WO2008137270A1 - Methods of diagnosing and monitoring of npy y5 based disorders - Google Patents

Abstract

The present invention relates to methods of selecting a patient population most responsive to treatment with a NPY Y5 receptor antagonist, and in particular, to the use of a challenge test of the HPA axis in said selection method. In one aspect of the invention, the challenge test involves measuring levels of cortisoi in a biological sample obtained from a subject. The invention further provides for methods of identifying, diagnosing and monitoring of a subject most responsive to said treatment The present invention is further directed to methods of treating a subject diagnosed with a hypercortisolism-related disorder. The invention further concerns methods of marketing a medicinal product comprising a NPY Y 5 receptor antagonist for the treatment of a hypercortisolism-related disorder. Additionally, the subject invention is directed to uses of a NPY Y5 receptor antagonist for the manufacture of a medicament for treating a hypercortisolisrn-related disorder, in one aspect of the invention, the hypercortisolism- reiated disorder is a stress-related disorder such as depression and anxiety,

Description

METHODS OF DIAGNOSING AND MONITORING OF NPY Y5 BASED DISORDERS

FIELD OF THE INVENTION

The present invention relates to methods of selecting a patient population most responsive to treatment with a NPY Y5 receptor antagonist for treating a hypercortisolism-related disorder, and in particular, to the use of a challenge test of the HPA axis in said selection method. In one aspect the invention, the hypercortisolism- related disorder is a stress-related disorder such as depression and anxiety. The subject invention further relates to uses of a NPY Y 5 receptor antagonist in the manufacture of a medicament for treating a hypercortisolism-disorder.

BACKGROUND OF THE INVENTION

Depression and anxiety disorders are highly prevalent forms of mental illness which are also considered stress-related disorders in that their symptoms are often precipitated by stressful life events or conditions. Psychosocial stress, for example, can affect the onset of a depressive disorder (Kendler, K. et al. Am. J. Psychiatry 2000, 157, 1243-1251; and Lewinsohn, P. et al. J. Abnorm. Psychol. 1999, 108, 483-489) as well as symptom severity (Hammen, C. Abnorm. Psychol. 1992, 101, 45-52) and the time course of the disease (Kendler, K. et al. Psychol. Med. 1997, 27, 107-117). Early life stress is another variable which is cited for increasing the probability of anxiety symptoms, substance abuse and depression in the adult (Heim, C. et al. Neuropsychopharmacology 2004, 29, 641-648).

Recent advances in psychotherapy and in the development of innovative psychopharmacological therapies for treating stress-related disorders such as depression and anxiety disorders have dramatically improved the quality of life for millions of people throughout the world. Many of the current psychopharmacological therapies to treat depression and anxiety disorders are directed to curing a deficiency in the monoamine system which includes the monoamine oxidase enzyme inhibitors (MAOI) and the selective serotonin/noreprinephrine reuptake inhibitors (SSRI/SNRI). Although many people respond to these treatments, a number of patients do not respond to currently available treatments. Thus, it would be beneficial to understand the mechanisms linking stress sensitivity and depression and anxiety symptoms, particularly as a means of identifying novel therapies.

For some decades now, researchers have been aware of the importance of the hypothalamus-pituitary-adrenal gland (HPA) axis in the physiological response to stress (Holsboer, F. Neuroendocrinology, 1997, 65, 216-222; Gold, P. W. and Chrousos, G.P. MoI Psychiatry 2002, 7, 254-275; and Antonijevic, I.A. Psychoneuroendocrinology 2006, 31, 1-15), and likewise the importance of the correlations between emotional and biological factors in stress related disorders. The normal function of the HPA axis is to enable an organism to adapt to various stressors whether they are physical, psychosocial or environmental. In normal subjects, the perception of stress activates the central nervous system (CNS) resulting in the release of corticotrophin-releasing hormone (CRH) and vasopressin (AVP) from the hypothalamus, followed by adrenal corticotrophic hormone (ACTH) from the anterior pituitary, followed by Cortisol from the adrenal cortex. The elevated Cortisol concentration in the plasma inhibits the HPA axis via negative feedback.

A dysregulation of the HPA axis was one of the first biological findings in depression- related disorders was described more than three decades ago (Carroll, B. et al. Arch. Gen. Psychiatry 1976, 33, 1039-1044; and Rubin, R. et al. Arch. Gen. Psychiatry 1987, 44, 328-336). For example, a significant number of patients with depression were noted to exhibit both hypersecretion of Cortisol (Gibbons, J. and McHugh, P. J. Psychiatr Res. 1962, 162-171) and its metabolites. Since then, a multitude of studies have described altered secretion of ACTH and Cortisol, an altered stress-responsivity and impaired feedback mechanisms of the HPA axis in subjects with depression and anxiety disorders (Holsboer, F. et al. New Engl. J. Med. 1984, 311, 1 127; Halbreich, U. et al. Arch. Gen. Psychiatry 1985, 42, 904-914; Pfohl, B. et al. Arch. Gen. Psychiatry 1985, 42, 897-903; and Young, E. et al. Arch. Gen. Psychiatry 1993, 50, 395-403). These alterations can be ascertained by analysis of basal activity over the 24 h period (Carroll, B. et al. Acta Psychiatrica. Scandinavica 2007, 115, 90-103) or by a pharmacological challenge to evaluate the negative feedback mechanism, such as the dexamethasone suppression test (Carroll, B. et al. Arch. Gen. Psychiatry 1981, 38, 15-22), and also by measuring the HPA response to a stressor (Heim, C. et al. Neuropsychopharmacology 2004, 29, 641- 648). HPA overdrive due to impaired negative feedback is of particular interest for its association with severe and in particular melancholic MDD; patients having this type of depression also tend to have insomnia, decreased appetite and weight loss and increased visceral fat deposition. The latter has also been associated with insulin resistance that is only normalized in those patients that show remission following antidepressant treatment (Weber-Hamann, B. et al. J. Clin. Psychiatry 2006, 67, 1856-1861). The metabolic changes associated with hypercortisolemic depression are considered a critical risk factor for cardiovascular disorders (Weber-Hamann, B. et al. Psychoneuroendocrinology 2006, 31, 347-354). Additionally, melancholic depressed subjects show non-suppression of Cortisol to dexamethasone (Halbreich, U. et al. Arch. Gen. Psychiatry 1985, 42, 904-914; and Linkowski, P. et al. J. Clin. Endocrinol. Metab. 1985, 61, 429-438). Studies have found an association between Cortisol non-suppression to dexamethasone and baseline Cortisol (Poland, R. et al. Arch. Gen. Psychiatry 1987, 44, 790-795) suggesting that increased Cortisol secretion is the primary defect responsible for dexamethasone feedback.

Recent data suggest that that there is a large subgroup of patients with anxious depression among the severely depressed patients (Fava, M. et al. Can. J. Psychiatry 2006, 51, 823-835). These patients have also been reported to present with a more chronic course and delayed response to treatment.

HPA hypoactivity, on the other hand, is associated with atypical depression, an apparently more heterogeneous grouping with a distinct constellation of symptoms, including increased sleep and increased appetite, an early onset of symptoms and a chronic course (Stewart, J. et al. Acta Psychiatr. Scand. 2007, 1 15, 58-71; and Stewart, J. et al. J. Affect. Disord. 2005, 86, 161-167). These differentiations may be clinically relevant in that they are associated with different responses to pharmacological treatment (Antonijevic, I.A. Psychoneuroendocrinology 2006, 31, 1-15). In particular, atypical depression has been associated with unsatisfactory response to SSRI as well as tricyclic antidepressants (Quitkin, F.M. et al. Arch. Gen.. Psychiatry 1990, 47, 935-941).

Possibly yet another group of depressed patients is characterized by an exaggerated stress response and adverse early life events (Heim, C. et al. JAMA 2000, 284, 592-597). These patients seem to respond better to psychotherapy than antidepressant medication (Heim, C. et al. Neuropsychopharmacology 2004, 29, 641-648), opening up the possibility that a novel drug target that normalizes the HPA sensitization would be of benefit for this subgroup of patients. Finally, even for those patients that do not fall into any of the above categories, non-response to current therapies or residual symptoms and/or side effect profile remain a substantial unmet medical need (Fava, M. et al. Can. J. Psychiatry 2006, 51, 823-835).

Potential drug therapies capable of restoring proper HPA axis function may target receptors for peptide modulators such as corticotropin releasing hormone (CRH) and neuropeptide Y (NPY). The latter is a 36 amino acid neurotransmitter that is well known for its ability to regulate multiple physiological processes including stress response and mood as well as food intake and adiposity. NPY is considered to exert a critical role for the homeostatic regulation of the HPA axis system. For example, an acute injection of NPY in the paraventricular nucleus (PVN) produces increases in circulating ACTH and Cortisol in both conscious and anesthetized rats (Walhlestedt, C. et al. Brain Res. 1987, 417, 33-38; Inoue, T. et al. Life ScL 1989, 44, 1043-1051; and Hanson, E. and Dallman, M. J. Neuroendocrinal. 1995, 7, 273-279). Chronic infusion of NPY into the cerebral ventricule in normal rats also causes hypercorticosteronemia (Zarjevski, N., et al. Endocrinology 1993, 133, 1753-1758).

Presently, five NPY receptor subtypes have been cloned: Yl (Larhammar, D. et al. J.

Biol. Chem. 1992, 267, 10935-10938); Y2 (Gerald, C. et al. J. Biol. Chem. 1995, 270, 26758-26761); Y4 (Bard, J. et al. J. Biol. Chem. 1995, 270, 26762-26765); Y5 (Gerald, C. et al. J. Biol. Chem. 1995, 270, 26758-26761); and y6 (Gregor, P. et al. J. Biol. Chem. 1996, 271, 27776-27781). All these receptor subtypes are expressed in several species except for the y6 subtype, which has been shown to be expressed in mouse and rabbit but not in rat and primate. A Y3 subtype has been proposed based on pharmacological data. However, the Y3 subtype has yet to be cloned and its existence remains to be fully established.

The Y5 receptor is of interest for its widespread distribution throughout the limbic system and along the stress axis, and its highly conserved expression pattern in human and rat brain (Nichol, K. et al. J. Neurosci. 1999, 19, 10295-10304). Y5-like immunoreactivity has been documented throughout the hypothalamus, particularly along the NPY projection path from arcuate to paraventricular (PVN) and supraoptic nuclei (Campbell, R. et al. Neuroendocrinology 2001, 74, 106-1 19). Y5 receptors in these nuclei are positioned to modulate the release of stress-related neurotransmitters such as CRF, vasopressin, oxytocin and/or urocortin, with subsequent effects on the HPA axis and other circuits important for depression and anxiety. In male Fischer 344 rats, it was shown that exogenous administration of a NPY YS specific agonist in undisturbed conscious rats produced increases in plasma ACTH and Cortisol and prior treatment with a NPY YS-selective receptor antagonist blocked these alterations (Kakui, N. and Kitamura, K. Endocrinology, published online March 15, 2007); however, this experiment does not address whether NPY Y5 overdrive is responsible for hypercortisolism observed in a disease state. Animal models of depression (chronic mild stress, maternally separated rats) were also shown to possess altered NPY expression in various brain regions including hypothalamus, where a relative increase has been reported (Husum, H. and Mathe, A. Neuropsychopharmacology 2002, 27, 756-764; and Sergeyev, V. et al. Psychopharmacology 2005, 178, 115-124). NPY levels in the rat arcuate nucleus can be elevated by dexamethasone, and Y 5 mRNA in rat hypothalamus can be elevated by chronic corticosterone administration.

In our laboratories, we evaluated selective NPY Y5 receptor antagonists in animal models predictive for antidepressant and antianxiolytic activity. It was discovered that these compounds produce effects similar to that observed by known antidepressants and antianxiolytics. Accordingly, it is expected that the administration of NPY receptor antagonists can treat the symptoms of stress-related disorders such as depression and anxiety in patients.

While it is expected that patients with stress-related disorders can be treated with a NPY Y5 receptor antagonist-based therapy, we theorized that since NPY Y5 drive in the HPA axis appears to be a contributing factor for stress-related disorders, a select population of patients with a dysfunction of the HPA axis would benefit most from said treatment. Moreover, we theorized that a NPY Y5 receptor antagonist-based therapy can be used to treat a disorder associated with hypercortisolism as well as a stress-related disorder. We theorized that the level of Cortisol could be analyzed from a biological sample to identify a patient most responsive to a NPY Y5 receptor antagonist-based therapy. To this end, we tested this hypothesis in the following two rodent models of HPA axis dysfunction: 1. the maternally separated rat model; and 2. the high anxiety bred/low anxiety bred rat model.

Herein, we demonstrate the efficacy of selective NPY Y5 receptor antagonists in two in- vivo animal models where the dysfunction of the HPA axis resembles that reported for various patient populations. In these disease state models, our evidence indicates that the NPY Y5 receptor is endogenously activated and therefore contributes to the output of the HPA axis. In one aspect, the HPA axis dysfunction in the models resembles those patients with HPA overdrive and impaired negative feedback, which includes patients with moderate to severe and/or melancholic depression and metabolic disturbances. In another aspect, the HPA axis dysfunction in the models resembles patients with a sensitized HPA response resulting in exaggerated Cortisol and ACTH secretion to a stressful stimulus, which includes patients with MDD and disorders resulting from stressful life events.

Accordingly, we propose that presenting to a challenge test of the HPA axis to a subject and analyzing a biological sample for the level of Cortisol would lead to the identification/selection of the most responsive population of patients for treating stress- related disorders with a NPY Y5 receptor antagonist-based therapy. Moreover, we propose that a NPY Y5 receptor antagonist-based therapy can be used to treat a disorder associated with hypercortisolism as well as a stress-related disorder.

SUMMARY OF THE INVENTION

A method of identifying a subject most responsive to the administration of a NPY Y5 receptor antagonist for treating a hypercortisolism-related disorder comprising (a) presenting a challenge test to the HPA axis on the subject; (b) obtaining a biological sample from the subject; and (c) analyzing said sample for the level of Cortisol.

A method of diagnosing and/or monitoring a subject's responsiveness to the administration of a NPY Y 5 receptor antagonist, wherein the subject is diagnosed with a hypercortisolism-related disorder comprising (a) presenting a challenge test to the HPA axis on the subject; (b) obtaining a biological sample from the subject; and (c) analyzing said sample for the level of Cortisol.

A method of treating a subject diagnosed with a hypercortisolism-related disorder comprising (a) presenting a challenge test to the HPA axis on the subject; (b) obtaining a biological sample from the subject; (c) analyzing said sample for the level of Cortisol; and (d) administering a NPY Y5 receptor antagonist.

A method of marketing a medicinal product comprising a NPY Y 5 receptor antagonist for the treatment of a hypercortisolism-related disorder, said marketing comprising the public spreading of the information that the level of Cortisol can be used to monitor the progress of said disorder.

Use of a NPY Y5 receptor antagonist in the manufacture of a medicament for treating a hypercortisolism-related disorder in a subject, wherein said subject is characterized by a Cortisol level above about 125 nniol/l following a dexamethasone based challenge test to the HPA axis.

A NPY Y5 receptor antagonist for use in treating a hypercortisolism-related disorder in a subject, wherein said subject is characterized by a Cortisol level above about 125 nmol/l following a dexamethasone based challenge test to the HPA axis. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 displays the ethanol consumption / session of rats treated with Compound X.

Figure 2 displays the open field, time spent in center before and after two weeks of treatment with Compound X.

Figure 3 illustrates the current understanding of a role for the NPY Y5 receptor after early life stress, chronic stress or disease. Figure 4 displays the effects of Compound Y on HAB rats in the elevated plus maze test. Figure 5 displays the effects of Compound Y on HAB rats in the forced swim test.

DETAILED DESCRIPTION

The present invention is based on the discovery that presenting a challenge to the HPA axis to a subject and analyzing the level of Cortisol in a biological sample can be used to select a target population of patients who would benefit most from the treatment of a NPY Y5 receptor antagonist-based therapy. The invention is explained in greater detail below but this description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. Hence, the following specification is intended to illustrate some embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Definitions

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

For the purposes of this invention, the phrase "NPY Y5 receptor antagonist" refers to a peptidyl or non-peptidyl compound which binds to, and decreases or inhibits the activity of, the NPY Y5 receptor in the presence of an agonist such as pancreatic polypeptide (PP) and peptide YY (PYY). The phrase "NPY Y5 receptor antagonist" further encompasses the corresponding pharmaceutically acceptable salt. Examples of NPY Y5 receptor antagonists are described below in example 4.

As used herein, a "hypercortisolism-related disorder" is selected from the group consisting of Cushing's syndrome; depression; major depression; moderate depression; severe depression; melancholic depression; anxious depression; anxiety; generalized anxiety disorder; post traumatic stress syndrome (PTSD); attention-deficit hyperactivity disorder (ADHD); a phobia; a panic disorder; stress following surgery; stress following fever-conditions; Alzheimer's Disease; aging preceding dementia; AIDS dementia; schizophrenia; a disorder related to behavioral inhibition and neuroticism; primary insomnia; chronic alcoholism; violent alcoholism; a disorder from alcohol exposure during fetal development; cocaine addiction; obesity; bulimia nervosa; binge eating disorder (BED); a functional gastrointestinal disorder (FGD) with depressed mood; arterial hypertension in the elderly; poorly controlled or uncontrolled diabetes; a metabolic syndrome; and herpes simplex virus-1 (HSV-I) encephalitis.

Furthermore, the disorders encompassed by a "hypercortisol ism-related disorder" may be grouped into subcategories such as a "stress-related disorder", a "metabolic-related disorder", a "cognitive disorder", an "addiction disorder" and the like.

For the purpose of this invention, a "stress-related disorder" is defined to include depression; major depression; moderate depression; severe depression; melancholic depression; anxious depression; anxiety; generalized anxiety disorder; post traumatic stress syndrome (PTSD); attention-deficit hyperactivity disorder (ADHD); a phobia; and a panic disorder.

As used herein, a "metabolic-related disorder" is defined to include obesity; bulimia nervosa; binge eating disorder (BED); poorly controlled or uncontrolled diabetes; and a metabolic syndrome.

For the purpose of this invention, a "cognitive-related disorder" is defined to include Alzheimer's Disease; aging preceding dementia; and AIDS dementia.

As used herein, the phrase "analyzing said sample for the level of Cortisol" is defined to include both the measuring of the level of Cortisol from a biological sample and comparing said level of Cortisol against a threshold level in which non-suppression is defined. The threshold level of Cortisol in which non-suppression is defined is dependent on the particular challenge test to the HPA axis that is used. For example, if a dexamethasone based challenge test is used to determine whether the subject would be most responsive to the treatment with a NPY Y5 receptor antagonist, the threshold level to compare against the level of Cortisol measured from the biological sample is about 125 nmol/1.

As used herein, the phrase "presenting a challenge to the HPA axis" refers to the initiation of a challenge test to the HPA axis to the subject. Challenge tests which may be used in the subject invention are described below in example 3.

A "biological sample" that may be tested in a method of the invention includes whole blood, blood serum or plasma, urine, saliva, cerebrospinal fluid (CSF) or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof.

The term "treating" or "treatment" as used herein refers to partially or completely ameliorating at least one symptom of, partially or completely treating or curing and/or preventing the development of a stress-related disorder such as depression or anxiety.

"Diagnosing" and "monitoring" methods of the invention can be used to monitor onset, progression, stabilization, amelioration and/or remission of a stress-related disorder.

Additionally, the invention further provides for certain embodiments of the present invention which are described below.

In one embodiment, the hypercortisol ism-related disorder is a stress-related disorder.

In one embodiment, the stress-related disorder is selected from the group consisting of depression; major depression; moderate depression; severe depression; and melancholic depression.

In another embodiment, the stress-related disorder is selected from the group consisting of anxious depression; anxiety; generalized anxiety disorder; post traumatic stress syndrome; attention-deficit hyperactivity disorder; a phobia and a panic disorder.

In yet another embodiment, the hypercortisol ism-related disorder is a metabolic-related disorder selected from the group consisting of obesity; bulimia nervosa; binge eating disorder (BED); poorly controlled or uncontrolled diabetes; and a metabolic syndrome

In one embodiment, the stress-related disorder results from chronic stress or disease.

In one embodiment, the stress-related disorder results from acute stress.

In yet another embodiment, the challenge test to the HPA axis is presented prior to treatment.

In one embodiment, the challenge test to the HPA axis is presented during and after treatment.

In one embodiment, the biological sample is a blood sample. In one embodiment, the challenge test is dexamethasone based.

Pharmaceutical Compositions and Dosage Regimes Pharmaceutical compositions comprising NPY Y5 receptor antagonists that may serve therapies to be tested by the methods of the present invention may be administered to the subject by any of several modes of delivery known in the art. For example, the pharmaceutical formulations for use in the novel methods of the present invention may be administered topically, subcutaneously, intramuscularly, orally, systemically and parenteral Iy.

Pharmaceutically useful compositions to be used by the methods of the present invention may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age.

NPY Y5 receptor antagonists to be used according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable. The compositions to be tested according to this invention can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

Advantageously, compounds to be used by the methods of the present invention may be administered to the subject in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.

Furthermore, compounds to be tested by the methods of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. For a therapeutic regime including combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

Diagnosing and Rating Psychiatric Disorders

In practicing the invention, any method (e.g., "rating scale") or protocol can be used to diagnose a stress-related disorder (e.g., depression and anxiety) or assess the progress of treatment for a stress-related disorder (e.g., depression and anxiety). The depression diagnosed in practicing the methods and compositions of the invention includes all diseases and conditions which are associated with depression, including those classified in the IDC-IO and Diagnostic and Statistical Manual IV (DSM-IV) rating scales. These diseases or disorders comprise major depression, dysthymic disorder, depressive episodes of bipolar disorders and depressive episodes associated with other mood disorders, including seasonal mood disorders and mood disorders due to a general medical condition and substance induced mood disorder. For example, in practicing the invention any rating scale can be used to measure the severity of a stress-related disorder (e.g., depression and anxiety) in a subject. For example, in depression, the most frequently used scales include the Hamilton Depression Rating (HAM-D) Scale, the Beck Depression Inventory (BDI), the Montgomery-Asberg Depression Rating Scale (MADRS), the Geriatric Depression Scale (GDS), and the Zung Self-Rating Depression Scale (ZSRDS). For anxiety, the most frequently used scales include the Hamilton Anxiety Rating (HAM-A) Scale, and the Beck Anxiety Inventory (BAI). These or any art-acceptable means to diagnose and/or access a stress-related disorder (e.g., depression and anxiety) in a subject can be used.

All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the methodologies and materials that are disclosed therein that might be used in connection with the present invention. Having described embodiments of the invention with reference to the accompanying examples and drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims

EXAMPLES

The invention will be further understood by reference to the examples provided below. The following examples are used to support and enable one skilled in the art to make, use and practice the methods and uses of the invention. Example 1 : Maternally Separated Rat Model

This preclinical model is the maternally separated rat model, in which the rat pup undergoes a sequence of daily separations (>= 180 min) from the mother during postnatal weaning and develops an anxious phenotype as an adult. The maternally separated rat is of particular interest as a model of early life stress / disease symptoms. These rats have increased hypothalamic NPY, plus an exaggerated Cortisol response after restraint stress. The HPA axis abnormality in this model is therefore one of hyper- reactivity. They also show increased ethanol consumption and reduced open field activity. The maternally separated rat can be referred to as HMS 180 (for handled maternally separated >= 180 min). Comparisons can be made with the animal facility reared control (AFR). Comparisons can also be made with the handled control HMS 15 (for handled maternally separated 15 min).

Procedure: The protocol for the maternally separated rat model experiments described herein can be found in Huot, R. et al. Psychopharmacology, 2001, 158, 366-373, the contents of which are herein incorporated by reference into the subject application. Compound X is N-{[4<4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2- ylamino)cyclohexyl]methyl} -methane sulfonamide and is disclosed as example 2 in U.S. 6,989,379.

Discussion: AFR, HMS- 15 and HMS- 180 rats exhibited very similar plasma levels of Cortisol under basal conditions, in the range of 16-22 ng/nil (Table 1). Exposure to 60 min restraint stress resulted in a dramatic rise in Cortisol levels in AFR (288 ng/ml) and HMS- 15 (280 ng/ml) groups. HMS-180 rats, however, showed a significantly greater elevation in plasma Cortisol (409 mg/ml). In HMS- 180 rats, the exaggerated Cortisol response to restraint was normalized to the level of that in the control groups following 28 day treatment with Compound X as a food formulation (10 mg/kg/day). The Cortisol response in the two control groups was unaffected by 28 day drug treatment (Table 1). Note also that Compound X was without effect on basal levels of Cortisol in any of the three groups. Table 1. Basal vs. stress- induced levels of plasma Cortisol (ng / ml) before treatment and after 28 days of treatment with Compound X.

In a separate experiment, the HMS 180 rats were observed to consume significantly larger amounts of ethanol compared to controls. This behavior in the HMS 180 has been interpreted as an attempt to minimize anxiety symptoms.

Results: HMS 180 rats exposed to the NPY Y5 receptor antagonist, Compound X, for 28 days showed a significant reduction in ethanol consumption, whereas consumption in the control groups was essentially unchanged (Figure 1). We propose based on these findings that treatment with an NPY Y 5 receptor antagonist is capable of producing anxiolytic-like effects specifically in the HMS 180 rat, thereby decreasing the drive for ethanol consumption.

In yet another experiment, the HMS 180 rats were tested in the elevated plus maze model as a measure of anxiety-like behavior and were observed to increase the amount of time spent in the open field arena.

Results: Consistent with above-identified proposal regarding ethanol consumption, treatment with Compound X also increased the amount of time that HMS 180 rats spent in the center of an open field arena (Figure 2) whereas the behavior in control rats was essentially unchanged.

Discussion: The data from the maternal separation rat model suggest that endogenous NPY Y5 receptors may contribute to a pathophysiology that develops into adulthood after early life stress. Especially during chronic stress or disease, susceptible individuals may have enhanced function of the NPY Y5 receptor, leading to enhanced output from the HPA axis and associated anxiety-like behaviors. For these individuals, a NPY Y5 receptor antagonist may therefore act as a brake on the system, and help to restore normal function. This represents a novel mechanism compared to existing therapies (Figure 3).

Example 2. High Anxiety Bred / Low Anxiety Bred Rat Model

The second preclinical model is the High Anxiety Bred / Low Anxiety Bred rat model, otherwise referred to as HAB / LAB. These are rats bred for either high or low anxiety as measured by the amount of time spent in the open arms of an elevated plus maze (defined as <5% of the time for HAB vs. > 45% of the time for LAB). The HAB rat shows elevated expression of vasopressin in the paraventricular nucleus of the hypothalamus. The HAB rat also displays an HPA axis dysfunction that can be described as "overdrive", in that the Cortisol release elicited by CRF is not suppressed by prior treatment with dexamethasone. This is interpreted as an insensitivity in the feedback loop.

Procedure: The protocol for the high anxiety bred / low anxiety bred rat model experiment can be found in Keck M.E. et al. Neuropsychopharmacology 2002, 26, 94- 105, the contents of which are herein incorporated by reference into the subject application. Compound Y is [(methylethyl)sulfonyl](trans-4-{[(4-(2-pyridyl)(l,3-thiazol-2- yl))amino]methyl}cvclohexyl)amine and is disclosed as example Ia in WO 07/002126.

Results: Acute administration of the NPY Y5 receptor antagonist, Compound Y, (1 mg/kg s.c.) to HAB rats produced antianxioly tic-like behavior in the elevated plus maze, with a significant increase in percentage of entries and percentage time spent in the open arms (Figure 4). Acute administration of Compound Y also produced antidepressant- like behavior in the forced swim test, with a significant reduction in immobility (floating) and increase in "struggling" behavior (Figure 5). The same dose of Compound Y was without effect in the LAB rats. These data indicate that in the HAB rat, endogenous NPY Y5 receptors may contribute specifically to a pathophysiology which is defined in part by HPA "overdrive" (e.g. lack of dexamethasone suppression).

Discussion: The data from the HAB rats suggest that in the context of an HPA overdrive that involves both elevated CRF and vasopressin secretion an acute administration of a NPY Y5 receptor antagonist reduces both anxiety- and depressive-like behavior. A possible alignment with depressive patients includes familial depression with early onset, which seems to affect more women than men (Zisook, S. et al. Acta Psychiatr.Scand. 2007, 115, 196-205). In addition, childhood depressive symptoms are associated with a higher BMI in adulthood and a higher incidence of binge-eating behavior, particularly in women (Hasler, G. et al. MoI. Psychiatry 2005, 10, 842-850). In this view, the effects seen with a NPY Y5 receptor antagonist in HAB rats are encouraging

As anxiety-like behavior is a core feature in HAB rats, the effects of the NPY Y5 receptor antagonist on both anxiety- and depressive-like behavior opens up the possibility that such treatment could also be efficacious in anxious major depressive disorder (MDD) (Fava, M. et a Can. J. Psychiatry 2006, 51, 823-835). In addition, offspring from high risk families, namely families with first rank relatives suffering from MDD, have a higher risk to develop depression than those from low risk families (Weissman, M. et al. Am. J. Psychiatry 2006, 163, 1001-1008). In such subjects at risk, the first symptoms are often heightened anxiety rather than depressive symptoms (Grillon, C. et al. Biol. Psychiatry 2005, 57, 953-960). In this regard, it seems justified to explore whether NPY Y5 receptor antagonist treatment in young patients with anxiety, particularly those from high-risk families, can reduce anxiety and prevent, or at least delay, the development of depression. It seems also warranted to examine whether such an early treatment intervention could reduce the risk of obesity and binge-eating disorder.

Example 3. Challenge Tests of the HPA axis

For illustrative purposes, but without limiting the scope of the invention, the following challenge tests are dynamic tests of the HPA axis which may be used in the present invention. It is noted that the below-identified challenge tests are well-known to one skilled in the art and their procedures are briefly summarized below.

Moreover, it is noted that the protocols described below are meant to be illustrative and not to limit the invention to the particular protocol itself. For example, the time periods in which to collect a biological sample are not meant to be rigid time points to follow but are only used to illustrate the particular procedure used. Example 3a. Dexamethasone based tests

Dexamethasone is a synthetic glucocorticoid that suppresses pituitary ACTH secretion through the negative feedback effect of corticosteroids on the hypothalamo-pituitary complex. Failure to suppress Cortisol concentrations after dexamethasone administration suggests hyperactivity and impaired feedback of the HPA axis.

Rapid screening dexamethasone suppression test

Protocol: Patients receive an oral dose of dexamethasone the night before the challenge test. The following day, the patients are served a standardized meal for breakfast and lunch. Around 13:00 h, the patients are to rest supine in bed. An intravenous catheter is placed and blood samples may be collected after around 2 h afterwards and at certain time periods intervals thereafter. For example, blood samples may be collected around 15:00, around 15:30, around 15:45, around 16:00 and around 16:15 and measured for Cortisol levels. In normal patients, Cortisol level may fall to less than about 138 nmol/1. In a separate embodiment, the Cortisol level may fall to less than about 125 nmol/1.

Dexamethasone perfusion test

This test allows the investigator to look at the rapid and delayed feed back responses.

Standard dexamethasone test

Protocol: Dexamethasone is administered approximately around every 6 h on days 3 and 4 at a dose of about 0.5 mg and at a higher dose of about 2.0 mg around every 6 h on days 5 and 6. This test allows one to distinguish between subjects of non-suppression and subjects with Cushing's syndrome. However, non-suppression may occur with concurrent ingestion of alcohol, barbiturates, carbamazepine, meprobamate and phenytoin. In contrast amphetamine, bezodiazepines and corticoids enhance suppression.

Example 3b. Dexamethasone/ Corticotrophin-Releasing Hormone (CRH) based test The dexamethasone-suppressed CRH test (Dex-CRH test) can be used to differentiate patients with Cushing's syndrome from those with pseudo-Cushing states, who have decreased ACTH responses to CRH because of negative feedback exerted by chronic hypercortisolism. Protocol: Patients receive an oral dose of dexamethasone the night before the CRH challenge. The following day, patients are served a standardized meal for breakfast and lunch. Around 13:00 h, the patients are to rest supine in bed. An intravenous catheter is placed and blood samples may be collected after around 2 h afterwards and at certain time periods intervals thereafter. For example, blood samples may be collected around 15:00, around 15:30, around 15:45, around 16:00 and around 16:15 and measured for Cortisol levels. About 2 min after the first collection of a blood sample, about 100 μg human CRH reconstituted in 1.0 ml 0.02% HCl in 0.9% saline is infused within approximately 30 sec. A cut-off value is defined as about 40ng/ml above which non-suppression is defined. Cortisol can also be measured in saliva.

Example 3c. ACTH based tests

The ACTH stimulation tests assess hypothalamic and pituitary function by measuring the functional integrity of the adrenal glands and their sensitivity to ACTH stimulation. These tests are implemented to assess the ability of the adrenal glands to secrete normal amounts of steroid hormones.

Rapid ACTH test Protocol: About 0.25 mg of ACTH is infused over around 30 s. A rapid increase in Cortisol within 30 min takes place. Cortisol samples are taken before stimulation and around 30 min and around 60 min after stimulation. Reference interval levels are about 591-1 1 13 nmol/l for peak Cortisol levels. This is a screening test and can not be used for partial secondary insufficiency in patients who can not maintain sufficient basal levels of ACTH to respond to stress or to hypoglycemia.

ACTH infusion test

Native ACTH is no longer used. This test is performed using tetracosactrin. Protocol: IV infusion of about 500 μg tetracosactrin is administered over a period of around 6 h. Maximum Cortisol are observed about 6-8 h with peak concentrations being about 1 105- 1380 nmol/l.

Long ACTH tests using depot tetracosactrin

Native ACTH is no longer used. This test is performed using tetracosactrin. Protocol: IM injections of about 0.5mg/m² are given around every 6 h. Blood samples before are taken around 12 h after the last injection. Urine samples can be taken for around 4 days.

Example 3d. ACTH/Growth Hormone Releasing Hormone (GHRHJ/arginine based test Protocol: About lmg/kg is given as an IV infusion at time zero followed by an infusion of about 30g L-arginine in 100 niL normal saline and about 250 μg ACTH for around 30 min. Blood samples are taken at time 0 and around 30, 45 and 60 min afterwards. The samples are analyzed for Cortisol.

Example 3e. Corticotrophin-Releasing Hormone (CRH) based test The CRH stimulation test may be used to assess the ability of the anterior pituitary gland to secrete ACTH. Protocol: About 1 μg/kg or about 100 μg of CRH is infused as a bolus and blood samples are taken for Cortisol measurements during around hour 1 and 2 after the administration of the bolus injection. Normal response is when the maximal rise is greater than fourfold increase in mean baseline concentrations of Cortisol.

Example 3f. Insulin based test

The aim of the insulin tolerance test is to assess the integrity of the hypothalamo- pituitary-adrenal axis. The stress of insulin induced hypoglycemia results in increased secretion of ACTH and hence of corticoids, but also of GH, prolactin and catecholamines. Cortisol is an indirect measure of ACTH secretion. Protocol: About 0.1 IU/kg is administered intravenously for provoking hypoglycaemia to decrease to less than about 2.2 mmol. Cortisol is then measured at around 15 min intervals for around 90 min after the stimulation. This is a reference test for evaluating the stability of the HPA axis and the reference value for peak plasma Cortisol is about 557-1015 nmol/L.

Example 3g. Lysine 8 vasopressin based test

Lysine 8 vasopressin directly stimulates the secretion of ACTH and related peptides. Protocol: About 3-5 pressor units are infused per hour for around 2 h and Cortisol is measured.

Example 3h. Arginine/vasopressin based test

Protocol: Pressor units of about 0.18 units/kg of arginine/vasopresin are given intramuscular and blood samples are taken before infusion and around every 15 min after the initial 90 min period. Cortisol and ACTH are measured.

Example 3 i. Metyrapone based test

Metyrapone blocks 1 1 -hydroxy lation and thus prevents normal synthesis of Cortisol.

This test assesses the feedback control of ACTH secretion. Protocol: About 750 mg is given around every 4 h for 2 days. Plasma and urine 11-deoxycorticosteroids or urinary 17-ketogenic steroids or 17-hydrocorticosteroids are measured and compared to baseline values. A simpler version involves the administration of Metyrapone (about 30 mg/kg) around midnight and blood may be drawn for deoxycortisol between around 8:00 and 9.30. Cortisol is to be measured and the reference value for Cortisol is about 50-640 nmol/1.

Example 3j. Yohimbine based test

Yohimbine, an a₂ adrenergic antagonist, increases the release of norepinephrine (NE) in the hippocampus and other brain areas through increased firing of the locus ceruleus. Yohimbine augments sympathetic outflow and blocks presynaptical alpha 2-adrenergic receptors releasing NE into the blood stream. Protocol: About 0.125 mg/kg IV bolus followed by about 0.001 mg/kg/min infusion for a total of around 15 min. Cortisol level is to be analyzed.

Example 3k. Naloxone based test Naloxone induces ACTH and Cortisol secretion by blocking opioid inhibitory tone directly at hypothalamic CRH neurons. Protocol: Sequential doses of naloxone (placebo, about 50, about 100, about 200 and about 400 μg/kg) dissolved in 0.9% saline are administered at approximately 30 min intervals. Baseline Cortisol and ACTH are obtained at around 15 min and immediately prior to placebo. Post placebo blood samples are taken around every 15 min for around 180 min. Cortisol is analyzed in the blood samples.

Example 31. Interleukin-6 (IL-6) based test

IL-6 is a cytokine that stimulates the HPA axis. Protocol: Doses from about 3.0-30 μg /kg are given.

Example 3m. Glucagone based test

Protocol: About 1.0 mg glucagone is given intramuscular and blood samples for Cortisol are analyzed.

Example 3n. Thyrotropin-releasing hormone based test

Protocol: About 500 μg of thyrotropin-releasing hormone (TRH) is infused over a period of around 30 sec. Blood samples are drawn before and around 15, 30, 60 and 90 min after infusion. TSH is measured in blood samples. Example 4. NPY Y5 Receptor Antagonists

Non-limiting examples of NPY Y5 antagonists include compounds of the Formula I that may found in U.S. 6,989,379, which is hereby incorporated by reference in its entirety.

wherein R₁ is independently H, F, Cl, Br, -CN, -OH, -NO₂, -NR₅R₆, -SO₂R₅, - (CH₂)_nORs, -(CH₂)_nCONR₅R6, -(CH₂)_nNR₅COR₅, perfluoroalkyl, polyfluoroalkyl, aminoalkyl, or straight chained or branched C₁-C₇ alkyl; wherein R₅ is independently H; or straight chained or branched C₁-C₇ alkyl; wherein R₆ is independently H; or straight chained or branched C₁-C₇ alkyl; wherein each n independently is an integer from 0 to 6 inclusive; wherein R₇ is independently straight chained or branched C₁-C₇ alkyl; wherein R₈ is

wherein R₉ is independently H; or straight chained or branched C₁-C₄ alkyl; wherein R₁₀ is independently H; or straight chained or branched C₁-C₄ alkyl; wherein R₁₁ is

wherein R₁₂ is H, straight chained or branched C₁-C₇ alkyl, - (CH₂)_uOR₁₇, or - O(CH₂)_UOR₁₇; wherein R₁₃ is independently H; - (CH₂)_UOR₅; - (CH₂)_tCONR₅R₆; - (CH₂)_UN R₅COR₅; - (CH₂)_tCOR₇ ; - (CH₂)_tCO₂R₅; - (CH₂)_uNR₅R6; - (CH₂)_uCN; straight chained or branched C₁-C7 alkyl; C₁-C7 alkyl in which the C₂-C7 atoms may be optionally substituted with one or more F or Cl; C₃-C₇ cycloalkyl-C|-C₇ alkyl; straight chained or branched C₂-C₇ alkenyl or alkynyl; or C3-C7 cycloalkyl; phenyl or C₁-Ce phenylalkyl; wherein the phenyl or C₁-C₆ phenylalkyl may be substituted with one or more of F, Cl, - CN, -NO₂, -NR₅R₆, -SO₂R₅, - (CH₂)_nCOR₇, - (CH₂)_nOR₅, - (CH₂)_nCONR₅R6, - (CH₂J_nNR₅COR₅, - (CH₂)_nCO₂R₅, - (CH₂X₁SO₂NR₅R₆, straight chained or branched Q- C 7 alkyl, perfluoroalkyl, polyfluoroalkyl, or aminoalkyl; or R₁₂ and Ru together with the amide linkage to which they are attached are pyrrolidinonyl, piperidonyl or oxazolidinonyl; wherein Ri₄ is H; straight chained or branched C₁-C₄ alkyl; F; or - (CH₂J_rOR₅; wherein R|₅ is H, straight chained or branched C₁-C₄ alkyl, or F; with the proviso that when Rn is -OH, Ri₅ cannot be F; wherein Ri₆ is perfluoroalkyl, unsubstituted straight chained or branched C₁-C7 alkyl, substituted straight chained or branched C₂-C₇ alkyl, wherein the C₂-C₇ alkyl may be substituted with one or more of F, Cl, -CN, -SO₂R₅, - (CH₂)_nCOR₇, - (CH₂)_nOR₅, - (CH₂)_nCONR₅R6, - (CH₂)_nNR₅COR₅, - (CH₂)_nCO₂R₅, - (CH₂)_nOCF₃, perfluoroalkyl, polyfluoroalkyl, or aminoalkyl, straight chained or branched C₂-C₇ alkenyl or alkynyl, or C₃-C7 cycloalkyl or cycloalkenyl; phenyl, heteroaryl, or C₁-C₇ phenylalkyl, wherein the phenyl, heteroaryl, or C₁-C₇ phenylalkyl may be substituted with one or more of F, Cl, Br, -CN, -NO₂, -NR₅R₆, - (CH₂)_nNR₅COR₅, -SO₂R₅, - (CH₂)_nCOR₇, - (CH₂)_nOR₅, - (CH₂J_nCONR₅R₆, - (CH₂)_nCO₂R₅, - (CH₂)USO₂NR₅R₆, ethylenedioxy, methylenedioxy, straight chained or branched C₁-C₇ alkyl, perfluoroalkyl, polyfluoroalkyl, or aminoalkyl, straight chained or branched C₂-C₇ alkenyl or alkynyl, or C₃-C₇ cycloalkyl or cycloalkenyl; quinolinyl, 1-napthyl, 2-napthyl, or 2,1,3-benzothiadiazolyl; wherein the quinolinyl, 1-napthyl, 2-napthyl, or 2,1,3-benzothiadiazolyl may be substituted with one or more of F, Cl, Br, -CN, -NO₂, -NR₅R₆, - (CH₂J_nNR₅COR₅, -SO₂R₅ - (CH₂J_nCOR₇, - (CH₂J_nOR₅, - (CH₂J_nCONR₅R₆, - (CH₂J_nCO₂R₅, - (CH₂J_nSO₂NR₅R₆, ethylenedioxy, methylenedioxy, straight chained or branched CpC₇ alkyl, perfluoroalkyl, polyfluoroalkyl, or aminoalkyl; with the proviso that when Rg is

Ri6 cannot be quinolinyl; wherein Rj₇ is H, straight chained or branched C₁-C₄ alkyl, perfluoroalkyl, or polyfluoroalkyl; wherein each p independently is an integer from 0 to 2 inclusive; wherein each r independently is an integer from 0 to 3 inclusive; wherein each s independently is an integer from 1 to 6 inclusive; wherein t is an integer from 1 to 4 inclusive; and wherein each u independently is an integer from 2 to 4 inclusive; or a pharmaceutically acceptable salt thereof.

Representative compounds of Formula I include, but are not limited, to the following: N-[6- (4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2-ylamino)hexyl]methane sulfonamide; N-{[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino) cyclohexyl]methyl} -methanesulfonamide; N 1 -[6-(4,5-dihydrobenzo[2,3]thiepino[4,5- d] [ 1 ,3]thiazol-2-ylamino)hexy I]- 1 -ethane sulfonamide; N 1 -[6-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-ylamino)hexy I]- 1 -ethane sulfonamide; N 1 - {[[trans-4-(4,5-Dihydrobenzo[2,3]tiiiepino[4,5-d][l,3]thiazol-2- ylamino)cyclohexyl]methyl}-l -ethanesulfonamide; N2-{ [[trans-4-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-ylamino)cyclohexyl]methyl } -2- thiophenesulfonamide; N l-[5-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino) pentyl]- 1 -ethane sulfonamide; N2-[5-(4,5-Dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2- ylamino)penty l]-2-thiophene sulfonamide; N4- [5-(4,5-dihydrobenzo[2,3]thiepino[4,5- d] [1 ,3]thiazol-2-ylamino)pentyl]- 1 -methyl- 1 H-4-imidazolesulfonamide; N4-[6-(4,5- dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)hexyl]-2,l,3-benzothiadiazole-4- sulfonamide; Nl-[6-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)hexyl]-2- methoxy-5-methyl-l -benzenesulfonamide; N 1 -[6-(4,5-dihγdrobenzo[2,3]thiepino[4,5- d][l,3] thiazol-2-ylamino)hexyl]-l -naphthalene sulfonamide; Nl-[6-(4,5-dihydrobenzo[2,3 ]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)hexy l]-2-nitro- 1 -benzene sulfonamide; N5-[6-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)hexyl]-6-chloroimidazo[2, 1 -b] [ 1 ,3]

26 thiazole-5-sulfonamide; N4-{ [[trans-4-{4,5-dihydrobenzo[2,3]thiepino[4,5-d][l ,3]thiazol-2- ylaminoJcyclohexyllmethylJ^jl^-benzothiadiazole^sulfonamidej Nl-ftltrans^^.S- dihydrobenzoP^Jthiepino^S-dltUSlthiazol^-ylaminoJcyclohexyπmethylJ^-methoxy-S- methyl-1 -benzenesulfonamide; N2-{[[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l ,3] thiazol-2-ylamino)cyclohexyl]methyl}-5-(2-pyridyl)-2-thiophenesulfonamide; N l-{[[trans- 4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l ,3]thiazol-2-ylamino)cyclohexyl]methyl}- 1 - naphthalenesulfonamide; N 1 - { [[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2- ylaminoJcyclohexyllmethylJ-S-CdimethylaminoJ-l-naphthalenesulfonamidei Nl-^^S- dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)pentyl]-5-(dimethylamino)-l- naphthalenesulfonamide; N 1 - { [[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2- y Iamino)cyclohexy l]methyl} -2-nitro- 1 -benzenesulfonamide; N4- { [[trans-4-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)cyclohexy l]methyl } - 1 -methyl- 1 h-4- imidazolesulfonamide; N2-{[[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2- ylaminoJcyclohexyllmethylJ-S^-isoxazolyl^-thiophenesulfonamidej Nl-tS^S- dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)pentyl]-l-naphthalene-sulfonamide; Nl-[5-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)penlyl]-2-fluoro-l- benzene; N2-[6-(4,5-dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-yIamino)hexyl]-5-(3- isoxazolyl)-2-thiophenesulfonamide; N 1 -[5-(4_>5-dihydrobenzo[2_>3]thiepino[4,5-d][ 1 ,3] thiazol-2-ylamino)pentyl]-2-nitro-l -benzene sulfonamide; Nl -[5-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)penty l]-2,6-dichloro- 1 - benzenesulfonamide; Nl-[5-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2- ylamino)pentyl]-2-bromo-6-methoxy-l -benzenesulfonamide; N-[5-(4,5- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)pentyl]pheny 1-methane sulfonamide; N 1 -[5-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2-ylamino)pentyl]-2- fluoro-6-methyl-l -benzenesulfonamide; N 1 -[4-(4,5-dihydrobenzo[2,3]thiepino[4,5- d][l,3]thiazol-2-ylamino)butyl]-2-fluoro-6-methyl-l-benzenesulfonamide; Nl-[5-(4,5- dihydrobenzo[2,3] thiepino[4,5-d][l,3]thiazol-2-ylamino)pentyl]-l-propane sulfonamide; N 1 -[5-(4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2-ylamino)pentyl]-2,4-difluoro-l - benzenesulfonamide; N- { [[trans-4-(4,5-dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2- ylaminoJcyclohexylJmethylJphenyl-methanesulfonamidej Nl-I^^S- dihydrobenzo[2,3]thiepino[4,5-d] [ 1 ,3]thiazol-2-y lamino)cyclohexyl]methyl } -2-cyano- 1 - benzenesulfonamide; Nl-{[4-(4,5-dihydrobenzo[2,3Ithiepino[4,5-d][l,3]thiazol-2- ylamino)cyclohexyl]methyl}-4-methyl-l -benzenesulfonamide; N8-{[4-(4,5- dihydrobenzoP^lthiepino^S-dllUSlthiazol^-ylaminoJcyclohexyllmethylJ-S-

27 quinolinesulfonamide; N 1 -{ [4-(4,5-dihydrobenzo[2,3] thiepino[4,5-d][ 1 ,3]thiazol-2- y Iamino)cyclohexy l]methyl }-2-fluoro-6-methy 1- 1 -benzenesulfonamide; N- { 5-[(9-fluoro- 4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-yl)amino]pentyl}methanesulfonamide; Nl-{5-[(9-fluoro-4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-yl)amino]pentyl}-2- methoxy-5-methy 1- 1 -benzenesulfonamide; N 1 - {5-[(9-fluoro-4,5 -dihydrobenzo[2,3] thiepino[4,5-d][ 1 ,3]thiazol-2-yl)amino]pentyl}-2-fluoro-l -benzenesulfonamide; N2-{5-[(9- fluoro-4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-yl)amino]pentyl}-2-thiophene- sulfonamide; trans-N-4-[(4,5-dihydrobenzo[2,3]thiepino[4,5-d][ 1 ,3]thiazol-2-ylamino) methyllcyclohexyl-N^-methoxyethyOi trans-N^-^-Fluoro^S-dihydrobenzoP^] thiepino-[4,5-d] [ 1 ,3]thiazol-2-y l)amino]methylcyclohexyl)-N-(2-methoxyethyl)formamide; trans-N-4-[(4,5-dihydrobenzo[2,3]thiepino[4,5-d][l,3]thiazol-2-ylamino)methyl] cyclohexyl-N-isopropylformamide; and N-(4-[(9-fluoro-4,5-dihydrobenzo[2,3]thiepino[4,5- d] [ 1 ,3]thiazol-2-y l)amino]methylcyclohexyl)-N-isopropy lformamide.

Further non-limiting examples of NPY Y5 receptor antagonists include the compounds of the Formula II that can be found in WO 07/002126, which is hereby incorporated by reference in its entirety.

o^/χXo ^m

Formula II wherein R¹ is H or Ci-Ce straight chained or branched alkyl; wherein R² is CpCe straight chained or branched alkyl; or wherein R¹, R² and the carbon to which they are attached may form C₃-C6 cycloalkyl; wherein R³ is H or methyl; wherein R⁴ is 2-pyridyl, 3-pyridyl or pyrazinyl, wherein the 2-pyridyl, 3-pyridyl or pyrazinyl may be substituted with methyl; wherein R⁵ is H or methyl; wherein m is an integer from 0 to 2 inclusive; and wherein n is an integer from 0 to 2 inclusive;

28 or a pharmaceutically acceptable salt thereof.

Representative compounds of Formula II include, but are not limited, to the following: [(methylethyl)sulfonyl](/rα«s-4- { [(4-(2-pyridyl)( 1 ,3-thiazol-2-yl))amino]methyl} cyclohexy l)amine; [(methylethyl)sulfonyl]( {/rαrø-4-[(4-(2-pyridyl)( 1 ,3-thiazol-2-yl))amino] cyclohexyl}methyl)amine; [(methylethyl)sulfonyl](/rⁱαns-4-{[(4-(3-pyridyl)(l,3-thiazol-2- yl))amino] methyl}cyclohexyl)amine; [(methylethyl)sulfonyl]({/rύfn$-4-[(4-(3-pyridyiχi,3- thiazol-2-yl))aniino] cyclohexyl}methyl)amine; [(methylethyl)sulfonyl][({/raws-4-{[4-(3- methylpyrazin-2-y 1)( 1 ,3-thiazol-2-yl))]amino} cyclohexyl)methyl]amine; [(methylethyl)sulfonyl]({/rfl/M-4-[(4-(2-pyridyl)(5-methyl-thiazol-2-yl))amino] cyclohexy 1} methy l)amine; [(methy lethyl)sulfonyl](/rύrns-4- { [(4-(2-methyl-pyridin-3-yl)( 1 ,3- thiazol-2-yl))amino]methyl} cyclohexyl)amine; [(methy lethyl)sulfonyl](*rβm-4-{[(4-(2- pyrazinyl)(l ,3-thiazol-2-yl))amino]methyl} cyclohexyl)amine;

[(methylethyl)sulfonyl]({/rσns-4-[(4-(6-rnethyl-pyridin-2-yl)(5-rnethyl-thiazol-2-yl))amino] cyclohexyl}methyl)amine; [(methy lethyl)sulfonyl]( {/røns-4-[(4-(4-methy l-pyridin-2-yl)(5- methyl-thiazol-2-yl))amino] cyclohexyl}methyl)amine; [(methylethyl)sulfonyl]({/rαns-4- [(4-(3-methyI-pyridin-2-yl)(5-methyl-thiazol-2-yl))amino]cyclohexyl}methyl)amine; (ethylsulfonyl)({ιrαns-4-[(4-(2-pyridyl)(l,3-thiazol-2-yl))amino] cycIohexyI}methyl)amine; (cyclopropylsulfonyl)( {/rαns-4-[(4-(2-pyridyiχ 1 ,3-thiazol-2-yl))amino] cyclohexyl}methyl)amine; (butylsulfonyl)({frans-4-[(4-(2-pyridyl)(l,3-thiazol-2-yl))amino] cyclohexyl}methyl)amine; and (butylsulfonyl)[({/ra«y-4-[4-(3-methylpyrazin-2-yl)(l,3- thiazol-2-yl)]amino}cyclohexyl)methyl]amine.

Further non-limiting examples of NPY Y5 antagonists include compounds of the Formula III that can be found in WO 01/14376, and in U.S. Patent Nos. 6,326,375, and 6,335,345, which are hereby incorporated by reference in their entirety.

Formula III

29 wherein A is selected from the group consisting of aryl or heteroaryl, wherein said aryl and heteroaryl groups may be optionally substituted on either the carbon or hetero atom, the substituent being selected from the group consisting of halogen, nitro, lower alkyl, halo (lower) alkyl, hydroxy (lower) alkyl, cyclo (lower) alkyl, lower alkenyl, lower alkoxy, halo (lower) alkoxy, lower alkylthio, carboxyl, lower alkanoyl, lower alkoxycarbonyl, lower alkylene optionally substituted with oxo, and a group represented by formula of-Q-D;

D is selected from the group consisting of aryl or heteroaryl, wherein said aryl and heteroaryl groups may be optionally substituted, the substituent being selected from the group consisting of halogen, cyano, lower alkyl, halo (lower) alkyl, hydroxy (lower) alkyl, hydroxy, lower alkoxy, halo (lower) alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyl and aryl; n is an integer from 0 to 1; Q is selected from the group consisting of a single bond or carbonyl;

T, U, V and W are each independently selected from the group consisting of nitrogen or a methylene group, said nitrogen or methylene group may be optionally substituted with a substituent selected from the group consisting of: halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from the group consisting of methylene or nitrogen;

Y is selected from the group consisting of nitrogen and oxygen, wherein said nitrogen may be optionally substituted with lower alkyl or oxygen; and the pharmaceutically acceptable salts and esters thereof. These compounds of Formula IV are further described and methods of preparing them can be found in International Publication Number WO 01/14376, and in US Patent Nos. 6,326,375, and 6,335,345, which are hereby incorporated by reference in their entirety.

Further non-limiting examples of NPY Y5 receptor antagonists include the compounds of the Formula IV that can be found in PCT/US2007/05563, which is hereby incorporated by reference in its entirety.

30 R⁵

^-JEJ-

- tP -Hi. "¹

Formula IV wherein R¹ is H or Ci-Ce straight chained or branched alkyl; wherein R² is Ci-Ce straight chained or branched alkyl; or wherein R¹, R² and the carbon to which they are attached may form C₃-C₆ cycloalkyl; wherein R³ is H or methyl; wherein R⁴ is 2-pyridyl or 3-pyridyl, wherein the 2-pyridyl or 3-pyridyl is substituted with one or more F, Cl, Br, I or C¥χ and optionally substituted with CH₃; wherein R⁵ is H or methyl; wherein m is an integer from 0 to 2 inclusive; and wherein n is an integer from 0 to 2 inclusive; or a pharmaceutically acceptable salt thereof.

Representative compounds of Formula IV include, but are not limited, to the following: [(methylethyl)sulfonyl](trans-4-{ [(4-(6-fluoro-pyridin-2-yl)( 1 ,3-thiazol-2- yl))amino]methyl}cyclohexyl)amine; [(methylethyI)sulfonyl]({trans-4-[(4-(6-fluoro-pyridin- 2-yl)(l ,3-thiazol-2-yl))amino]cyclohexyl} methyl)amine; [(methylethyl)sulfonyl]({trans-4- ^^-methyljό-fluoro-pyridin-S-ylJfl^-thiazol^-yl^aminoJcyclohexylJmethylJaminej and (methylethyl)sulfonyl](trans-4-{[(4-(2-fluoro-pyridin-3-yl)(l,3-thiazol-2- yOJaminolmethylJcyclohexyOaminej KmethylethyOsulfony^ttrans^-^^-fluoro-pyridin- 3-yl)(l,3-thiazol-2-yl))amino]cyclohexyl}methyl)amine; [(methylethyl)sulfonyl]({trans-4- ^-(ό-fluoro-pyridin-S-ylXS-methyl-l^-thiazol^-yl^aminoJcyclohexylJmethyOaminej and (methylethyl)sulfonyl]({trans-4-[(4-(2-fluoro-pyridin-3-ylK5-methyl-l,3-thiazol-2- yl))amino] cyclohexyl} methyl)amine; [(methylethyl)sulfonyl]({trans-4-[(4-(6- trifluoromethyl-pyridinO-ylXS-methyl-US-thiazol^-yOJaniinoJcyclohexylJmethyOamine; and [(methylethyl)sulfonyl] (trans-4-{[(4-(6-bromopyridin-2-yiχi,3-thiazol-2- yl))amino]methyl}cyclohexyl)amine. Further non-limiting examples of NPY Y5 receptor antagonists include the compounds of the Formula V that can be found in US 60/914,832, which is hereby incorporated by reference in its entirety.

Formula V wherein each R¹ independently is F, Cl, Br, I, -CN, -COR³, -CO₂R³, straight chained or branched C₁-C7 alkyl or Ci -C₇ perfluoroalkyl, straight chained or branched C₁-C7 alkoxy or Ci -C₇ perfluoroalkoxy, or phenyl optionally substituted with one or more R⁴; wherein R² is C3-C6 cycloalkyl, straight chained or branched C₁-C7 alkyl or phenyl optionally substituted with one or more R⁵; wherein each R³ independently is H or straight chained or branched C₁-C7 alkyl; wherein each R⁴ independently is F, Cl, Br, I or straight chained or branched C₁-C7 alkyl; wherein each R⁵ independently is F, Cl, Br, I, straight chained or branched Ci -C₇ alkyl or straight chained or branched C₁-C7 alkoxy; wherein A is

^■ a ^■

A¹ A" A'" wherein n is an integer from O to 4 inclusive; wherein p is an integer from O to 2 inclusive; and wherein q is an integer from O to 2 inclusive; or a pharmaceutically acceptable salt thereof.

Further non-limiting examples of NPY Y5 antagonists include the following compounds: r-(4-t-butyl-pyridylcarbamoyl)-spiroisobenzofiiran- 1 ,4'-piperidine-3-one; 1 '-{4-isopropyl- pyridylcarbamoyl)-spiroisobenzofiiran-l,4'-piperidine-3-one; r-(4-trifluoromethyl- pyridylcarbamoyl)-spiroisobenzofiιran-l,4'piperdine-3-one; l'-(lH-benzimidazol-2-yl)- spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-cyano- 1 H-benzimidazol-2-y l)-spiro

32 [isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-acety 1- 1 H-benzimidazol-2-yl)-spiro [isobenzofuran- l,4'-piperidin]-3-one; I¹- H-benzimidazol-2-yl)-spiro[isobenzofuran-l, 4- piperidin]-3-one methyl ester; l'-^'-pyridin-S-y 1-1 H-benzimidazol-2-yl)-spiro [isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-methyl-l H-benzimidazol-2-yl)-spiro [isobenzofuran- l,4'-piperidin]-3-one; l'-(5-niethoxy-lH-benzimidazol-2-yl)-spiro [isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-chloro- 1 H-benzimidazol-2-yl)-spiro [isobenzofuran- l,4'-piperidin]-3-one; r-H-benzimidazol-2-yl)-spiro[isobenzofuran-l,4'- piperidin]-3-one; 1 - (5-trifluoromethyl-l H-benzimidazol-2-yl)-spiro[isobenzofuran-l, 4'- piperidin]-3-one; r-(6-trifluoromethyl-3-H-imidazo[4,5-b]pyridine-2-yl)-spiro [isobenzofuran- l,4'-piperidin]-3-one; r-(7-chloro-IH-benzimidazol-2-yl)-spiro

[isobenzofuran- 1 ,4'-piperidin]-3-one; T-(I H-benzimidazol-2-y l)-spiro[isobenzofuran- 1 ,4'- piperidin]-3-one; r-(5-n-propylsulfonyl-lH-benzimidazol-2-yl)-spiro[isobenzofurarn-l,4'- piperidin]-3-one; l'- (5 cyano-1 H-benzimidazol-2-yl)-spiro[isobenzofuran-l, 4'-piperidin] - 3-one; r-(5-acetyl-l-H-benziιnidazol-2-yl)-spiro[isobenzofuran-l ,4'-piperidin]-3-one; 1 '-H- benzimidazol-2-yl)-spiro[isobenzofiiran- 1 ,4'-piperidin]-3-one, methyl ester;

•'^■(S'pyrazin^-yl-lH-benzimidazol^-yO-spiroCisobenzofiiran-l^'-piperidinl-S-one; !¹- (5'pyridin-3-yl-l H-benzimidazol-2-yl)-spiro[isobenzofiιran-l, 4'-piperidin]-3-one; V-(S- trifluorometoxy-1 H-benzimidazol-2-yl)-spiro[isobenzofuran-l, 4'-piperidin] -3-one; l'-(5- methyl- 1 H-benzirnidazol-2-yl)-spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-benzoyl- 1 H-benzimidazol-2-yI)spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5- methoxy-1 H-benzimidazol-2-yl)-spiro [isobenzofiiran-l, 4'-piperidin]-3-one; 1'- (5-chloro- 1 H-berizirnidazol-2-yl)-spiro[isobenzo-uran-l,4^l-piperidin]-3-one;6-bromo-7-chloro-2-(spiro [isobenzofuran- l,4'-piperidin]-3-one-3H-imidazo [4,5- b] pyridine; 1'- (5-fluoro-l H- benzimidazol-2-yl)-spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-methy 1- 1 H- benzimidazol-2-yl)-spiro[isobenzoniran-l,4'-piperidin]-3-one; l'-(5-methylsulfonyl-l H- benzimidazol^-ylJ-spirotisobenzofuran-l^'-piperidinJ-S-one; l'-(5-oxazol-2-yl-l H- benzirnidazol-2-yl)-spiro [i$obenzofiiran-l,4'-piperidin]-3-one; r-(5,6-difluoro-l H- benzimidazol-2-yl)-spiro [isobenzofuran- 1 ,4'-piperidin] -3-one; 1 '-(5pheny l-IH-imidazo[4,5- b]pyrazin-2-yl)-spiro[isobenzofiiran-l,4'-piperidin]-3-one; l'-(5-trifluoromethyl-lH- benzimidazol-2-yl)-spiro[isobenzofiιran- 1 ,4'-piperidin]-3-one; 1 '-(5,7-dichloro- 1 H- benzimidazol-2-yl)-spiro[isobenzofuran-l,4'-piperidin]-3-one; l'-(5,6-dimethoxy-lH- benzimidazol-2-yl)-spiro[isobenzofiιran-l,4'-piperidin]-3-one; T- (5-trifluoromethylsulfonyl- 1 H-benzimidazol-2-y l)-spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-(5-(3,5-dimelthyl- isoxazol-4-y I)- 1 H-benzimidazol-2-y l)-spiro[isobenzofuran- 1 ,4'-piperidin]-3-one; 1 '-H-

33 benzimidazol-2-yl)-spiro[isobenzofiiran-l,4'-piporidin]-3-one; and 5-chloro-2- (spiro[isobenzofliran-l, 4'-piperidin]-3-one-3H-imidazo [4,5-b] pyridine. The compounds described in the immediate paragraphs can be prepared by the synthetic methods described and referred to in WO 02/48152, which is hereby incorporated by reference herein in its entirety.

Further non-limiting examples of NPY Y5 antagonists can be found in International Publication Number WO 00/27845, and U. S. Patent Nos. 6,191,160, and 6,313,298, which are hereby incorporated by reference in their entirety. Non-limiting examples of NPY Y5 receptor antagonists further include compound L- 152,804.

Example 5: Screening Methods to Identify NPY Y5 Receptor Antagonists

The pharmacological properties of the compounds of the present invention were evaluated at the cloned human NPY Y5 receptor using the protocols disclosed in U.S. Patent No. 6, 124,331 , the contents of which are hereby incorporated by reference. Using this protocol, the binding by the compound to a radiolabeled ligand (¹²⁵I-labeled PYY or an alternative radioligand such as ^l25I-labeled NPY) to membranes of cloned human NPY Y5 receptors expressed in COS-7 cells was determined in vitro.

NPY Y5 Receptor B inding Assay

Membrane suspensions were diluted in binding buffer supplemented with 0.1 % bovine serum albumin to yield an optimal membrane protein concentration so that ¹²³I-PYY bound by membranes in the assay was less than 10 % of ¹²⁵I-PYY delivered to the sample (100,000 dpm/ sample = 0.08 nM for competition binding assays). ¹²⁵I-PYY and small molecule ligand competitors were also diluted to desired concentrations in supplemented binding buffer. Individual samples were then prepared in 96-well polypropylene microtiter plates by mixing ¹²⁵I-PYY, competing peptides or supplemented binding buffer (25 μL), and finally, membrane suspensions (200 μL). Samples were incubated in at 30 °C for 120 min. Incubations were terminated by filtration over Whatman GF/C filters (pre-coated with 1% polyethyleneimine and air- dried before use), followed by washing with 5 mL of ice-cold binding buffer. Filter- trapped membranes were impregnated with MeltiLex solid scintillant (Wallac, Turku, Finland) and counted for ¹²⁵I-PYY in a Wallac Beta-Plate Reader. Alternatively,

34 incubations were carried out in GF/C filter plates (pre-coated with 1 % polyethyleneimine and air-dried before use), followed by vacuum filtration and three washes of 300 μL of ice-cold binding buffer. SO μL of UltimaGold (Packard) scintillant were added and counted for ¹²⁵I-PYY in a Wallac MicroBeta Trilux. Non-specific binding was defined by 300 nM human PYY. Specific binding in time course and competition studies was typically 80 %; most non-specific binding was associated with the filter. Binding data were analyzed using nonlinear regression and statistical techniques available in the GraphPAD Prism package (San Diego, Calif.).

NPY Y5 Receptor Functional Assay

The functional potency of NPY Y5 receptor antagonists was determined using various assays which measure inhibition of second messenger pathways. NPY increases intracellular Ca²⁺ concentration via activation of Y5 receptors through coupling to Gaqi5. The potency of a NPY Y5 receptor antagonist in blocking NPY mediated Ca²⁺ increase was used as a measure of its functional antagonist activity. For example, CHO cells expressing both NPY Y5 receptors and Gaqi5 were seeded into 96-well plate 24 hr before assay. Cells were loaded for 1 h with a Ca²⁺ sensitive fluorescent dye, Fluo-4-AM in assay buffer (Hank's Balanced Salts Solution (HBSS) containing 20 mM HEPES, 0.5 % BSA and 2.5 mM probenecid, pH 7.4), washed 3 times with the assay buffer, then returned to the incubator for 1 hr before assay on a fluorometric imaging plate reader, FLIPRTM (Molecular Device, California). The NPY-induced maximum change in fluorescence over baseline was determined and the dose which induces a 50% increase in fluorescence is defined as the EC50 dose for NPY. To evaluate Y5 antagonists, the assay was repeated with the EC50 dose of NPY in the presence of various concentrations of a NPY Y5 antagonist to generate a functional IC50. The concentration-response curves are fitted using GraphPAD Prism package (San Diego, Calif.).

The above-identified compounds were found to bind to the NPY Y5 receptor. Useful NPY5 receptor antagonists would also have to posses other characteristics such as selectivity over the other NPY receptors, good systemic exposure, sufficient half-life and brain penetration.

35

Claims

WHAT IS CLAIMED:

1. A method of identifying a subject most responsive to the administration of a NPY Y5 receptor antagonist for treating a hypercortisolism-related disorder comprising: (a) presenting a challenge test to the HPA axis on the subject;

(b) obtaining a biological sample from the subject; and

(c) analyzing said sample for the level of Cortisol.

2. A method of diagnosing and/or monitoring a subject's responsiveness to the administration of a NPY Y5 receptor antagonist, wherein the subject is diagnosed with a hypercortisolism-related disorder comprising:

(a) presenting a challenge test to the HPA axis on the subject;

(b) obtaining a biological sample from the subject; and

(c) analyzing said sample for the level of Cortisol.

3. A method of treating a subject diagnosed with a hypercortisolism-related disorder comprising:

(a) presenting a challenge test to the HPA axis on the subject;

(b) obtaining a biological sample from the subject; (c) analyzing said sample for the level of Cortisol; and

(d) administering a NPY Y5 receptor antagonist.

4. A method of marketing a medicinal product comprising a NPY Y5 receptor antagonist for the treatment of a hypercortisolism-related disorder, said marketing comprising the public spreading of the information that the level of Cortisol can be used to monitor the progress of said disorder.

5. Use of a NPY Y5 receptor antagonist in the manufacture of a medicament for treating a hypercortisolism-related disorder in a subject, wherein said subject is characterized by a Cortisol level above about 125 nmol/1 following a dexamethasone based challenge test to the HPA axis.

6. A NPY Y5 receptor antagonist for use in treating a hypercortisolism-related disorder in a subject, wherein said subject is characterized by a Cortisol level

36 above about 125 nmol/1 following a dexamethasone based challenge test to the HPA axis.

7. The method of anyone of claims 1-4, wherein the hypercortisolism-related disorder is a stress-related disorder.

8. The method of claim 7, wherein the stress-related disorder is selected from the group consisting of depression; major depression; moderate depression; severe depression; and melancholic depression.

9. The method of claim 7, wherein the stress-related disorder is selected from the group consisting of anxious depression; anxiety; generalized anxiety disorder; post traumatic stress syndrome; attention-deficit hyperactivity disorder; a phobia and a panic disorder.

10. The method of anyone of claims 1-4, wherein the hypercortisolism-related disorder is a metabolic-related disorder selected from the group consisting of obesity; bulimia nervosa; binge eating disorder; poorly controlled or uncontrolled diabetes; and a metabolic syndrome

11. The method of anyone of claims 7-9, wherein the stress-related disorder results from chronic stress or disease.

12. The method of anyone of claims 7-9, wherein the stress-related disorder results from acute stress.

13. The method of anyone of claims 1-4, wherein the challenge test to the HPA axis is presented prior to treatment.

14. The method of anyone of claims 1-4, wherein the challenge test to the HPA axis is presented during and after treatment.

15. The method of anyone of claims 1-4, wherein the biological sample is a blood sample.

16. The method of anyone of claims 1 -4, wherein the challenge test is dexamethasone based.

37

17. The use of claim 5 or 6, wherein the hypercortisolism-related disorder is a stress- related disorder.

18. The use of claim 17, wherein the stress-related disorder is selected from the group consisting of depression; major depression; moderate depression; severe depression; and melancholic depression.

19. The use of claim 17, wherein the stress-related disorder is selected from the group consisting of anxious depression; anxiety; generalized anxiety disorder; post traumatic stress syndrome; attention-deficit hyperactivity disorder; a phobia and a panic disorder.

20. The use of anyone of claims 5 or 6, wherein the hypercortisolism-related disorder is a metabolic-related disorder selected from the group consisting of obesity; bulimia nervosa; binge eating disorder; poorly controlled or uncontrolled diabetes; and a metabolic syndrome.

21. The use of anyone of claims 17-19, wherein the disorder results from chronic stress or disease.

22. The use of anyone of claims 17-19, wherein the disorder results from acute stress.

38