Method for diagnosing diseases and disorders associated with altered orexin-a levels

Abstract

The present invention relates to in vitro methods for diagnosing or prognosing diseases and disorders associated with altered orexin-A levels. It further relates to in vitro methods for monitoring the effect of a treatment or the evolution of a disease or disorder associated with altered orexin-A levels. It also relates to the use of orexin-A fragment as biomarker for the in vitro diagnosis of diseases and disorders associated with altered orexin-A levels.

Description

METHOD FOR DIAGNOSING DISEASES AND DISORDERS ASSOCIATEDWITH ALTERED OREXIN-A LEVELSFIELD OF INVENTION

[0001] The present invention relates to in vitro methods for diagnosing or prognosing diseases and disorders associated with altered orexin-A levels. It further relates to in vitro methods for monitoring the effect of a treatment or the evolution of a disease or disorder associated with altered orexin-A levels. It also relates to the use of orexin-A fragment as biomarker for the in vitro diagnosis of diseases and disorders associated with altered orexin-A levels.BACKGROUND OF INVENTION

[0002] Orexin, also known as prepro-orexin or preprohypocretin, is a precursor protein which gives rise to two mature neuropeptide isoforms: orexin-A, also known as hypocretin- 1, and orexin-B, also known as hypocretin-2. The orexin-A and orexin-B peptides bind to the two 7-transmembrane G-protein coupled receptors: orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R); with orexin-A binding to both OX1R and OX2R with high affinity, while orexin-B binds selectively to OX2R with a similar high affinity. The orexin peptides have roles in regulating feeding and drinking behavior, metabolism, the sleep-wake cycle, and the endocrine system.

[0003] It has been demonstrated that an orexin deficiency results in the sleep disorder narcolepsy in many mammalian species, including mice, dogs, and humans, suggesting that the orexin system is particularly important for normal regulation of sleep / wakefulness states, and especially for maintenance of wakefulness. On the opposite, it has been demonstrated that increased orexin levels are associated to Alzheimer’s disease and mild cognitive impairment, suggesting a role of the orexin system in neuropsychiatric diseases as well.

[0004] The standard technique for quantifying orexin-A, which is used to diagnose type 1 narcolepsy, implies measuring the levels of orexin-A in the cerebrospinal fluid of patients by I125radioimmunoassay (RIA). However, this technique suffers from numerous disadvantages such as interferences due to cross-reactions with matrix constituents, high variability between batches, low precision and accuracy, but most of all, it requires special precautions for the use and disposal of radioactive material.

[0005] More recently, a quantitative liquid chromatography combined with mass spectrometry (LC-MS) assay of orexin-A was developed, based on a multiple reaction monitoring (MRM) approach, and demonstrated correlation with the RIA method (Hirtz et al., Sci Rep, 2016, 6:25162). This method was based on the use of the human orexin- A peptide (with a pyroglutamic acid as N-terminal amino acid, an amidated leucine as C- terminal amino acid, and disulfide bridges at positions C6-C12 and C7-C14) as standard. However, it also possesses some drawbacks such as low levels of orexin-A peptide itself compared to its different metabolites, which are also measured by RIA. In addition, this method demonstrated major discrepancies as compared to the RIA method, making it unreliable for diagnosing patients.

[0006] Therefore, novel and improved methods are needed to better quantify orexin-A levels in patients and allow an accurate diagnosis of the numerous diseases and disorders associated with altered orexin-A levels, such as sleep disorders and neuropsychiatric diseases.

[0007] The Inventors of the present invention identified a novel fragment of human orexin-A, which corresponds to the major form of orexin-A, that can be for example detected in the RIA assay. Using for example a method involving liquid chromatography coupled to mass spectrometry, they were able to precisely quantify the concentration of this novel fragment of orexin-A. In addition, the Inventors demonstrated that this method offered a good correlation with the standard RIA assay, in analyzing cerebrospinal fluid samples from a cohort of patients.

[0008] Thus, the present invention provides valuable methods for diagnosing or prognosing a disease or disorder associated with altered orexin-A levels in a subject, formonitoring the effect of a treatment for a disease or disorder associated with altered orexin-A levels in a subject, for monitoring the evolution of a disease or disorder associated with altered orexin-A levels in a subject, and for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels.SUMMARY

[0009] The present invention relates to an in vitro method for diagnosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), if said subject is affected by said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0010] The present invention also relates to an in vitro method for prognosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), if said subject is at risk of developing said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5

[0011] The present invention further relates to an in vitro method for monitoring the effect of a treatment on a subject diagnosed with a disease or disorder associated with altered orexin-A levels wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) assessing, from the comparison of step b), if said subject is responsive to the treatment for said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5

[0012] The present invention also relates to an in vitro method for monitoring the evolution of a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) assessing, from the comparison of step b), the evolution of said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5

[0013] The present invention also relates to an in vitro method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, andc) determining, from the comparison of step b), the appropriate treatment to be administered to the subject, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5

[0014] The sample previously obtained from the subject may be a bodily fluid.

[0015] The bodily fluid may be selected from cerebrospinal fluid, blood, plasma, serum, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat and alveolar macrophages, preferably the bodily fluid is cerebrospinal fluid.

[0016] The level or concentration of orexin-A fragment may be measured by immunohistochemistry, immunoassay, enzyme-linked immunosorbent assay (ELISA), enzymatic methods, western-blot, mass spectrometry, liquid chromatography, or flow cytometry, preferably by liquid chromatography coupled to mass spectrometry (LC-MS).

[0017] The reference level or concentration of orexin-A fragment may be derived from the measurement of the level or concentration of orexin-A fragment in a sample from a reference subject or in samples from a population of reference subjects.

[0018] The level or concentration of orexin-A fragment in the sample from the reference subject or in samples from the population of reference subjects and the level or concentration of orexin-A fragment in the sample previously obtained from the subject may be measured by the same method, preferably by liquid chromatography coupled to mass spectrometry (LC-MS).

[0019] The sample previously obtained from the subject and the sample from the reference subject or the samples from the population of reference subjects may be samples from the same bodily fluid, preferably cerebrospinal fluid.

[0020] The present invention also relates to the use of orexin-A fragment as a biomarker for the in vitro diagnosis of a disease or disorder associated with altered orexin-A levels,wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0021] The disease or disorder associated with altered orexin-A levels may be a sleep disorder with orexin-A deficiency, or a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0022] The sleep disorder with orexin-A deficiency may be selected from type I narcolepsy disease, insomnia, hypersomnia, parasomnia, restless legs syndrome, and sleep apnea.

[0023] The neuropsychiatric and metabolic disease with orexin-A deficiency may be selected from appetite disorders, depression, anxiety, mood disorders, addictions, dementia, Alzheimer’s disease and related disorders, multiple sclerosis, epilepsy, Parkinson’s disease and related disorders, schizophrenia, type 2 diabetes, migraine, autism, and post-traumatic stress disorder (PTSD).DEFINITIONS

[0024] In the present invention, the following terms have the following meanings:

[0025] “A”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0026] “About”, preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0027] “Diagnosis” or “diagnosing” refers to medical diagnosis, i.e., the process of identifying or determining a pathological state, disease or condition, such as a disease or disorder associated with altered orexin-A levels.

[0028] “Level” refers to the measured amount, level, quantity or concentration, whether relative or absolute, of a biomarker in a sample from a subject. The level of a biomarkercan be determined relative to a control molecule in a sample, or relative to the level of the same biomarker in a reference population (i.e., relative to a reference level).

[0029] “Measuring” or “measurement”, or alternatively “detecting” or “detection”, mean assessing the presence, absence, level, quantity, or amount of a given substance, e.g., orexin-A fragment. “Measuring” or “measurement”, or alternatively “detecting” or “detection” as used herein include the derivation of the qualitative or quantitative concentration of said substance, in particular said orexin-A fragment.

[0030] “Orexin” refers to a hypothalamic neuropeptide precursor protein that gives rise to two mature neuropeptides: orexin-A and orexin-B, by proteolytic processing. Human orexin typically refers to the protein referenced as NP_001515 in the NCBI databases on July 1st, 2024. In the NCBI databases (https: / / www.ncbi.nlm.nih.gov), the reference human orexin gene sequence corresponds to NCBI Gene ID: 3060, as updated on September 18, 2024. The human orexin gene consists of two exons on chromosome 17q21.2. Orexin transcript encompasses 577 nucleotides and encodes a 131 amino acid protein. The reference human orexin protein sequence corresponds to SEQ ID NO: 1. Alternatives names for orexin include “hypocretin”, “Hypocretin Neuropeptide Precursor”, “HCRT”, “PPOX”, “OX”, “Prepro-Orexin”, “Preprohypocretin”, “Hypocretin Neuropeptide”, “Orexin precursor”, “NRCLP1”, “PPORX”, as non-limiting examples.

[0031] “Orexin-A” also known as “hypocretin- 1” refers to a hypothalamic neuropeptide protein which regulates arousal, wakefulness, and appetite. Human orexin-A corresponds to amino acid residues 34 to 66 of human orexin. Human orexin-A typically refers to the protein referenced as 1RO2_A in the NCBI databases on December 1st, 2020. The reference human orexin-A protein is a 33 amino acid protein characterized by a pyroglutamic acid residue at position 1, an amidated leucine residue at position 33, and two disulfide bridges between the cysteine residues at position 6 and 12, and between the cysteine residues at position 7 and 14. The reference human orexin-A protein sequence corresponds to SEQ ID NO: 2.

[0032] “Prognosis” refers to the likelihood or expected progression of a disease or disorder associated with altered orexin-A levels, including whether the signs and symptoms will improve or worsen (and how quickly) or remain stable over time; expectations of quality of life, such as the ability to carry out daily activities; the potential for complications and associated health issues; and the likelihood of survival (including life expectancy). Accordingly, a “good prognosis” or “positive prognosis” refers to a beneficial clinical outcome such as long-term survival; and a “bad prognosis” or “negative prognosis” refers to a negative clinical outcome such as worsening of the disease or death.

[0033] “Reference level” refers to the amount, level, quantity or concentration of a biomarker in a sample from a reference subject or to the mean or median level of a biomarker in samples from several subjects in a reference population. A reference level can be a normal reference level or a disease-state reference level. A normal reference level is the level of a biomarker in a substantially healthy subject or in several substantially healthy subjects in a reference population, such as a subject (or several subjects) who is / are not suffering from or otherwise was / were not diagnosed with, a disease or disorder associated with altered orexin-A levels such as a sleep disorder with orexin-A deficiency, or a neuropsychiatric and metabolic disease with orexin-A deficiency. A disease-state reference level is the level of a biomarker in a diseased subject or in several diseased subjects in a reference population, such as a subject (or several subjects) who is / are suffering from or otherwise was / were diagnosed with, a disease or disorder associated with altered orexin-A levels such as a sleep disorder with orexin-A deficiency, or a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0034] “Sample” refers to any biological material obtained via suitable methods known to the person skilled in the art from a subject. The sample may be collected in a clinically acceptable manner, e.g., in a way that cells, nucleic acids (such as DNA and RNA), and proteins are preserved. A “sample” may be a body tissue and / or a bodily fluid, preferably a bodily fluid. Examples of bodily fluids include, but are not limited to, cerebrospinal fluid, blood, plasma, serum, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovialfluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat and alveolar macrophages. The sample may be cerebrospinal fluid.

[0035] “Subject” refers to an animal, preferably a mammal, more preferably a human. The subject may be an animal, preferably a mammal, more preferably a primate. The subject may be a human. The subject may be a male. The subject may be a female. The subject may be a child, an adolescent or an adult. The subject may be a patient, i.e., a recipient of health care services.

[0036] “Substantially healthy”, with reference to a subject or a population of subjects, means that said subject (or subjects in the population) is / are not suffering from or otherwise was / were not diagnosed with, a disease or disorder associated with altered orexin-A levels such as a sleep disorder with orexin-A deficiency, or a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0037] “Treating” or “treatment” refers to a therapeutic treatment, to a prophylactic (or preventive) treatment, or to both a therapeutic treatment and a prophylactic (or preventive) treatment; wherein the object is to prevent or slow down or lessen a disease or disorder associated with altered orexin-A levels, its associated symptoms and / or complications. A subject may be successfully "treated" if, after receiving treatment as described herein, including adapted care as described herein, the subject shows at least one of the following: relief to some extent of one or more of the symptoms and / or complications associated with the disease or disorder associated with altered orexin-A levels, and / or improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the symptoms and / or complications associated with the disease or disorder associated with altered orexin-A levels are readily measurable by routine procedures familiar to a physician.DETAILED DESCRIPTION

[0038] The present invention relates to an in vitro method for diagnosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises or consists of the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), if said subject is affected by said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0039] As used herein “disease or disorder associated with altered orexin-A levels’’ refers to diseases or disorders in which the level or concentration of orexin-A differs from the level or concentration of orexin-A in a substantially healthy subject or a population of substantially healthy subjects. In particular, the disease or disorder associated with altered orexin-A levels may be a sleep disorder with orexin-A deficiency, in which the level of orexin-A fragment may be lower than the level of orexin-A fragment measured in a substantially healthy subject. Alternatively, the disease or disorder associated with altered orexin-A levels may be a neuropsychiatric and metabolic disease with orexin-A deficiency, in which the level of orexin-A fragment may be lower or higher than the level of orexin-A fragment measured in a substantially healthy subject.

[0040] Another object of the invention is an in vitro method for prognosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises or consists of the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, andc) determining, from the comparison of step b), if said subject is at risk of developing said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0041] Another object of the invention is an in vitro method for monitoring the effect of a treatment on a subject diagnosed with a disease or disorder associated with altered orexin-A levels, wherein said method comprises or consists of the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) assessing, from the comparison of step b), if said subject is responsive to the treatment for said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0042] Another object of the invention is an in vitro method for monitoring the evolution of a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises or consists of the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, c) assessing, from the comparison of step b), the evolution of said disease or disorder associated with altered orexin-A levels in said subject, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0043] Another object of the invention is an in vitro method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises or consists of the steps of:a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, c) determining, from the comparison of step b), the appropriate treatment to be administered to the subject, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0044] The orexin-A fragment may consist in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 3. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 4. The orexin-A fragment may consist in the amino acid sequence as set forth in SEQ ID NO: 5.

[0045] The orexin-A fragment may consist in the full amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 3. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 4. The orexin-A fragment may consist in the full amino acid sequence as set forth in SEQ ID NO: 5.

[0046] The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for diagnosing a disease or disorder associated with altered orexin-A levels in a subject. The amino acid sequence as set forthin any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for prognosing a disease or disorder associated with altered orexin-A levels in a subject. The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for monitoring the effect of a treatment on a subject diagnosed with a disease or disorder associated with altered orexin-A levels. The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels in a subject.

[0047] The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for diagnosing the same disease or disorder associated with altered orexin-A levels in a subject. The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for prognosing the same disease or disorder associated with altered orexin-A levels in a subject. The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for monitoring the effect of a treatment on a subject diagnosed with the same disease or disorder associated with altered orexin-A levels. The amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may be used alternatively for determining the appropriate treatment to be administered to a subject diagnosed with the same disease or disorder associated with altered orexin-A levels in a subject.

[0048] The method may comprise a step of providing a sample from a subject.

[0049] The sample may be a bodily fluid. The sample may be a bodily fluid selected from cerebrospinal fluid, blood, plasma, serum, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat and alveolar macrophages.The sample may be a bodily fluid selected from cerebrospinal fluid, blood, plasma, serum, saliva, tears. The sample may be cerebrospinal fluid.

[0050] The sample may have been previously taken from the subject, i.e., the methods as described herein may not comprise an active step of recovering a sample from the subject. Consequently, the methods as described herein may be non-invasive methods, i.e., the methods as described herein may be in vitro methods.

[0051] The method as described herein may comprise a step of processing the sample. This step of processing the sample may apply to any sample as described herein, i.e., the sample previously obtained from the subject, the sample obtained from the subject at an earlier timepoint, the sample from the reference subject, and the samples from the population of reference subjects

[0052] The step of processing the sample may comprise centrifuging the sample at 10,000 g for 10 minutes and collecting the supernatant for analysis.

[0053] The method as described herein comprises a step of measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject.

[0054] The level or concentration of orexin-A fragment may be measured by any conventional method known to the one skilled in the art. Such methods include for example immunohistochemistry, immunoassay (including radioimmunoassay (RIA)), enzyme-linked immunosorbent assay (ELISA), enzymatic methods, western-blot, mass spectrometry including chromatography-assisted mass spectrometry (such as gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography coupled to mass spectrometry (LC-MS)), liquid chromatography including high- performance liquid chromatography (HPLC), or flow cytometry.

[0055] The level or concentration of orexin-A fragment may be measured by immunohistochemistry, immunoassay, enzyme-linked immunosorbent assay (ELISA), enzymatic methods, western-blot, mass spectrometry, liquid chromatography, or flow cytometry. The level or concentration of orexin-A fragment may be measured by liquid chromatography coupled to mass spectrometry (LC-MS).

[0056] It will be readily understood by one skilled in the art that the level or concentration of orexin-A fragment may vary depending on how the sample is processed. Any methods to measure the level or concentration of orexin-A fragment, and any associated reference levels described herein, are given for exemplary purposes. However, one skilled in the art will understand that both the level or concentration of orexin-A fragment, and the reference level or concentration of orexin-A fragment shall be measured using the same method to have comparable data.

[0057] The disease or disorder associated with altered orexin-A levels may be a sleep disorder with orexin-A deficiency or a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0058] The sleep disorder with orexin-A deficiency may be selected from type I narcolepsy disease, insomnia, hypersomnia, parasomnia, restless legs syndrome, and sleep apnea. The sleep disorder with orexin-A deficiency may be type I narcolepsy disease.

[0059] The neuropsychiatric and metabolic disease with orexin-A deficiency may be selected from appetite disorders, depression, anxiety, mood disorders, addictions, dementia, Alzheimer’s disease and related disorders, multiple sclerosis, epilepsy, Parkinson’s disease and related disorders, schizophrenia, migraine, type 2 diabetes, autism, and post-traumatic stress disorder (PTSD).

[0060] The method as described herein comprises a step of comparing the level or concentration of orexin-A fragment in said sample from the subject with a reference level or concentration of orexin-A fragment.

[0061] The reference level or concentration of orexin-A fragment may be derived from the measurement of the level or concentration of orexin-A fragment in a sample from a reference subject. The reference level or concentration of orexin-A fragment may be derived from the measurement of the level or concentration of orexin-A fragment in samples from a population of reference subjects. The reference level or concentration of orexin-A fragment may be derived from the measurement of the level or concentration oforexin-A fragment in a sample from a reference subject, or in samples from a population of reference subjects.

[0062] As used herein, a “reference population” is a population comprising at least 2, preferably at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100 or more reference subjects.

[0063] The reference subject or the population of reference subjects may be substantially healthy subject(s). The reference subject or the population of reference subjects may be subject(s) not suffering from and / or not diagnosed with a disease or disorder associated with altered orexin-A levels.

[0064] The reference subject or the population of reference subjects may be diseased subjects or patients. The reference subject or the population of reference subjects may be subjects suffering from a disease which is not a disease or disorder associated with altered orexin-A levels. The reference subject or the population of reference subjects may be subjects suffering from a disease or disorder associated with altered orexin-A levels.

[0065] The reference subject or the population of reference subjects may be a subject suffering from a disease or disorder associated with altered orexin-A levels that has not received a treatment for a disease or disorder associated with altered orexin-A levels.

[0066] The reference subject or the population of reference subjects may be a subject suffering from a disease or disorder associated with altered orexin-A levels that receives a treatment for a disease or disorder associated with altered orexin-A levels.

[0067] Alternatively, the reference level or concentration of orexin-A fragment may be derived from the measurement of the level or concentration of orexin-A fragment in a sample obtained from the subject at an earlier timepoint.

[0068] The sample previously obtained from the subject and the sample from a reference subject or the samples from a population of reference subjects may be samples from the same bodily fluid. The sample previously obtained from the subject and the sample from a reference subject or the samples from a population of reference subjects may be samples from cerebrospinal fluid.

[0069] The sample previously obtained from the subject and the sample obtained from the subject at an earlier timepoint may be samples from the same bodily fluid. The sample from the subject and the sample obtained from the subject at an earlier timepoint may be samples from cerebrospinal fluid.

[0070] The level or concentration of orexin-A fragment in the sample from the reference subject or in samples from the population of reference subject and the level or concentration of orexin-A fragment in the sample previously obtained from the subject may be measured by the same method, preferably by liquid chromatography coupled to mass spectrometry (LC-MS).

[0071] Based on a quantification using I125radioimmunoassay (RIA) with the kit from Phoenix Pharmaceuticals, an orexin-A concentration above 200 pg / mL is considered a normal orexin-A concentration, an orexin-A concentration below 110 pg / mL is considered a low orexin-A concentration, and an orexin-A concentration below 50 pg / mL is considered an absence of orexin-A.

[0072] The level or concentration of orexin-A measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects may be of about 200 pg / mL, as determined by I125RIA with the kit from Phoenix Pharmaceuticals .

[0073] The level or concentration of orexin-A measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be lower than 110 pg / mL, as determined by I125RIA with the kit from Phoenix Pharmaceuticals, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0074] The level or concentration of orexin-A measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be higher than 200 pg / mL, as determined by I125RIA with the kit from Phoenix Pharmaceuticals, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0075] The level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects may be of about 0.23, as determined by mass spectrometry.

[0076] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0077] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be lower than about 0.23, such as about 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, or 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0078] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be comprised between about 0.14 and about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0079] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be higher than about 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels may be a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0080] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be higher than about 0.14, such as about 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22,0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38,0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54,0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70,0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.,77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, or 1.10 as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels may be a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0081] The level or concentration of orexin-A fragment measured in a sample from a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be comprised between about 0.14 and about 1.1, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels may be a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0082] Comparing the level or concentration of orexin-A fragment with a reference level or concentration of orexin-A fragment may include a comparison “by eye”.

[0083] Comparing the level or concentration of orexin-A fragment with a reference level or concentration of orexin-A fragment may include one or more forms of statistical analysis.

[0084] Examples of such statistical analysis include, but are not limited to, regression analysis, univariate analysis, multivariate analysis, variation calculations, best-fit analysis, curve fitting, extrapolation, interpolation, least squares, mean calculations, simulation analysis, logrank test, Kaplan-Meier estimator, and the like.

[0085] Comparing the level or concentration of orexin-A fragment with a reference level or concentration of orexin-A fragment may comprise or consist of comparing a statistical or mathematical representation (such as, e.g., absolute values, mean, median, or regression) of the level or concentration of orexin-A fragment with a statistical or mathematical representation (such as, e.g., absolute values, mean, median, or regression) of the reference level or concentration of orexin-A fragment.

[0086] The difference between the level or concentration of orexin-A fragment and the reference level or concentration of orexin-A fragment may be different with statistical significance.

[0087] By “different with statistical significance”, it is meant that, in a statistical analysis, the difference between the level or concentration of orexin-A fragment, and the reference level or concentration of orexin-A fragment shows a p value at or below 0.05, 0.04, 0.03, 0.02, 0.01, or less.

[0088] Comparing the level or concentration of orexin-A fragment with a reference level or concentration of orexin-A fragment may be carried out manually or computer-assisted. Thus, comparing the level or concentration of orexin-A fragment with a reference level or concentration of orexin-A fragment may be carried out by a computing device. The values of the measured level or concentration of orexin-A fragment in the sample obtained from the subject and the reference level or concentration of orexin-A fragment may be compared to each other and said comparison can be automatically carried out by a computer program executing an algorithm for the comparison. The computer program carrying out said evaluation will provide the desired assessment in a suitable output format. For a computer-assisted comparison, the value of the measured level or concentration of orexin-A fragment may be compared to values corresponding to suitable reference levels or concentration of orexin-A fragment which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e., automatically provide the desired assessment in a suitable output format.

[0089] Appreciation of the difference between the level or concentration of orexin-A fragment, and the reference level or concentration of orexin-A fragment may be left to the physician who is able to interpret a level or concentration of orexin-A fragment, in comparison with a reference level or concentration of orexin-A fragment.

[0090] The method for diagnosing a disease or disorder associated with altered orexin-A levels in a subject as described herein comprises a step of determining if the subject is affected with a disease or disorder associated with altered orexin-A levels.

[0091] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease ordisorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0092] The term “substantially lower” denotes a sufficiently high degree of difference between the level or concentration of orexin-A fragment measured in the sample from the subject and the reference level or concentration of orexin-A fragment, such as, different with statistical significance.

[0093] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0094] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0095] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0096] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, such as substantially lower than about 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16,0.15, or 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0097] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is comprised between about 0.14 and about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0098] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0099] The term “substantially higher” denotes a sufficiently high degree of difference between the level or concentration of orexin-A fragment measured in the sample from the subject and the reference level or concentration of orexin-A fragment, such as, different with statistical significance.

[0100] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0101] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in asample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0102] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0103] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is comprised between about 0.14 and about 1.1, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels may be a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0104] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the reference level or concentration of orexin-A fragment.

[0105] The term “substantially similar” denotes a sufficiently low degree of difference between the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject and the reference level or concentration of orexin- A fragment.

[0106] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population.

[0107] The subject may be diagnosed as being affected with a disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a subject suffering from a disease or disorder associated with altered orexin-A levels, or samples from a population of subjects suffering from a disease or disorder associated with altered orexin-A levels.

[0108] A subject with a level or concentration of orexin-A fragment of about 0.23, as determined by mass spectrometry, may be considered as not affected with a disease or disorder associated with altered orexin-A levels.

[0109] The method for prognosing a disease or disorder associated with altered orexin-A levels in a subject as described herein comprises a step of determining if the subject is at high or low risk of developing said disease or disorder associated with altered orexin-A levels.

[0110] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0111] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0112] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower thanthe level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0113] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0114] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, such as substantially lower than about 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, or 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0115] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is comprised between about 0.23 and about 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0116] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0117] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragmentmeasured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0118] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0119] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0120] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0121] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0122] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0123] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0124] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0125] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0126] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher thanabout 0.14, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0127] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is comprised between about 0.14 and about 1.1, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0128] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0129] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a reference subject or samples from a reference population, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0130] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0131] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0132] The subject may be determined at high risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0133] The subject may be determined at low risk of developing said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0134] The method for monitoring the effect of a treatment on a disease or disorder associated with altered orexin-A levels in a subject as described herein comprises a step of assessing if the subject is responsive to the treatment for said disease or disorder associated with altered orexin-A levels.

[0135] The subject may be considered as responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the reference level or concentration of orexin-A fragment, wherein saiddisease or disorder associated with altered orexin-A levels is a sleep disorder with orexin- A deficiency.

[0136] The subject may be considered as responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0137] The subject may be considered as responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0138] The subject may be considered as responsive to the treatment for said disease or disorder associated with altered orexin-A levels if after receiving treatment as described herein, the subject shows improvement in the symptoms and / or complications associated with the disease or disorder associated with altered orexin-A levels and / or improvement in quality-of-life issues.

[0139] The subject may be considered as responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0140] Alternatively, the subject may be considered as non-responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the reference level or concentration of orexin-A fragment.

[0141] The subject may be considered as non-responsive to the treatment for said disease or disorder associated with altered orexin-A levels if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially similar to the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint.

[0142] The method for monitoring the evolution of a disease or disorder associated with altered orexin-A levels in a subject as described herein comprises a step of assessing the evolution of said disease or disorder associated with altered orexin-A levels in the subject.

[0143] The disease or disorder associated with altered orexin-A levels may be considered as progressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0144] The disease or disorder associated with altered orexin-A levels may be considered as progressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0145] The disease or disorder associated with altered orexin-A levels may be considered as regressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0146] The disease or disorder associated with altered orexin-A levels may be considered as regressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0147] The disease or disorder associated with altered orexin-A levels may be considered as progressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0148] The disease or disorder associated with altered orexin-A levels may be considered as progressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment measured in a sample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0149] The disease or disorder associated with altered orexin-A levels may be considered as regressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0150] The disease or disorder associated with altered orexin-A levels may be considered as regressing if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher or substantially lower than the reference level or concentration of orexin-A fragment measured in asample obtained from the subject at an earlier timepoint, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0151] The method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels comprises a step of determining the appropriate treatment to be administered to the subject.

[0152] The method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels may comprise a step of determining if a subject is a good responder to said appropriate treatment.

[0153] The method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels may comprise a step of determining personalized care for the subject.

[0154] The appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels may be selected from orexin receptor agonists and orexin receptor antagonists.

[0155] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0156] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0157] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0158] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower that about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0159] The appropriate treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0160] The appropriate treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0161] The appropriate treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a substantially healthysubject or in samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0162] The appropriate treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0163] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0164] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0165] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0166] The appropriate treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0167] The orexin receptor agonist may be selected from firazorexton, suntinorexton, TAK 360, ALKS-2680 and ORX750.

[0168] As used herein, “orexin receptor agonist” refers to substances that bind to and functionally activate orexin receptors OX1R and / or OX2R.

[0169] Firazorexton, also known as TAK-994, is an orexin type 2 receptor (OX2R) agonist, registered under CAS number 2274802-95-6. Firazorexton is a small molecule, which chemical name is N-[(2S,3S)-2-[[3-(3,5-difhiorophenyl)-2-fluorophenyl]methyl]- l-(2-hydroxy-2-methylpropanoyl)pyrrolidin-3-yl]methanesulfonamide.

[0170] Suntinorexton, also known as TAK-861, is a selective agonist of the orexin 0X2 receptor, registered under CAS number 2274802-89-8. Suntinorexton is a small molecule, which chemical name is N-[(2S,3S)-2-[[2-fluoro-3-(3-fluorophenyl)phenyl]methyl]-l- (2-hydroxy-2-methylpropanoyl)pyrrolidin-3-yl]ethanesulfonamide.

[0171] As used herein, “orexin receptor antagonist” refers to substances that bind to and inhibit the action of orexin receptors 0X1R and / or OX2R.

[0172] Orexin receptor antagonists may be selected from daridorexant, suvorexant, and lemborexant.

[0173] Daridorexant, also known as ACT-541468, Quviviq™ or nemorexant, is a selective dual antagonist of orexin receptors OX1R and OX2R, registered under CAS number 1505484-82-1. Daridorexant is a small molecule, which chemical name is [(2S)- 2-(5-Chloro-4-methyl-lH-benzimidazol-2-yl)-2-methylpyrrolidin-l-yl]-[5-methoxy-2- (triazol-2-yl)phenyl] methanone .

[0174] Suvorexant, also known as MK-4305 or Belsomra®, is a selective dual antagonist of orexin receptors OX1R and OX2R, registered under CAS number 1030377-33-3. Suvorexant is a small molecule, which chemical name is [(7R)-4-(5-chloro-l,3- benzoxazol-2-yl)-7-methyl- 1,4-diazepan- 1-yl] [5-methyl-2-(2H- 1,2,3-triazol- 2-yl)phenyl] methanone .

[0175] Lemborexant, also known as E-2006 or Dayvigo®, is a dual antagonist of the orexin receptors OX1R and OX2R, registered under CAS number 1369764-02-2. Lemborexant is a small molecule, which chemical name is (lR,2S)-2-[(2,4- Dimethylpyrimidin-5-yl)oxymethyl]-2-(3-fluorophenyl)-N-(5-fluoropyridin-2- yl)cyclopropane- 1 -carboxamide.

[0176] Another object of the present invention is a method for treating a disease or disorder associated with altered orexin-A levels in a subject in need thereof, said method comprising the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, c) determining, from the comparison of step b), if said subject is affected by said disease or disorder associated with altered orexin-A levels, d) administering a treatment to said subject,wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0177] Administering a treatment to the subject determined as being affected by said disease or disorder associated with altered orexin-A levels may comprise or consist of providing an adapted care.

[0178] The method of treatment comprises a step of determining if said subject is affected by said disease or disorder associated with altered orexin-A levels. Said step may be conducted as described herein above.

[0179] The treatment to be administered to the subject may be selected from orexin receptor agonists and orexin receptor antagonists as described herein.

[0180] The subject to be treated is administrated at least once with the therapeutically effective amount of an orexin receptor agonist or an orexin receptor antagonist as described herein.

[0181] The terms “therapeutically effective amount” refers to an amount of the orexin receptor agonist or an orexin receptor antagonist as described herein, effective to achieve a particular biological result.

[0182] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0183] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0184] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0185] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency.

[0186] The treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0187] The treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0188] The treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or insamples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0189] The treatment to be administered to the subject may be an orexin receptor antagonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0190] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0191] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0192] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0193] The treatment to be administered to the subject may be an orexin receptor agonist if the level or concentration of orexin-A fragment measured in the sample previouslyobtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency.

[0194] Another object of the present invention is an orexin receptor agonist or an orexin receptor antagonist as described herein for use in the treatment of a disease or disorder associated with altered orexin-A levels.

[0195] The orexin receptor agonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin- A fragment.

[0196] The orexin receptor agonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin A fragment measured in a sample from a reference subject or in samples from a population of reference subjects.

[0197] The orexin receptor agonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects.

[0198] The orexin receptor agonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a sleep disorder with orexin-A deficiency, if thelevel or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry.

[0199] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the reference level or concentration of orexin-A fragment.

[0200] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects.

[0201] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects.

[0202] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially higher than about 0.23, as determined by mass spectrometry.

[0203] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the reference level or concentration of orexin-A fragment.

[0204] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a reference subject or in samples from a population of reference subjects.

[0205] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than the level or concentration of orexin-A fragment measured in a sample from a substantially healthy subject or in samples from a population of substantially healthy subjects.

[0206] The orexin receptor antagonist may be used in the treatment of a disease or disorder associated with altered orexin-A levels in a subject, wherein said disease or disorder associated with altered orexin-A levels is a neuropsychiatric and metabolic disease with orexin-A deficiency, if the level or concentration of orexin-A fragment measured in the sample previously obtained from the subject is substantially lower than about 0.23, as determined by mass spectrometry.

[0207] Another object of the present invention is an orexin receptor agonist or an orexin receptor antagonist as described herein for use as a medicament.

[0208] The medicament may comprise, may consist essentially of or may consist of orexin receptor agonists or orexin receptor antagonists as described herein.

[0209] Another object of the present invention is the use of orexin receptor agonists or orexin receptor antagonists as described herein, for the manufacture of a medicament for the treatment of a subject, wherein the subject is diagnosed with disease or disorder associated with altered orexin-A levels as described herein.

[0210] Another object of the present invention is a pharmaceutical composition for the treatment of a disease or disorder associated with altered orexin-A levels, comprising an orexin receptor agonist or an orexin receptor antagonist as described herein.

[0211] The pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient.

[0212] The pharmaceutical composition may comprise, may consist essentially of or may consist of an orexin receptor agonist or an orexin receptor antagonist as described herein and at least one pharmaceutically acceptable excipient.

[0213] Pharmaceutically acceptable excipients that may be used in the pharmaceutical composition of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as, for example, human serum albumin, buffer substances such as, for example, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as, for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, poly acrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0214] As used herein, the term “consisting essentially of’, with reference to pharmaceutical composition or medicament, means that the orexin receptor agonist or an orexin receptor antagonist is the only one therapeutic agent or agent with a biologic activity within said pharmaceutical composition or medicament.

[0215] The present invention also relates to the use of orexin-A fragment as a biomarker for the in vitro diagnosis of a disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0216] The present invention also relates to the use of orexin-A fragment as a biomarker for the in vitro prognosis of a disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.BRIEF DESCRIPTION OF THE DRAWINGS

[0217] Figure 1 is a graph showing the concentration of Hcrt-1 detected by radioimmunoassay (RIA) across the fraction collection from ImL of cerebrospinal fluid. During the separation of proteins from a cerebrospinal fluid pool on a C18 column, fractions were collected every minute. These fractions were evaporated and resuspended in the orexin RIA kit buffer. The orexin assay is performed using the same RIA kit. A strong concentration of orexin was detected in fractions 23 and 24, and a weaker concentration was detected in fractions 29 and 33.

[0218] Figure 2 is a graph showing the concentration of Hcrt-1 detected by radioimmunoassay (RIA) across the fraction collection from ImL of cerebrospinal fluid and spiked with orexin standard. The same cerebrospinal fluid pool as in Figure 1 was spiked with standard chemically synthetized orexin-A. This resulted in a sharp increase in the concentration detected in fraction 33.

[0219] Figure 3 is a graph showing the liquid chromatography-mass spectrometry (LC- MS) analysis of fraction 24 by Mascot software. An ion at 610.6026m / z was fragmented at 47.6 minutes and could be correlated to a truncated form of orexin A using Mascot software.

[0220] Figure 4 is a graph showing an annotation of monoisotopic masses of tricharged precursor ion of fragmented orexin-A. Ions of monoisotopic mass were in perfect agreement with the Skyline software theoretical mass predictions.

[0221] Figure 5 is a graph showing a Parallel Reaction Monitoring (PRM) chromatogram of orexin-A fragment detected in 750 pL of fractionated Cerebrospinal Fluid.

[0222] Figure 6 is a scheme of the annotated ions monitored in PRM mode.

[0223] Figure 7 is a graph showing the linear regression of Radioimmunoassay concentration and orexin-A fragment by LC-MS ratio.

[0224] Figure 8 is a graph showing the comparison between Orexin-A detection using the Orexin-A Fragment Mass Spectrometry (MS) method and the Orexin-A quantification via Radioimmunoassay (RIA).

[0225] Figure 9 is graph showing the correlation between orexin-A measured by Radioimmunoassay (reference method) and orexin-A fragment measured by mass spectrometry in a patient cohort.

[0226] Figure 10A-D is a combination of graphs showing the comparison of Radioimmunoassay (RIA) and Orexin-A Fragment Mass Spectrometry (MS) for detecting orexin deficiency in patients with narcolepsy type 1 (NT1) (Fig. 10A), narcolepsy type 2 (NT2) (Fig. 10B), idiopathic hypersomnia (Fig. 10C), and a patient group with various neurological disorders excluding NT1 (NSH-others) (Fig. 10D).

[0227] Figure 11 is a graph showing the comparison between Orexin-A detection with Mass Spectrometry (MS) using the Orexin-A Fragment having the SEQ ID NO: 4 with disulfide bridges (XPLPDCCRQKTCSCRL) wherein X is a pyroglutamic acid, the Orexin-A Fragment having the SEQ ID NO: 6 with disulfide bridges (XPLPDCCRQKTCSCRL) wherein X a glutamine (Q) and the Orexin-A Fragment having the SEQ ID NO: 7 XPLPDCCRQKTCSCRL without disulfide bridges and wherein X is a glutamine (Q). MS areas are an average on CSF of 5 healthy patients.TABLE OF SEQUENCESEXAMPLES

[0228] The present invention is further illustrated by the following examples.1: Identification of orexin-Ain thefluidMaterials and MethodsStandards

[0229] Human orexin-A standard(pyroEPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL-NH2) (SEQ ID NO: 2) with two disulfide bridges C6-C12 and C7-C14 was purchased from Phoenix Scientific. Fragment of Human orexin-A standard (pyroEPLPDCCRQKTCSCRL) (SEQ ID NO: 4) comprising two isotope-labelled leucin amino acid residues containing 13C6 and two disulfide bridges C6-C12 and C7-C14 with a purity of > 90% by High-Performance Liquid Chromatography (HPLC), was purchased from Eurogentec.

[0230] Purchased standards of orexin-A were dissolved in water (water ULC-Mass Spectrometry (MS), ref. 23214102) / acetonitrile (acetonitrile ULC-Mass Spectrometry (MS) (ACN) ref.01204101) / formic acid (formic Acid ULC-Mass Spectrometry (MS) (FA) ref. 069141A8) (66.2 / 33.8 / 0.1, v / v / v) at 200 pg / mL. The solution was then separated into aliquots of 50 pL in LoBind tubes (Protein LoBind tube 1.5mL, ref. 022431081 Eppendorf, Le Pecq, France) and stored at -80 °C.Characteristics of subjects

[0231] In order to test the analytical method, a pool of Cerebrospinal Fluid (CSF) samples from patients (n=20) with various neurological disorders was used.

[0232] To test the relationship between the orexin-A fragment Mass Spectrometry (MS) detection and the Radioimmunoassay (RIA), 39 patients with diverse diagnosis and remaining Cerebrospinal Fluid (CSF) volume after Radioimmunoassay (RIA) orexin-Aquantification were also analyzed with the Mass Spectrometry (MS) method dedicated to orexin-A fragment detection.

[0233] To validate the interest of the orexin fragment detection, the following clinical population samples were set up:1) 52 drug-free patients (8 females, mean age: 27.7+ 18.7 and 14 men, mean age: 24.5 + 12.7) with typical type 1 narcolepsy (NT1) defined by the presence of sleepiness, clearcut cataplexy, human leukocyte antigen (HLA) DQB 1*0602 positivity, at least two sleep onset REM (rapid eye movement) periods and a mean sleep latency below 8 min during the multiple sleep latency test. All these patients underwent a lumbar puncture prior to the study and were found to have low Cerebrospinal Fluid (CSF) Orex-A / Hcrt-1 levels (<110 pg / ml) as determined by performing direct 1125 Radioimmunoassay (RIA);2) 6 type 2 narcolepsy (NT2);3) 13 idiopathic hypersomnia; and4) 44 drug-free patients (16 females, mean age: 38.1+ 16.4 and 6 men, mean age: 42.8 + 13.9) with various neurological disorders but not type 1 narcolepsy. These patients underwent a lumbar puncture prior to the study and were found to have Cerebrospinal Fluid (CSF) Hcrt-1 levels > 200 pg / ml as determined by performing direct 1125 Radioimmunoassay (RIA).

[0234] All the 115 patients gave their informed consent to having their samples / specimens stored in a registered biological collection (#DC-2008-417) at the Montpellier CHRU’s certified NFS 96-900 biobank (Ref: BB-0033-00031 www.biobanques.eu) to be measured in this study. Cerebrospinal Fluid (CSF) was collected in polypropylene tubes under standardized conditions. Each Cerebrospinal Fluid (CSF) sample was sent to the laboratory within 4 hours of being collected and centrifuged at 1000g for 10 minutes at 4°C. Cerebrospinal Fluid (CSF) was aliquoted into 1.5-mE polypropylene tubes and stored at -80°C until further analysis.Cerebrospinal Fluid (CSF) fractionation

[0235] Cerebrospinal Fluid (CSF) pool was centrifuged at 10,000 g for 10 minutes and the supernatant was used for analysis. One ml of Cerebrospinal Fluid (CSF) wereseparated by High-Performance Liquid Chromatography (HPLC) (Nexera 20AD, Shimadzu) at a flow rate of 1 ml / min and linear gradient of 1-60% acetonitrile / 0.1% formic acid over 40 minutes. Samples were separated by pBondapak C18 column 3.9 x 300 mm, 10 pM 125 A, (Waters Corporation, Milford, MA). Detection was performed with UV detector at 214nm. Fractions were collected by fraction collector (FRC-10A, Shimadzu) every 0.8 minute between 3 and 41 minutes and were dried up using a vacuum centrifuge system (SpeedVac, Labconco). Samples were stored at -20 °C until use.

[0236] In order to confirm the authentic hcrt-1 peptide, 400ng of orexin-A standard was spiked into Cerebrospinal Fluid (CSF) sample before Cerebrospinal Fluid (CSF) fractionation.Radioimmunoassay

[0237] Orexin-A quantification was performed in duplicate from Cerebrospinal Fluid (CSF) samples in all patients using 1125 Radioimmunoassay (RIA) kits from Phoenix Pharmaceuticals (Belmont, CA) following manufacturer’s instructions (ref: RK-003-30). Cerebrospinal Fluid (CSF) Orexin-A levels below 110 pg / ml were considered as low, intermediate between 110 and 200, and normal when over 200pg / ml. All values were back-referenced to Stanford reference samples (HHMI Stanford University Center for Narcolepsy, Palo Alto CA).Mass Spectrometry Analysis - Discovery

[0238] Dried samples were resuspended with lOpL of 0.1% formic acid and 2% acetonitrile. 7pL of samples were injected on nanoElute (Bruker Daltonics, Massachusetts, USA). NanoFlow LC was coupled to Q-TOF Mass Spectrometry (MS) instrument (Impact II, Bruker Daltonics, Massachusetts, USA) through captive spray ion source (1200V, dry gas: 31 / min at 150°C) operating with nanobooster (0.2 Bar of Nitrogen boiling in acetonitrile). In the LC part, samples were desalted and pre-concentrated online on a PepMap u-precolumn (300 pm x 5 mm, C18 PepMap 100, 5 pm, 100 Angstrom, ThermoFisher, Massachusetts, USA). To perform separation, peptides were transferred to analytical column (75 pm x 500 mm; Acclaim Pepmap RSLC, C18, 2pm, 100Angstrom, ThermoFisher, Massachusetts, USA). A gradient consisting of 5-26% B for 192 min and 90% B for 10 min (A = 0.1% formic acid, 2% acetonitrile in water; B = 0.1% formic acid in acetonitrile) at 400 nL / min, 50°C, was used to elute peptides from the reverse-phase column.

[0239] For peptides identification, data dependent acquisition (DDA) was performed with a lock-mass as internal calibrator (m / z 1222, Hexakis ‘’ 1H, 1H, 4H- hexafluorobutyloxy” phosphazine, Agilent Technologies, Santa Clara, USA). Using Instant Expertise software (Bruker Daltonics, Massachusetts, USA), the most intense ions per cycle of 3 seconds were selected and then active exclusion was used (after 1 spectrum for 2 minutes unless the precursor ion exhibited intensity higher of 3 times than the previous scan).

[0240] For peptide confirmation, mass of interest (610.6020 m / z) was targeted with Parallel Reaction Monitoring (PRM) mode. Mass Spectrometry (MS) analyzer focus on the mass of interest only with a collision energy of 35eV and 20000 summation by cycle.Orexin fragment identification

[0241] All Mass Spectrometry (MS) / Mass Spectrometry (MS) spectra were searched against the Nextprot database (2018-01-17) by using the Mascot v 2.6.0 algorithm (Matrix Science, http: / / www.matrixscience.com / ) with the following settings: (1) enzyme: None, (2) variable modifications: oxidation (M), Xlink:Disulfide (C); Glu->pyro-Glu (E, Q) and deamidated (N,Q), (3) missed cleavages: 2, (4) instrument type CID: ESI-QUAD-TOF, (5) peptide tolerance: 10.0 ppm, (6) Mass Spectrometry (MS) / Mass Spectrometry (MS) tolerance: 0.05 Da, (7) peptide charge: 1+, 2+ and 3+, (8) mass: monoisotopic, (9) C13: 1, (10) peptide decoy: ON, (11) ions score cut-off: 12.Mass Spectrometry Analysis - Quantification

[0242] LC-Mass Spectrometry (MS) acquisitions were performed on Evosep One using 8cm x 150pm, 3pm (EV1064, Evosep) with 60SPD method coupled to TIMass Spectrometry (MS) TOF HT (Bruker Daltonics) through a captive spray ion source. Ion source parameters were 1500V on capillary with 3.0L / min at 180°C for the drying gas.DDA-PASEF method was used in positive ion mode. Mass Spectrometry (MS)1 range was 400-1700 m / z. TIMass Spectrometry (MS) settings were 1 / K0 0.75-1.25, Ramp and Accumulation time of 100ms. At Mass Spectrometry (MS)2 level, PRM mode was used to target orexin-A fragment with an isolation window of 2.5Da, at retention time target of 7.7 minutes with a window of 4 minutes. Ion mobility scan was between 0.78 and 0.90 V.s / cm2.Orexin-A fragment extraction from Cerebrospinal Fluid (CSF)

[0243] Solvents used were purchased to Biosolve (Dieuze, France).

[0244] The samples (300 pL) were mixed with orexin-A fragment standard (66.6 pg / mL) and shaken 13 min at 1000 rpm. 200 p L of 3% (v / v) formic acid were added and shaken 7 min at 1000 rpm and then centrifuged at 18,360g for 5 min at 4 °C.

[0245] Oasis HLB 96-well pElution Plate, 2 mg Sorbent per Well, 30 pm (186001828BA, Waters) were used as SPE cartridges to extract orexin-A fragment. At each step, liquid was added on the top of the phase, wait few second, and aspirate with a limited vacuum.

[0246] SPE plate was conditioned with 350 pL MeOH, then 350 pL H2O. Acidified sample was loaded into an HLB pElution plate. SPE was washed in three steps: (1) washing with 200 pL H2O, (2) washing with 200 pL methanol / water / NH4OH mixture (30:65:5, v / v), and (3) washing with 200 pL H2O.

[0247] Elution was performed with 2x25 pL of acetonitrile / water / formic acid (80:20:0.1 , v / v / v). The supernatant was partially evaporated at room temperature.

[0248] For the last step, sample was diluted to 100 pL of 0.1% formic acid and loaded on Evotip Pure (Evosep, Denmark).Software

[0249] Skyline software (University of Washington), version 23.1, was used for data visualization and peak integration.Statistical analysis

[0250] For comparison of orexin concentrations between clinical groups, the concentrations were represented as mean ± standard deviation, and comparisons were made using a t-test. A two-sided a-level of 0.05 was used for significance testing. Correlations between orexin levels obtained using different methods were analyzed using Pearson's correlation method and Bland- Altman analysis.Results

[0251] Cerebrospinal Fluid (CSF) fractionation was performed according to the known method of Sakai et al. (doi: 10.1038 / s41598-018-36942-8). Fractions of 0.8min were collected, dried to remove solvent, reconstituted with Radioimmunoassay (RIA) kit buffer and analyzed using the orexin Radioimmunoassay (RIA) kit.

[0252] For cerebrospinal fluid pool fractionation, a strong radioimmunoassay (RIA) signal, indicating a high concentration of orexin-A, was detected in fractions 23 / 24 and a weaker signal, indicating a low concentration of orexin-A, was detected in fractions 29 and 33 (Figure 1).

[0253] Then, the same CSF pool was spiked with standard chemically synthesized orexin-A. The signal detected in fractions 23 / 24 was still detected. However, the signal detected at fraction 33 strongly increased (Figure 2). These data suggest that fraction 33 corresponds to the chemically synthesized orexin-A of known sequence and fraction 24 to a shorter form of this protein naturally present in the CSF.

[0254] Fractions 23 and 24 were analyzed by LC-Mass Spectrometry (MS) to identify the most abundant form of orexin-A present in CSF samples and to characterize the truncated form of endogenous orexin-A. NanoElute LC was coupled to Q-TOF Mass Spectrometry (MS) instrument (Impact II, Bruker Daltonics, Massachusetts, USA) to perform data dependent acquisition.

[0255] An ion at 610.6026m / z was fragmented at 47.6 minutes and could be correlated to a truncated form of orexin-A using Mascot software. The sequence pyroEPLPDCCRQKTCSCRL (SEQ ID NO: 4) was assigned from the numerous y and bions assigned in the MS / MS spectrum. The fragment of orexin-A was identified with Mascot search (Figure 3). This molecule was detected with a tri charged precursor ion. Ions of monoisotopic mass are in perfect agreement with the Skyline software theoretical mass predictions (Figure 4).

[0256] To fully validate the orexin-A fragment, the deconvolute sequence was synthetized and analyzed with the same PRM method. Using this synthetized standard, a targeted assay method, PRM Parallel Reaction Monitoring, was developed. The [y 15]2+; [yl4]2+; [yl4]3+; [yl3]2+; [yl3]3+; [yl2]2+; [yl2]3+; [yl l]2+; [yl l]3+ fragment ions were followed for simultaneous identification and quantification. Once again, the ratio of transitions between the species discovered and the standard synthesized confirmed this molecule (Figures 5 and 6).

[0257] These results indicate that the radioimmunoassay (RIA) quantification assay measures both forms of orexin-A (fragment and endogenous). Figure 1 shows that the amount of the cleaved form (fraction 23 / 24) represents the majority of orexin-A measured by RIA, as compared to fraction 33.Example 2: Validation of new method for measuring orexin-A fragments present in the Cerebrospinal fluid (CSF).Materials and Methods

[0258] See Materials and methods of Example 1.Results

[0259] To move to orexin-A fragment quantification in Cerebrospinal Fluid (CSF) of patients, sample preparation and EC-Mass Spectrometry (MS) system were changed. Indeed, the previous method, which involved starting from 1 mF of cerebrospinal fluid (CSF), performing an offline separation, collecting different fractions, drying these fractions and resuspending them before analysis, was a lengthy process with a risk of low reproducibility performances.

[0260] A direct extraction of orexin-A fragment by solid phase extraction (SPE) was developed. In addition, the nanoLC-Mass Spectrometry (MS) acquisition of 120 min was transferred to a microLC-Mass Spectrometry (MS) system with higher flow rate, allowing higher reproducibility and shorter analysis time (20 min).

[0261] All these compromises could be a problem for the sensitivity of the orexin-A fragment, but in this configuration the detectability was still performant.

[0262] In addition, the new microLC-mass spectrometry (MS) system has an additional dimension of separation with ion mobility. The orexin-A standard has an ion mobility of 0.83 V.s / cm2 and the endogenous form of the new orexin-A species also has the same ion mobility value.

[0263] The initial CSF volume was then assessed by testing 500 / 250 / 100 / 50 pL of the CSF pool. The endogenous form was still detectable at 50 pF.

[0264] The PRM method for the quantification of cleaved orexin-A was applied to a cohort of patients. The same cohort was tested using the Phoenix peptide orexin-A kit.

[0265] 39 samples from patients with different diagnoses (as indicated in the Materials and methods above) and CSF volumes remaining after radioimmunoassay (RIA) orexin-A quantification were also analyzed by mass spectrometry (MS) dedicated to orexin-A fragment detection.

[0266] A very good linear correlation (0.785) was observed between radioimmunoassay (RIA) and mass spectrometry (MS) detection of the orexin-A fragment (Figure 7).

[0267] As indicated above, the RIA method detects both forms of orexin-A. However, as shown on Figure 1, orexin-A fragment is the predominant form detected by RIA method. The mass spectrometric quantification of the orexin-A fragment was highly correlated with the RIA assay, confirming the hypothesis that the orexin-A fragment is the predominant form of orexin-A present in CSF samples.

[0268] Even though a multiple reaction monitoring (MRM) assay was developed, as well as a new high-resolution mass spectrometry (HRMS) method, the detection of orexin-Aby mass spectrometry is currently too inconsistent to be analytically validated. Although the two species have different ionization responses, it can be seen in the present results that the proportion of orexin-A is very low compared to the new orexin-A fragment.

[0269] In this new mass spectrometry (MS) assay, orexin-A was also monitored. There were many missing values because the detection was very low (Figure 8). Detection in samples started when the radioimmunoassay (RIA) value was closer to 200 pg / mL.

[0270] Therefore, the detection and quantification of this newly identified orexin-A fragment represents a promising alternative to the orexin-A RIA assay.

[0271] The cohort of interest was analyzed with 52 drug-free patients with typical type 1 narcolepsy, 6 type 2 narcolepsy, 13 idiopathic hypersomnia and 44 patients with various neurological disorders but no type 1 narcolepsy. The correlation between radioimmunoassay (RIA) reference method and orexin-A fragment was plotted for all patients (Figure 9).

[0272] Cohort analysis showed a good correlation between radioimmunoassay (RIA) and orexin-A fragment quantification by mass spectrometry (Figure 7). Looking in details at the extreme values (lower values or higher values), the radioimmunoassay (RIA) assay showed a lot of missing values for the type 1 narcolepsy group (low range of orexin-A), and a limited concentration range in the highest concentrations compared to orexin-A fragment intensities (Figures 10A-D).

[0273] Quantification of orexin-A by radioimmunoassay is very convenient for clinicians, with very clear cut-off values. An absence of orexin-A is considered below 50 pg / mL, a low orexin-A concentration is considered below 110 pg / mL, while a normal orexin-A concentration is considered above 200 pg / mL.

[0274] The cut-off values obtained by mass spectrometry show that a ratio of 0.14 corresponds to an orexin-A concentration of 110 pg / mL and a ratio of 0.23 corresponds to an orexin-A concentration of 200 pg / mL.Example 3: Validation of the specificity and sensibility of the new method for measuring orexin-A fragments present in the Cerebrospinal fluid (CSF).Materials and Methods

[0275] See Materials and methods of Example 1. Results

[0276] The result demonstrates that using MS, it is not possible to detect orexin-A fragments having the amino acid sequence of SEQ ID NO: 6 or amino acid sequence of SEQ ID NO: 7, wherein glutamine is the first amino acid in N-Terminal in comparison with orexin-A fragments having a amino acid sequence of SEQ ID NO: 4 with pyroglutamic acid in N-Terminal, on CSF of 5 healthy patients (Figure 11).

[0277] The result demonstrates that XPLPDCCRQKTCSCRL fragment wherein X is a pyroglutamic acid, allows to obtain better results for detecting and consequently diagnosing and / or for prognosing and / or for monitoring the evolution of a disease or disorder associated with altered orexin A levels in a subject, in comparison with comparative fragment wherein X is a glutamine.

Claims

CLAIMS1. In vitro method for diagnosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), if said subject is affected by said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

2. In vitro method for prognosing a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), if said subject is at risk of developing said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

53. In vitro method for monitoring the effect of a treatment on a subject diagnosed with a disease or disorder associated with altered orexin-A levels wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, andc) assessing, from the comparison of step b), if said subject is responsive to the treatment for said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

54. In vitro method for monitoring the evolution of a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) assessing, from the comparison of step b), the evolution of said disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

55. In vitro method for determining the appropriate treatment to be administered to a subject diagnosed with a disease or disorder associated with altered orexin-A levels in a subject, wherein said method comprises the steps of: a) measuring the level or concentration of orexin-A fragment in a sample previously obtained from the subject, b) comparing the level or concentration of orexin-A fragment in said sample with a reference level or concentration of orexin-A fragment, and c) determining, from the comparison of step b), the appropriate treatment to be administered to the subject, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

56. The method according to any one of claims 1 to 5, wherein the sample previously obtained from the subject is a bodily fluid.

7. The method according to claim 6, wherein the bodily fluid is selected from cerebrospinal fluid, blood, plasma, serum, saliva, tears, lymph, ascetic fluid, cysticfluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat and alveolar macrophages, preferably the bodily fluid is cerebrospinal fluid.

8. The method according to any one of claims 1 to 7, wherein the level or concentration of orexin-A fragment is measured by immunohistochemistry, immunoassay, enzyme-linked immunosorbent assay (ELISA), enzymatic methods, western-blot, mass spectrometry, liquid chromatography, or flow cytometry, preferably by liquid chromatography coupled to mass spectrometry (LC-MS).

9. The method according to any one of claims 1 to 8, wherein the reference level or concentration of orexin-A fragment is derived from the measurement of the level or concentration of orexin-A fragment in a sample from a reference subject or in samples from a population of reference subjects.

10. The method according to claim 9, wherein the level or concentration of orexin-A fragment in the sample from the reference subject or in samples from the population of reference subjects and the level or concentration of orexin-A fragment in the sample previously obtained from the subject are measured by the same method, preferably by liquid chromatography coupled to mass spectrometry (LC-MS).

11. The method according to claim 9 or claim 10, wherein the sample previously obtained from the subject and the sample from the reference subject or the samples from the population of reference subjects are samples from the same bodily fluid, preferably cerebrospinal fluid.

12. Use of orexin-A fragment as a biomarker for the in vitro diagnosis of a disease or disorder associated with altered orexin-A levels, wherein the orexin-A fragment consists in the amino acid sequence as set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

13. The method according to any one of claims 1 to 10, or the use according to claim 11, wherein the disease or disorder associated with altered orexin-A levels is a sleepdisorder with orexin-A deficiency, or a neuropsychiatric and metabolic disease with orexin-A deficiency.

14. The method or the use according to claim 12, wherein the sleep disorder with orexin-A deficiency is selected from type I narcolepsy disease, insomnia, hypersomnia, parasomnia, restless legs syndrome, and sleep apnea.

15. The method or the use according to claim 12, wherein the neuropsychiatric and metabolic disease with orexin-A deficiency is selected from appetite disorders, depression, anxiety, mood disorders, addictions, dementia, Alzheimer’s disease and related disorders, multiple sclerosis, epilepsy, Parkinson’s disease and related disorders, schizophrenia, type 2 diabetes, migraine, autism, and post-traumatic stress disorder (PTSD).

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