and ␤) are expressed in limited subcortical areas of the rodent brain (e.g. lateral septum) and in non-neural structures (e.g. choroid plexus, cerebral blood vessels) [54, 71,72]. In humans, however, CRHR2 exist in three different functional splice variants [40]. Given the evidence that the neuropeptide CRH, when hypersecreted in rats, produces numerous behavioral changes resembling the cardinal symptoms of depression and anxiety (e.g. [52,79]), the most straightforward therapeutic approach is to downregulate its receptors within a distinct relevant brain region [24]. One strategy for delineating which specific CRH receptor subtype mediates CRH-induced psychopathology consists in using antisense probes against the corresponding mRNA [44,62]. Liebsch et al. [48] infused an antisense oligodeoxynucleotide corresponding to the CRHR1 mRNA bilaterally into the central amygdala of rats for four days prior to subjecting the animals to social defeat and subsequent testing for their anxiety-related behavior. This approach revealed a significant anxiolytic effect of CRHR1 downregulation. In a subsequent study, the effects of an antisense probe corresponding to CRHR1 mRNA were studied extensively in vitro and in vivo [88]. The antisense probe reduced CRH binding and function, as measured by ACTH secretion, in primary rat anterior pituitary cells and in clonal mouse pituitary cells (AtT20), and provided evidence that its action is associated with cytoplasmic uptake of the probe. Intracerebroventricular infusion of the antisense probe confirmed that inhibition of CRHR1 mRNA translation reduces CRH-elicited anxiety-related behavior in rats. This effect was associated with decreased CRH binding in the hypothalamus and cortex [88]. However, the localization of CRHR2 suggests that these receptors are also involved in the mediation of CRH-induced behavioral changes. Therefore, the effects of antisense probes directed against CRHR1 and CRHR2 were compared [18,47]. As expected, there was an anxiolytic effect in animals treated with CRHR1 antisense oligodeoxynucleotides, whereas no such effect was observed after treatment with CRHR2 antisense. However, the CRHR2 antisense treatment increased immobility in the forced swim test, which suggests that the CRHR2 plays a role in modulating acute stress coping behavior [47].

3. Genetic targeting of HPA system function: genetically engineered mice In order to complement and extend the studies with antisense probes targeted against CRHR1, mice with a truncated CRHR1 protein were generated [96]. These mice express deficient CRHR1 which are unable to activate cAMP in response to CRH. In cultured pituitary cells obtained from heterozygous mutants and homozygous mutants CRH stimulated a much weaker ACTH response than in pituitary cells from wildtype animals, whereas forskolin, which directly activates the catalytic subunit of adenylyl cyclase, evoked a much more pronounced activation of

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Fig. 2. Anxiety-related behavior under basal conditions in mice lacking CRHR1 function (⫺/⫺) as measured in the dark-light box (left panel) and in CRHR2 null mutant mice (⫺/⫺) as measured in the open field (right panel). Mice that express deficient CRHR1 show significantly reduced anxiety-related behavior (i.e. decreased latency to enter the lit compartment). In contrast, CRHR2 null mutant mice display increased anxietyrelated behavior (i.e. decreased time spent in the inner square of the open field apparatus). *P ⬍ 0.05; **P ⬍ 0.01 vs. wildtype mice (⫹/⫹). Data are means ⫹ SEM [2,96].

ACTH. In contrast to wildtype mice, in homozygous and heterozygous mutants CRH did not elicit a marked cAMP increase, which suggests that the mutation specifically impaired CRH/CRHR1 signaling. However, in contrast to a markedly blunted stress-induced ACTH release, basal plasma ACTH concentrations were indistinguishable between homozygous and heterozygous mutants and wildtype mice in vivo [96]. Similar findings have been reported by other authors [89]. This discrepancy in basal and stressinduced ACTH secretion could be attributed to the increased synthesis of other ACTH secretagogues such as AVP under in vivo conditions [59] and further compensatory mechanisms rendering the adrenal cortex more sensitive to ACTH. At the behavioral level, mice lacking CRHR1 function showed significantly reduced anxiety-related behavior according to a variety of behavioral assays (Fig. 2; [89,96]). During the severe stress of alcohol withdrawal, a gene/dosage effect of the CRHR1-mediated anxiety-related behavior could be shown with homozygous mice displaying significantly less anxiety compared to heterozygous and wildtype animals [96]. The analysis of CRHR2 null mutant mice provided more ambiguous information regarding the role of these receptors in conveying anxiety-like behavior. While Coste et al. [9] failed to observe any changes in anxiety-like behavior, two other reports found a role of CRHR2 in anxiety-like behaviors [2,39]. However, in contrast to Kishimoto et al. [39] who observed this difference in anxiety-like behavior only when employing the dark/light box test, Bale et al. (Fig. 2; [2]) detected differences between wildtype and mutant mice in both the elevated plus-maze paradigm and open field test, but not in the dark/light emergence task. Moreover, in one of the studies [39] increased anxiety-like behavior was only observed among male, but not female CRHR2 null mutants. Within the limits of neuroendocrine HPA regulation it

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seems clear that corticosteroids restrain CRH and AVP expression through activation of hypothalamic glucocorticoid receptors (GR) [12]. The mechanism underlying HPA hyperdrive in depression is not yet firmly established, but clinical studies in patients and probands with high genetic risk are consistent with decreased GR function, rendering the cortisol-mediated negative feedback on CRH expression insufficient (Fig. 1; [25,56,57]). Therefore, to further elucidate the role of impaired GR signaling in depression, a transgenic mouse expressing antisense to GR mRNA was generated [70]. The transgene was driven by a neurofilament promoter and was therefore mainly active in neuronal tissue leading to a marked reduction in brain GR mRNA expression. Accordingly, HPA axis regulation in the antisense GR transgenic mice is diminished, as shown by a reduced glucocorticoid negative feedback efficiency [3,31, 90,92], and enhanced CRH- and stress-induced increases in plasma ACTH and adrenocortical hyperresponsiveness to ACTH [3,31,58]. Paradoxically, expression of the CRH gene is not increased but rather decreased in the PVN and the external zone of the median eminence [13]. Interestingly, as determined by in vivo microdialysis technique, free corticosterone levels in the brain during the morning were enhanced whereas during the afternoon and evening they were markedly lower in transgenic mice than in control animals [51,85]. As the diurnal rise in HPA system activity is mainly driven by CRH, this result is consistent with the observed reduction in CRH peptide expression in the hypothalamic-pituitary system of antisense GR transgenic mice [13]. The mutant mice also display an impaired cognitive performance as revealed by a decreased immobility in the forced swim test, a decreased olfactory-cued short-term memory, and an increased escape latency in the Morris water maze [58]. Intriguingly, the transgenic mice showed reduced anxiety-related behavior as measured in the elevated plus-maze paradigm. To investigate whether the decreased anxiety might be secondary to cognitive disturbances, the mice were treated with moclobemide, a selective reversible inhibitor monoamine oxidase type A. After longterm treatment almost all behavioral deficits disappeared and the HPA system hyperactivity following stress returned to normal [58]. It therefore appears that cognitive rather than anxiety-related symptoms are present in these mutants [92]. This is in line with the finding that CRH secretion from the hypothalami of these mice is reduced and that CRHR1 receptors are hypersensitive, resembling the neuroendocrine changes seen in Alzheimer’s disease rather than those accompanying depression [13]. Decreased CRH neuronal activity in Alzheimer’s disease is supported by several findings, advocating the use of drugs that release CRH from CRH-binding protein [4]. In support of this, first evidence has recently been provided that CRH might act as an endogenous neuroprotectant against oxidative cell death in addition to its function in the HPA system [46]. Thus, taken together, the GR transgenic mouse model presents with some alterations of depression (i.e. cognitive alterations and

peripherally enhanced HPA system activity), but the overlap is limited. However, it is of interest to note that these genetically modified mice are potentially useful models to predict antidepressive drug effects, herewith dissecting face from predictive validity [58].

4. Environmental effects on HPA system function Stress probably represents one of the main factors that lead to sustained hyperactivity of the HPA system and has been closely related to the etiology of depression. Stressful experience during early brain development or exposure to chronic unpredictable stress during adulthood has been repeatedly shown to induce profound alterations in neuroendocrine and behavioral mechanisms of adaptation [8]. Thus, chronically stressed animals show both the neurochemical abnormalities and at least some of the behavioral alterations seen in humans with activated HPA system activity and in depressed patients (review: [53]). Rats that were postnatally traumatized by repeated maternal separation had increased anxiety, a decreased number of corticotropic CRH receptors, and increased transcription of the genes encoding CRH in the hypothalamic PVN and in the median eminence [41,66,73]. These changes in CRH pathways have been repeatedly shown to be manifested by enhanced basal and stress-induced plasma ACTH concentrations, which, if extrapolated to humans traumatized in early life, can increase the vulnerability to develop stress-related affective disorders [41,66,73,97]. Moreover, nonhuman primates that were exposed to adverse rearing conditions in infancy had persistently elevated cerebrospinal fluid concentrations of CRH [8]. Similarly, prenatal stressors have been shown to affect the HPA system lifelong: Immunostimulation of pregnant rats changes fetal brain development in a way that results in persistent HPA hyperactivity in offspring [81]. Correspondingly, other studies showed that prenatally stressed rats have increased CRH concentrations within the amygdala in adulthood [10]. It is of note however that also the effects of early trauma upon various kinds of developmental changes is partly genetically influenced [64]. Patchev et al. [66] examined the potential role of endogenously secreted steroids that might counteract the adverse effects of stress: Chronic treatment with the steroid 3,21dihydropregnan-20-one (tetrahydrodeoxycorticosterone, THDOC) during early development has been shown to counteract behavioral and neuroendocrine dysregulation induced by adverse early life events such as emotional stress [66]. So called neuroactive steroids, chemically characterized by a reduced steroid ring structure [82], were shown to modulate the activity of GABAA receptors, thereby inducing anxiolysis and HPA system attenuation. Accordingly, tetrahydroprogesterone (allopregnanolone; THP), another endogenous neuroactive steroid, could be shown to suppress the gene expression of hypothalamic CRH; these findings represent the first demonstration of genomic effects of

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neurosteroids that were hitherto considered to act solely by altering chloride conductance [67,68]. It is of note that Stro¨hle et al. [93] recently observed that antidepressants over time can elevate plasma levels of THP which may lead to a suppression of CRH gene expression. Provided that disturbed regulation of CRH neuronal circuitries plays a key role in producing cardinal signs and symptoms of depression, these behavioral studies in animals demonstrate that early trauma may increase the vulnerability to develop an affective disorder later in life. In humans, epidemiological studies also suggest an important role of life events in triggering a depressive episode [6,69]. Therefore, it may be hypothesized that the stress system is shaped by genetic factors and life experiences, and that predisposed individuals will be more susceptible to chronic stress or acute life events, subsequently leading to clinical manifestation of depression. Recently, it has been suggested that genetic factors influence the kinds of life events to which people expose themselves [38]. Therefore, those with inherited premorbid HPA dysfunction might be more vulnerable to expose themselves to harmful life events, resulting in insufficiently restrained HPA activation. This, in turn, may subsequently lead to depression and possibly other stressrelated clinical conditions through excessive CRH synthesis and release. Future results from the Munich Vulnerability Study will reveal whether those individuals who are at risk because of a high genetic load for depression and who show aberrant HPA symptoms will exhibit a manifestation of depression in later life [27,45].

5. Selectively bred rat lines: genetic and environmental effects It has long been known that most major psychiatric disorders, including depression, cluster in families [37]. Yet it is important to appreciate that expression of genes conferring disease susceptibility may not be altered to the extent necessary to precipitate the clinical phenotype unless environmental factors amplify gene effects. In studies of gene-environmental interactions, the HPA system plays a pivotal role in transducing external stressors into neural adaptations [25]. As outlined above, it has been documented in numerous studies that behavioral and neuroendocrine responses to stress depend on conditions of neonatal rearing [41,66,73,97] or stressors experienced by the fetus in utero [81]. Although such neuroendocrine sensitization plays a crucial role in changing the expression pattern of genes relevant for psychopathology, it is important to recognize that genetic susceptibility is a prerequisite. As such genetic predisposition does not necessarily lead to the manifestation of psychopathology, animal models are needed that fulfill the criterion of susceptibility when exposed to environmental perturbations. Because the risk to develop affective disorders is determined by genetic susceptibility at several loci, and in most

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cases also by non-genetic environmental factors it is necessary to complement studies using mice where one or two genes are mutated with the analysis of animals carrying more complex allelic variants. Ideally, such animal models should provide face and predictive validity [16]. A pragmatic approach is to identify those behaviors that are frequently disturbed in the clinical condition and that can be studied confidently in animals (e.g. [23,99]). Such behaviors include anxiety-related behavior and stress coping strategies. Another important criterion of a valid animal model of psychopathology concerns the neurobiological mechanisms underlying the behavioral disturbances [98], i.e. in the case of depression and anxiety HPA system dysregulation. An animal model of altered emotionality, which has been selected on the basis of behavior on the elevated plus-maze, are two Wistar rat lines selectively bred for high (HAB) and low (LAB) anxiety-related behavior at the Max Planck Institute of Psychiatry in Munich [49,50]. These two rat lines differ not only in their inborn anxiety, but also in their stress coping strategies, and their reactivity to benzodiazepine and antidepressant drug treatment [19,43,49,50]; Keck et al. unpublished observation). HAB rats do not significantly differ from LAB rats in terms of basal or CRH-induced ACTH and corticosterone activity [49], but show an increased HPA system susceptibility when exposed to the same external stressor, pointing towards an increased CRH release from PVN neurons [43]. In contrast, it has been shown that basal levels of both ACTH and corticosterone are increased in pregnant HAB rats, supporting the possibility that this increase in basal HPA activity during pregnancy in turn could also influence the emotional development of the offspring [63]. Interestingly, HAB but not LAB rats show a pathological outcome of the dexamethasone/CRH challenge-test [36], herewith displaying striking neuroendocrine similarities to depressed patients [22]. Taken together, this animal model reflects significant psychopathological and neuroendocrine features of human depression. Despite the fact that HAB and LAB rats provide an animal model with remarkable face (e.g. acute stress coping behavior, anxiety-like behavior) and predictive (e.g. response to treatment with the antidepressant paroxetine or repetitive transcranial magnetic stimulation; [34]) validity, the genetic differences underlying the neuroendocrine and behavioral changes are yet to be elucidated. Consistent with the view of impaired glucocorticoid receptor function as one key mechanism are preliminary data where CRH mRNA is enhanced in selected brain areas [74] indicating that corticosterone-mediated control over CRH gene expression is disturbed.

6. The therapeutic approach Both clinical and preclinical neuroendocrine studies strongly suggest that dysregulation of the HPA system plays a causal role in the development and course of depression

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[24]. The reason for HPA hyperactivity and, in particular, for enhanced synthesis and release of CRH in depression is not yet clear. Genetic and experience-related factors may interact to induce manifold changes in corticosteroid receptor signaling, finally resulting in hypersecretion of CRH and AVP (review: [12]). A considerable amount of evidence has been accumulated suggesting that normalization of the HPA system might be the final step necessary for stable remission of the disease [25,101]. However, the complex regulation of the HPA system provides multiple levels for intervention. In rats it could be shown that different classes of antidepressant drugs potently reduce HPA responsiveness to stressful stimuli after longterm treatment via a differentiated upregulation of GR and mineralocorticoid receptors [78,80]. Importantly, the time period required to precipitate such action (i.e. 2 to 5 weeks) is similar to that observed for HPA system normalization and clinical improvement in depressed patients (review: [26]). Similarly, long-term application of the putative antidepressant treatment of repetitive transcranial magnetic stimulation resulted in a blunted HPA response to stress in rats [32,34]. As these effects take considerable time, a promising strategy to shorten the time span until conventional antidepressant treatment strategies act by finally suppressing CRH gene activation and release is the blockade of CRH receptors [24]. Combining molecular genetics with behavioral pharmacology, studies with antisense probes that selectively reduce CRH receptor subtype levels and transgenic mouse models have indicated that CRHR1 may be the primary target at which selective nonpeptide compounds should be directed [47,48,88,96]. Several drug companies have taken up this concept and employed high-speed screening of compound libraries for specific CRHR1 antagonists. One compound recently examined is R121919 (formerly called NBI 30775) developed by Neurocrine Biosciences (La Jolla, USA). This compound binds with high affinity to cloned human and rat CRHR1 and inhibits CRHR1-mediated signal transduction in transfected cells whilst binding to other receptors known to be present in the central nervous system is extremely low. In preclinical studies with the two Munich rat lines, in high anxiety-related behavior (HAB) animals R121919 reduced anxiety-related behavior in a dose-dependent manner (Fig. 3), whereas it had virtually no behavioral effect in low anxiety-related behavior (LAB) rats. At the highest dose tested, HAB rats reached the performance level of LAB animals [35]. In contrast, the stress-induced activity of the HPA system, as determined by simultaneously measured plasma ACTH levels, was similarly blunted by R121919 in both HAB and LAB animals. This finding suggests that the behavioral effects were mediated via cerebral CRHR1 independently of the peripheral secretion of ACTH [35]. Despite the differences in the behavioral responses to R121919 treatment in HAB and LAB rats, R121919 similarly reduced 125 I-oCRH binding in all brain regions in both rat lines. However, CRH gene expression was observed to be elevated in the locus coeruleus of HAB rats [74], a brain area

Fig. 3. Effects of subcutaneous administration of R121919 on elevated plus-maze behavior in rats selectively bred for high anxiety-related behavior (HAB). HAB rats treated with 20 mg/kg body weight R121919 (n ⫽ 7) made significantly more entries into the open arms (left panel) and spent more time on the open arms (right panel) than the hyper-anxious vehicletreated animals (n ⫽ 10) or animals treated with 2 (n ⫽ 6) and 5 (n ⫽ 6) mg/kg R121919. Similar effects were obtained with 1 mg/kg diazepam injected intraperitoneally (n ⫽ 6). *P ⬍ 0.05; **P ⬍ 0.01 vs. vehicle. Data are means ⫹ SEM [35].

known to be richly innervated with CRH immunoreactive fibers linked to the norepinephrine system. Because activation of this locus is associated with stress and fear in rats, the anxiolytic effect of R121919 in HAB rats overexpressing CRH in the locus coeruleus is consistent with an action of the CRHR1 antagonist at this brain stem nucleus. The lack of effect of R121919 in LAB rats is consistent with the hypothesis submitted by Tomas Ho¨kfelt that neuropeptides are only released at significant quantities under potentially pathogenic conditions. Therefore only HAB but not LAB rats hypersecrete CRH, rendering the animals of the latter breeding line nonresponsive to R121919. Clinical studies have provided indirect evidence that CRH also accounts for some of the sleep-electroencephalographic (EEG) changes seen in depression [29]. Specifically, CRH was suspected to decrease slow wave sleep activity in humans and rats, which prompted us to investigate the sleep EEG effects of R121919 in HAB and LAB rats after stress exposure. We found that HAB and LAB rats differed in their sleep-wake behavior and that the sleep EEG was more markedly affected by stress exposure in HAB rats. Following administration of R121919, these differences disappeared. Particularly, the suppressive effect of stress-elicited CRH effects on slow wave sleep was avoided when R121919 was administered [42]. These promising preclinical effects led us to conduct an open-label trial in patients suffering from major depression where R121919 was administered to 20 patients primarily in order to investigate whether its endocrine mode of action compromises the stress hormone system or whether other safety and tolerability concerns may arise. A dose escalation strategy was employed which led to a 50% reduction in depressive symptoms comparable to that obtained with the selective serotonin reuptake inhibitor paroxetine (Fig. 4). Moreover, those patients receiving higher dosages had better responses and discontinuation of the drug led to wors-

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Fig. 4. Changes of 21-item Hamilton Depression Rating Scale (HAMD) and the self-rating Beck Depression Inventory (BDI) during treatment with R121919 and paroxetine. During the screening period all psychoactive medication was stopped for a minimum of 5 days. Patients were enrolled in two dose-escalation panels. In panel one the dose range increased from 5– 40 mg (diamonds), and in panel two from 40 – 80 mg (circles) within 30 days each. Panel 2 resulted in a response rate (50% reduction of initial depression severity) that matched the selective serotonin reuptake inhibitor paroxetine (squares). In both panels HAMD and BDI rating scales worsened after drug discontinuation. Data are means ⫹ SEM [100].

ening of depressive psychopathology. Such effects were achieved at dosages that did not hamper the ACTH and cortisol response to CRH stimulation [100]. There is clearly a need for new antidepressant and anxiolytic drugs that are pharmacologically distinct from the existing therapeutic agents in possessing fewer side effects and a greater rapidity of response. Our preclinical and clinical findings suggest that the pharmacological principle of CRHR1 antagonism has considerable therapeutic potential in the treatment of a variety of clinical conditions involving exaggerated stress responses resulting from CRH hyperactivity, such as anxiety, depression, eating disorders, drug withdrawal and disturbed sleep [17,24]. References [1] Antoni FA. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front Neuroendocrinol 1993;14:76 – 122. [2] Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, Vale WW, Lee KF. Mice deficient for corticotropinreleasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nature Genet 2000;24:410 – 4. [3] Barden N, Stec ISM, Montkowski A, Holsboer F, Reul JMHM. Endocrine profile and neuroendocrine challenge test in transgenic mice expressing antisense RNA against the glucocorticoid receptor. Neuroendocrinology 1997;66:212–20. [4] Behan DP, Heinrichs SC, Troncoso JC, Liu XJ, Kawas CH, Ling N, de Souza EB. Displacement of corticotropin releasing factor from its binding protein as a possible treatment for Alzheimer’s disease. Nature 1995;378:284 –7.

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