WO2010064030A1

WO2010064030A1 - Biomarkers

Info

Publication number: WO2010064030A1
Application number: PCT/GB2009/051614
Authority: WO
Inventors: Sabine Bahn; Marlis Huebner; Natalia Krzyszton
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2008-12-01
Filing date: 2009-11-30
Publication date: 2010-06-10
Anticipated expiration: 2011-06-01
Also published as: GB0821912D0

Abstract

The invention relates to a method of diagnosing or monitoring bipolar disorders, in particular bipolar I and bipolar II disorders.

Description

BIOMARKERS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bipolar disorder is a psychiatric disease that describes a category of mood disorders defined by the presence of one or more episodes of abnormally elevated mood clinically referred to as mania or, if milder, hypomania. Individuals who experience manic episodes also commonly experience depressive episodes or symptoms, or mixed episodes in which features of both mania and depression are present at the same time. Such individuals also experience a decreased quality of life. These episodes are usually separated by periods of "normal" mood, but in some individuals, depression and mania may rapidly alternate, known as rapid cycling. Extreme manic episodes can sometimes lead to psychotic symptoms such as delusions and hallucinations. The disorder has been subdivided into bipolar I, bipolar II, cyclothymia, and other types, based on the nature and severity of mood episodes experienced; the range is often described as the bipolar spectrum.

Bipolar I disorder is characterised by manic episodes; the "high" of the manic- depressive cycle . Genera l ly, th is man ic period is fol l owed by a period of depression, although some bipolar I individuals may not experience a major depressive episode. Mixed states, where both manic or hypomanic symptoms and depressive symptoms occur at the same time, also occur frequently with bipolar I patients (for example, depression with the racing thoughts of mania). Also, dysphoric mania is common and is mania characterised by anger and irritability.

Bipolar II disorder is characterised by major depressive episodes alternating with episodes of hypomania, a milder form of mania. Hypomanic episodes can be a less disruptive form of mania and may be characterised by low-level, non-psychotic symptoms of mania, such as increased energy or a more elevated mood than usual . It may not affect an individual's ability to function on a day to day basis. The criteria for hypomania differ from those for mania only by their shorter duration (at least 4 days instead of 1 week) and milder severity (no marked impairment of functioning, hospitalisation or psychotic features).

If the depressive and manic symptoms last for two years and do not meet the criteria for a major depressive or a manic episode then the diagnosis is classified as a cyclothymic disorder, which is a less severe form of bipolar affective disorder. Cyclothymic disorder is diagnosed over the course of two years and is characterised by frequent short periods of hypomania and depressive symptoms separated by periods of stability.

Rapid cycling occurs when an individual's mood fluctuates from depression to hypomania or mania in rapid succession with little or no periods of stability in between. One is said to experience rapid cycling when one has had four or more episodes in a given year that meet criteria for major depressive, manic, mixed or hypomanic episodes. Some people who rapid cycle can experience monthly, weekly or even daily shifts in polarity (sometimes called ultra rapid cycling).

To date, no empirical diagnostic tests are available, making diagnosis a subjective evaluation which often leads to misdiagnosis and delay in accurate treatment. When symptoms of mania, depression, mixed mood or hypomania are caused directly by a medical disorder, such as thyroid disease or a stroke, the current diagnosis is Mood Disorder Due to a General Medical Condition.

In a manic mood brought about through an antidepressant, ECT or through an individual using street drugs, the diagnosis is Substance-Induced Mood Disorder, with Manic Features.

Diagnosis of bipolar has been used to categorise manic episodes which occur as a result of taking an antidepressant medication, rather than occurring spontaneously. Confusingly, it has also been used in instances where an individual experiences hypomania or cyclothymia (i.e. less severe mania) without major depression.

SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided the use of one or more first peptides selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Purine nucleoside phosphorylase, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase, Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin-IXb, Myosin-7, Myosin-11, Myosin-10, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG) and Natural killer cell-enhancing factor A (NKEF-A)) and Uncharacterized protein C12orf35, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

According to a second aspect of the invention, there is provided the use of two or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor and Leucine zipper protein 1, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

According to a third aspect of the invention, there is provided a method of diagnosing or monitoring bipolar I or bipolar II disorders, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject , one or more of the first peptide biomarkers defined herein. According to a fourth aspect of the invention, there is provided a method of diagnosing or monitoring bipolar I or bipolar II disorders, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, two or more of the second peptide biomarkers defined herein.

According to a fifth aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to bipolar I or bipolar II disorders, comprising detecting and/or quantifying, in a sample from said subject, one or more of the first peptide biomarkers defined herein.

According to a sixth aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to bipolar I or bipolar II disorders, comprising detecting and/or quantifying, in a sample from said subject, two or more of the second peptide biomarkers defined herein.

A further aspect of the invention provides ligands, such as naturally occurring or chemically synthesised compounds, capable of specific binding to the peptide biomarker. A ligand according to the invention may comprise a peptide, an antibody or a fragment thereof, or an aptamer or oligonucleotide, capable of specific binding to the peptide biomarker. The antibody can be a monoclonal antibody or a fragment thereof capable of specific binding to the peptide biomarker. A ligand according to the invention may be labelled with a detectable marker, such as a luminescent, fluorescent or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag.

A biosensor according to the invention may comprise the peptide biomarker or a structural/shape mimic thereof capable of specific binding to an antibody against the peptide biomarker. Also provided is an array comprising a ligand or mimic as described herein. Also provided by the invention is the use of one or more ligands as described herein, which may be naturally occurring or chemically synthesised, and is suitably a peptide, antibody or fragment thereof, aptamer or oligonucleotide, or the use of a biosensor of the invention, or an array of the invention, or a kit of the invention to detect and/or q uantify the peptide . In these uses, the detection and/or quantification can be performed on a biological sample such as from the group consisting of CSF, whole blood, blood serum, plasma, urine, saliva, or other bodily fluid, breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof.

Diagnostic or monitoring kits are provided for performing methods of the invention. Such kits will suitably comprise a ligand according to the invention, for detection and/or quantification of the peptide biomarker, and/or a biosensor, and/or an array as described herein, optionally together with instructions for use of the kit.

A further aspect of the invention is a kit for monitoring or diagnosing bipolar I or bipolar II disorders, comprising a biosensor capable of detecting and/or quantifying one or more of the first peptide biomarkers as defined herein.

A further aspect of the invention is a kit for monitoring or diagnosing bipolar I or bipolar II disorders, comprising a biosensor capable of detecting and/or quantifying two or more of the second peptide biomarkers as defined herein.

Biomarkers for bipolar I or bipolar II disorders are essential targets for discovery of novel targets and drug molecules that retard or halt progression of the disorder. As the level of the peptide biomarker is indicative of disorder and of drug response, the biomarker is useful for identification of novel therapeutic compounds in in vitro and/or in vivo assays. Biomarkers of the invention can be employed in methods for screening for compounds that modulate the activity of the peptide.

Thus, in a further aspect of the invention, there is provided the use of a ligand, as described, which can be a peptide, antibody or fragment thereof or aptamer or oligonucleotide according to the invention; or the use of a biosensor according to the invention, or an array according to the invention; or a kit according to the invention, to identify a substance capable of promoting and/or of suppressing the generation of the biomarker.

Also there is provided a method of identifying a substance capable of promoting or suppressing the generation of the peptide in a subject, comprising administering a test substance to a subject animal and detecting and/or quantifying the level of the peptide biomarker present in a test sample from the subject.

DETAILED DESCRIPTION OF THE INVENTION

According to a first particular aspect of the invention, there is provided the use of one or more first peptides selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Leucine zipper protein 1, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Purine nucleoside phosphorylase, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase, Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin- IXb, Myosin-7, Myosin-11, Myosin-10, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG) and Natural killer cell-enhancing factor A (NKEF-A)) and Uncharacterized protein C12orf35, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

According to a second particular aspect of the invention, there is provided the use of two or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic nonspecific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor) and Receptor-type tyrosine-protein phosphatase S precursor, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

The term "biomarker" means a distinctive biological or biologically derived indicator of a process, event, or condition. Peptide biomarkers can be used in methods of diagnosis, e.g . clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.

It will be readily apparent to the skilled person that the first and second peptides listed herein are known and have been described in the literature, however, for completeness, full characterising information for these peptides is provided in Table 1 :

Table 1: Characterising Information of the First and Second Peptides of the Invention

In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Phosphatidylinositol 4-kinase alpha. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Eukaryotic translation initiation factor 3 subunit A. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Tubulin alpha-lA chain. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG) and Natural killer cell-enhancing factor A (NKEF-A)). In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Purine nucleoside phosphorylase. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Zinc finger CCCH domain-containing protein 13. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Dedicator of cytokinesis protein 7.

In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-2. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-Va. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-4. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-13. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-IXb. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-7. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-11. In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is other than Myosin-10.

In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Purine nucleoside phosphorylase, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase and Uncharacterized protein C12orf35.

In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Leucine zipper protein 1, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Purine nucleoside phosphorylase, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase and Uncharacterized protein C12orf35.

In one embodiment of any of the previously mentioned aspects of the invention, the first peptide is selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase and Uncharacterized protein C12orf35.

In a further embodiment of any of the previously mentioned aspects of the invention, the first peptide is selected from : Transitional endoplasmic reticulum ATPase (VCP), Alpha-protein kinase 2, Stress-70 protein (mitochondrial precursor) and Myosin-Va.

In one embodiment of the use of any of the previously mentioned aspects of the invention, additionally comprises the use of one or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation- associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochond rial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor and Leucine zipper protein 1.

In one embodiment of the use of any of the previously mentioned aspects of the invention, additionally comprises the use of one or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation- associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor) and Receptor-type tyrosine-protein phosphatase S precursor.

In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Phosphatidylinositol 4- kinase alpha. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Eukaryotic translation initiation factor 3 subunit A. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Tubulin alpha-lA chain. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG) and Natural killer cell-enhancing factor A (NKEF-A)). In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Purine nucleoside phosphorylase. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Zinc finger CCCH domain- containing protein 13. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Dedicator of cytokinesis protein 7.

In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-2. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-Va. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-4. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-13. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-IXb. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-7. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-11. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second peptides additionally comprise Myosin-10.

According to a further aspect of the invention, there is provided the use of two or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Endoplasmin precursor HSP90B1 Grp94, Heat shock protein HSP 90-beta, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG), Natural killer cell-enhancing factor A (NKEF-A)), Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin-IXb, Myosin-7, Myosin-11 and Myosin-10, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

According to a further aspect of the invention, there is provided the use of two or more second peptides selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Endoplasmin precursor HSP90B1 Grp94, Heat shock protein HSP 90-beta, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor, Leucine zipper protein 1, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation- associated gene protein (PAG), Natural killer cell-enhancing factor A (NKEF-A)), Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin-IXb, Myosin-7, Myosin-11, Myosin-10, Purine nucleoside phosphorylase, Zinc finger CCCH domain-containing protein 13 and Dedicator of cytokinesis protein 7, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

In a further embodiment of any of the previously mentioned aspects of the invention, one of said second peptides is Aconitate hydratase 2 (mitochondrial precursor).

In one embodiment, one or more of the biomarkers may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.

As used herein, the term "biosensor" means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein.

Biosensors according to the invention may comprise a ligand or ligands, as described herein, capable of specific binding to the peptide biomarker. Such biosensors are useful in detecting and/or quantifying a peptide of the invention.

Diagnostic kits for the diagnosis and monitoring of bipolar I or bipolar II disorders are described herein. In one embodiment, the kits additionally contain a biosensor capable of detecting and/or quantifying a peptide biomarker.

Monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration and/or remission.

In methods of diagnosing or monitoring according to the invention, detecting and/or quantifying the peptide biomarker in a biological sample from a test subject may be performed on two or more occasions. Comparisons may be made between the level of biomarker in samples taken on two or more occasions. Assessment of any change in the level of the peptide biomarker in samples taken on two or more occasions may be performed. Modulation of the peptide biomarker level is useful as an indicator of the state of the bipolar I or bipolar II disorders or predisposition thereto. An increase in the level of the biomarker, over time is indicative of onset or progression, i.e. worsening of this disorder, whereas a decrease in the level of the peptide biomarker indicates amelioration or remission of the disorder, or vice versa.

A method of diagnosis of or monitoring according to the invention may comprise quantifying the peptide biomarker in a test biological sample from a test subject and comparing the level of the peptide present in said test sample with one or more controls.

The control used in a method of the invention can be one or more control(s) selected from the group consisting of: the level of biomarker peptide found in a normal control sample from a normal subject, a normal biomarker peptide level; a normal biomarker peptide range, the level in a sample from a subject with bipolar I or bipolar II disorders, or a diagnosed predisposition thereto; bipolar I or bipolar II disorders biomarker peptide level, or bipolar I or bipolar II disorders biomarker peptide range.

In one embodiment, there is provided a method of diagnosing bipolar I or bipolar II disorders, or predisposition thereto, which comprises:

(a) quantifying the amount of the peptide biomarker in a test biological sample; and

(b) comparing the amount of said peptide in said test sample with the amount present in a normal control biological sample from a normal subject.

A higher level of the peptide biomarker in the test sample relative to the level in the normal control is indicative of the presence of bipolar I or bipolar II disorders, or predisposition thereto; an equivalent or lower level of the peptide in the test sample relative to the normal control is indicative of absence of bipolar I or bipolar II disorders and/or absence of a predisposition thereto.

The term "diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of bipolar I or bipolar II disorders, or predisposition thereto. By predisposition it is meant that a subject does not currently present with the disorder, but is liable to be affected by the disorder in time. Methods of monitoring and of diagnosis according to the invention are useful to confirm the existence of a disorder, or predisposition thereto; to monitor development of the disorder by assessing onset and progression, or to assess amelioration or regression of the disorder. Methods of monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

Efficient diagnosis and monitoring methods provide very powerful "patient solutions" with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), reducing "downtime" and relapse rates.

Also provided is a method of monitoring efficacy of a therapy for bipolar I or bipolar II disorders in a subject having such a disorder, suspected of having such a disorder, or of being predisposed thereto, comprising detecting and/or quantifying the peptide present in a biological sample from said subject. In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control (s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the level of the biomarker(s) in test samples taken on different occasions.

The invention provides a method for monitoring efficacy of therapy for bipolar I or bipolar II disorders in a subject, comprising : (a) quantifying the amount of the peptide biomarker; and

(b) comparing the amount of said peptide in said test sample with the amount present in one or more control(s) and/or one or more previous test sample(s) taken at an earlier time from the same test subject. A decrease in the level of the peptide biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject is indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder, suspected disorder or predisposition thereto.

Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.

Suitably, the time elapsed between taking samples from a subject undergoing diagnosis or monitoring will be 3 days, 5 days, a week, two weeks, a month, 2 months, 3 months, 6 or 12 months. Samples may be taken prior to and/or during and/or following an anti-depressant therapy. Samples can be taken at intervals over the remaining life, or a part thereof, of a subject.

The term "detecting" as used herein means confirming the presence of the peptide biomarker present in the sample. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the peptide biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.

In alternative aspects of the invention, the presence of the peptide biomarker is assessed by detecting and/or quantifying antibody or fragments thereof capable of specific binding to the biomarker that are generated by the subject's body in response to the peptide and thus are present in a biological sample from a subject having bipolar I or bipolar II disorders or a predisposition thereto.

Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of the peptide biomarker in the sample or samples. Biological samples that may be tested in a method of the invention include cerebrospinal fluid (CSF), whole blood, blood serum, plasma, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

Detection and/or quantification of peptide biomarkers may be performed by detection of the peptide biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

The biomarker may be directly detected, e.g. by SELDI or MALDI-TOF. Alternatively, the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.

For example, detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TO F), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used.

Methods of diagnosing or monitoring according to the invention may comprise analysing a sample of cerebrospinal fluid (CSF) by SELDI TOF or MALDI TOF to detect the presence or level of the peptide biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment.

Detecting and/or quantifying the peptide biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the peptide biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the peptide biomarkers is performed using two antibodies which recognize different epitopes on a peptide biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent a ss a ys ( E LI SA ) , e n z y m e immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtitre plate or strip format.

Immunological methods in accordance with the invention may be based, for example, on any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insol uble agg regate. Immunoprecipitation reactions may be q ual itative or quantitative.

In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction . This allows rapid and sensitive detection of the biomarker.

In immunonephelometry, the interaction of an antibody and target antigen on the biomarker results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen, i.e. biomarker, concentration can be determined within minutes of the reaction.

Radioimmunoassay (RIA) methods employ radioactive isotopes such as I¹²⁵ to label either the antigen or antibody. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.

Enzyme (EIA) immunoassays were developed as an altern ati ve to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA). ELISA methods may use two antibodies one of which is specific for the target antigen and the other of which is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemiluminescent or fluorescent signal.

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector. Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of peptide biomarkers of the invention.

The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (hapten, antigen, ligand, aptamer, antibody, enzyme etc) is labelled with biotin and the other partner (surface, e.g. well, bead, sensor etc) is labelled with avidin or streptavidin. This is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one. For example a biotinylated ligand (e.g. antibody or aptamer) specific for a peptide biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the peptide biomarker in order to detect and/or q ua ntify a peptide biomarker of the i nvention . Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.

The term "antibody" as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies,

Fab fragments and F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.

The immunoglobulin molecules of the invention can be of any class (e. g., IgG,

IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

The identification of key biomarkers specific to a disease is central to integration of diagnostic procedures and therapeutic regimes. Using predictive biomarkers appropriate diagnostic tools such as biosensors can be developed, accordingly, in methods and uses of the invention, detecting and quantifying can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor may incorporate an immunological method for detection of the biomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker(s) at the anticipated concentrations found in biological samples.

Thus, according to a further aspect of the invention there is provided an apparatus for diagnosing or monitoring schizophrenia or other psychotic disorders which comprises a biosensor, microanalytical, microengineered, microseparation and/or immunochromatography system configured to detect and/or quantify any of the biomarkers defined herein.

The biomarker(s) of the invention can be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code" or array configurations.

In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker. On exposure, the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram. The test result read-out can be a change in the optical brightness, image, colour and/or position of the image. For qualitative and semi-quantitative applications, a sensor hologram can be read by eye, thus removing the need for detection equipment. A simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor. The format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible.

Suitably, biosensors for detection of one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal. Biosensors can be adapted for "alternate site" diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.

Biosensors to detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the one or more biomarkers of the invention.

Methods involving detection and/or q uantification of one or more peptide biomarkers of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g . in the physician's office or at the patient's bedside. Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-neuromedicine.

Any suitable animal may be used as a subject non-human animal, for example a non-human primate, horse, cow, pig, goat, sheep, dog, cat, fish, rodent, e.g . guinea pig, rat or mouse; insect (e.g. Drosophila), amphibian (e.g. Xenopus) or C. elegans.

The test substance can be a known chemical or pharmaceutical substance, such as, but not limited to, an anti-depressive disorder therapeutic; or the test substance can be a novel synthetic or natural chemical entity, or a combination of two or more of the aforesaid substances.

There is provided a method of identifying a substance capable of promoting or suppressing the generation of the peptide biomarker in a subject, comprising exposing a test cell to a test substance and monitoring the level of the peptide biomarker within said test cell, or secreted by said test cell. The test cell could be prokaryotic, however a eukaryotic cell will suitably be employed in cell-based testing methods. Suitably, the eukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibian cell (e.g. from Xenopus), C. elegans cell or is a cell of human, non-human primate, equine, bovine, porcine, caprine, ovine, canine, feline, piscine, rodent or murine origin.

In methods for identifying substances of potential therapeutic use, non-human animals or cells can be used that are capable of expressing the peptide.

Screening methods also encompass a method of identifying a ligand capable of binding to the peptide biomarker according to the invention, comprising incubating a test substance in the presence of the peptide biomarker in conditions appropriate for binding, and detecting and/or quantifying binding of the peptide to said test substance.

High-throughput screening technologies based on the biomarker, uses and methods of the invention, e.g. configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g. from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.

Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.

The invention further provides a substance, e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention. Such substances may be capable of inhibiting, directly or indirectly, the activity of the peptide biomarker, or of suppressing generation of the peptide biomarker. The term "substances" includes substances that do not directly bind the peptide biomarker and directly modulate a function, but instead indirectly modulate a function of the peptide biomarker. Ligands are also included in the term substances; ligands of the invention (e.g. a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, suitably specific binding, to the peptide.

The invention further provides a substance according to the invention for use in the treatment of bipolar I or bipolar II disorders, or predisposition thereto.

Also provided is the use of a substance according to the invention in the treatment of bipolar I or bipolar II disorders, or predisposition thereto.

Also provided is the use of a substance according to the invention as a medicament.

Yet further provided is the use of a substance according to the invention in the manufacture of a medicament for the treatment of bipolar I or bipolar II disorders, or predisposition thereto.

A kit for diagnosing or monitoring bipolar I or bipolar II disorders, or predisposition thereto is provided. Suitably a kit according to the invention may contain one or more components selected from the group: a ligand specific for the peptide biomarker or a structural/shape mimic of the peptide biomarker, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

The identification of biomarkers for bipolar I or bipolar II disorders permits integration of diagnostic procedures and therapeutic regimes. Currently there are significant delays in determining effective treatment and hitherto it has not been possible to perform rapid assessment of drug response. Traditionally, many antidepressant therapies have required treatment trials lasting weeks to months for a given therapeutic approach. Detection of a peptide biomarker of the invention can be used to screen subjects prior to their participation in clinical trials. The biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels. The biomarkers may be used to provide warning of adverse drug response. Biomarkers are useful in development of personalized brain therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions. Thus by monitoring a biomarker of the invention, patient care can be tailored precisely to match the needs determined by the disorder and the pharmacogenomic profile of the patient, the biomarker can thus be used to titrate the optimal dose, predict a positive therapeutic response and identify those patients at high risk of severe side effects.

Biomarker-based tests provide a first line assessment of 'new' patients, and provide objective measures for accurate and rapid diagnosis, in a time frame and with precision, not achievable using the current subjective measures.

Furthermore, diagnostic biomarker tests are useful to identify family members or patients at high risk of developing bipolar I or bipolar II disorders. This permits initiation of appropriate therapy, or preventive measures, e.g. managing risk factors. These approaches are recognised to improve outcome and may prevent overt onset of the disorder.

Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder, poor patient compliance or substance abuse. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy or of substance abuse.

The following study illustrates the invention.

This study aimed to identify protein biomarkers in peripheral blood mononuclear cells (PBMCs) obtained from euthymic bipolar disorder I and bipolar disorder II patients. Proteome profiling was performed on cytosolic- and membrane-protein enriched cellular fractions using a label-free mass spectrometry approach and differences in protein expression profiles between patients and controls established. In the cytosolic-enriched fraction, 49 proteins were detected with significantly altered expression between patient and control subjects. The membrane-enriched fraction showed 6 proteins with significantly altered expression. Pathway analyses showed that these proteins were assigned predominantly to signalling pathways associated with cell death, suggesting a dysregulation of this function.

Methodology

Bipolar sample collection

PBMCs from euthymic bipolar disorder patients (according to DSM-IV code 296.89 and 296.4) and sex, age and race matched controls were obtained from Dr. Markus Leweke, University of Cologne, Germany. PBMCs were isolated by a density gradient using Ficoll-Paque PLUS and frozen at -80⁰C in foetal calf serum (FCS) containing 10% dimethyl sulphoxide (DMSO).

Cell preparation for MS

1. PBMCs were thawed in RPMI-1640 medium supplemented with 10% FCS and 1% glutamine, penicillin and streptomycin, counted and washed in DPBS. 10*10⁶ cells were transferred into protein low binding tubes and pelleted for 4 min at 10,000 rpm. PBMCs were stored at -80⁰C until fractionation.

2. The ProteoExtract^® Subcellular Proteome Extraction Kit (Merck, UK) was applied to obtain cytosolic- and membrane-enriched protein fractions. The procedure was performed according to the manufacturers' specifications with minor changes. In brief, these included halving the volumes of all four buffers and the protease inhibitor cocktail . Centrifugation forces chosen were 1,000 x g, 6,000 x g and 9,000 x g, respectively.

3. The protein concentration (Biorad Protein Assay) of the cytosolic-enriched fraction was measured after fractionation, whereas the membrane fraction was concentrated for 1 h at 30 ⁰C using the Concentrator Plus machine before protein concentration measurement.

4. In order to remove all non-protein impurities, fractions were precipitated using the ProteoExtract^® Protein Precipitation Kit (Merck, UK). The precipitation was carried out as stated in the manufacturers' specifications.

5. For digestion, the ProteoExtract^® All-in-One Trypsin Digestion Kit (Merck, UK) was used. Per 100 μg precipitated protein, 30 μl_ extraction buffer I was added for resuspension and vortexed for 20 min. The pipetting and vortexing steps were repeated. Afterwards, 25 μl_ of digestion buffer and 1 μl_ of reducing agent were added and samples were incubated for 10 min at 37°C. Afterwards, samples were cooled to room temperature and 1 μl_ of blocking agent was added for further 10 min incubation. After addition of 8 μl_ trypsin (0.4 μg/μL, Promega, USA), the samples were incubated for 17 h at 37°C with shaking. Finally, the reaction was stopped by adding 1.1 μ l_ 8.8 M hydrochloric acid. The samples were stored at -80⁰C until nanoLC/MS^E analysis.

6. A total of 0.6 μg digested protein sample was loaded onto the nanoLC column, diluted in H₂O + 0.1% formic acid if necessary. Samples were injected three times followed by an injection of a blank or a digested protein standard.

7. Among the clinical samples quality controls obtained from one PBMC donor were prepared and run. These samples were included to monitor the reproducibility of the preparation and LC/MS^E performance.

Label-free nanoLC/MSE

The samples were separated using the split-less nano Ultra Performance LC (nanoUPLC) system from Waters (10 kpsi nanoAcquity, Waters, UK). Buffers used were: A) H₂O + 0.1% formic acid and B) Acetonitril + 0.1% formic acid (Fisher Scientific, UK). Prior to separation, the samples were desalted for 1 min with 100% buffer A using an online RP C18 trapping column (180 μm i.d., 20 mm length and 5 μm particle size, Waters, UK). Subsequently, the samples were separated using a C18 nanocolumn (75 μm i.d., 200 mm length, 1.7 μm particle size, Waters, UK). A two step gradient at a flow rate of 300 nL/min was used starting with 97% buffer A and 3% buffer B and ending with 5% buffer A and 95% buffer B. The nanoUPLC was coupled through a nanoESI online emitter (7 cm length and 10 μm tip; New Objective, USA) to the Q-TOF Premier™ (Waters, UK). Data were acquired in positive ion-, V- and MS^E mode. In the MS^E mode the collision cell switches every 0.6 s from low (4 eV) to high collision energy (20-43 eV) whereas the quadrupole is set to transfer all ions. As this mode generates fragments of peptides, it allows not only relative quantitative measurement of the peptides but also their identification. Using a LockSpray, mass accuracy was maintained throughout the analysis time. Glu-Fibrinopeptide B

(Sigma, Germany) served as reference compound and was infused and scanned all 30 s using the LockSpray apparatus. The whole system was operated using the MassLynx software v4.1 provided by Waters.

Data Processing

The generated raw data were processed with the ProteinLynx Global Server software v2.3 (Waters, UK). Databank searching was conducted using the human SwissProt v55 protein database (SIB, Switzerland). The total ion current (TIC) was used for data normalization.

Data Analysis

Processed data were exported to the software package R for filtering and analysis. Filtering guaranteed that only high confidence peptides were included in the analysis. The filtering criteria required the appearance of a peptide in at least two out of three injections per sample and in at least 80% of samples in any one of the groups. In addition, calculation of protein intensities was based on correlating peptides for each corresponding protein as described previously. Peptides were considered as correlating if the correlation coefficient was greater than 0.6 (Pearson's correlation). The peptide intensities of proteins passing the filtering criteria were summed in order to obtain the corresponding protein intensities. After log-transformation of the data to obtain an approximately normal distribution, a two-tailed t-test (unequal variances) was used to reveal statistically significant differentially-expressed proteins (p < 0.05) between the disease and control group. Because several proteins were measured in each sample, multiple hypotheses testing problems were accounted for by controlling the false discovery rate (FDR) according to Benjamini and Hochberg. Furthermore, protein intensity data were exported to SIMCA-P+ 10.5 (Umetrics, Sweden) to perform principal component analysis (PCA) on the cytosolic- and membrane-fraction derived data sets.

Results

This study aimed to find differences in protein expression of PBMCs between patients having bipolar I, bipolar II disorder and healthy controls. In order to identify the maximum possible number of proteins the proteome of these PBMCs was fractionated into a cytosolic- and membrane-enriched sub-proteome. Qualitative and relative quantitative information was obtained using a label-free nanoLC/MS^E approach. Statistically significant differences in protein expression between patients and controls were obtained by two-tailed t-test (unequal variances). The significance level was set to 5% (p < 0.05). Furthermore, multiple hypothesis testing was applied to control the FDR (q-value) according to Benjamini and Hochberg.

Cytosolic-Enriched Fraction After nanoLC/MS^E, data processing and data analysis, 1437 peptides were identified. These were assigned to 295. Twenty-nine percent of these were identified to be cytosolic proteins, 8% membrane proteins and 61% had another subcellular localisation or could not be assigned. Out of the 295 proteins, 49 showed statistically significant differences in expression. In Table 2, all 49 proteins are listed with their corresponding p- and q-values.

Table 2 Statistically significant proteins of the cytosolic-enriched fraction

Each of the 49 proteins is indicated by the corresponding SwissProt accession number and name. A p-value of 0.05 was set as significance level. The q-value refers to the FDR. FC is the fold change for each protein with a FC > 1, up-regulated in disease group and a FC < 1 down-regulated in disease group.

Membrane-Enriched Fraction

6 proteins indicated in the above mentioned analysis showed statistically significant differences in expression. These proteins are indicated in Table 3 by the corresponding SwissProt accession number and name. A p-value of 0.05 was set as significance level. The q-value refers to the FDR. FC is the fold change for each protein with a FC > 1, up-regulated in disease group and a FC < 1 down-regulated in disease group. The proteins marked by an asterisk (*) indicate those which were also detected in the cytosolic fraction listed in Table 2.

Table 3 Statistically significant proteins of the membrane-enriched fraction

Claims

1. Use of one or more first peptides selected from : Spliceosome RNA helicase BATl, Zinc finger CCCH domain-containing protein 13, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Purine nucleoside phosphorylase, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Dedicator of cytokinesis protein 7, Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase, Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin- IXb, Myosin-7, Myosin-11, Myosin-10, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG) and Natural killer cell-enhancing factor A (NKEF-A)) and Uncharacterized protein C12orf35, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

2. Use as defined in claim 1, wherein the first peptide is selected from : Spliceosome RNA helicase BATl, Transitional endoplasmic reticulum ATPase (VCP), Sterile alpha motif domain-containing protein 9-like, Rho guanine nucleotide exchange factor 17, Nuclear mitotic apparatus protein 1, Peptidyl-prolyl cis-trans isomerase A, Clathrin heavy chain 1, Uncharacterized protein KIAA1529, Genetic suppressor element 1, Stress-70 protein (mitochondrial precursor), Alpha-protein kinase 2, DNA polymerase nu, Proteasome activator complex subunit 1, Histone demethylase JARIDlA, M-phase phosphoprotein 1, Triosephosphate isomerase and Uncharacterized protein C12orf35.

3. Use as defined in claim 1, wherein the first peptide is selected from : Transitional endoplasmic reticulum ATPase (VCP), Alpha-protein kinase 2, Stress- 70 protein (mitochondrial precursor) and Myosin-Va.

4. Use as defined in any preceding claims, additionally comprising the use of one or more second peptides selected from: Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non- specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-l a ctate d e hyd rog e n ase A ch a i n , 60 k Da h eat sh ock p rotei n (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor and Leucine zipper protein 1.

5. Use of two or more of the second peptides as defined in claim 4, as a biomarker for bipolar I or bipolar II disorders, or predisposition thereto.

6. Use as defined in claim 5 wherein the second peptides are selected from : Heterogeneous nuclear ribonucleoprotein F, Signal-induced proliferation-associated 1-like protein, Phosphoglycerate kinase 1, Annexin A6, Desmuslin, Myomesin-1, Cytosolic non-specific dipeptidase, Uncharacterized protein C22orf30, Structural maintenance of chromosomes protein 2, Kinesin-like protein KIF7, Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain, 60 kDa heat shock protein (mitochondrial precursor), Heat shock protein HSP 90-beta, Endoplasmin precursor HSP90B1 Grp94, Malate dehydrogenase (mitochondrial precursor), Aconitate hydratase 2 (mitochondrial precursor), Aspartate aminotransferase (mitochondrial precursor), Receptor-type tyrosine-protein phosphatase S precursor, Phosphatidylinositol 4-kinase alpha, Eukaryotic translation initiation factor 3 subunit A, Tubulin alpha-lA chain, Peroxiredoxin-1 (also known as Proliferation-associated gene protein (PAG), Natural killer cell-enhancing factor A (NKEF-A)) Myosin-2, Myosin-Va, Myosin-4, Myosin-13, Myosin-IXb, Myosin-7, Myosin-11, Myosin-10, Leucine zipper protein 1, Purine nucleoside phosphorylase, Zinc finger CCCH domain-containing protein 13 and Dedicator of cytokinesis protein 7.

7. Use as defined in any of claims 4 to 6, wherein one of said second peptides is Aconitate hydratase 2 (mitochondrial precursor).

8. Use as defined in any of claims 1 to 7, wherein one or more of the biomarkers may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.

9. A method of diagnosing or monitoring bipolar I or bipolar II disorders, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, one or more of the first peptide biomarkers as defined in any of claims 1 to 3.

10. A method as defined in claim 9, additionally comprising detecting and/or quantifying, in a sample from a test subject, one or more of the second peptide biomarkers as defined in any of claims 4 to 7.

11. A method of diagnosing or monitoring bipolar I or bipolar II disorders, predisposition thereto, comprising detecting and/or quantifying, in a sample form a test subject, two or more of the second peptide biomarkers as defined in any of claims 4 to 7.

12. A method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to bipolar I or bipolar II disorders, comprising detecting and/or quantifying, in a sample from said subject, one or more of the first peptide biomarkers as defined in any of claims 1 to 3.

13. A method as defined to claim 12, additionally comprising detecting and/or quantifying, in a sample from said subject, one or more of the second peptide biomarkers as defined in any of claims 4 to 7.

14. A method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to bipolar I or bipolar II disorders, comprising detecting and/or quantifying, in a sample from said subject, two or more of the second peptide biomarkers as defined in any of claims 4 to 7.

15. A method as defined in any of claims 9 to 14, which is conducted on samples taken on two or more occasions from a test subject.

16. A method as defined in any of claims 9 to 15, further comprising comparing the level of the biomarker present in samples taken on two or more occasions.

17. A method as defined in any of claims 9 to 16, comprising comparing the amount of the biomarker in said test sample with the amount present in one or more samples taken from said subject prior to commencement of therapy, and/or one or more samples taken from said subject at an earlier stage of therapy.

18. A method as defined in any of claims 9 to 17, further comprising detecting a change in the amount of the biomarker in samples taken on two or more occasions.

19. A method as defined in any of claims 9 to 18, comprising comparing the amount of the biomarker present in said test sample with one or more controls.

20. A method as defined in claim 19, comprising comparing the amount of the biomarker in a test sample with the amount of the biomarker present in a sample from a normal subject.

21. A method as defined in any of claims 9 to 20, wherein samples are taken prior to and/or during and/or following therapy for bipolar I or bipolar II disorders.

22. A method as defined in any of claims 9 to 21, wherein samples are taken at intervals over the remaining life, or a part thereof, of a subject.

23. A method as defined in any of claims 9 to 22, wherein quantifying is performed by measuring the concentration of the peptide biomarker in the or each sample.

24. A method as defined in any of claims 9 to 23, wherein detecting and/or quantifying is performed by one or more methods selected from SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC or other LC or LC-MS-based technique.

25. A method as defined in any of claims 9 to 24, wherein detecting and/or quantifying is performed using an immunological method.

26. A method as defined in any of claims 9 to 25, wherein the detecting and/or quantifying is performed using a biosensor or a microanalytical, microengineered, microseparation or immunochromatography system.

27. A method as defined in any of claims 9 to 26, wherein the biological sample is cerebrospinal fluid, whole blood, blood serum, plasma, urine, saliva, or other bodily fluid, or breath, condensed breath, or an extract or purification therefrom, or dilution thereof.

28. A kit for monitoring or diagnosing bipolar I or bipolar II disorders, comprising a biosensor capable of detecting and/or quantifying one or more of the first peptide biomarkers as defined in any of claims 1 to 3.

29. A kit as defined in claim 28, additionally comprising a biosensor capable of detecting and/or quantifying one or more of the second peptide biomarkers as defined in any of claims 4 to 7.

30. A kit for monitoring or diagnosing bipolar I or bipolar II disorders, comprising a biosensor capable of detecting and/or quantifying two or more of the second peptide biomarkers as defined in any of claims 4 to 7.