The corticotropin releasing factor CRF

The corticotropin-releasing factor (CRF) also referred to as corticotropin-releasing hormone, is a key mediator of mammalian endocrine, behavioral, autonomic, and immune responses to stress (Vale et al., 1981, Owens and Nemeroff, 1991, Bale and Vale, 2004). Within the hypothalamic–pituitary–adrenal axis, CRF is the principal regulator of pituitary adrenocorticotropin hormone (ACTH) and adrenal glucocorticoid secretion in response to stressful stimuli. Basic and clinical research studies indicate that elevated central CRF levels are involved in the etiology of stress-related psychiatric, physiological and behavioral disorders like depression and anxiety (Holsboer et al., 1984, Nemeroff et al., 1984, Linthorst et al., 1997). In the rat and human brain, CRF acts at two distinct G-protein coupled receptors, CRF1 and CRF2, each having a distinct anatomical localization and pharmacology (Perrin and Vale, 1999, Dautzenberg and Hauger, 2002). Recent studies have been conducted to establish the respective roles of CRF1 and CRF2 in stress-related physiological and behavioral processes (Risbrough et al., 2004, De Groote et al., 2005). Although there is strong evidence that CRF1 is intimately involved in anxiety-related behavior (Ayala et al., 2004), a role for CRF2 cannot be excluded and according to some, seems to be underestimated (Takahashi, 2002). Some evidence indicates that anxiety behavior mediated by CRF1 activation is reduced when CRF2 is stimulated (Bale et al., 2002, Valdez et al., 2002). Nevertheless, other evidence indicates that CRF2 is also capable of inducing anxiety-like behavior (Pelleymounter et al., 2002). These findings have led to the NAD/NADH Quantitation Colorimetric Kit that the CRF1 and CRF2 receptors could become novel therapeutic targets and that CRF1 and CRF2 antagonists could be evaluated in the treatment of anxiety disorders associated with a maladaptive stress response (Nemeroff, 1998, Holmes et al., 2003). One approach to testing that hypothesis is to determine whether clinically established anxiolytic compounds behave as CRF1 and CRF2 antagonists. This approach was used in the present study with etifoxine as the test compound. Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride, Stresam®), a molecule structurally unrelated to benzodiazepines has demonstrated anxiolytic properties in rodents (Boissier et al., 1972, Verleye and Gillardin, 2004) and in humans in the treatment of adjustment disorder with anxiety in humans (Servant et al., 1998), a disorder described in Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, Fourth ed., 1995). In animals, anxiolytic-like properties of etifoxine, at doses ranging between 12.5 and 50mg/kg by intraperitoneal route, were evidenced in classical anxiety tests, e.g. the Vogel's conflict test (Schlichter et al., 2000) and in the stress-induced hyperthermia paradigm in rats (Verleye and Gillardin, 2004). We have conducted a number of studies on the mechanism of action of etifoxine showing an enhancement of GABAergic synaptic transmission (Verleye et al., 1999, Verleye et al., 2002, Schlichter et al., 2000). Recent binding and electrophysiological experiments have demonstrated that etifoxine binds to γ-amino butyric acidA (GABAA) receptors via an allosteric site near or on the chloride channel, which differs from that of benzodiazepines (Verleye et al., 1999, Verleye et al., 2002). Recently, it was been shown that the etifoxine enhancing effects of the GABAergic synaptic transmission are mediated by the β2 and β3 subunits composing the GABAA receptor (Hamon et al., 2003). However, it is not known whether this anxiolytic interacts directly with the CRF1 and CRF2 receptors. The pathophysiology of anxiety disorders is complex: it is linked to abnormal regulation of several neurobiological components including GABA, monoamines and the CRF system. Although effective treatments exist, there is still considerable room for improvement. An anti-anxiety drug with multiple actions could be beneficial in the treatment of different pathological anxious states involving more than a single neurotransmitter system dysfunction (Bourin and Lambert, 2002; see review by Millan, 2003). In the present study, the hypothesis that etifoxine might behave as a CRF1 and CRF2 antagonist was evaluated using in vivo and in vitro approaches. In vivo studies were conducted in rat and mouse models based on the fact that central injections of CRF reproduce the patterns of behavioral and autonomic changes induced by stress (Williams et al., 1987, Habib et al., 2000). In the rat, CRF induces an increase in grooming associated with a decrease of exploration when the animal is exposed to an unfamiliar environment (Dunn and Berridge, 1990, Koob, 1999). In the mouse, central administration of CRF induces stress-related disturbances in gastrointestinal motor function such as an inhibition of gastric emptying of a solid meal (Sheldon et al., 1990, Martinez et al., 2002, Zorrilla et al., 2003). In vitro binding studies explored the interactions between etifoxine and the CRF1 and CRF2 receptors in rat brain membrane preparations. The functional counterpart of this binding was studied concurrently in vitro by testing the effects of etifoxine on CRF-induced stimulation of cAMP in human neuroblastoma SH-SY5Y cells. The CRF receptor antagonist, α-helical CRF9–41 (α-h-CRF), which binds to both subtypes of CRF receptors (Rivier et al., 1984), was used for comparison.