WO2011140607A1

WO2011140607A1 - A method of treatment and diagnosis

Info

Publication number: WO2011140607A1
Application number: PCT/AU2011/000560
Authority: WO
Inventors: Charlotte L. Keating
Original assignee: Keating Charlotte L
Priority date: 2010-05-14
Filing date: 2011-05-13
Publication date: 2011-11-17
Also published as: AU2011252763B2; AU2011252763A1

Abstract

The present disclosure relates generally to the field of pathobiology. More particularly, the present disclosure contemplates the use of hormonal agonists and antagonists in the treatment or management of a range of pathophysiological conditions including cancer, menopause, puberty, reproductive function and psychological conditions which manifest with adverse behavioral, physiological and/or clinical symptoms. Diagnosis of such pathologies is also taught herein.

Description

A METHOD OF TREATMENT AND DIAGNOSIS

FILING DATA [0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2010902058, filed on 14 May 2010, entitled "A method of treatment and diagnosis", the entire contents of which, are incorporated herein by reference. FIELD 9

[0002] The present disclosure relates generally to the field of pathobiology. More particularly, the present disclosure contemplates the use of hormonal agonists and antagonists in the treatment or management of a range of pathophysiological conditions including cancer, menopause, puberty, reproductive function and psychological conditions which manifest with adverse behavioral, physiological and/or clinical symptoms. Diagnosis of such pathologies is also taught herein.

BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Pathobiology is the study of physiological and psychological processes which can present in a broad spectrum of conditions which have a variety of behavioral and clinical manifestations. These manifestations extend from mild to heavily debilitating symptoms as well as death. Conditions such as menopause, puberty, reproduction, cancer and a range of psychological issues can lead to quite debilitating consequences.

[0006] For example, there is a social significance of psychological disorders which is highlighted by a gender bias in their onset. For example, both depression and anorexia nervosa are more prevalent in females with major depression occurring twice as often (Burt and Quezada, Canadian Journal of Clinical Pharmacology 16(l) e6-e\4, 2009; Doris et al, Lancet 554: 1369-1375, 1999; Fava and Kendler, Neuron 25:3350-341 , 2000) and anorexia nervosa presents in up to 95% of female cases (Barbarich-Masteller, Central Nervous system Agents in Medicinal Chemistry 7(7^:35-43, 2007). The gender bias is proposed to arise from various factors including genetically inherent alterations in mood linked to changing hormones during reproductive transitions such as puberty, paramenstruum, pregnancy, postpartum and perimenopause (Angold et al, Psychological Medicine 25:51-61, 1998; Burt and Quezada, 2009 supra; Freeman et al, Archives of General Psychiatry 63 (¾):375-382, 2006). Anorexia nervosa is characterized by a fear of weight gain, below minimal normal body weight and disturbance body shape and size perception. Amenorrhea is currently a diagnostic for anorexia nervosa but its clinical utility is questionable (Abraham et al, Journal of Psychosomatic Obstetric Gynecology 24:185-193, 2003). This illness presents with the greatest risk of mortality of any other psychiatric condition (see Kaye et al, Nature Reviews Neuroscience 7(7:573-584, 2009; Sullivan, American Journal of Psychiatry 152(7): 1073- 1074, 1995).

J0007] The etiology of major depression has been explained in large part by the 5- hydroxytryptamine (5-HT) hypothesis. This hypothesis proposes that the illness is initially underscored by.reduced availability of synaptic 5-HT or decreased activity of the 5-HT system (Biller and Montigny, Neuropsychopharmacology 2/:91s-98s, 1999; Lesch and Heils, International Journal of Neuropsychopharmacology 3: 67-79, 2000; Venstra- VanderWeele et al, European Journal of Pharmacology 470:165-181, 2000). In addition to the 5-HT system, depressive episodes have been linked to other neurochemical and neurohormonal abnormalities, which includes the stress system, and consistent with this, pathophysiological studies have revealed dysfunction in various end-points of the reproductive axis, the hypothalamopituitary gonadal axis (HPG)-axis, including changes (elevations) in Cortisol levels (Gold and Chrousos, Molecular Psychiatry 7:254-275, 2002). Abnormalities in the HPG-axis are present in up to 80% of patients, with most reporting hyperactivity of the axis. Abnormalities in the stress-linked system, the hypothalamopituitary adrenal (HP A) axis, have been shown to normalize following treatment and may predict relapse-risk in patients with depression (Schule, Journal of Neuroendocrinology 19:213-226, 2006).

[0008] Specific substrates within the 5-HT system responsible for aspects of major depression have been extensively investigated and reported, at the receptor level and neurotransmitter level. In brief, brain imaging studies (e.g. Bhagwagar et al, American Journal of Psychiatry 163(9): 1580- 1587, 2006) involving Positron Emission Tomography (PET) have directly demonstrated abnormalities including abnormally elevated binding potential of the 5HT2A receptor as well as elevated 5-HT 1 A receptor binding during an illness episode, which may also predict poorer response to treatment (Parsey et al, Neuropsychopharmacology 57:1745-17 '49, 2006).

[0009] Central concentrations of 5-HT in patients have revealed abnormalities in patients relative to healthy participants. In males and females, abnormally elevated illness-state 5- HT levels were reported (Barton et al, Archives of General Psychiatry 65(1):38-Λ6, 2008) with a twofold increase in concentrations correlating with those genotyped with the "s" allele of the 5-HT transporter (5-HTT) relative to those genotyped with the "I" allele (Barton et al, 2008 supra). Functionality of the transporter has been linked to response to treatment. The "s" or short allele and the "1" allele correspond to polymorphisms in the 5- HTTLPR (serotonin-linked-pOlymorphic region) region of the transporter gene. The "1" allele has further been defined as the LA or LQ, such that the latter behaves similarly to the "s" allele and is, therefore, often grouped during analyses of 5-HTT function. Following treatment with selective serotonin re-uptake inhibitors (SSRIs) patients showed significant reductions to healthy levels. Reduced recovery levels of 5-HT (relative to the illness state) seem to contradict the basic action of SSRIs. In other research, however, cerebral spinal fluid (CSF) levels of 5-HT were elevated (e.g. as indexed via 5 -hydroxy indolacetic acid, 5-HIAA measures) in patients with depression and patients with depression and comorbid panic disorder (Gjerris et al, Journal of Affected Disorders 12(1 ^: 13-22, 1987; Sullivan et al, International Journal of Neuropsychopharmacolog 9(50):547-556, 2006; respectively).

[0010] The 5-HTT is also posited to contribute to the pathophysiology of depression. Located within the receptor, 5-HTT is responsible for the re-uptake of 5-HT into the neuron. Patients presenting as homozygous 1/1 (or L_A) for the 5-HTT have been shown to respond more favorably to SSRI treatment than those who are carriers of the "s" (or LG) allele (Hu et al, Archives of General Psychiatry <¾(7j:783-792, 2007). Genotype of the 5-HTT has also been associated with adverse effects of treatment in patients with depression (Hu et al, 2007 supra). Mechanisms by which variations in the combination of the alleles impact treatment response as well as the potential for gender-specific impact of the 5-HTT may further understanding of the potential for a predictive relationship between polymorphisms, gender and treatment in illnesses such as depression. [0011] The stress endocrine system has also been extensively investigated in major depression. For example, aberrant activity in the hypothalamic pituitary adrenal axis (HPA-axis), particularly hyperactivity has been linked to major depression and has been one of the most common findings (Gold and Chrousos, 2002 supra). Enhanced activity of the HPA-axis shown in a significant percentage of patients and has been demonstrated via increased levels of Cortisol in saliva, plasma and urine and increased size (and activity and responsivity) of the pituitary and adrenal glands (Nemeroff and Vale, Journal of Clinical Psychiatr ^> 66(Suppl) 5-\3, 2005).

[0012] These studies suggest potential mechanisms that may contribute to abnormal HPA- axis activity in patients with major depression. Hyperactivity of the HPA-axis is widely reported in the pathophysiology of the illness and its normalization has been linked to clinical response to anti-depressant drugs (at least in some males) [Binder et al, Psychoneuroendocrinolgy 34:99-109, 2009; Young et al, Psychoneuroendocrinology 29(9): 1 198-1204, 2004; Juruena et al, Br J Psychiatry 794(^:342-349, 2009]. As the majority of patients with major depression present with over-active HPA-axes, and in some patients, overactivity of the HPA-axis precludes a response to anti-depressants, potential physiological mechanisms responsible for hyperactivity and treatment adjuncts, which aim to attenuate over-activity of the HPA-axis, require further exploration. See Keating et al.. Int. J. Neuropsychopharm. 74:553-566, 201 1.

[0013] Gonadotropin inhibiting hormone (GnlH) is a dodecapeptide belonging to a family of FRamide peptides with a carboxy terminal COOH-Pro-Xaa-Arg-Phe-NH₂ motif. The gene encoding the precursor protein for GnlH in Japanese quail, chickens and white- crowned sparrows has been cloned and all share a high degree of homology (Satake et al. The Biochemical Journal, 354(2):379-385 2001 ; Osugi et al, Journal of Endocrinology, 182(1), 33-42, 2004; Ikemoto and Park, Journal of Reproduction and Development, 51(3), 359-377: 2005). It has also been cloned from mammals including hamsters, rats and mice (Kriegsfeld et al, Proceedings of the National Academy of Sciences 703(7 :24100-2415, 2006).

[0014] GnlH is principally involved in the negative regulation of reproduction and has been shown to have seasonal regulatory effects on the hypothalamic pituitary gonadal axis (HPG-axis) in several species (Bentley et al, Journal of Ornithology, 148 (suppl.) S521 - S536:2007; Ciccone et al, Journal of Neuroendocrinology 16(12).999-} 06, 2007) as well as mammals (Johnson et al, Hormones and Behavior 5/( :171-180, 2007; Kriegsfeld et al, 2006 supra). Released from the parvocellular neurons of the hypothalamus, GnlH (unlike gonadotropin releasing hormone [GnRH]) functions by negatively controlling the release of gonadotropins (perhaps via inhibition of GnRH; Kriegsfeld et al, 2006 supra), follicle stimulating hormone (FSH) and lutenizing hormone (LH) from the anterior pituitary. Both FSH and LH are necessary for stimulating pro-ovulatory hormones, estrogen and progesterone, released from the ovaries. The consequence of FSH and LH inhibition is a suppression of synthesis and secretion of estrogen and progesterone which in turn results in the inhibition of ovulation, and hence cessation of reproductive function. In addition to its role in reproduction, GnlH has been shown to be located in multiple areas of the brain related to disparate behaviors including feeding behavior in chickens (Tachibana et al, Brain Research 1050(1-2), 94-100: 2005) and sexual behavior in white-crowned sparrows (Bentley et al, 2007 supra). [0015] With respect to GnlH central (brain) identification, GnlH-immunoreactive neurons are exclusively found in the hypothalamic paraventricular nucleus and fibers are found throughout the brain including the median eminence in Japanese qual (Ukena et al, Journal of Endocrinology, 178(2),31 1-318: 2003) and sparrows (Bentley et al, Journal of Neuroendocrinology, 15(8), 794-802: 2003). In addition to avian species, GnlH homologs have been shown centrally in hamsters, rats and mice (Kriegsfeld et al, 2006 supra) in which GnlH cell body distribution have been localized in the mediobasal hypothalamus with projections and terminals throughout the central nervous system. [0016] With respect to the periphery, there is evidence to demonstrate the presence of GnlH in the ovaries of avian species including chickens in addition to which there have been GnlH receptors identified on the placenta and pituitary (Hinuma et al, Nature Cell Biology, 2(10), 703-708: 2000; Smith & Clarke, Trends in Endocrinology and Metabolism, 21(4), 255-260, 2010).

[0017) There is an intertwine of complex stress-linked and reproductive-linked biological systems which are associated with a range of pathophysiological conditions which impact on wellbeing and pharmacoefficacy. An understanding of these systems enables the development of treatment and diagnostic protocols for a range of pathophysiological conditions such as cancer, menopause, puberty, reproduction and psychological illnesses.

SUMMARY

[0018] Enabled herein is the use of agonists and antagonists of stress-linked and/or reproductive-linked biological systems alone or in combination with other medicaments in the treatment of pathophysiological aspects of medical and mental conditions. Reference to "medical conditions" includes cancer such as a gynecological cancer which encompasses ovarian cancer among other hormone related cancers as well as menopause, puberty or reproductive status. Components within the stress-linked and reproductive- linked systems are also proposed to be diagnostic targets instructive as to particular pathophysiological conditions and/or their states.

[0019] Reference to "mental condition" includes "neuropsychological illness" which encompasses a neurological, psychological and/or psychiatric condition, state, disorder or sub-threshold form thereof. Such conditions include (but are not limited to) depression, anxiety and schizophrenia. "Depression" includes major depression and postnatal depression among other depressive disorder subtypes (ie., bipolar disorder). It is proposed herein to manage or ameliorate symptoms of pathophysiological conditions by administering to subjects a GnlH agonist or antagonist alone or in combination with one or more of (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive- linked biological system; (iii) an estrogen or an estrogen analog or agonist and/or an agent which induces estrogen production or a selective estrogen receptor modulator (SERM); (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an antidepressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer.

[0020] In an embodiment, a human subject diagnosed with symptoms of depression is treated by the administration of an effective amount of a GnlH antagonist for a time and under conditions sufficient to ameliorate the symptoms of depression. The subject method also includes the combination therapy of effective amounts of a GnlH antagonist and an anti-depressant such as selected from an SSRI, SSNRI, a tri-cyclic anti-depressant and a tetra-cyclic anti-depressant. An example of a GnlH antagonist is a GnRH agonist. Examples of an SSRI include agomelatine, escitalopram, sertraline, citalopram and fluoxetine. Examples of an SSNRI include esmertazapine, venlafaxine, desvenlafaxine and duloxetine. Examples of a tri-cyclic anti-depressant include amitriptyline and imipramine. Examples of a tetra-cyclic anti-depressant include amoxapine and mirtazapine.

[0021] When two or more agents are used, they may be administered simultaneously or sequentially and in any order. When sequentially administered, the period of time between administration of each drug may be nanoseconds, milliseconds, seconds, a minute or minutes, hours or days. The drugs may be administered via the same or different routes.

[0022] The present disclosure further contemplates a pathophysiological medicament comprising a GnlH agonist or antagonist, alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive- linked biological system; (iii) an estrogen or an estrogen analog or agonist or selective estrogen receptor modulator (SERM) and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an antidepressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer; for use in treating or managing a pathophysiological condition.

[0023] Pathophysiological conditions include cancer, menopause, puberty, reproduction, estrogen-induced thymus degradation, and psychological conditions including depression, anxiety, schizophrenia and eating disorders. [0024] Psychological conditions contemplated herein include eating disorders (such as anorexia nervosa and bulimia nervosa), depression (including major depression and ppstnatal depression among other depressive subtypes such as treatment resistant depression and chronic depression), bipolar disorder, anxiety disorders, addiction, dementia, epilepsy, schizophrenia, Tourette's syndrome, obsessive compulsive disorder (OCD), panic disorder, PTSD, phobias, acute stress disorder, adjustment disorder, agoraphobia without history of panic disorder, alcohol dependence (alcoholism), amphetamine dependence, brief psychotic disorder, cannabis dependence, cocaine dependence, cyclothymic disorder, delirium, delusional disorder, dysthymic disorder, hallucinogen dependence, nicotine dependence, opioid dependence, paranoid personality disorder, Parkinson's disease, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder, schizophrenia, schizotypal personality disorder, sedative dependence, shared psychotic disorder, smoking dependence and social phobia.

[0025] The drugs may also be given as part of another therapeutic program and/or behavioral modification therapy or counseling. [0026] The present disclosure further provides for the use of a GnlH agonist or antagonist alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist, in the manufacture of a medicament for the treatment or prophylaxis of a pathophysiological condition; and/or (vi) a mood stabilizer. "Prophylaxis" in this context is particularly useful in pre-emptive treatment of at risk individuals or individuals who have a genetic predisposition for a pathophysiological condition or whose lifestyle may result in such a condition.

[0027] Aspects of the enabled herein include a method for treating a subject with a pathophysiological condition, phenotype, state, condition or sub-threshold form thereof, the method comprising administering to the subject a GnlH agonist or antagonist alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti- depressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer in amounts and under conditions sufficient to ameliorate symptoms of the pathophysiological condition, illness, phenotype, state, condition or sub-threshold form thereof. [0028] The present disclosure further provides diagnostic assays instructive as to the level of reproductive wellbeing, the presence of cancer or a psychological illness and the like. [0029] The present disclosure teaches a range of biomarkers of pathophysiological conditions or states. The biomarkers include the levels of activity of GnlH or GnRHalone or in combination with a component within (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an antidepressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer. Collectively, these are referred as "biomarkers" or "pathophysiological condition markers" or "markers of a pathophysiological condition". ^;

[0030] Accordingly, another aspect enabled herein provides an assay for determining the presence or state of a pathophysiological condition in a subject, the assay comprising determining the concentration or activity of biomarkers in a biological sample from the subject selected from GnlH alone or in combination with a biomarker selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist; (vi) a mood stabilizer wherein an alteration in the levels or activity of the biomarkers relative to a control provides an indication of the presence or state of the pathophysiological condition.

[0031] Abbreviations used in the present specification are defined in Table 1. Table 1

Abbreviations

Abbreviation Definition

ACTH Adrenocorticotrophin hormone

CRH Corticotropin releasing hormone

DHEAS Dehydroepiandrosterone sulfate

ER Estrogen receptor

FSH Follicle stimulating hormone

GnlH Gonadotropin inhibitory hormone

GnRH Gonadotropin releasing hormone

HPA-axis Hypothalamic pituitary adrenal axis

HPG-axis Hypothalamic pituitary gonadal axis

HRT Hormone replacement therapy

IUGR Intrauterine growth restriction

JNK/AP-1 c-Jun N-terminal kinase/activator protein- 1

LH Lutenizing hormone

LHRH Lutenizing hormone releasing hormone

OCa Ovarian cancer

OSE Ovarian surface epithelium

PgR Progesterone receptor

PKC Protein kinase C

PTSD Post-traumatic stress disorder

PvFRP-3 Avian homolog of GnlH

SERM Selective estrogen receptor modulator

SSNRI Selective serotonin and noradrenergic re-uptake inhibitor

SSRI Selective serotonin re-uptake inhibitor BRIEF DESCRIPTION OF THE FIGURES

[0030] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0031] Figure 1 is a diagrammatic representation of reciprocal interaction between GnlH and HPA axes; stress results in upregulation of GnlH tone which feeds back to maintain upregulation of HPA-axis. This relationship is further mediated by sympathoadrenal system input. Sympathoadrenal input may mediate the reciprocity between HPA-axis and GnlH.

[0032] Figure 2 is a diagrammatic representation of reciprocal interaction between GnlH and HPA axes; stress results in upregulation of GnlH tone which fees back to maintain upregulation of HPA-axis. This relationship is further mediated by sympathoadrenal system input. Sympathoadrenal input may mediate the reciprocity between HPA-axis and GnlH. Further to this relationship, GnlH likely regulates kisspeptin, an RFamide that responds to the levels of sex steroids by positively regulating GnRH. [0033] Figure 3 is a diagrammatic representation of mean (+SEM) plasma concentrations of RFRP3 in internal jugular and arterial samples before and following treatment with selective serotonin reuptake inhibitor (SSRI). Comparisons were also made to healthy subjects Pre-SSRI. [0034] Figure 4 is a diagrammatic representation of mean (+SEM) plasma concentrations of RFRP3 in internal jugular and arterial samples before and following treatment with selective serotonin reuptake inhibitor (SSRI) in males and females with major depression. DETAILED DESCRIPTION

[0035] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers are method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0036] The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a condition" includes a single condition as well as two or more conditions; reference to "an agonist" or "an antagonist" includes a single agonist or antagonist as well as two or more agonists or antagonists; reference to "the embodiment" includes single or multiple embodiment; and so forth. [0037] Taught herein is an empirically-based protocol based on the use of GnlH agonists or antagonists alone or in combination with anti-psychotic agents and anti -depressant agents or mood stabilizers to treat or manage a pathophysiological condition. By "pathophysiological condition" means a medical condition or situation such as a cancer, menopause, puberty or pregnancies and a psychological or mental condition. The term "cancer" includes any or a range of oncological situations and includes a gynecological cancer such as ovarian cancer and related and other conditions. By "psychological or mental condition" is meant a psychological, neurological and psychiatric illness, disorder, phenotype, state or sub-threshold form thereof in which a subject exhibits a behavioral or clinical phenotype characterized by abnormalities in reproductive- or stress-linked biological systems or dopamine or serotonin pathways. The term "psychological or mental condition" encompasses all such neuropsychological and neuropsychiatric phenotypes. The present disclosure contemplates a method of ameliorating the symptoms of or managing a pathophysiological condition such as an anomaly associated with a medical or mental condition, Terms which are also applicable to these phenotypes include oncological, cancerous, tumor, psychiatric, psychological and neurological illnesses or condition or state. The term "illness" does not necessarily mean that a subject is medically incapacitated. Rather, it includes the full spectrum of pathophysiological conditions, disorders, phenotypes, states or sub-threshold forms thereof which manifest in from complete debilitation to minor behavioral, physiological and/or clinical abnormalities.

[0038] In an embodiment, a human subject diagnosed with symptoms of depression is treated by the administration of an effective amount of a GnlH antagonist for a time and under conditions sufficient to ameliorate the symptoms of depression. The subject method also includes the combination therapy of effective amounts of a GnlH antagonist and an anti-depressant such as selected from an SSRI, SSNRI, a tri-cyclic anti-depressant and a tetra-cyclic anti-depressant. The combination therapy includes a GnlH antagonist, with or without anti -depressant and one or both of oxytocin and/or estrogen. An example of a GnlH antagonist is a GnRH agonist. Examples of an SSRI include agomelatine, escitalopram, sertraline, citalopram and fluoxetine. Examples of an SSNRI include esmertazapine, venlafaxine, desvenlafaxine and duloxetine. Examples of a tri-cyclic antidepressant include amitriptyline and imipramine. Examples of a tetra-cyclic anti- depressant include amoxapine and mirtazapine.

[0039] Examples of psychological illnesses contemplated by the present disclosure include conditions related to dopamine pathway function and the function of the associated neurotransmitters GABA, glutamate and/or serotonin including but are not limited to eating disorders (such as anorexia nervosa and bulimia nervosa), depression (including major depression, postnatal depression, treatment resistant depression, chronic depression), biopolar disorder, anxiety disorders, addiction, dementia, epilepsy, schizophrenia, Tourette's syndrome, obsessive compulsive disorder (OCD), panic disorder, post-traumatic stress disorder (PTSD), phobias, acute stress disorder, adjustment disorder, agoraphobia without history of panic disorder, alcohol dependence (alcoholism), amphetamine dependence, brief psychotic disorder, cannabis dependence, cocaine dependence, cyclothymic disorder, delirium, delusional disorder, dysthymic disorder, hallucinogen dependence, nicotine dependence, opioid dependence, paranoid personality disorder, Parkinson's disease, schizoaffective disorder, schizoid personality disorder, schizophreniform disorder, schizotypal personality disorder, sedative dependence, shared psychotic disorder, smoking dependence and social phobia. [0040] Particular conditions contemplated herein include eating disorders (such as anorexia nervosa and bulimia nervosa), bipolar disorder, anxiety disorders (such as OCD and PTSD) and depression (such as major depression) and postnatal depression and schizophrenia. Reference to "anxiety disorder" includes "generalized anxiety disorder".

[0041] Hence, the present disclosure teaches the use of a GnlH agonist or antagonist, alone or in combination with anti-psychotic and anti-depressant drugs, mood stabilizer or an agent which is from or targets a stress-linked or reproductive-linked biological system in the treatment or prophylaxis or clinical or behavioral management of subjects with or at risk of developing a pathophysiological condition. It is proposed herein that the GnlH agonist or antagonist is used alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or selective estrogen receptor modulator (SERM) and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer.

[0042] The disclosure enables provides a selection of pharmapathophysiological agents based on an understanding of the interaction these drugs have on stress-linked and reproductive-linked biological systems. If a combination of drugs is used, the combination may be considered synergistic, since the combination of the drugs has more efficacious outcomes than the use of the single drugs above. Hence, the combination is functionally synergistic.

[0043] Reference to a "dopamine antagonist" and a "selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant" alternatively includes an agent which modulates a downstream signaling target within the stress-linked or reproductive_^ linked biological system. By "stress-linked" includes the HPA-axis. By "reproductive- linked" includes the HPG-axis. [0044] Accordingly, an aspect taught herein is a method for treating a subject with a pathophysiological condition, illness, phenotype, state, condition or sub-threshold form thereof, the method comprising administering to the subject a GnlH agonist or antagonist alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer in amounts and under conditions sufficient to ameliorate symptoms of the pathophysiological condition, illness, phenotype, state, condition or sub-threshold form thereof.

[0045] Another aspect enabled by the present disclosure is a method of treating a human subject diagnosed with symptoms of depression, the method comprising administering to the subject an effective amount of GnlH antagonist for a time and under conditions sufficient to ameliorate the symptoms of depression. The subject may further be provided with an anti-depressant such as selected from an SSRI, SSNRI, tri-cyclic anti-depressant and a tetra-cyclic anti-depressant. The subject may also be provided with a GnlH antagonist and one or more of an anti-depressant, oxytocin and/or estrogn.

[0046] It is proposed herein that GnlH or an agonist thereof is useful in treating or preventing hormone-dependent cancer growth and development such as ovarian cancer or other gynecological malignancy. Furthermore, a GnlH or an agonist or antagonist thereof may be indicated for a female subject during pregnancy, such as at the end of the pregnancy. GnlH levels or activity may directly or indirectly be measured and if levels or activity is reducing during the pregnancy, GnlH or its agonist or antagonist may be required to be administered. In addition, GnlH or its agonist may be required to induce menopause for study purposes or to arrest a gynecological cancer. In the postnatal period, GnlH or its antagonist may be used to alter mood. [0047] Reference to a GnlH agonist includes agents which down-regulate gonadotropin releasing hormone (GnRH) and kisspeptin. GnlH itself is also regarded herein as a GnlH agonist. [0048] GnlH antagonists include agents which up-regulate levels of GnRH, kisspeptin, LH, FSH, estrogen and progesterone. Antagonists include genetic antagonists such as anti- sense RNA, microRNA, sense nucleic acid molecules, RNAi constructs and other regulatory RNAs such as double stranded and single stranded RNAs, hairpin RN As, short interfering RNAs and the like. Aptamers may also be regarded as an expression antagonist.

[0049] Agents which target the endocrine system include agents which modulate the HPA- axis. [0050] GnlH antagonists are proposed to be useful in managing stress, depression, anxiety, schizophrenia, to induce puberty or to delay menopause.

[0051] In an embodiment, a method is contemplated herein for the treatment or prophylaxis of an ovarian cancer condition in a subject, the method comprising administering to the subject cancer cell arresting effective amount of GnlH or an agonist thereof for a time and under conditions to prevent or reduce cancer development.

[0052] Reference to "ovarian cancer" includes epithelial ovarian cancer and a stage of epithelial ovarian cancer.

[0053] In an embodiment, a super-antagonist of GnlH at the ovaries is employed, or a super-agonist is employed in a psychological or a super-antagonist or super-agonist in other physiological conditions. [0054] An agonist of GnlH includes GnlH itself and an antagonist of GnRH. U2011/000560

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[0055] In an embodiment, the GnlH or its agonist is provided via a nasal spray, injection or oral medicaments and may include transdermal patches.

[0056] Still another aspect enabled herein is a method for managing stress in a subject, the method comprising administering to the subject suffering from stress or exposed to a potentially stressful situation, an amount of an antagonist of GnlH effective to reduce stress or its development.

[0057] Reference to "stress" includes anxiety or the presence or development of an anxious situation or feeling, where the experience may be acute and/or for treatment where symptoms are chronic.

[0058] The GnlH antagonist may conveniently be administered vr^'a nasal spray, injection or oral medicament and may also be used in as a transdermal patch including (but not limited to) longer-term treatment management.

[0059] Yet another aspect taught herein is a method for managing a pregnancy in a female subject, the method comprising optionally monitoring GnlH levels or activity in the subject or other indicator of GnlH levels or activity and providing to the subject GnlH or an agonist or antagonist, thereof if required at any stage of pregnancy and postpartum.

[0060] Even yet another aspect taught herein is a method for managing depression including postnatal depression in a subject, the method comprising administering to the subject an effective amount of an antagonist of GnlH for a time and under conditions to ameliorate the symptoms of depression.

[0061] Another aspect enabled herein is a method for modulating intrauterine growth restriction (IUGR) in a subject, the method comprising administering to the subject an effective amount of a GnlH antagonist to reduce IUGR or GnlH or an agonist thereof to enhance IUGR. [0062] Still another aspect enabled herein is a method for transiently inducing menopause in a female subject, the method comprising administering to the subject an effective amount of GnlH or an agonist thereof. Inducing menopause is useful as a medical model as well as in the treatment of a gynecological cancer. Similarly, delaying menopause to extend reproductive fertility may be achieved by use of an antagonist of GnlH.

[0063] Yet another aspect described herein a method for managing puberty in a subject, the method comprising administering to the subject GnlH, an agonist thereof or a GnlH antagonist for a time and under conditions sufficient to induce or delay puberty in the subject.

[0064] In a related embodiment, GnlH or an agonist thereof or a GnlH antagonist is used to modulate kisspeptin function. [0065] Even yet another aspect provides a method for suppressing estrogen-induced degradation of the thymic gland in a subject, the method comprising administering to the subject GnlH or an agonist thereof for a time and under conditions sufficient to reduce or suppress thymus degradation. [0066] Another aspect disclosed herein is the use of GnlH or its agonists or antagonists to modulate stem cell growth and maintenance in culture or to facilitate or control stem cell therapies.

[0067] GnlH or agonist or antagonist may be provided alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress- linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SER and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer. [0068] Reference to a "selective serotonin and/or noradrenergic re-uptake inhibitor" includes a selective serotonin re-uptake inhibitor (SSRI) and a selective serotonin and noradrenergic re-uptake inhibitor (SSNRI). Examples of SSRIs and SSNRIs include citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline as well as pharmacologically acceptable derivatives, analogs, homologs and formulated forms thereof.

[0069] Other serotonin inhibitors or serotonin or noradrenergic uptake inhibitors include 1 -

(2-methoxyphenyl)-4-(4-(2-phthalimido)butyl)piperazine; 2-((2- (dimethylamino)ethyl)thio)-3-phenylquinoline; 4-(2'-methoxyphenyl)-l-(2'-(N-(2"- pyridinyl)-4-iodobenzamido)ethyl)piperazine; 6-nitroquipazine; alosetron; amperozide; azasetron; bemesetron; binospirone mesylate; bufotenin; cianopramine; cinanserin; clovoxamine; clozapine; CQ 32085; cyanopindolol; cyproheptadine; deramciclane; dolasetron mesylate; dotarizine; DuP 34; EEDQ; fananserin; fenclonine; GR 113808; GR 127935; granisetron; iprazochrome; ketanserin; LY 53857; lysergic acid diethylamide;

McN 5652; MDL 100907; metergoline; methiothepin; methysergide; mianserin; N,N- dimethyltryptamine; nafronyl; ondansetron; palonosetron; pindolol; pipamperone; pizotyline; ramosetron; renzapride; risperidone; ritanserin; sarpogrelate; SB 204070A; SB

206553; SDZ 205-557; setoperone; SR 46349B; tolfenamic acid; tropisetron; UH 301 ; WAY 100135; WAY 100635; wortmannin; xylamidine; zacopride; zatosetron; ziprasidone; alaproclate; amoxapine; citalopram; clomipramine; duloxetine; femoxetine; fenfluramine; fluoxetine; fluvoxamine; indalpine; milnacipran; norfenfluramine; olanzapine; paroxetine; sertraline; trazodone; venlafaxine and zimeldine. [0070] Selective serotonin and noradrenergic re-uptake inhibitors include psychoactive drugs which antagonize various adrenergic and serotonin receptors and include esmirtazapine, mianserin, mirtazapine, setiptiline, such agents are also referred to as "tetracyclic anti-depressants". Other anti-depressants include tricyclic anti-depressants. Such agents include serotonin and/or norepinephrine re-uptake inhibitors including amitriptyline, amitriptylinoxide, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, dimetacrine, dosulepin/dothiepin, doxepin, imipramine, imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine, nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline, and quinupramine.

[0071] Naturally occurring synthetic modified, and steroidal and non-steroidal forms of estrogens are contemplated for use in accordance with the present disclosure. Examples include 2,3-bis(3'-hydroxybenzyl)butane-l ,4-diol; 2,3-bis(3'-hydroxybenzyl)butyrolactone;

4-octylphenol; 8-prenylnaringenin; biochanin; bisphenol A; chlorotrianisene; coumestrol; daidzein; dienestrol; diethylstilbestrol; diethylstilbestrol dipropionate; epimestrol; equol; estradiol; estrogenic steroids, alkylated; estrogens, conjugated (USP); estrogens, conjugated synthetic A; estrogens, conjugated synthetic B; estrogens, esterified (USP); estrone; ethinyl estradiol; formononetin; genistein; glycitein; hexestrol; infecundin; mestranol; ο,ρ'-DDT; polyestradiol phosphate; quinestrol; secoisolariciresinol; zearalenone and zeranol. Selective estrogen receptor modulators (SERMs) include: clomifene, femerelle, ormeloxifene, raloxifene, tamoxifen and toremifene or their functional homologs and equivalents.

[00721 Other agents contemplated herein include agents which target or act within the sympathetic nervous system or the monaminergic, catecholaminergic, glutametergic, gabaergic, noradrenergic/epinephriergic and opioidergic systems.

[0073] Reference to a "dopamine receptor antagonist" includes a partial antagonist and a drug or medicament comprising a full or partial dopamine receptor antagonist and all encompass an antagonist of the D2 and/or D3 receptor. By "antagonist" means an agent which reduces fully or partially dopamine neurotransmission. It is proposed herein that antagonism of dopamine neurotransmission reduces behaviors linked to reward stimuli. A dopamine receptor antagonist is consistent with an anti-psychotic drug which tends to antagonize both dopamine and serotonin. Examples of D2 and D2/D3 antagonists include first generation anti-psychotics (typical anti-psychotics) and second generation antipsychotics (atypical anti-psychotics). Pimozide is also a dopamine receptor antagonist but this drug is contraindicated with selective serotonin re-uptake inhibitors. Other dopamine receptor antagonists include amisulpride (D2/D3 antagonist), nemonapride, remoxipride (D2 antagonist), sultropride and triapride as well as pharmacologically acceptable derivatives, analogs, homologs, salts and formulated forms thereof and any typical or atypical anti-psychotics with full or partial dopamine antagonist actions.

[0074] Other dopamine antagonists include (5,6-dimethoxyindan-2-yl)dipropylamine; 1 -methyl- 1, 2,3 ,4-tetrahydroisoquinoline; l-methyl-4-(2'-methylphenyl)- 1 ,2,3,6- tetrahydropyridine; . 3-((4-(4-chlorophenyl)piperazin-l-yl)methyl)-lH-pyrrolo(2,3- b)pyridine; 3-iodo-2-hydroxy-6-methoxy-N-(( 1 -ethyl-2-pyrrolidinyl)methyl)benzamide; 5- methoxy-l-methyI-2-(n-propyIamino)tetralin; 8-iodo-2,3,4,5-tetrahydro-3-methyl-5- phenyl-lH-3-benzazepine-7-ol; acepromazine; amoxapine; azaperone; benperidol;

bromopride; butaclamol; ϋΙιΙοφΓοη^ίηε; ΛΙοφΓθΐΙΰχεηε; clopenfhixol; domperidone; droperidol; EEDQ; eticlopride; FLB 457; flupenthixol; flupenthixol decanoate; fluphenazine; fluspirilene; haloperidol; loxapine; mesoridazine; methotrimeprazine; metoclopramide; nafadotride; nemonapride; penfluridol; perazine; peφhenazine; pimozide; ρΓθϋη1θ εΓ3ζίηβ; promazine; raclopride; remoxipride; risperidone; Sch 39166; SK^A&F 83566; spiperone; spiroxatrine; stepholidine; sulpiride; sultopride; tetrahydropalmatine; thiethylperazine; thioridazine; thiothixene; tiapride; trifluoperazine; trifluperidol; triflupromazine; UH 232; ziprasidone; dopamine uptake inhibitors may also be used which include (lR-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; l-(2-(diphenylmethoxy)ethyl)-4- (3-phenyl-2-propenyl)piperazine; amphetamine; benzphetamine; benztropine; bupropion; cocaethylene; cocaine; dextroamphetamine; duloxetine; mazindol; methamphetamine; methylphenidate; nomifensine; and vanoxerine.

[0075] Reference to mood stabilizers includes inter alia antimania, anticonvulsants, anti- psychotics and antihypertensives. Examples include lithium carbonate, divalproex sodium, tiagabine, levetiracetam, lamotrigine, gabapentin, carbamazepine, oxcarbazepine, topiramate, zonisamide, olanzapine, verapamil, clonidine, propranolol, mexiletine and guanfacine. [0076] GnlH or its antagonist may also be used to alter a mood. [0077] The drugs may be administered singularly or co-administered simultaneously or sequentially. Hence, the drugs may be provided in a single formulation or in a multipart form in which the contents are admixed prior to the administration. Where sequential administration occurs, the drugs may be provided within nanoseconds, milliseconds, seconds, a minute or minutes, hours or days apart. The present disclosure teaches a therapeutic or medicament kit comprising a GnlH agonist or antagonist alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (iii) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an antidepressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer. GnlH itself is also regarded herein as a GnlH agonist. A GnRH agonist encompasses a GnlH antagonist.

[0078] The formulations may also be in a form requiring reconstitution or admixing with a pharmaceutically acceptable carrier, diluent or excipient prior to use.

[0079] The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. [0080] Pharmaceutical compositions enabled herein for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0081] The drugs described herein may be administered orally, parenterally (including by subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intraperitoneial injection or infusion techniques), topically such as via ophthalmic and mucus membranes, by inhalation, intranasal spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, excipients, diluents and/or other vehicles. [0082] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, .lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

[0083] When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

[0084] Intravenous administration includes both bolus and infusion when administered by injection, the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

[0085] When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

[0086] The effective dosage of the agents employed in therapy may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Thus, the dosage regimen utilizing the compounds herein described is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or clinician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on th& kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

c

[0087] Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual agents, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or six monthly or as required Persons of ordinary skill in the art can readily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the drugs are administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, weekly, monthly, six monthly or as required. Variations include daily, twice daily, thrice daily, multiple weekly and dosages may be given in International units such as from 1 to 1000 International units.

[0088] The pharmaceutical formulations described herein, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general* the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0089] The compositions taught herein may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0090] The drugs proposed to be used in accordance with the present disclosure may also be referred to as medicaments, agents, therapeutics, actives, compounds and the like. Reference to a "medicament", "agent", "therapeutic", "active" and "compound" includes a single drug or a combination of two or more drugs. The drugs may also be referred to as neuTopsychopharmacological agents. This term is not to exclude agents which modulate signaling targets within the reproductive-linked biological system such as estrogen. Such signaling targets or other agents include inter alia follicle stimulatory hormone (FSH), luteinizing hormone (LH), dehydroplandrosterone (5-DHEA), prolactin (PRL) [also known as leuteotropichormone (LTH)], progesterone, gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin (HCG) and catecholamine.

[0091] The subject contemplated herein is generally a human subject and may also be referred to as a patient, individual or recipient. The human subject may be an infant, child, adolescent, teenager, young adult, adult or elderly adult of male or female gender. Notwithstanding, the present disclosure extends to the use of the drug combinations in a variety of small to large animal models such as mice, rats, guinea pigs, hamsters, cats, dogs, pigs, sheep, cattle, horses, camels, monkeys and other non-human primates. A suitable animal model includes the Forced Swim Test (or Portsolt Test). Such an animal model is particularly useful for testing agents or combinations of agents in acute and chronic therapies.

[0092] The present disclosure further provides for the use of a GnlH agonist or antagonist alone or in combination with a drug selected from (i) an agent which modulates a downstream signaling target within a stress-linked biological system; (ii) an agent which stimulates downstream signaling of a reproductive-linked biological system; (ii i) an estrogen or an estrogen analog or agonist or SERM and/or an agent which induces estrogen production; (iv) a selective serotonin and/or noradrenergic re-uptake inhibitor and/or an anti-depressant; (v) a dopamine receptor antagonist; and/or (vi) a mood stabilizer in the manufacture of a medicament for the treatment or prophylaxis of a pathophysiological condition.

[0093] By "treatment" includes aspects of prevention (prophylaxis) such as in subjects at risk of developing a pathophysiological condition. Such as genetically or environmentally predisposed individuals. [0094] The present disclosure further contemplates the use of GnlH itself as a diagnostic indicator for a range of pathophysiological conditions. For example, treatments may be monitored for the effectiveness of GnlH agonists or antagonists. GnlH inhibits hormones such as FSH, LH and downstream effectors such as estrogen and progesterone. In an example, GnlH levels will have an effect on estrogen-induced epithelial ovarian cancer. Monitoring localized or systemic levels of GnlH in cancer patients is proposed to be useful in monitoring for disease progression.

[0095] Diagnostic assays . may be protein-based such as using antibodies or a chromatographic procedure and/or nucleic acid based such as measuring mRNA expression levels.

[0096] Aspects of the present disclosure provide numerical values in various ranges. Slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, these ranges are intended as a continuous range including every value between the minimum and maximum values. In addition, the present disclosure extends to ratios of two or more markers providing a numerical value associated with a state of pathophysiological condition. [0097] The present disclosure further provides an assay for the identification and monitoring of a pathophysiological condition and to determine pharmacoefficacy of medicaments. In an embodiment, the assay enables early detection or monitoring of ovarian cancer. Notwithstanding, the present disclosure is not limited to just the early detection of ovarian cancer since the assay may be used at any stage of a gynecological disease hormone related condition, or its treatment or any complication arising therefrom as well as a range of other conditions such as menopause, puberty and psychological conditions such^' as anxiety, depression, schizophrenia stress and reproductive state.

[0098] Reference to a "cancer" with respect to a "gynecological condition" includes ovarian cancer as well as a sub-type of ovarian cancer such as mucinous or endometrial ovarian cancer or a stage of ovarian cancer such as stage I, II, III or IV. Terms such as "ovarian cancer", "epithelial ovarian cancer" and an "ovarian malignancy" may be used interchangeably herein. _, The assay described herein is particularly useful when applied to the diagnosis of symptomatic women, but may equally be applied to the diagnosis of asymptomatic women and/or women at high risk of developing a gynecological condition. [0099] A range of biomarkers of pathophysiological conditions or states are taught herein. The biomarkers include the levels of activity of GnlH alone or in combination with a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer. Collectively, these are referred as "biomarkers" or "pathophysiological condition markers" or "markers of a pathophysiological condition".

[0100] Reference to any one or more of the^' biomarkers includes a modified or homolog form thereof. A modified form includes a derivative, polymorphic variant, truncated form (truncate) and aggregated or multimeric forms or forms having expansion elements (e.g. amino acid expansion elements). For brevity, such modified and homolog forms are included by reference to any or some or all of the biomarkers.

[0101] Accordingly, an assay is taught herein for determining the presence or state of a pathophysiological condition in a subject, the assay comprising . determining the concentration or activity of biomarkers in a biological sample from the subject selected from GnlH alone or in combination with a biomarker selected a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer wherein an alteration in the levels or activity of the biomarkers relative to a control provides an indication of the presence or state of the pathophysiological condition.

[0102] In an alternative embodiment, an assay is enabled for determining the presence or state of a pathophysiological condition in a subject, the assay comprising determining the concentration or activity of biomarkers in a biological sample from the subject selected- from GnlH alone or in combination with a biomarker selected from a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine and/or or a dopamine receptor pathway; subjecting the levels to an algorithm generated from a first knowledge base of data comprising the levels of the same biomarkers from a subject of known status with respect to the condition wherein the algorithm provides an index of probability of the subject having or not having the condition or having a particular state of the condition. Reference to the "algorithm" is an algorithm which performs a multivariate or univariate analysis function. [0103] In an embodiment, the present disclosure contemplates an assay for determining the presence of a pathophysiological condition in a subject, the assay comprising determining the concentration or activity of GnlH in a biological sample from the subject alone or in combination ^"with a biomarker selected from a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer wherein an altered concentration or activity in the biomarker is indicative of the subject having a pathophysiological condition. [0104] The first knowledge base of data may also come from multiple subjects.

[0105] An "altered" level means an increase or elevation or a decrease or reduction in the concentrations or activities of the biomarker. [0106] The determination of the concentrations or levels of the biomarkers enables establishment of a diagnostic rule based on the concentrations relative to controls. Alternatively, the diagnostic rule is based on the application of a statistical and machine learning algorithm. Such an algorithm uses relationships between biomarkers and disease status observed in training data (with known pathophysiological status) to infer relationships which are then used to predict the status of patients with unknown status. An algorithm is employed which provides an index of probability that a patient has a particular pathophysiological condition or state. The algorithm performs a multivariate or univariate analysis function.

[0107] Hence, in an embodiment, the a diagnostic rule is based on the application of statistical and machine learning algorithms. Such an algorithm uses the relationships between biomarkers and disease status observed in training data (with known disease status) to infer relationships which are then used to predict the status of patients with unknown status. Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the subject assay.

[0108] The present disclosure contemplates the use of a knowledge base of training data comprising levels of biomarkers from a subject with a pathophysiological condition or state to generate an algorithm which, upon input of a second knowledge base of data comprising levels of the same biomarkers from a patient with an unknown state of a pathophysiological condition, provides an index of probability that predicts the nature of the pathophysiological condition.

[0109J The term "training data" includes knowledge of levels of biomarkers relative to a control. A "control" includes a comparison to levels of biomarkers in a subject devoid of the gynecological condition or cured of the condition or may be a statistically determined level based on trials. The term "levels" also encompasses ratios of levels of biomarkers.

[0110] The "training data" also include the concentration or activity of GnlH alone or in combination with one or more biomarkers selected from a component within (i) a stress- linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer. The data may comprise information on an increase or decrease in the biomarker(s). [0111] A panel of biomarkers is taught herein for the detection of a pathophysiological condition or state in a subject, the panel comprising agents which detect biomarkers selected from GnlH alone or in combination with a biomarker selected from a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer which determine levels of one or more biomarkers and then subjecting the levels to an algorithm generated from a first knowledge base of data comprising the levels of the same biomarkers from a subject of known status with respect to the condition wherein the algorithm provides an index of probability of the subject having or not having the condition. [0112] The levels or concentrations of the biomarkers provide the input test data referred to herein as a "second knowledge base of data". The second knowledge base of data either is considered relative to a control or is fed into an algorithm generated by a "first knowledge base of data" which comprises information of the levels of biomarkers in a subject with a known pathophysiological condition. The second knowledge base of data is from a subject of unknown status with respect to a condition. The output of the algorithm is a probability or risk factor, referred to herein as an index of probability, of a subject having a particular pathophysiological condition or state or not having the condition or state. [0113] The agents which "specifically bind" to the biomarkers generally include an immunointeractive molecule such as an antibody or hybrid, derivative including a recombinant or modified form thereof or an antigen-binding fragment thereof. The agents may also be a receptor or other ligand. These agents assist in determining the level of the biomarkers. Information on the level is input data for the algorithm.

[0114] Hence, the present disclosure describes a panel of immobilized ligands to GnlH alone or in combination with ligands to a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer.

[0115] Another aspect described herein is directed to a kit for diagnosing the presence or absence of a pathophysiological condition, the kit comprising a composition of matter comprising ligands or other diagnostic agents for GnlH alone or one or more of a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer wherein the kit further comprising reagents to facilitate determination of the concentration or activity of biomarker binding to a ligand. In use, the kit facilitates the determination of the biomarkers. The levels are then compared to a control or subjected to an algorithm generated from a first knowledge base of data comprising the levels of the same biomarkers from a subject of known status with respect to the condition wherein the algorithm provides an index of probability of the subject having or not having the condition.

[0116] The ligands, such as antibodies specific to each of the biomarkers, enable the quantitative or qualitative detection or determination of the level of the at least two or more biomarkers. Reference to "level" includes concentration as weight per volume, activity per volume or units per volume or other convenient representative as well as ratios of levels.

[0117] The "sample" is generally blood, plasma or serum, ascites, lymph fluid, tissue exudate, mucus, urine or respiratory fluid, reproductive fluid, placental or ovary fluid or tissue extract. Alternatively, the sample is a tissue sample which is being histologically examined.

[0118] As indicated above, the "ligand" or "binding agent" and like terms, refers to any compound, composition or molecule capable of specifically or substantially specifically (that is with limited cross-reactivity) binding to an epitope on the biomarker. The "binding agent" generally has a single specificity. Notwithstanding, binding agents having multiple specificities for two or more biomarkers are also contemplated herein. The binding agents (or ligands) are typically antibodies, such as monoclonal antibodies, or derivatives or analogs thereof, but also include, without limitation: Fv fragments; single chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments; humanized antibodies and antibody fragments; camelized antibodies and antibody fragments;^' and multivalent versions of the foregoing. Multivalent binding reagents also may be used, as appropriate, including without limitation: monospecific or bispecific antibodies; such as disulfide stabilized Fv fragments, scFv tandems [(scFv)₂ fragments], diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e. leucine zipper or helix stabilized) scFv fragments. "Binding agents" also include aptamers, as are described in the art.

[0119] Methods of making antigen-specific binding agents, including antibodies and their derivatives and analogs and aptamers, are well-known in the art. Polyclonal antibodies can be generated by immunization of an animal. Monoclonal antibodies can be prepared according to standard (hybridoma) methodology. Antibody derivatives and analogs, including humanized antibodies can be prepared recombinantly by isolating a DNA fragment from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technology is described in the literature and permit in vitro clonal amplification of antigen-specific binding reagents with very affinity low cross-reactivity. Phage display reagents and systems are available commercially, and include the Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida. Aptamer technology is described for example and without limitation in US Patent Nos. 5,270,163; 5,475,096; 5,840,867 and 6,544,776.

[0120] ECLIA, ELISA and Luminex LabMAP immunoassays are examples of suitable assays to detect levels of the biomarkers. In one example a first binding reagent antibody is attached to a surface and a second binding reagent/antibody comprising a detectable group binds to the first antibody. Examples of detectable-groups include, for example and without limitation: fluorochromes, enzymes, epitopes for binding a second binding reagent (for example, when the second binding reagent/antibody is a mouse antibody, which is detected by a fluorescently-labeled anti-mouse antibody), for example an antigen or a member of a binding pair, such as biotin. The surface may be a planar surface, such as in the case of a typical grid-type array (for example, but without limitation, 96-welI plates and planar microarrays) or a non-planar surface, as with coated bead array technologies, where each "species" of bead is labeled with, for example, a fluorochrome (such as the Luminex technology described in U. S. Patent Nos. 6,599, 331,6, 592,822 and 6,268, 222), or quantum dot technology (for example, as described in U. S. Patent No. 6,306. 610). Such assays may also be regarded as laboratory information management systems (LIMS):

[0121) In the bead-type immunoassays, the Luminex LabMAP system can be utilized. The LabMAP system incorporates polystyrene microspheres that are dyed internally with two spectrally distin'ct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of 1 OOHifferent microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface. Microspheriss are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.

[0122] As used herein, "immunoassay" refers to immune assays, typically, but not exclusively sandwich assays, capable of detecting and quantifying a desired biomarker, namely one or more of GnlH and a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and or (vi) a mood stabilizer.

[0123] Data generated from an assay to determine fluid or tissue levels of the biomarkers can be used to determine the likelihood of or progression of a pathophysiological condition in the subject. The input of data comprising the levels or activity of the biomarker(s) is compared with a control or is put into the algorithm which provides a risk value of the likelihood that the subject has, for example, ovarian cancer or a psychological illness. A treatment regime can also be monitored as well as a likelihood of a relapse. 0

- 36 -

[0124] In context of the present disclosure, "fluid" includes any blood fraction, for example serum or plasma, that can be analyzed according to the methods described herein. By measuring blood levels of a particular biomarker, it is meant that any appropriate blood fraction can be tested to determine blood levels and that data can be reported as a value present in that fraction. Other fluids contemplated herein include ascites, tissue exudate, urine, lymph fluid, mucus and respiratory fluid, placental fluid and ovary fluid or tissue biopsy. [0125] As described above, methods for diagnosing a pathophysiological condition by determining levels of specific identified biomarker(s) and using these levels as second knowledge base data in an algorithm generated with first knowledge base data or levels of the same biomarkers in patents with a known disease. Also provided are methods of detecting pre-symptomatic or pre-clinical conditions comprising determining the presence and/or velocity of specific identified biomarkers in a subject's sample. By "velocity" it is meant the change in the concentration of the biomarker in a patient's sample over time.

[0126] As indicated above, a gynecological condition includes cancer or a compilation thereof. The term "cancer" as used herein includes all cancers generally encompassed by a "gynecological cancer". In one embodiment, a gynecological cancer, including, but not limited to, tubal metaplasia, ovarian serous borderline neoplasms, serous adenocarcinomas, low-grade mucinous neoplasms and endometrial tumors. In a specific embodiment, the gynecological cancer is an ovarian neoplasm, undergoing aberrant Mullerian epithelial differentiation. Other gynecological conditions contemplated herein include inflammatory disorders such as endometriosis.

[0127] The term "sample" as used in the context of cancer means any sample containing cancer cells- that one wishes to detect including, but not limited to, biological fluids (including blood, plasma, serum, ascites), tissue extracts, freshly harvested cells, and lysates of cells which have been incubated in cell cultures. In a particular embodiment, the sample is gynecological tissue, blood, serum, plasma or ascites. - 3.7 -

[0128] The term "control sample" includes any sample that can be used to establish a first knowledge base of data from subjects with a known disease status.

[0129] The method described herein is used in the diagnosis and staging of a pathophysiological condition such as a gynecological cancer including ovarian cancer or a psychological illness. The present assay may also be used to monitor the progression of a condition and to monitor whether a particular treatment is effective or not. In particular, the method can be used to confirm the absence or amelioration of the symptoms of the condition such as following surgery, chemotherapy, and/or radiation therapy. The methods can further be used to monitor chemotherapy and aberrant tissue reappearance. Behavior modification may also be required for psychological conditions.

[0130] In an embodiment, a method is taught for monitoring the progression of a pathophysiological condition in a patient, comprising:

(a) providing a sample from a patient;

(b) determining the level of GnlH alone or in combination with one ore more of a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; and/or (v) dopamine or a dopamine receptor pathway; and/or (vi) mood stabilizer and subjecting the levels to an algorithm to provide an index of probability of the patient having a pathophysiological condition; and

(c) repeating steps (a) and (b) at a later point in time and comparing the result of step (b) with the result of step (c) wherein a difference in the index of probability is indicative of the progression of the condition in the patient.

[0131] In particular, an increased index of probability of a disease condition at the later time point may indicate that the condition is progressing and that the treatment (if applicable) is not being effective. In contrast, a decreased index of probability at the later time point may indicate that the condition is regressing and that the treatment (if applicable) is effective.

[0132] As indicated above, antibodies may be used in any of a number of immunoassays which rely on the binding interaction between an antigenic determinant of the biomarker and the antibodies. Examples of such assays are radioimmunoassay, enzyme immunoassays (e.g. ECLIA, ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. The antibodies may be used to detect and quantify the level of the biomarker in a sample in order to determine its role in the pathophysiological condition and to diagnose the pathophysiological condition.

[0133] In particular, the antibodies described herein may also be used in immunohistochemical analyses, for example, at the cellular and subcellular level, to detect a biomarker, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.

(0134] Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect the biomarker. Generally, an antibody of the present disclosure may be labeled with a detectable substance and a biomarker protein may be localized in tissues and cells based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following : radioisotopes (e.g. ³H, ¹⁴C ³⁵S, ^l25I, ¹³ Ί), fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g. horseradish peroxidase, beta-ga}actosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g. streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains; epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy. [0135] The antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip) Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against biomarker protein. By way of example, if the antibody having specificity against biomarker protein is a rabbit IgG antibody, the second antibody may be goat anti- rabbit gamma-globulin labeled with a detectable substance as described herein.

[0136] Where a radioactive label is used as a detectable substance, the biomarker may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.

[0137] Labeled antibodies against biomarker proteins may be used in locating tumor tissue in patients undergoing surgery i.e. in imaging. Typically for in vivo applications, antibodies are labeled with radioactive labels (e.g. iodine-123, iodine-125, iodine-131 , gallium-67, technetium-99, and indium-I l l). Labeled antibody preparations may be administered to a patient intravenously in an appropriate carrier at a time several hours to four days before the tissue is imaged. During this period unbound fractions are cleared from the patient and the only remaining antibodies are those associated with tumor tissue. The presence of the isotope is detected using a suitable gamma camera. The labeled tissue can be correlated with known markers on the patient's body to pinpoint the location of the , tumor for the clinician. [0138] The methods described herein may also be performed using microarrays, such as oligonucleotide arrays, cDNA arrays, genomic DNA arrays, or tissue arrays.

[0139] In an embodiment, the subject method involves the detection of expression of nucleic acid molecules encoding the biomarkers and to determine the level of biomarkers based on level of expression. Those skilled in the art can construct nucleotide probes for use in the detection of mRNA sequences encoding the biomarker in samples. Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least five sequential amino acids from regions of the biomarker, preferably they comprise 15 to 30 nucleotides. A nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as ³²P, ³H, ^C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, Molecular Cloning, A Laboratory Manual. (2nd ed), 1989. The nucleic acid probes may be used to detect genes, preferably in human cells, that encode the biomarker. The nucleotide probes may also be useful in the diagnosis of disorders involving a biomarker, in monitoring the progression of such disorders, or in monitoring a therapeutic treatment. In an embodiment, the probes are used in the diagnosis of, and in monitoring the progression of a gynecological cancer such as ovarian cancer.

[0140] The probe may be used in hybridization techniques to detect expression of genes that encode biomarker proteins. The technique generally involves contacting and incubating nucleic acids (e.g. mRNA) obtained from a sample from a patient or other cellular source with a probe under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non- annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.

[0141] The detection of mRNA may involve converting the mRNA to cDNA and/or the amplification of specific gene sequences using an amplification method such as polymerase chain reaction (PCR), followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable primers can be routinely designed by one of skill in the art.

[0142] Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of expression of genes encoding the biomarker. For example, RNA may be isolated from a cell type or tissue known to express a gene encoding the biomarker, and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques referred to herein. The techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing. The techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcripts detected in normal individuals relative to those individuals exhibiting symptoms of a cancer involving a biomarker protein or gene. [0143] The primers and probes may be used in the above described methods in situ i.e. directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.

[0144] Accordingly, a method is enabled herein for detecting a pathophysiological condition in a patient comprising:

(a) providing a sample from the patient;

(b) extracting nucleic acid molecules comprising mRNA from a biomarker gene or portion thereof from the sample;

(c) amplifying the extracted mRNA using the polymerase chain reaction;

(d) determining the level of mRNA encoding the biomarker; and

(e) subjecting the levels of two or more biomarkers to an algorithm which provides an index of probability of the patient having cancer.

[0145] The methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising the necessary reagents to perform any of the methods of the disclosure. For example, the kits may include at least one specific nucleic acid or antibody described herein, which may be conveniently used, e.g, in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing cancer. The kits may also include nucleic acid primers for amplifying nucleic, acids encoding the biomarker in the polymerase chain reaction. The kits can also include nucleotides, enzymes and buffers useful in the method of the disclosure as well as electrophoretic markers such as a 200 bp ladder. The kit also includes detailed instructions for carrying out the methods of the present disclosure.

[0146] An algorithm-based screening assay is taught herein to screen samples from patients. Generally, input data are collected based on levels of two or more biomarkers (or levels of expression of genes encoding two or more biomarkers) and subjected to an algorithm to assess the statistical significance of any elevation or reduction in levels which information is then output data. Computer software and hardware for assessing input data are encompassed by the present disclosure. [0147] Another aspect taught herein is a method of treating a patient with a pathophysiological condition such as ovarian cancer or a psychological illness the method comprising subjecting the patient to a diagnostic assay to determine an index of probability of the patient having the condition, the biomarkers selected from GnlH alone or with one or more of a component within (i) a stress-linked biological system; (ii) a reproductive- linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer and where there is a risk of the patient having the condition, subjecting the patient to a treatment or behavioral change; and then monitoring index of probability over time.

'

[0148] The assay described herein permits integration into existing or newly developed pathology architecture or platform systems. For example, the method described herein allows a user to determine the status of a subject with respect to a pathophysiological condition, the method including:

(a) receiving data in the form of levels or concentrations of GnlH alone or in combination with one or more of a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway, and/or (vi) a mood stabilizer; from the user via a communications network;

(b) processing the subject data via an algorithm which provides a disease index value; (c) · determining the status of the subject in accordance with the results of the disease index value in comparison with predetermined values; and

(d) transferring an indication of the status of the subject to the user via the communications network.

[0149] Conveniently, the method generally further includes:

(a) having the user determine the data using a remote end station; and

(b) transferring the data from the end station to the base station via the communications network.

[0150] The base station can include first and second processing systems, in which case the method can include:

(a) transferring the data to the first processing system;

(b) transferring the data to the second processing system; and

(c) causing the first processing system to perform the algorithmic function to generate the disease index value.

[0151] The method may also include:

(a) transferring the results of the algorithmic function to the first processing system; and

(b) causing the first processing system to determine the status of the subject.

[0152] In this case, the method also includes at lest one of:

(a) transferring the data between the communications network and the first processing system through a first firewall; and

(b) transferring the data between the first and the second processing systems through a second firewall.

[0153] The second processing system may be coupled to a database adapted to store predetermined data and/or the algorithm, the method include:

(a) querying the database to obtain at least selected predetermined data or access to the algorithm from the database; and (b) comparing the selected predetermined data to the subject data or generating a predicted probability index.

[0154] The second processing system can be coupled to a database, the method including storing the data in the database.

[0155] The method can also include having the user determine the data using a secure array, the secure array of elements capable of determining the level of biomarker and having a number of features each located at respective position(s) on the respective code. In this case, the method typically includes causing the base station to:

(a) determine the code from the data;

(b) determine a layout indicating the position of each feature on the array; and

(c) determine the parameter values in accordance with the determined layout, and the data.

[0156] The method can also include causing the base station to:

(a) determine payment information, the payment information representing the provision of payment by the user; and

(b) perform the comparison in response to the determination of the payment information.

[0157] The present assay also enables a base station for determining the status of a subject with respect to a pathophysiological condition, the base station including:

(a) a store method;

(b) a processing system, the processing system being adapted to:

(i) receive subject data from the user via a communications network, the data including levels or concentrations of GnlH alone or in combination with one or more of a component within (i) a stress-linked biological system; (ii) a reproductive-linked biological system; (iii) estrogen or estrogen receptor pathway or SERM; (iv) a serotonin and/or noradrenergic pathway; (v) dopamine or a dopamine receptor pathway; and/or (vi) a mood stabilizer from a subject;

(ii) performing an algorithmic function including comparing the data to predetermined data;

(iii) determining the status of the subject in accordance with the results of the algorithmic function including the comparison; and

(c) output an indication of the status of the subject to the user via the communications network.

[0158J The processing system can be adapted to receive data from a remote end station adapted to determine the data. [0159] The processing system may include:

(a) a first processing system adapted to:

(i) receive the data; and

(ii) determine the status of the subject in accordance with the results of the algorithmic function including comparing- the data; and

(b) a second processing system adapted to:

(i) receive the data from the processing system;

(ii) perform the algorithmic function including the comparison; and

(iii) transfer the results to the first processing system. [0160] The base station typically includes: .

(a) a first firewall for coupling the first processing system to the communications network; and

(b) a second firewall for coupling the first and the second processing systems. [0161] The processing system can be coupled to a database, the processing system being adapted to store the data in the database.

[0162] Reference to an "algorithm" or "algorithmic functions" as outlined above includes the performance of a multivariate analysis function. A range of different architectures and platforms may be implemented in addition to those described above. It will be appreciated that any form of architecture suitable for implementing the present disclosure may be used. However, one beneficial technique is the use of distributed architectures. [0163] It will also be appreciated that in one example, the end stations can be hand-held devices, such as PDAs, mobile phones, or the like, which are capable of transferring the subject data to the base station via a communications network such as the Internet, and receiving the reports.

[0164] In the above aspects, the term "data" means the levels or concentrations of the biomarkers. The "communications network" includes the internet. When a server is used, it is generally a client server or more particularly a simple object application protocol (SOAP).

[0165] Aspects taught herein are further described by the following non-limiting Examples. These Examples take the form of an empirical review of the literature culminating in the selection of a drug or a combination of drugs proposed to be useful in accordance with the practice of the methods and assays herein described.

EXAMPLE 1

Role of GnlH and GnlH agonists in the treatment of estrogen-induced epithelial cancer

Epidemiology and Etiology

[0166] Ovarian carcinoma (OCa) is the leading cause of death due to gynecologic malignancies. The vast majority of OCa (85%) is derived from the ovarian surface epithelium (OSE) and its cystic derivatives (Leung and Choi, Human Reproduction Update 73^:143-162, 2007), The remainder of OCa is derived from granulosa cells or, rarely, stroma or germ cells (Leung and Choi, 2007 supra).

. ,

[0167] With respect to a link between GnlH and ovarian cancer, the gonadotrophin hypothesis proposes that risk for the illness is increased by increased levels of pituitary ganadotropins (LH and FSH) which stimulate the surface epithelium of the ovary. Further to this contention, evidence strongly suggests that steroid hormones, primarily estrogens and progesterone, are implicated in ovarian carcinogenesis. Furthermore, with respect to the hormone-dependent mechanisms at the site of cancerous tumor, on the ovaries of chickens, it has been demonstrated that there are GnlH, estrogen and progesterin, FSH and LH receptors (Bentley et al, 2007 supra). [0168] Support for a hormone-dependent mechanism in OCa progression is stipulated by the link between contraceptive use and development of OCa. For example, the sequential use of estrogen followed by progestin results in an increased risk for the development of OCa, whereas contraception containing both estrogen and progestin used continuously confers no increased risk to OCa. Further evidence for a role for a cancer-inducing role for estrogen comes from data that estrogen rich hormone replacement therapeutic (HRT) preparations are linked to the development of OCa (The Lancet, 24 January, 2008). Taken together, indicate it is proposed here that hormonal balance is important with respect to illness development and that progesterone preparations need to be present at the same time or before estrogen, to prevent or protect from estrogen-induced damage that confers risk to OCa development. Estrogen is carcinogenic and progesterone is protective

[0169] Validation of a role for estrogen linked to OSE cancer progression is provided by recent convincing data showing that whilst estrogens favor neoplastic transformation of the OSE, progesterone offers protection against OCa development (Ho, Reproductive Biology and Endocrinology 1 (73), 2003). The potentially^" carcinogenic involvement of estrogen and conversely protective influence of progesterone underscores the utility of GnlH in OCa treatment due to its apparent selectivity for LH suppression, with little influence over FSH secretion. [0170) With respect to estrogen and OCa, estrogens, particularly those present in ovulatory follicles, are both genotoxic and mitogenic to OSE cells (Ho, 2003 supra). In contrast, pregnancy-equivalent levels of progesterone are highly effective as apoptosis inducers for OSE and OCa cells (Ho, 2003 supra). In this regard, high-dose progestin has been suggested to exert an exfoliation effect and rid an aged OSE of pre-maligriant cells. A limited number of clinical studies have demonstrated efficacies of aromatase inhibitors (which prevent the conversion of androgen to estrogen), progestins alone or in combination with chemotherapeutic drugs, as well as the use of anti-estrogens in the treatment of OCa. It is proposed here that there is a protective role for progesterone against cancer development and that estrogen-induced proliferation of cells which may be carcinogenic.

[0171] In patients, levels of oestrogen receptor (ER) and progesterone receptor (PgR) in OCa tissue have been examined with regard to their prognostic importance for survival (Sevelda et al, British Journal of Obstertrics and Gynaecology 706-712, 2008). In 179 patients diagnosed with primary epithelial ovarian cancer stage III or IV, response to postoperative chemotherapy was significantly correlated with progesterone receptor content (80% responders in the group with progesterone receptor positive tumors and only 61% responders in the group with progesterone negative tumors) suggesting a protective role for patients with increased progesterone levels, such that among protective mechanisms progesterone receptor content was an independent prognostic factor for survival in advanced epithelial ovarian carcinoma. [0172] In particular, within this group of with post-operative residual tumor mass less than or equal to 2cm, those who were progesterone positive had a 2-year survival probability of 83% compared with only 51 % in the progesterone-negative group. It is proposed here that this provides clinical evidence of the importance or the protective value of progesterone in patients with epithelial OCa. In terms of a link with GnlH, it inhibits LH and, hence, estrogen function, with little influence over FSH.

Treatment of ovarian epithelial cancer via GnRH antagonists and superagonists

[01731 Indirect validation for a role of GnlH in anti-cancerous application is via efficacy of the antagonist of the opposing reproductive hormone, GnRH. There have been identified 2 subtypes of GnRH peptides, ih human malignant tumors (Chien et al, International Journal of Gynecological Cancer, 2004). It has been demonstrated that the expression of GnRH (GnRH-I, LHRH) (luteinizing hormone releasing hormone) and its receptor as a part of an autocrine (self-regulatory) system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the ovary (Chien et al, International Journal of Gynecological Cancer, 2004). Several GnRH analogs have been used for the treatment of breast, prostate and ovarian cancers (OCa) (Chien et al, International Journal of Gynecological Cancer, 2004). The proliferation of human OCa cell lines is time- and dose-dependently reduced by superagonistic analogs of GnRH, however, the classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of anti-proliferative effects of GnRH analogs in these cancer cells (Grundker and Emons, Reproductive Biological Endocrinology 65, 2003). The GnRH receptor interacts with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in downregulation of cancer cell proliferation. Furthermore GnRH induces activation of the c-Jun N-terminal kinase/activator protein- 1 (JNK/AP-1) pathway independent of the known AP-1 activators, protein kinase C (PKC) or mitogen activated protein kinase (MAP /ERK). In addition GnRH activates nuclear factor kappa-light chain beta (NF- β) and protects the cancer cells from apoptosis. These findings add support for the potential importance for apoptotic properties of GnlH on cancers induced by GnRH activity. [0174] It has been shown that human OCa cells express a putative second GnRH receptor specific for GnRH type II (GnRH-II). The proliferation of these cells is dose- and time- dependently reduced by GnRH-II in a greater extent than by GnRH-I (GnRH, LHRH) superagonists. It has also been demonstrated that in OCa cell lines except for the EFO-27 cell line GnRH-I antagonist, Cetrorelix, has comparable antiproliferative effects as GnRH- I agonists indicating that the dichotomy of GnRH-I agonists and antagonists might not apply to the GnRH-I system in cancer cells. It is proposed herein to also be the case for GnlH. After GnRH-I receptor knock down the antiproliferative effects of GnRH-I agonist, Triptorelin, were abrogated while the effects of GnRH-I antagonist Cetrorelix and GnRH- II were still existing. In addition, in the OCa cell line EFO-27, GnRH-I receptor but not putative GnRH-II receptor expression was found. These data are proposed herein to indicate that in OCa cells, the antiproliferative effects of GnRH-I antagonist, Cetrorelix, and GnRH-II are not mediated through the GnRH-I receptor. This is important to investigate in terms of the role of GnlH in a receptor specific manner.

Rationale for treatment

[01751 GnlH upregulation is postulated to have an arresting effect on cancerous epithelial ovarian cells via the eventual suppression of GnRH and reduction in estrogen production at the ovaries. The proposed mechanism of action, therefore, is proliferation induced arrest via GnlH activation including agonism or super-antagonism at the ovaries. In addition, OCa is linked to the development of brain mestases, hence, in addition to targeted treatment at the ovarian level and central level (hypothalamus) will prevent potential malignancy and disease progression. [0176] Hence, it is proposed herein to upregulate GnlH in targeting the pathophysiological development of OCa. The utility of application of potential therapeutics in a double-arrest manner by arresting both central (hypothalamic) as well as peripheral (ovarian) mediators of the sex-steroid hormones involved in maintenance of the tumor and development of secondary metastases. Unlike site specific laser treatment proposed by other radiotherapy based treatments, a further consideration of this approach is that it may have reversible effects on reproductive functioning and, therefore, conserve the capacity to reproduce. [0177] A GnlH agonist nasal spray or an oral pill (to cause tonic enhancement of GnlH) and/or tumor-site specific GnlH slow-release agonist is proposed as two forms of treatment. EXAMPLE 2

Reciprocal interaction between GnlH and the HPA-axis, and mediation by the sympathoadrenal system: Implications for illness related to stress and reproduction

Link between Stress, Reproduction and the sympathoadrenal system

[0178] Stress is a physiological mechanism responsible for integrating diverse processes including both physiological and behavioral responses to situations rendering a challenge to an organism's state of homeostasis. Under conditions of chronic stress for example, reproduction has been shown to be compromised. Despite this, a link between hormones of the reproductive axis (GnlH) and stress hormones of both the HPA*axis and sympathoadrenal system have not yet been demonstrated.

[0179] Studies have shown a link between rat GnlH homolog and peripheral secretion of numerous hormones, including stress hormone, Cortisol. For example, it has been demonstrated that central administration of RFRP-3 (avian homolog of GnlH) in the rat significantly reduces plasma levels of LH but increased growth hormone regardless of the time of day. At no time, however, did RFRP-3 alter plasma levels of FSH, thyroid hormone, pr Cortisol (Johnson et al, Hormones and Behavior, 2007). In particular, this later finding may infer that under homeostatic conditions, there may be no activation of the HPA-axis in the presence of GnlH. In light of the above findings, GnlH may not be activated without physiological/stress challenge. Hence, the relationship between GnlH and stress in so far as which axis or mechanism is responsible for the others regulation is yet to be determined. Fibrous tracts between the HPA-axis and GnlH are yet to be characterized and would be required to determine the direct nature of the relationship between these axes. In addition, apposition between GnlH and HPA-axis and direct links to the sympathoadrenal system (cortex or medulla) needs to be characterized. Rationale of treatment

[0180] Evolutionarily, reproductive success is usually best in the absence of psychosocial stress or stressors. However, it is postulated that there is reciprocal control between negative regulation of reproduction and stress that is mediated via sympathoadrenal input. Mediation of this relationship by the sympathoadrenal system is postulated on the basis that it may specifically exert control over GnlH during stress, preventing GnlH upregulation in an effort to maintain reproductive capacity at all costs. But, under conditions where stress becomes chronic, such a stronghold may dissipate in which case GnlH would be disinhibited and hence upregulated. So despite upregulation of the HPA- axis during stress (and concomitant elevation of Cortisol) perhaps adaptively or evolutionarily, there would be a lack of GnlH upregulation. This underscores the notion that sympathoadrenal input is the contingency upon which, under behaviorally induced physiological response to stress, a rise in GnlH would or would not be seen (proposed interaction, Figure 1).

[0181] Extrapolating this hypothetical framework to a clinically relevant situation/humans (and animals) that are predisposed to stress-induced illnesses (be it medical or mental such as depression) may be likely as a result of inefficient sympathoadrenal system input to the HPG-axis, as well as HPA-axis, and thus, the organism would become more susceptible to the impact of stress on reproduction or associated illnesses. The net result of chronic stress may be sustained GnlH-HPA-axis synergy, and ultimate failure for sympathoadreno- medullary abrogation of stress-induced negative impact on reproduction, among other illnesses. [0182] It is proposed herein that there is a reciprocal interaction between GnlH and HPA- axis mediated by the sympathoadrenal system in pathology linked to stress and reproductive disorders.

[0183] The present disclosure provides a double-approach to targeting multiple axes (stress and reproductive) as potential treatment of both reproductive-linked physiological and/or depression-related (and other psychological) disorders. EXAMPLE 3

GnlH is upregulated during pregnancy

Hormones and reproduction

[0184] Reproductive function (including pregnancy), in mammals is characterized in terms of hormonal aspects that mediate its progression. Under conditions of normal menstrual cycling there is evidence that GnlH controls the maturation and production of ovarian follicles in the chicken (Bentley et al, 2007 supra). For example, GnlH serves as a signaling pathway for sex steroid regulation of the reproductive axis such that in vivo GnlH administration rapidly inhibits luteinizing hormone secretion (Bentley et al, 2007 supra). However, a role for GnlH during pregnancy has yet to be clearly characterized.

[0185] Cessation of ovulation (due to fertilization of ovum) and successful pregnancy therefore requires shut down of the ovaries. Hypothetically, ovarian shut down may be driven by and maintained by a gradual (but yet to be determined quantitatively) increase in GnlH tone, until such time that the placenta is ready to take over the role of production of sex steroids (estrogen and progesterone) necessary for maintenance of pregnancy. This transition from ovarian production of sex steroids to placental production of sex steroids is known to be achieved in humans by approximately 8 weeks gestation. At this point, placental production of these hormones then form the negative feedback loop to the anterior pituitary, important in maintaining inhibition of gonadotrophins. In order to maintain the suppression of gonadotrophins, the HPA-axis (which is upregulated during pregnancy) may reciprocally up-regulate GnlH in order to maintain suppression of GnRH in the paraventricular nucleus of the hypothalamus. Importantly, GnlH would be expected to be maximally upregulated (by 8 weeks gestation) in order to control maintained ovarian shut-down as well as enable (lead to) an environment that triggers placental production of estrogen and progesterone necessary for pregnancy.

[0186] The upregulation of the stress axis in the later stages of pregnancy may be due to placental release of large amounts of CRH a stress-related hormone which stimulates the pituitary glands of both mother and her fetus to secrete ACTH, (Cortisol) which acts on their adrenal glands causing them to release, the estrogen precursor dehydroepiandrosterone sulfate (DHEAS). The functional significance of upregulated CRH and ACTH is important for ultimate parturition.

[0187] In addition to a role in parturition, upregulated HPA^axis could be important for maintaining GnlH upregulation (via reciprocity) during pregnancy. The functional importance of this relationship is so that GnRH remains shut off. (via GnlH induced suppression) and so too the peripheral ovaries. Importantly, GnlH receptors have been identified on the placenta and anterior pituitary (Hinuma et al, Nature Cell Biology, 2(10), 703-708: 2000; Smith & Clarke, Trends in Endocrinology & Metabolism, 21(4), 255-260, 2010), permitting the potential for autocrine regulation of placental regulation of sex steroids and potentially influencing the stress axis, whether or not the placenta in itself acts as a autocrine system or relays directly to central mechanisms, remains to be explored.

Rationale of diagnosis

[0188] In so far as GnlH has been shown to have receptors co-localized with GnRH, it is likely that the former regulates the later ( riegsfeld et al, 2006 supra). Within this context, there would be expected a down regulation of GnRH to reduce the production of sex steroids from the ovaries and an upregulation of GnlH to suppress GnRH-induced sex steroid production. Following this, the sex steroids secreted from the placenta become responsible for negative feedback actions on the gonadotrophin releasing hormones in the brain if indeed the systems are distinct.

[0189] It is proposed herein that GnlH is upregulated in pregnancy to suppress GnRH induced stimulation of FSH/LH and subsequent estrogen/progesterone secretion from the ovaries. There is probably feedback between the placenta (estrogen and progesterone) and brain (hypothalamus) that maintains inhibition of GnRH, facilitating GnlH stronghold. In addition to which, reciprocity between the HPA-axis (unregulated during pregnancy) and GnlH axes may be necessary to the maintenance of pregnancy. GnlH is proposed, therefore, as a diagnostic indicator of the stage and/or health of a pregnancy. GnlH or an agonist may need to be administered to a female subject during the pregnancy such as towards the end of the pregnancy if levels of GnlH begin to drop. EXAMPLE 4

Use of GnlH antagonist for postnatal depression and postnatal blues Epidemiology

[0190] Postnatal depression has been reported to afflict 10-13% of mothers in recent reports. In addition to this statistic, reports further suggest that 80% of women experience postnatal 'blues' - a less severe, sub-clinical form of postnatal depression.

Etiology and treatment

[0191] The etiology of postnatal depression is yet to be determined, however, there are established links between mothers who experience postnatal depression and; premenstrual syndrome as well as difficulties during pregnancy (stressors) and many with a previous history of major depressive disorder (a disorder in which up to 80% of patients experience HPA-axis dysfunction). Such an association underscores the hormonal-dependent contribution to these disorders. The diagnostic criteria for postnatal depression are similar to that of major depression and treatment options, though limited with respect to efficacy, include anti-depressants.

Hormones and postnatal depression

[0192] In addition to dysfunctional response to stress-challenge (indicative of HPA-axis dyregulation), it is believed that patients with postnatal depression have a deficiency or imbalance of sex hormones (Lawrie et al, Cochrane database of systematic reviews (Online: Update software) (2) pp.CD001690, 2000), perhaps as a cause or major contributor to the pathophysiology of the disorder. Accordingly, supplementation with either progesterone or oestrogen is believed be of prophylactic and/or therapeutic value. Despite this, research into hormonal prophylaxis and treatment of postnatal depression (PND) is limited (Karuppaswamy and Vlies, Journal of Obstetrics and Gynaecology 23(4):34\-346, 2003). A recent review of the literature from 1970 to 2002 examined the prophylactic and treatment value of both oestrogen and progesterone (Karuppaswamy and Vlies, 2003 supra). Some uncontrolled studies reported the benefit of progesterone in preventing postnatal depression. In addition, the value of oestrogen in preventing and treating this disorder was also suggested by some articles but most studies were undermined by methodological shortcomings.

[0193] It is proposed herein that pregnancy, involving acute changes in hormone levels, leads to the development of mood disorders including postnatal depression.

Hormones that mediate mood during pregnancy and may be precursory to postnatal depression

[0194] With respect to change in hormones likely to mediate changes in mood during pregnancy; when the fetus reaches term and is ready for parturition, the internal environment is characterized by a surge in numerous hormones, which are believed to be essential for parturition. The placenta releases large amounts of corticotrophin releasing hormone, CRH a stress-related hormone which stimulates the pituitary glands of both mother and her fetus to secrete adrenocorticotrophin hormone ACTH, (Cortisol) which acts on their adrenal glands causing them to release the estrogen precursor dehydroepiandrosterone sulfate (DHEAS). Perhaps in those are at risk for the development of postnatal depression, there is a greater sensitivity to the upregulation of these hormones (and the HPA-axis). In addition, the increase in estrogen is responsible for driving up of the HPA-axis, which may be responsible in some for predisposing them to the illness via the interaction between stress and reproductive hormones.

Hormone changes that precipitate postnatal depression

[0195] Post-pregnancy, there is a drop in estrogen and progesterone as a result of the placenta being passed, and hence the source of both estrogen and progesterone all but ceases. Importantly, this is an indication that the production of sex steroids ceases, until central control (hypothalamic) is again turned on and the ovaries are disinhibited, enabling sex steroidal production (and normal cycling) to resume. Perhaps, in the short term following parturition, it takes quite some time before the balance between GnlH and GnRH function returns. This may explain why the symptoms of PND tend to persist, often worsening before improving. Mechanistically, there may be a stress-hormone (HPA-axis) stronghold on GnlH upregulation, preventing the resumption of normal menstrual cycling. Rationale of treatment

[0196] It is, proposed herein that following parturition, the balance between GnlH and GnRH becomes pivotal with respect to turning on or triggering normal ovulatory/menstrual function. Whilst GnRH up-regulation is required to stimulate gonadotrophin release from the anterior pituitary, it is possible that GnlH remains competitively upregulated as a result of stress-induced actions of the HPA-axis during pregnancy (ie., due to placental CRH secretion etc) and whilst follicular development may ensue, the balance of hormones may be dysregulated enough to influence mood (as is the case in premenstrual dysphoria). [0197] Elevated GnlH inhibits re-establishment of GnRH/GnlH balance required for normal menstrual cycles. Hence, it is proposed that a GnlH antagonist would break the HPA-axis-GnlH signaling aspect.

EXAMPLE 5

Use of GnlH to treat for Intrauterine Growth Restriction (IUGR)

Etiology

[0198] IUGR involves placental insufficiency such that there is a reduction in blood carrying nutrients and essential gases across the placenta to the fetus. Stress is a risk factor for development of IUGR. It is known that stress leads selectively to vasoconstriction and may therefore cause IUGR. In terms of its pathophysiological underpinnings, IUGR has been linked to an increase in stress-releasing hormone corticotrophin releasing hormone (CRH), released both from the placenta and the fetus. Placental overproduction of CRH may lead to an increase in adrenal release of Cortisol in the mother (whom may also be sensitive to fetal CRH release) which positively up-regulates CRH expression as well as HPA-axis drive.

[0199] When stress posses a threat to maternal survival, reproductive function may be compromised. For example, it has been shown that maternal food restriction (a stressor) is a risk factor for IUGR. Hence, stress and reproduction may be linked such that enhanced stress (HPA-axis drive) is linked to enhanced GnlH upregulation. Within this context, stress-induced GnlH-activity may in fact focus HPA-axis driven selective vasoconstriction of the placenta and thereby cause IUGR.

[0200] Further to this, the relationship between HPA-axis and GnlH may be mediated by sympathoadrenal activity, the later of which is a putative mechanism in the response to , stress as well being a functional mediator of pregnancy. Stress-induced IUGR may therefore be an evolutionarily conserved mechanism such that stress during pregnancy (combined with individual vulnerability ie., sympathoadrenal susceptibility) may lead to compromise of fetus for maternal survival. GnlH may be concomitantly^' up-regulated via reciprocity with the HPA-axis, and mediated via adrenal system-input. The increase in CRH is further associated with increase in estrogen, the later of which may drive up (abnormally) the HPA-axis in combination with enhanced GnlH tone. The upregulation of GnlH is proposed to focus HPA-axis induced vasoconstriction of the placenta. EXAMPLE 6

GnlH is up-regulated in menopause and is key to transient (reversible) induction of menopause relevant to scientific modeling and reproductive dormancy as an alternative to cancer treatment GnlH in the control of menopause

[0201] Menopause is characterized as a stage of the human female reproductive cycle whereby the ovaries stop producing estrogen, causing the reproductive system to gradually shut down. This period is characterized by changes in mood as the body adapts to the changing levels of natural hormones. In addition to which there are symptoms such as hot flushes, vaginal dryness, increased depression and anxiety, and increasingly scanty and erratic menstrual periods.

[0202] Physiologically, the ovaries are no longer able to develop an egg for ovulation. Ovarian production of estrogen goes into a permanent decline, and progesterone is no longer produced. The lining of the uterus thins, since it isn't being stimulated by high estrogen levels each month, and monthly bleeding stops. Menopause has occurred. The postmenopausal ovary, however, continues to produce hormones even after ovulation ends. producing some estrogen . and also androgens (male hormones) including testosterone. Some of the androgens are converted to estrogen (estrone) in a woman's fat tissue. The down regulation of primary sex hormones however; is strong support for the involvement of GnlH, the negative regulator of reproduction, in shutting down the reproductive axis via suppressing the production of gonadotropins, the precursors to sex-steroid production.

GnlH: Inducing transient and reversible menopause: A scientific Model

[0203] In addition to its role in menopause progression, GnlH may be able to be used to transiently induce menopause in otherwise reproductively virile women. This application is an important consideration because, in women suffering from cancer, their treatment modality can result in the induction of early menopause; as a result of radiation or hormone therapy following removal of the tumor. There are believed to be key consequences of reduction of sex steroids in terms of cognitive function among other processes as a result of menopause as well as therapeutically-induced menopause.

[0204] The current approved therapeutics used to suppress sex steroid production following surgical removal or radiation-targeted destruction of tumors (as they present a risk factor for re-development of sex-steroid positive or responsive tumors) are called aromatase inhibitors. The key action of these drugs is to prevent the enzyme, aromatase, from converting androgens to estrogens. Other kiiown treatments include. GnRH antagonists. Aromatase inhibitors also prevent ovulation. Hence GnlH may be an alternative to aromatase inhibitors for the treatment of cancers, such as breast cancer among others, but also a means by which to induce transient menopause. GnlH may therefore be used to determine the influence of transient menopause (unconfounded by the influence of time) on cognition. In addition, it could be used to compare efficacy-profiles with aromatase inhibitors and other GnRH antagonists among other currently approved therapies. Importantly, this offers a new model for transient induction of human menopause that takes advantage of the bodies natural physiological mechanism responsible for inducing menopause Rationale of treatment

[0205] GnlH agonists at high doses are proposed herein to transiently induce menopause. Importantly, menopausal induction in women otherwise reproductively virile will be reversible.

[0206] The proposed physiological model of transient menopause will enable the understanding of the impact of menopause on cognition and whether the level of estrogen impacts this. In addition, it offers a therapeutic approach to other cancers (than ovarian cancer) as such breast cancer and other reproductive linked cancers.

EXAMPLE 7

GnlH regulates GnRH; and GnlH further regulates Kisspeptin-modulation of GnRH

[0207] Kisspeptin, like GnlH, is a member of the same RF amide group of brain peptides. The principle role for kisspeptin is a positive, feed-forward action on reproductive peptide, GnRH. When sex-steroid levels are low (i.e. estrogen), kisspeptin appears to stimulate GnRH to release precursory gonadotrophs to produce sex steroids.

[0208] In addition, kisspeptin functionally important in 'switching-on' puberty via the same mechanism above described. However; what remains yet to be established, is the mechanism that regulates kisspeptin.

[0209] There are two possibilities: that it is regulated via feedback from the gonads to the brain or that it is regulated by a higher-functioning peptide. To prove brain-derived control of kisspeptin, the ability of kisspeptin to regulate GnRH in the absence of gonads would need to be established. This would be unequivocal that kisspeptin functions independently of the sex steroid input from the gonads. However, given kisspeptin's central localization and the fact it has been shown to be closely apposed with GnRH in several brain centres, itis proposed herein that it is regulated by the master negative regulator of reproduction, GnlH. [0210] Further to this relationship, it is proposed herein that kisspeptin is also controlled via stress-related inputs from the HPA-axis and sympathoadrenal systems (Figure 2).

[0211] The notion that kisspeptin may not be the primary .regulator of puberty, that in fact it may be that it is under control of GnlH, hence that GnlH is likely the primary controller of puberty and kisspeptin function.

EXAMPLE 8

The role of GnlH in suppressing estrogen-induced degradation of the thymus gland

[0212] GnlH controls the reproductive state of an individual. It is yet to be determined when GnlH becomes active but, it is possible that it is expressed from birth until GnRH, the pro-regulator of reproduction is switched on. [0213] Hence, it is proposed that GnlH could be the switch to GnRH that facilitates the degradation of the thymus.

EXAMPLE 9

Central and peripheral plasma concentrations of RFRP3 in major depression before and following treatment with serotonin reuptake inhibitors

Statistical analysis

[0214] Data were checked for normality and outliers and were reasonable. Repeated measures analysis of Variance was run on patients, with major depression with Jugular Pre- and Post-SSRI and Arterial Pre- and Post-SSRI as within subjects factors. The analysis was re-run with gender as a between subjects factor. Males and females were then separately filtered out, to see what this achieved.

[0215] Univariate ANOVA was used to compare arterial concentration with healthy controls Pre- and Post-SSRI. Univariate ANOVA was also used to compare central and peripheral concentrations. [0216] Correlations were conducted between Pre-SSRI concentrations centrally and peripherally and Hamilton D and Anxiety Trait parameters.

Outcome of analysis

[0217] Overall, there was a significant (P=0.011) arterial effect but no effect on jugular and no interaction. When gender was included as a between subjects factor there was a significant (P=0.049) arterial*gender interaction. When males were filtered out there was a significant (P=0.003) arterial effect for females. When females were filtered out the arterial effect for males just failed to reach statistical significance (P=0.056).

[0218] The reduction in peptide concentrations Pre- to Post-SSRI was 16.7% in females and 44.5% in males.

[0219] When univariate ANOVA was used to compare arterial concentrations with healthy controls Pre- and Post-SSRI, overall, there was no significant effect Pre-SSRI but there was a significant (P=0.041) Post-SSRI with the depressed patients having lower levels relative to healthy controls. Gender was then put in as a factor and it made no difference.

[0220] There were no significant correlations between GnlH concentrations Pre-SSRI and Hamilton D or Anxiety Trait. There are, however, few, if any, studies in schizophrenia demonstrating a relationship between estrogen levels and any illness symptoms, yet modulation of estrogen achieves significant symptom remission.

[0221] The results are shown in Figures 3 and 4.

EXAMPLE 10

Treatment of depression

[0222] Using methods described in the above examples, a clinical trial is conducted where subjects diagnosed with symptoms of depression are divided into groups and are given:

(i) GnlH antagonist alone;

(ii) GnlH antagonist + anti-depressant (e.g. an SSRI, SSNRI, a tri-cyclic antidepressant or a tetra-cyclic anti-depressant);

(iii) GnlH antagonist + oxytocin;

(iv) GnlH antagonist + estrogen;

(v) GnlH antagonist + oxytocin + anti-depressant;

(vi) GnlH antagonist + estrogen + anti-depressant;

(vii) GnlH antagonist + oxytocin + estrogen; or

(viii) GnlH antagonist + anti-depressant + oxytocin + estrogen.

[0223] Examples of an SSRI include agomelatine, escitalopram, sertraline, citalopram and fluoxetine. Examples of an SSNRI include esmertazapine, venlafaxine, desvenlafaxine and duloxetine. Examples of a tri-cyclic anti-depressant include amitriptyline and imipramine. Examples of a tetra-cyclic anti-depressant include amoxapine and mirtazapinc.

[0224] Symptoms of depression are then monitored over time.

[0225] In preparation for the treatment of human patients, an animal model may be used. An example of a suitable animal model is the Forced Swim Test (or Porsolt Test). Such an animal model is particularly useful in testing drugs in acute and chronic treatments. [0226] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

0560

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Claims

CLAIMS:

1. A method of treating a human subject diagnosed with symptoms of depression, said method comprising administering to said subject an effective amount of a GnlH antagonist for a time and under conditions sufficient to ameliorate the symptoms of depression.

2. The method of Claim 1 further comprising the administration of an anti -depressant selected from a selective serotonin re-uptake inhibitor (S SRI), a selective serotonin and noradrenergic re-uptake inhibitor (SSNRI), a tri-cyclic anti-depressant and a tetra-cyclic anti-depressant.

3. The method of Claim 1 or 2 further comprising the administration of oxytocin,

4. The method of Claim 1 or 2 or 3 further comprising the administration of estrogen.

5. The method of Claim 1 or 2 or 3 or 4 wherein the GnlH antagonist is a GnRH agonist.

6. The method of Claim 7 wherein the SSRI is selected from escitalopram, sertraline, citalopram, fluoxetrine and agomelatine.

7. The . method of Claim 2 wherein the SSNRI is selected from esmirtazapine, venlafaxine, desvenlafaxine and duloxetine.

8. The method of Claim 2 wherein the tri-cyclic anti-depressant is selected from amitriptyline and imipramine.

9. The method of Claim 2 wherein the tetra-cyclic anti-depressant is selected from amoxapine and mirtazapine.

10. The method of Claim 5 wherein the GnRH agonist is selected from leuprolide, buserelin, nafarelin, histrelin, goserelin, deslorelin and triptorelin.

11. The method of any one of Claims 1 to 10 performed in conjunction with behavioral modification therapy.

12. A pharmaceutical composition comprising a GnlH antagonist and an antidepressant selected from an SSRI, SSNRI, a tri-cyclic anti-depressant and a tetra-cyclic anti-depressant.

13. The pharmaceutical composition of Claim 12 further comprising oxytocin.

14. The pharmaceutical composition of Claim 12 or 13 further comprising estrogen.

15. The pharmaceutical composition of Claim 12 or 13 or 14 wherein the SSRI is selected from escitalopram, sertraline, citalopram, fluoxetine and agomelatine; the SSNRI is selected from esmirtazapine, venlafaxine, desvenlafaxine and duloxetrine; the tri-cyclic anti-depressant is selected from amitriptyline and imipramine; and the tetra-cyclic antidepressant is selected from amoxapine and mirtazapine.

16. The pharmaceutical composition of Claim 12 or 13 or 14 or 15 further comprising a pharmaceutically acceptable carrier, diluent or excipient.

17. The pharmaceutical composition of any one of Claims 12 to 16 in the form of a sustained release or slow release formulation.

18. Use of GnlH in the manufacture of a medicament in the treatment of a subject with symptoms of depression.

19. Use of Claim 18 further comprising use of an anti-depressant selected from an SSRI, SSNRI, a tri-cyclic anti-depressant and a tetra-cyclic anti-depressant.

20. Use of Claim 18 or 19 further comprising use of oxytocin and/or estrogen.

21. Use of Claim 18 or 19 or 20 wherein the SSRI is selected from escitalopram, sertraline, citalopram, fluoxetine and agomelatine; the SSNRI is selected from esmirtazapine, venlafaxine, desvenlafaxine and duloxetrine; the tri-cyclic anti-depressant is selected from amitriptyline and imipramine; and the tetra-cyclic anti-depressant is selected from amoxapine and mirtazapine.

22. An agent for use in treating a subject with symptoms of depression, said agent comprising a GnlH antagonist.

23. The agent of Claim 22 further comprising one or more of an anti -depressant selected from an SSRI, SSNRI, a tri-cyclic anti-depressant and a tetra-cyclic antidepressant, oxytocin and/or estrogen.

24. The agent of Claim 22 wherein the SSRI is selected from escitalopram, sertraline, citalopram, fluoxetine and agomelatine; the SSNRI is selected from esmirtazapine, venlafaxine, desvenlafaxine and duloxetrine; the tri-cyclic anti-depressant is selected from amitriptyline and imipramine; and the tetra-cyclic anti-depressant is selected from amoxapine and mirtazapine.